Available Projects - University of Birmingham

Transcription

College of Medical and Dental Sciences
Intercalated BMedSc Clinical Sciences
Research projects 2015/16
Please state your preferences for up to six research projects from this booklet on
your application form. You are expected to consult the supervisor(s) before
submitting your application form.
Application forms are available online:
http://www.birmingham.ac.uk/students/courses/undergraduate/med/Cli
nicalScienceBMedSc-IntercalatedDegree.aspx and should be returned to the
course Administrator: [email protected] or via post to:
Miss Yvonne Palmer
Room 220 Teaching Office
School of Cancer Sciences
University of Birmingham
Vincent Drive
Edgbaston
Birmingham
B15 2TT
Ethical considerations
We understand that students' ethical standards and convictions vary and may
influence the type of work you feel you can conduct throughout the course. Students
should be assured that all projects have ethics approval if required. However it is
possible that personal convictions may be brought into conflict with the nature of the
work you are conducting. For example some projects involve the use of cell lines that
have been derived from human embryonic tissue. Other products involve use of
animal matter and animal testing. Please talk directly to your supervisor from the
outset about the exact nature of the materials, methods and procedures in the
projects you are interested in.
Home Office ‘Working with Animals’ Licence
If your project requires you to attend this course, please ask your supervisor to book
a place for you on the next course. Payment for a place on the course can be made
from an existing account (your supervisor can confirm an account code) and
reconciled once confirmation of the lab consumables account is received.
Home Office Course Contact: [email protected]
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College of Medical and Dental Sciences
Intercalated BMedSc in Clinical Sciences
Research projects for 2015/2016
Lead Supervisor:
Dr Zubair Ahmed
Contact Email:
Telephone:
[email protected]
0121 4148858
Co Supervisor:
Prof Ann Logan
Project Title:
PI3K/Akt pathway-initiation of regeneration of the dorsal
column projection of dorsal root ganglion neurons.
Department:
Neurobiology, CEM
Will the project require a Home Office working with animals licence?
Yes or
No
Is the Project Cancer related? No
Project Outline
Recent work has demonstrated a key role for mTOR signalling in axonal regeneration
and the potential therapeutic utility in neurotrauma patients of next generation siRNA
drugs that modulate the signalling pathway. Activation of PI3K/PDK/Akt pathway
regulates axon growth cone dynamics through GSK3β, axogenic protein synthesis
through mTORC1 and neuronal survival by activating anti-apoptotic pathways.
Deletion of the phosphatase, PTEN, activates the PI3K pathway and leads to survival
of axotomised neurons and axogenesis in the injured visual system1-3, corticospinal
tracts (CST) in the cord4,5 and peripheral nerves5. This suggests that modulation of
the mTOR pathway might be beneficial for promoting CNS axon regeneration after
trauma. To date, there are no data on PTEN, Akt, mTOR and GSK3β expression in
dorsal column (DC) axotomised DRGN, and the effects of siRNA knockdown of
signalling molecules in the above pathways on DC axon regeneration have not been
studied. In this project, we hypothesise that activation of the mTOR pathway is
critical
for DC axon regeneration. Therefore, we will modulate key components of the mTOR
pathway both in vitro and in vivo using therapeutic siRNAs and monitor their effects
on DC axon regeneration. Transganglionic tracers will detect regenerating axons in
adult rats after DC injury as well as various markers to identify the injury response in
longitudinal sections of the spinal cord. Confirmation of the pathways involved will be
evaluated by microarray analysis of mRNA extracted from affected spinal ganglion
neurons. These studies will yield important data on the use of therapeutic siRNAs to
modulate the mTOR pathway and promote DC axon regeneration.
References
1. Park KK, Liu K, Hu Y, Smith PD, Wang C, Cai B, Xu B, Connolly L, Kramvis I, Sahin
M, He Z. (2008) Promoting axon regeneration in the adult CNS by modulation of the
PTEN/mTOR pathway. Science 322:963-966.
2. Kurimoto T, Yin Y, Omura K, Gilbert H, Kim D, Cen L-P, Moko L, Kügler S,
Benowitz LI. (2010) Long-distance axon regeneration in the mature optic nerve:
Contributions of oncomodulin, cAMP, and pten gene deletion. J Neurosci 30:1565415663.
3. Leibinger M, Müller A, Gobrecht P, Diekmann H, Andreadaki A, Fischer D. (2013)
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Interleukin-6 contributes to CNS axon regeneration upon inflammatory stimulation.
Cell Death Dis. 4:e609;doi:1038/cddis.2013.126.
4. Liu K, Lu Y, Lee JK, Samara R, Willenberg R, Sears-Kraxberger I, Tedeschi A, Park
KK, Jin D, Cai B, Xu B, Connolly L, Steward O, Zheng B, He Z. (2010) PTEN deletion
enhances the regeneration ability of adult corticospinal neurons. Nature Neurosci
13:1075-1081.
5. Zukor K, Belin S, Wang C, Keelan N, Wang X, He Z. (2013) Short hairpin RNA
against PTEN enhances regenerative growth of corticospinal tract axons after spinal
cord lesions. J. Neurosci. 33:15350-14361.
How are you planning to ensure adequate supervision?
Students will be supervised by Dr Ahmed on a day-to-day basis and will receive input
from Prof Logan through weekly progress meetings. Students will also be paired up
with other postdocs and senior PhD students who perform all of the routine
techniques required for this project.
The student role.
The student will cut the sections of the spinal cord using a cryostat and then proceed
to perform immunohistochemistry, microarray analysis and western blot. The student
will also perform microscopy and analyse collected images using Image analysis
software. Students will interact with other members of the laboratory who are
working on related molecules involved in DC axon regeneration and partake in
weekly lab meetings, where they will have a chance to present their work to
colleagues from the Neurotrauma Research Group.
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Lead Supervisor:
Dr. Francesca Barone
Contact Email:
Telephone:
[email protected]
07726899695/ 01213713248
Co Supervisor:
Dr. Anne Fletcher and Dr. Saba Nayar
Project Title:
Lymphoid stromal cells: friends or foes?
Department:
Rheumatology Research Group
Yes
Project Outline
In secondary lymphoid organs (SLOs) such as lymph nodes and spleen,
lymphoid stromal cells provide lymphocyte survival signals (such as IL-7,
BAFF) and chemokine (CXCL13, CCL19, CCL21) cues. These allow migration
of T and B cells and maintain SLO architecture and function(1, 2). Several
distinct stromal cell populations have been identified in SLOs, including
fibroblastic reticular cells (FRC) that sustain T cell survival and organization,
follicular dendritic cells (FDC) that regulate antigen presentation and the
germinal center (GC) reaction and marginal reticular cells (MRC) whose role
remains unclear. These cell populations are characterized by different
combinations of cell surface markers (gp38/Podoplanin, MAdCAM-1, VCAM-1,
ICAM-1 and RANK-L) and differential expression of lymphoid chemokines and
survival factors (3).
Stromal cells can influence the size and shape of the immune repertoire, not
only by modulating the availability of the survival factors, but also inducing
deletion or expansion of specific cell clones. This process, ultimately aimed to
induce peripheral tolerance, is achieved by stromal cells’ presentation of a
range of peripheral tissue restricted antigens (4). Recent reports demonstrate
that LN stromal cells also induce CD4+ T cell tolerance (5, 6). These data
suggest that induction of tolerance is not achieved by a single cell type but by
different stromal cells toward different peripheral tissue antigens. Indeed
lymph node FRCs, upon inflammation, can produce nitric oxide (NO) a natural
immunosuppressant, which influences T cell expansion and priming (7-9).
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TLOs are accumulations of lymphoid cells that share similar cellular
compartments, spatial organization, vasculature, chemokines and function to
secondary lymphoid organs (10-14). Chronic antigenic stimulation is believed
to be required for TLO maintenance, while tolerance seems not to occur. This
suggests that locally activated TLO lymphoid like stromal cells might have lost
the capacity to present antigens and express NO to induce tolerance and
immunosuppression unlike their lymph node counterparts.
Project objective:
To explore immunosuppressive functions of lymphoid stromal cells in TLOs
(tertiary lymphoid organs) compared to reactive lymph node stromal cells.
Plan of Investigation:
In order to compare the immunosuppressive potency of TLO-associated
lymphoid stromal cells with lymphoid stromal cells from reactive secondary
lymphoid organs particularly lymph nodes (LNs) we will isolate and study
lymphoid like stromal cells from LN and TLO that form in the salivary glands
of wild type mice infected with a replication deficient adenovirus.
Adenovirus induced formation of TLOs in C57/BL6 SG (cannulation model).
This model involves delivery of replication-defective adenovirus 5 (AdV5) via
retrograde cannulation of submandibular gland excretory ducts in C57BL/6
mice (15). Within 15 days post cannulation TLO form that present T/B cell
segregation and expression of lymphoid cytokines and chemokines.
Mice will be immunized with NP-CGG Alum+Bordetella pertussis. This will
involve injecting the paw-pad with a commonly used antigen [(4-hydroxy-3nitrophenyl)-acetyl
conjugated
to
Chicken
Gamma
Globulin]
NP-CGG
Alum+Bordetella pertussis to activate local draining lymph nodes (LNs).

TLO-associated lymphoid stromal cells will be isolated using a protocol
developed in-house to isolate stromal cells from various organs such as
salivary glands and lymph nodes. Phenotypical differences between TLO and
SLO stroma will be studied by FACS analysis prior to sorting.

On isolated, sorted stromal cells we will perform gene expression profile
analysis for an array of immunosuppressive genes such as NOS2 (also known
as iNOS i.e. inducible nitric oxide synthase), Arginase-1, IDO (indoleamine
2,3-dioxygenase), PD-L1, TGFβ, cyclooxygenases (COX-1 and COX2) and
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lipooxygenases (LOX-5 and LOX-15).

Once we have established the ex vivo immunosuppressive phenotype,
stromal cells will be isolated from either (TLOs or LNs) and co-cultured with
activated lymphocytes to assess the immunosuppressive function of stromal
cells. As an indicator of immunosuppressive ability of stromal cells, T cell
proliferation and the levels of inflammatory cytokine production by T cells will
be determined. At the same time, culture supernatants will be used to
evaluate the production of immunosuppressive molecules (such as Nitric
oxide (NO), TGFβ, IL-10 and prostaglandin E2) from stromal cells.
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References
Mueller SN, Germain RN. 2010. Stromal cell contributions to the homeostasis
and functionality of the immune system. Nature Reviews Immunology
Roozendaal R, Mebius RE. 2011. Stromal Cell–Immune Cell Interactions.
Annual Review of Immunology 29: 23-43
Kain MJ, Owens BM. 2013. Stromal cell regulation of homeostatic and
inflammatory lymphoid organogenesis. Immunology 140: 12-21
Fletcher AL, Malhotra D, Turley SJ. 2011. Lymph node stroma broaden the
peripheral tolerance paradigm. Trends in Immunology 32: 12-8
Dubrot J, Duraes FV, Potin L, Capotosti F, Brighouse D, et al. 2014. Lymph
node stromal cells acquire peptide-MHCII complexes from dendritic cells and
induce antigen-specific CD4(+) T cell tolerance. J Exp Med 211: 1153-66
Abe J, Shichino S, Ueha S, Hashimoto S, Tomura M, et al. 2014. Lymph node
stromal cells negatively regulate antigen-specific CD4+ T cell responses. J
Immunol 193: 1636-44
Lukacs-Kornek V, Malhotra D, Fletcher AL, Acton SE, Elpek KG, et al. 2011.
Regulated release of nitric oxide by nonhematopoietic stroma controls
expansion of the activated T cell pool in lymph nodes. Nat Immunol 12:
1096-104
Siegert S, Huang HY, Yang CY, Scarpellino L, Carrie L, et al. 2011. Fibroblastic
reticular cells from lymph nodes attenuate T cell expansion by producing
nitric oxide. PLoS ONE 6: e27618
Khan O, Headley M, Gerard A, Wei W, Liu L, et al. 2011. Regulation of T cell
priming by lymphoid stroma. PLoS ONE 6: e26138
Aloisi F, Pujol-Borrell R. 2006. Lymphoid neogenesis in chronic inflammatory
diseases. Nat Rev Immunol 6: 205-17
Pitzalis C, Jones GW, Bombardieri M, Jones SA. 2014. Ectopic lymphoid-like
structures in infection, cancer and autoimmunity. Nature reviews.
Immunology 14: 447-62
Carragher DM, Rangel-Moreno J, Randall TD. 2008. Ectopic lymphoid tissues
and local immunity. Seminars in Immunology 20: 26-42
Neyt K, Perros F, GeurtsvanKessel CH, Hammad H, Lambrecht BN. 2012.
Tertiary lymphoid organs in infection and autoimmunity. Trends Immunol 33:
297-305
Link A, Hardie DL, Favre S, Britschgi MR, Adams DH, et al. 2011. Association
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15.
of T-Zone Reticular Networks and Conduits with Ectopic Lymphoid Tissues in
Mice and Humans. The American Journal of Pathology 178: 1662-75
Bombardieri M, Barone F, Lucchesi D, Nayar S, van den Berg WB, et al. 2012.
Inducible tertiary lymphoid structures, autoimmunity, and exocrine
dysfunction in a novel model of salivary gland inflammation in C57BL/6 mice.
J Immunol 189: 3767-76
Adequate daily supervision in the lab will be provided by Dr. Saba Nayar, a Post. Doc
working with Dr. Barone that has a large experience in the TLO model and in the
analysis of stromal cells subsets described in the application. Dr. Barone and Dr.
Fletcher will set up regular meetings to discuss progresses and development of the
project.
The student role.
The student will be involved in tissue dissection, digestion and processing.
The student will be learning FACS analysis, use of confocal microscopy, cell sorting
and PCR analysis.
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Lead Supervisor:
Dr. Francesca Barone
Contact Email:
Telephone:
[email protected]
07726899695/ 01213713248
Co Supervisor:
Dagmar Scheel-Toellner
Project Title:
RANK-ligand positive B cells: a new pathogenic
population in autoimmunity
Department:
Yes
Project Outline
Background:
RANK/RANK-L interaction is critical for lymph node establishment in
embryonic life. More recently this pathway has been involved in the formation
of lymphoid tissue at ectopic sites, a phenomenon that often occur in chronic
inflammatory conditions.
Sjogren’s’ syndrome (SS) is a chronic inflammatory autoimmune disease
which affects 0.1-0.4% of the UK adult population. It involves loss of function
of the exocrine glands and systemic manifestations due to B cell hyperactivation. In 20-40% of patients the lymphocytes infiltrating the salivary
glands accumulate to form structures resembling lymph nodes. These tertiary
lymphoid organs (or TLOs) contain organized T and B cell aggregates with
reticular networks of Podoplanin/gp38+ stromal cells and formation of
follicular dendritic cell network. TLOs can in some cases develop into
malignant Mucosal Associated Lymphoid Tissue (MALT) lymphoma.
We have recently described a proinflammatory B cell population in
rheumatoid arthritis that expresses RANK-L and is involved in disease
pathogenesis. These cells express a unique surface protein that, in humans, is
also expressed by MALT lymphoma B cells. We have recently detected these
cells in glands of SS patients and we have data showing a similar
proinflammatory phenotype to that observed in rheumatoid arthritis. In mice
we can study this proinflammatory B cell population taking advantage of their
expression of RANK-L.
Project objective:
In this project we use an inducible model of SS in salivary glands of wild type
mice by delivery of a replication deficient adenovirus. We will investigate the
presence and function of RANK-L+ B cells in relationship with the
development of the TLO.
Plan of Investigation:
Adenovirus induced formation of TLOs in C57/BL6 SG (cannulation model).
This model involves delivery of replication-defective adenovirus 5 (AdV5) via
retrograde cannulation of submandibular gland excretory ducts in C57BL/6
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mice. Within 15 days post cannulation TLO form that present T/B cell
segregation and expression of lymphoid cytokines and chemokines. TLO
analysis will be performed during the whole time course post cannulation (day
2,5,8,15 and 23 post cannulation) assessing by immunofluorescence (IF)
follicle size, degree of T/B cell segregation and area, FDC formation and
expression and distribution of lymphoid chemokines.
Analysis of RANK-L+ B cells
Using flow cytometry on digested tissue we will analyse the different cellular
components of the aggregates both in terms of number and phenotype of
DCs, T and B cells. On isolated lymphocytic populations we will assess the
expression RANK-L by flow cytometry. B cells will be also sorted and PCR
analysis will be performed to confirm the protein data and to investigate, in
this population the expression of proinflammatory cytokines. Using confocal
microscopy we will address the presence and distribution of RANK-L+ B cells
within the TLO and their interaction with local immune cells.
References
Yeo L, Lom H, Juarez M, Snow M, Buckley CD, Filer A, Raza K, Scheel-Toellner D.
Expression of FcRL4 defines a pro-inflammatory, RANKL-producing B cell subset in
rheumatoid arthritis. Ann Rheum Dis. 2014 Jan 15
Bombardieri M, Barone F, Lucchesi D, Nayar S, van den Berg WB, et al. 2012.
Inducible tertiary lymphoid structures, autoimmunity, and exocrine dysfunction in a
novel model of salivary gland inflammation in C57BL/6 mice. J Immunol 189: 376776
Mueller SN, Germain RN. 2010. Stromal cell contributions to the homeostasis and
functionality of the immune system. Nature Reviews Immunology
Pitzalis C, Jones GW, Bombardieri M, Jones SA. 2014. Ectopic lymphoid-like
structures in infection, cancer and autoimmunity. Nature reviews. Immunology 14:
447-62
Neyt K, Perros F, GeurtsvanKessel CH, Hammad H, Lambrecht BN. 2012. Tertiary
lymphoid organs in infection and autoimmunity. Trends Immunol 33: 297-305
Link A, Hardie DL, Favre S, Britschgi MR, Adams DH, et al. 2011. Association of TZone Reticular Networks and Conduits with Ectopic Lymphoid Tissues in Mice and
Humans. The American Journal of Pathology 178: 1662-75
Adequate daily supervision in the lab will be provided by Ms. Joana Campos and Dr.
Saba Nayar, a third year PhD student and a post doctoral fellow working with Dr.
Barone. Both have a large experience in the induction and analysis of the TLO model
described in the application. Dr. Barone and Dr. Sheel-Toellner will have regular
meetings to discuss progresses and development of the project.
The student role.
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The student will be involved in induction of the model, tissue dissection, digestion
and processing. The student will be learning FACS analysis, use of confocal
microscopy, cell sorting and PCR analysis.
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Lead Supervisor:
Andrew Beggs (Cancer Sciences)
Contact Email:
Telephone:
[email protected]
0121 414 7458
Co Supervisor:
Aditi Kadhere (School of Biosciences)
Project Title:
Investigation of the biological role of the long non-coding
RNA OR3A4 in the development of oesophageal cancer
Department:
Schools of Cancer Sciences & Biosciences
Yes or
No
Is the Project Cancer related? Yes
Project Outline
Oesophageal adenocarcinoma (OADC) is a cancer rapidly increasing in incidence,
with the UK having the highest rate in Western Europe. Typically OADC is driven
by TP53 mutation however the events that initiate OADC are unclear. It appears
to start in a pre-malignant lesion, known as Barretts Oesophagus (BO) however
the molecular drivers that cause this to progress to OADC are unclear. We have
recently carried out an epigenome wide association study that has highlighted the
role of expression of the non-coding RNA OR3A4 in the progression of BO to
OADC. What is unclear, however, is what biological role OR3A4 plays in
carcinogenesis, given its primary role as a non-coding olfactory receptor
pseudogene. We hypothese that it has a novel carcinogenic function and wish to
explore its biological relationships. We aim to knock down OR3A4 in a cell line
model and ascertain the pathways affected by this using gene expression studies.
We will employ a new technique, individual-nucleotide resolution CLIP, to
ascertain the mechanism behind its action.
References
1)Kretz M et al. Control of somatic tissue differentiation by the long non-coding RNA
TINCR. Nature. 493(7431): 231–235.
2) Broughton JP, Pasquinelli AE. Identifying Argonaute binding sites in C. elegans
using iCLIP. Methods 63 (2013) 119–125.
Regular supervisory meetings with student with both supervisors
Technical support staff in both labs will carry out day to day supervision.
The student role.
siRNA knockdown of OR3A4 & gene expression via RT-QPCR to produce OR3A4 null
cell line; Knockdown and wildtype line will then be subjected to RNA/protein
interaction mapping via iCLIP technique and products RNA sequenced.
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Lead Supervisor:
Glenn Matthews (Cancer Sciences)
Contact Email:
Telephone:
[email protected]
0121 414 7458
Co Supervisor:
Andrew Beggs (Cancer Sciences)
Project Title:
Validation of novel biomarkers for progression of Barrett’s
oesophagus to adenocarcinoma
Department:
Schools of Cancer Sciences
Yes or
No
Project Outline
Oesophageal adenocarcinoma (OADC) is a cancer rapidly increasing in incidence,
with the UK having the highest rate in Western Europe. Typically OADC is driven by
TP53 mutation however the events that initiate OADC are unclear. It appears to start
in a pre-malignant lesion, known as Barretts Oesophagus (BO) however the
molecular drivers that cause this to progress to OADC are unclear. We have recently
carried out an epigenome wide association study that has highlighted multiple
differentially methylated loci of significance in the transition from BO to OADC.
We now wish to test a subset of these loci in a new sample set of patients with BO
to see whether they continue to be significantly differentially methylated. This work
will allow us to decide which markers should be taken forward into a prospective
clinical trial.
References
1. Dilworth MP, Beggs AD et al. A novel methylation biomarker for oesophageal
adenocarcinoma. European Journal of Surgical Oncology 40 (11), S24
2. Leong KJ et al. Biomarker‐based treatment selection in early‐stage rectal
cancer to promote organ preservation. British Journal of Surgery 101 (10),
1299-1309.
Regular supervisory meetings with student with both supervisors
Technical support staff in both labs will carry out day to day supervision.
The student role.
DNA extraction of validation samples, DNA quantification, pyrosequencing assay
design and bisulphite pyrosequencing. Statistical analysis of results.
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Lead Supervisor:
Dr. Fedor Berditchevski
Contact Email:
Telephone:
[email protected]
0121 -414 2801
Co Supervisor:
Dr.Abeer Shaaban
Project Title:
Histological markers of response/resistance to
neoadjuvant therapy in breast cancer
Department:
Cancer Sciences
Will the project require a Home Office working with animals licence? No
Project Outline
Breast cancer is the commonest cancer in females (1:8 females will develop
breast cancer during their lifetime). One of the modalities of treatment is
neoadjuvant chemotherapy (NACT). Some patients show complete response
with no residual tumour tissue following NACT while others show partial or no
response. The practice of using neoadjuvant chemotherapy has evolved over
the last 10 years and now is an opportune time to review what we have learnt
and re-define criteria for advising patients about this approach to breast
cancer treatment. Previous reported trials do not take into account the impact
of Herceptin (HC) in appropriate patients. There is limited data in the
literature on factors that determine response/resistance to NACT.
Tetraspanins comprise a large family of evolutionarily conserved, fourtransmembrane domain proteins. The tetraspanin proteins are known to
facilitate the assembly of specialized molecular aggregates on plasma and
intracellular membranes known as tetraspanin-enriched microdomains1. These
microdomains also include adhesion receptors (integrins) and receptor
tyrosine kinases2. Tetraspanins are thought to influence tumour migration and
also tumour proliferation 3
We have previously analysed the expression of those markers in tumours of
various tissues including the breast4, and have in vitro evidence of a link
response to HC treatment. However, the relation between tetraspanins and
response to neoadjuvant HC has not been studied before.
The project aims to identify whether expression of tetraspanin proteins
predicts response/resistance to neoadjuvant HC treatment by relating tumour
features and molecular marker expression in pre-treatment core biopsy to
tumour response. Where there is residual invasive carcinoma, these will also
be analysed for expression of tetraspanins and other markers of interest.
Expression profiling will be performed using Laser Capture Microdissection
(LCM) and qPCR. Slides of patients who underwent neoadjuvant Herceptin
treatment will be selected, paraffin blocks retrieved and sections cut. The
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tissues will be used for extracting RNA from tumour tissues and subsequent
preparation of the templates for quantitative PCR (qPCR). Where appropriate
immunohistochemical staining will be performed using standard protocols.
Analyses will be performed on core biopsy pre-treatment samples and posttreatment residual material where available.
References
1.
2.
3.
4.
5.
Hemler, 2008 Nat Rev Drug Discov, 7:747-758
Novitskaya et al., 2013 Oncogene, doi:10.1038/onc.2013.231
Romanska and Berditchevski, 2011 J Pathol, 223:4-14
Shaaban et al., 2012 Breast Cancer Res Treat, 133:949-58
Fend and Raffeld, 2000 J Clin Pathol
This project involves two supervisors: Dr Berditchevski is based in the School of
Cancer Sciences and he will be able to supervise the student in pPCR protocols , and,
if required, in biochemical and cell biology experiments. The student will be
supervised on a daily basis by experienced post-docs in Berditchevski’s laboratory.
Dr.Shaaban is based in the Pathology Department, and she will supervise the
student’s work involving immunohistochemical and histology techniques. Both
supervisors will liaise regularly to ensure that the student is gaining adequate
training and guidance to complete the project.
The student role.
The student will be mainly based in the School of Cancer Sciences for qPCR work
where s/he will be working on breast cancer sections identified by Dr.Abeer
Shaaban. All the protocols and techniques are established in the department- and
the student will initially be taught these techniques followed by focussing on the
specific tetraspanin molecules which have been investigated in the past in analogous
studies (e.g. CD151, Tspan6). The experiments involving IHC staining will be
performed at the Department of Pathology, QEHB. The student will attend lab
meetings throughout their training and will be given an opportunity for a short
presentation at the end of the project.
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Lead Supervisor:
Contact Email:
Telephone:
Roy Bicknell
[email protected]
0121 414 4085
Co Supervisor:
Zsuzsanna Nagy
Project Title:
Mechanistic study of a prostate cancer gene
Department:
Immunity and Infection
Project Outline
Prostate cancer associated transcript 19 (PCAT19) has recently been associated with
aggressive prostate cancer and increased mortality by two large (20,000 patients)
independent Genome-wide association studies (GWAS) studies. One in UK (1), the
other in the USA (2). PCAT19 is of particular interest to our group because we
identified it as being restricted to the vasculature (3), but until now no function has
been ascribed to it
We have determined that the upregulation of PCAT19 blocks the progression of the
cell cycle through G2/M phase and regulates the expression of CBX5 (chromobox
homolog 5), a mediator of gene silencing (unpublished data). However the
mechanism by which this regulation occurs is not yet known.
References
1. Amin Al Olama, A. Kote-Jarai, Z. Schumacher, FR. et al. 2013. A metaanalysis of genome-wide association studies to identify prostate cancer
susceptibility loci associated with aggressive and non-aggressive disease.
Human Molecular Genetics. 22(2):408-15.
2. Shui, IM. Lindström, S. Kibel, AS. et al. 2014. Prostate cancer (PCa) risk
variants and risk of fatal PCa in the National Cancer Institute Breast and
Prostate Cancer Cohort Consortium. European Urology. 65:1069-1075.
3. Huminiecki, L. Bicknell, R. 2000. In silico cloning of novel endothelial-specific
genes. Genome Research. 10(11):1796-806.
One-to-one meetings at least once per month, plus fortnightly group meetings.
The student role.
The aims of this project are:
1. To experimentally validate the interaction between PCAT19 and CBX5
2. Modify the expression of PCAT19 and explore it's association with the cell
cycle.
16
Lead Supervisor:
Drs Sarah Blair-Reid and Jo Morris
Contact Email:
Telephone:
[email protected]
44033
Co Supervisor:
N/A
Project Title:
Generation and characterisation of cell line bearing
cancer-predisposition gene changes. Understanding
early-stage breast cancer.
Cancer sciences
Department:
No
Project Outline
Understanding the initiation of cancer development has historically been difficult to
study in a human context, and researchers have relied on genetic manipluation of
mice to study it. Now new technology of genome editing has changed this and
specific genetic changes can be placed in ‘normal’ human cells in order to examine
cell changes.
We have developed novel normal human breast epitheial cells that have gene
changes associated with breast cancer predisposition They possess changes in p53
or 53BP1. These changes can also modulate how responsive breast tumors are to
treatment.
You will:
 Aid in the generation of cell lines carrying a variety of mutations, introduced
using genome editing technology (CRISPR)
 Establish the survival characteristics of these cells, using colony formation
assays
 Determine whether these cells have perturbed responses to DNA damaging
agents and address their ability to repair DNA using a variety of techniques
Charaterisation of these cells will give us new insights into the very early faults that
arise in human cells once a mutation occurs but before the cell has transformed into
a cancer cell.
References
Hyongbum Kim and Jin-Soo Kim, A guide to genome engineering with programmable
nucleases. Nature Reviews Genetics, 2014. 15: 321-334
Zimmermann, M. and T. de Lange, 53BP1: pro choice in DNA repair. Trends in
cell biology, 2013.
Carvajal, L.A. and J.J. Manfredi, Another fork in the road--life or death
decisions by the tumour suppressor p53. Embo Reports, 2013. 14(5): p. 41421
17
The student will be supervised by post-doc Dr Sarah Blair-Reid on a day to day basis.
Dr Blair-Reid has experience working with a wide variety of cell lines and is familiar
with genomic editing technology and the assays to be used. Dr Morris will meet with
the student weekly for a one-to-one session to discuss progress and planning of the
write-up. In addition the student will present to members of the Morris Lab.
The student role.
The student will be expected to be in the laboratory for the majority of the time on
project and to bring a professional and determined approach.
The project will involve mammalian cell culture, the use of chemotherapeutic agents
in tissue culture, dual colour immunofluorescence and microscopy, and colony
assays.
The student will be expected to do a literature search and small write-up around
Christmas, to prepare and plan their laboratory work, in consultation with Drs BlairReid and Morris, to articulate their data and results at weekly meetings, keep
accurate and up-to-date lab books and to prepare a 40 minute talk on their project
at its beginning and end.
18
Lead Supervisor:
Constanze Bonifer
Co Supervisor:
Justin Loke
Contact Email:
Telephone:
[email protected]
07947352222
Project Title:
Identification of common and distinct epigenetic
reprogramming properties of core-binding factor
fusion proteins
Department:
No
Project Outline
Mutations involving the transcription factor RUNX1, a member of the core binding
factor (CBF) family, are one of the most frequent causes of acute myeloid leukaemia
(AML) but how these molecules block differentiation is only poorly understood.
RUNX1 activity can be altered as a result of translocations. This results in targeting
ectopic activities to RUNX1 binding sites by fusing its DNA-binding (RUNT) domain to
those of other protein domains. Currently, it is not known whether different RUNX1
translocation products bind to similar targets and deregulate similar pathways.
We have recently shown that expression of the translocation RUNX1-ETO leads to
the reprogramming of the epigenetic landscape, and to alterations of transcription
factor binding at thousands of genomic sites (1, 2). Knock-down of RUNX1-ETO
largely reverses reprogramming. We are currently studying a second RUNX1
translocation, the t(3;21), which fuses the RUNT domain to the entire EVI-1 gene,
resulting in the expression of the fusion protein RUNX1-EVI-1. Using genome-wide
assays, we will determine the RUNX1-EVI-1 specific cistrome and compare it to that
of RUNX1-ETO. We can show a functional siRNA specifically depleting RUNX1-EVI-1
results in phenotypic changes indicative of differentiation due to changes in gene
expression of key transcription factors.
DNA is packaged into chromatin structures involving histones. Post translational
modification of histone subunits mediate DNA processes such as transcription. We
aim to identify common and distinct epigenetic reprogramming properties between
the two core-binding factor fusion proteins. One histone mark that is likely to have a
different distribution, between the two leukaemic subtypes, is histone 3 lysine 9
trimethylation (H3K9me3), a defining feature of transcriptionally repressive
heterochromatin structure(3). EVI-1 has been shown to mono-methylate H3K9 (4)
and can interact with SUV39H1 and G9a (5), enzymes which can produce H3K9me3.
References
1.
Ptasinska A, Assi Salam A, Martinez-Soria N, Imperato Maria R, Piper J, Cauchy P, et
al. Identification of a Dynamic Core Transcriptional Network in t(8;21) AML that Regulates
Differentiation Block and Self-Renewal. Cell Reports. 2014;8(6):1974-88.
2.
Ptasinska A, Assi SA, Mannari D, James SR, Williamson D, Dunne J, et al. Depletion of
RUNX1/ETO in t(8;21) AML cells leads to genome-wide changes in chromatin structure and
transcription factor binding. Leukemia. 2012;26(8):1829-41.
3.
Bannister AJ, Kouzarides T. Regulation of chromatin by histone modifications. Cell
Res. 2011;21(3):381-95. Epub 2011/02/16.
19
4.
Pinheiro I, Margueron R, Shukeir N, Eisold M, Fritzsch C, Richter F, et al. Prdm3 and
Prdm16 are H3K9me1 Methyltransferases Required for Mammalian Heterochromatin Integrity.
Cell. 2012;150(5):948-60.
5.
Spensberger D, Delwel R. A novel interaction between the proto-oncogene Evi1 and
histone methyltransferases, SUV39H1 and G9a. FEBS Letters. 2008;582(18):2761-7.
1) Day to day working with clinical research fellow, Justin Loke
2) Supervision meetings with Prof Constanze Bonifer
This is also a dynamic and friendly lab with a number of post-doctoral researchers,
PhD students and technicians, all of whom are very accessible.
The student role.
1) Investigate whether RUNX1-EVI-1 can interact with SUV39H1 and G9a by coimmunoprecipitation.
2) We are currently transducing an inducible RUNX1-EVI-1 shRNA into a cell line with
the t(3;21) translocation. Select clones of RUNX1-EVI-1 shRNA t(3;21) cell line.
Show that the shRNA specifically targets RUNX1-EVI-1 by qPCR and Western blot
3) Investigate by ChIP whether RUNX1-EVI-1 binding sites are associated with
H3K9me1/3 and whether these marks are reversible upon knock down of RUNX1EVI-1.
4) Compare patterns of H3K9me3 between RUNX1-EVI-1 and RUNX1-ETO cell lines.
The student would also be expected to attend the weekly journal clubs, lab meetings
and departmental talks.
20
Lead Supervisor:
Dr S. John Curnow
Contact Email:
Telephone:
[email protected]
Co Supervisor:
Dr Mike Douglas
Project Title:
Repertoire and phenotype of T and B cells in
patients with early multiple sclerosis
Department:
Immunity & Infection
No
Project Outline
Although the central nervous system (CNS) is considered to be immune privileged
there is a distinct population of CCR7+ central memory T cells present in the
cerebrospinal fluid (CSF) in healthy individuals. When inflammation occurs (e.g. in
multiple sclerosis) there is an increase in the number of T cells found in the CSF.
However, the most distinct difference is the appearance of antibody-secreting B cells
and consequent presence of antibody in the CSF. There is also excess free light chain
which provides a sensitive and specific test to support the diagnosis of MS.
Research within the group is currently addressing the repertoire and phenotype of
the T and B cells in patients with early MS. The project would align to these research
aims with the specifics depending on the progress of the ongoing project. Cells will
be analysed using flow cytometry and real-time PCR for a range of molecules
involved in the selection of the T/B cell repertoire. Samples (CSF and blood) from
patients with early disease (clinically isolated syndrome) will be compared to those
who have already experienced a relapse of disease (relapsing-remitting MS).
Additional blood samples at follow-up will also be analysed.
References
Hassan-Smith G. High sensitivity and specificity of elevated cerebrospinal fluid kappa
free light chains in suspected multiple sclerosis. 2014 J. NeuroImmunol 276:175–
179.
Vafaii, P. Detection of B-cell populations with monotypic light chain expression in
cerebrospinal fluid specimens from patients with multiple sclerosis by polychromatic
flow cytometry. Cytometry B Clin Cytom, 2014. 86(2): p. 106-10.
Mullen KM. Expression of CCR7 and CD45RA in CD4+ and CD8+ subsets in
cerebrospinal fluid of 134 patients with inflammatory and non-inflammatory
neurological diseases. 2012 J Neuroimmunol 249:86-92.
The student will be trained in the lab by Dr Curnow’s technician and receive
additional support from other members of the group. Dr Curnow holds (at least)
weekly meetings with each student and the group meets once a week to discuss
data, future plans and the scientific literature. The labs at the Centre for
Translational Inflammation Research in the QE Hospital and fully equipped and
provide an excellent environment for the research.
The student role.
21
The student will prepare CSF and peripheral blood samples from patients with MS as
well as disease and healthy controls. They will analyse expression using flow
cytometry and real-time PCR. Data will be collated with any clinical information.
22
Lead Supervisor:
Dr Nick Davies
Contact Email:
Telephone:
[email protected]
ext 46823
Co Supervisor:
Prof T Stankovic
Project Title:
Assessment of potential of ATR inhibitor as a novel
therapeutic for treatment of diffuse large B cell
lymphoma
Cancer Sciences
Department:
Yes or
No
Project Outline
Diffuse large B cell lymphoma (DLBCL) is the most common tumour of
lymphoid tissues.1 One of the characteristics of DLBCL is impaired DNA
damage responses (DDR) as a result of inactivation or loss of p53 expression
due to overexpression of transcriptional repressor BCL6.2-4 Furthermore,
DLBCL with p53 mutations show a particularly poor prognosis.5, 6
Ataxia Telangiectasia Related (ATR) is a regulator of the cell cycle and a key
mediator of cellular responses to replication stress and DNA damage.7
Recently, we have demonstrated that chronic lymphocytic leukaemia tumours
with p53 inactivation are sensitive to inhibition of ATR due to accumulation of
intolerable levels of DNA double strand breaks (DSBs).8 Furthermore we have
observed that ATR inhibition synergises with conventional chemotherapies to
improve cell killing.
Our hypothesis is that a subset of DLBCL tumours will be sensitive to ATR
inhibition due to the loss of their p53 function.
To test this hypothesis a cohort of DLBCL cell lines will be screened for TP53
mutations, ATR, p53 and BCL6 expression as well as for DDR. A selection of
these cell lines with and without a clear p53 defect will then be used to assess
efficacy of ATR inhibition in vitro, both as a single agent and in combination
with conventional chemotherapies as well as BCL6 inhibitors. We anticipate
that inhibition of ATR will provide a novel therapeutic target in DLBCL,
particularly in combination with other agents due to its reduced DDR
response.
References
23
1.
2.
3.
4.
5.
6.
7.
8.
Carbone A, Gloghini A, Kwong YL, Younes A. Diffuse large B cell
lymphoma: using pathologic and molecular biomarkers to define
subgroups for novel therapy. Ann Hematol 2014;93(8):1263-77.
Young KH, Leroy K, Moller MB, Colleoni GW, Sanchez-Beato M,
Kerbauy FR, et al. Structural profiles of TP53 gene mutations predict
clinical outcome in diffuse large B-cell lymphoma: an international
collaborative study. Blood 2008;112(8):3088-98.
Phan RT, Saito M, Basso K, Niu H, Dalla-Favera R. BCL6 interacts with
the transcription factor Miz-1 to suppress the cyclin-dependent kinase
inhibitor p21 and cell cycle arrest in germinal center B cells. Nat
Immunol 2005;6(10):1054-60.
Jardin F, Ruminy P, Bastard C, Tilly H. The BCL6 proto-oncogene: a
leading role during germinal center development and
lymphomagenesis. Pathol Biol (Paris) 2007;55(1):73-83.
Winter JN. Prognostic markers in diffuse large B-cell lymphoma: Keys
to the underlying biology. Curr Hematol Malig Rep 2007;2(4):235-41.
Ichikawa A, Kinoshita T, Watanabe T, Kato H, Nagai H, Tsushita K, et
al. Mutations of the p53 gene as a prognostic factor in aggressive Bcell lymphoma. N Engl J Med 1997;337(8):529-34.
Fokas E, Prevo R, Hammond EM, Brunner TB, McKenna WG, Muschel
RJ. Targeting ATR in DNA damage response and cancer therapeutics.
Cancer Treat Rev 2014;40(1):109-17.
Kwok M, Davies N, Agathanggelou A, Smith E, Yates E, Brown J, et al.
ATR inhibition exacerbates replication stress in TP53 or ATM deficient
CLL cells and enhances sensitivity to chemotherapy and targeted
therapy. Manuscript in preparation.
I am a senior post-doc in Professor Stankovic’s group and am regularly present in the
lab and will supervise/ train the student on a daily basis until the student feels
comfortable with the techniques. We will have regular meetings to discuss results,
further experiments and the direction that the project might take. Furthermore, the
Stankovic group has weekly meetings where work is presented and ongoing projects
are discussed, providing an additional platform for the training.
The student role.
The student will be expected to show an enthusiastic approach to the project. The
student will learn a number of techniques and hopefully enjoy his/her time in the lab.
At the later stages the student will be expected to present the data at the Stankovic
group meeting as well as have some input in the experimental design and decisions
on the direction that the project might take.
24
Lead Supervisor:
Dr Jo Morris
Contact Email:
Telephone:
[email protected] or [email protected]
x4143016
Co Supervisor:
Dr Ruth Densham
Project Title:
Characterising patient variants of the Breast and
ovarian cancer predisposition gene, BRCA1.
Department:
Project Outline
An increased risk of breast and ovarian cancer is associated with inheritance of a
faulty copy of the Breast Cancer Susceptibility Gene 1 (BRCA1). While BRCA1 has
many roles in the cell, the only intrinsic enzymatic activity of the protein is found
within the highly conserved N-terminus. Clinically relevant missense variants found
within this region suggest a role for this activity in tumour suppression1.
Our recent unpublished data has identified a novel regulatory region in BRCA1 that
enhances the enzymatic activity of the protein. We have also shown that patient
variants that alter the protien in this new region also reduce the enzymatic activity of
BRCA1. These exciting new results suggest that the enzyme activity of BRCA1 may
be important to its ability to suppress cancer development. Now we want to
characterise whether these patient variants change the cellular function of BRCA1,
and, ultimately, relate to tumour development.
Using cutting edge genome editing techniques2 that we have optimised in our lab,
the student will generate a series of BRCA1 patient missense variants within a model
human cell line. We will then characterise the role of these variants in the response
to chemotherapy treatments and in tumour progression using cell survival assays,
fluorescent microscopy and standard lab techniques.
References
1. Morris JR et al. Genetic analysis of BRCA1 ubiquitin ligase activity and its
relationship to breast cancer susceptibility. Hum Mol Genet. (2006) 15(4):599-606.
2. Kim H, Kim JS. A guide to genome engineering with programmable nucleases. Nat
Rev Genet. (2014) 15(5):321-34.
Our lab is experienced in mentoring students. There will be weekly 1-2-1 meetings
with the Group Leader and hands-on lab training from experienced post-docs.
The student role.
The student will learn: CRISPR genome editing, tissue culture, survival assays, DNA
damage response assays, basic microscopy, western blotting, PCR.
In addition, the student will be expected to attend and participate in weekly lab
meetings, general lab rotas, and maintain a clear record of all experiments.
25
Lead Supervisor:
Dr. Anne Fletcher
Contact Email:
Telephone:
[email protected]
Collaborator:
Dr. Mark Cobbold, Dr. Konstantin Knoblich
Project Title:
Lymph node stromal cells create a metastasis
friendly microenvironment
Department:
Yes
Project Outline
Lymph nodes (LNs) are sites where immune responses are robustly initiated, and
a prevailing scientific paradigm states that lymph nodes are hostile environments for
tumour cells, due to the risk of immunogenicity (1). However, for many cancers, LNs
are the first observable site for metastasis, and decades of clinical evidence would
suggest tumour propagation is in fact well-tolerated within LNs (2).
Recent evidence suggests LNs are environments where fibroblastic reticular cells
(FRCs) prevent newly activated T cells from acquiring effector functions, regardless
of specificity (3-5). FRCs also express self-antigens native to various tissues, then
delete specific reactive T cell clones (6-9). The relevance of these normal tolerogenic
functions to anti-tumour immunity has not been assessed. This project tests the
hypothesis that FRCs systematically shut down CD8+ T cell responses
against tumour cells that reach the LN. Preliminary data shows that activated
anti-tumour CD8+ T cells do not respond normally to tumour cells in the presence of
FRCs.
Objectives:
1. To test how FRCs impair T cell rejection of tumour cells in vitro, using
sophisticated in vitro co-cultures and both mouse (melanoma) and human (colorectal
carcinoma) experimental systems with mouse and human anti-tumour T cells.
2. To examine the fate of anti-tumour T cells after being activated in the presence of
tumour cells and FRCs. We will explore whether T cells are temporarily or
permanently silenced, and whether the suppression can be overcome.
3. To test the effect of FRCs on tumour cell killing, using in vitro and in vivo systems.
We will utilise europium release assays and transgenic mice that permit deletion of
FRCs.
Techniques: The student will be taught relevant cell culture techniques, flow
cytometry, confocal microscopy, and skills required to study in vivo cancer models.
Outcomes:
We are hopeful (but cannot guarantee for obvious reasons!) that the work produced
by the student will be used in presentations and ideally submitted for publication
when a body of work comes together.
References and further reading
26
1.
2.
3.
4.
5.
6.
7.
8.
9.
O. Preynat-Seauve, E. Contassot, P. Schuler, V. Piguet, L. E. French, B. Huard, Extralymphatic
tumors prepare draining lymph nodes to invasion via a T-cell cross-tolerance process. Cancer Res
67, 5009-5016 (2007).
S. A. Stacker, M. G. Achen, L. Jussila, M. E. Baldwin, K. Alitalo, Lymphangiogenesis and cancer
metastasis. Nat Rev Cancer 2, 573-583 (2002).
O. Khan, M. Headley, A. Gerard, W. Wei, L. Liu, M. F. Krummel, Regulation of T cell priming by
lymphoid stroma. PLoS One 6, e26138 (2011).
V. Lukacs-Kornek, D. Malhotra, A. L. Fletcher, S. E. Acton, K. G. Elpek, P. Tayalia, A. R. Collier, S.
J. Turley, Regulated release of nitric oxide by nonhematopoietic stroma controls expansion of the
activated T cell pool in lymph nodes. Nat Immunol 12, 1096-1104 (2011).
S. Siegert, H. Y. Huang, C. Y. Yang, L. Scarpellino, L. Carrie, S. Essex, P. J. Nelson, M.
Heikenwalder, H. Acha-Orbea, C. D. Buckley, B. J. Marsland, D. Zehn, S. A. Luther, Fibroblastic
reticular cells from lymph nodes attenuate T cell expansion by producing nitric oxide. PLoS One
6, e27618 (2011).
A. L. Fletcher, V. Lukacs-Kornek, E. D. Reynoso, S. E. Pinner, A. Bellemare-Pelletier, M. S. Curry,
A. R. Collier, R. L. Boyd, S. J. Turley, Lymph node fibroblastic reticular cells directly present
peripheral tissue antigen under steady-state and inflammatory conditions. J Exp Med 207, 689697 (2010).
A. L. Fletcher, D. Malhotra, S. J. Turley, Lymph node stroma broaden the peripheral tolerance
paradigm. Trends Immunol 32, 12-18 (2011).
D. Malhotra*, A. L. Fletcher*, J. Astarita, V. Lukacs-Kornek, P. Tayalia, S. F. Gonzalez, K. G.
Elpek, S. K. Chang, K. Knoblich, M. E. Hemler, M. B. Brenner, M. C. Carroll, D. J. Mooney, S. J.
Turley, Transcriptional profiling of stroma from inflamed and resting lymph nodes defines
immunological hallmarks. Nat Immunol 13, 499-510 (2012).
A. L. Fletcher, J. S. Elman, J. Astarita, R. Murray, N. Saeidi, J. D'Rozario, K. Knoblich, F. D. Brown,
F. A. Schildberg, J. M. Nieves, T. S. Heng, R. L. Boyd, S. J. Turley, B. Parekkadan, Lymph node
fibroblastic reticular cell transplants show robust therapeutic efficacy in high-mortality murine
sepsis. Sci Transl Med 6, 249ra109 (2014).
The student and I will have formal weekly meetings, but as I am often in the lab
myself, there will be many informal chances to discuss data, methods, progress,
science, and anything else that comes up. The student will also present regularly to
our small group for feedback on results, as well as getting practice at presenting
scientific findings. I have supervised students for this length of project several times
before, and understand that close supervision and hands-on practical training is
required in the beginning, but that the student may wish to explore a little more
scientific independence by the end of the project. However, no one supervisory style
fits all and for this type of project I try to work with the student in a way tailored to
their strengths and challenges.
The student role.
The student’s role is to work through a series of experiments, and learn step by step
how to execute certain important assays. Some assays may require a few practices
before they are mastered, and some planned experiments may change depending on
previous results obtained. In this case, I would work with the student’s input to
decide what experiment to do next. The student should be able to interpret results,
think critically about what they mean with reference to literature, and present and
discuss data for friendly peer review within the laboratory.
27
Lead Supervisor:
Dr Aga Gambus
Contact Email:
Telephone:
[email protected]
01214149237
Co Supervisor:
Project Title:
How is the replisome disassembled at the end of
replication once it has done its job?
Department:
Cancer Sciences
No
Is the Project Cancer related?
Yes
Project Outline
We all have about 2m of DNA in every one of our cells. Every time cells
in our body want to divide they need to first duplicate their genome - their
DNA. Ever since the discovery od DNA structure scientists are trying to
describe how this process of DNA replication works in detail, as its perfect
execution is essential to maintain genomic stability and prevent cancer
formation. We know that one of the main causes of mutations that lead to
cancer development are unrepaired mistakes that arise during the process of
duplication. Thus, a lot of work had been done towards understanding the
synthesis of DNA and the initiation stage of DNA replication. However, very
little is known as to how this process finishes, which is the subject of our
paper.
Our recent work has shown a first glimpse of the mechanism that
unloads the replication machinery at the end of replication. The replication
machinery (replisome) is formed of about 150 proteins and built around a few
key organising centers. One of them is a replicative helicase, which is the
protein complex that can unwind double stranded DNA to open it to create
the templates for DNA synthesis. The helicases are loaded onto DNA in
hundreds of thousands, and about 30-50 thousand of them are activated per
cell during DNA replication. They then go through DNA, unwinding it, until
they meet a helicase coming from the opposite direction. These helicases,
once working, are very precious as if they get taken off DNA by mistake they
cannot be put back on. This means however that at the end, when they meet
each other they have to be taken off by some sophisticated mechanism. We
have shown in our work that one of the components of this helicase (Mcm7
subunit) is modified at the final stage of replication by attachment of a small
protein modifier called ubiquitin. This modification allows it to be recognised
by a protein remodeler, which takes it off DNA allowing the whole process to
complete.
As this is the first time we start to learn about this stage of replication
there is of course a lot more work that needs to be done to characterise it in
more detail and to learn the identity of all the essential factors needed for its
28
execution. The proposed project aims to understand better how the modified
Mcm7 (and the rest of the replisome) is taken off DNA by the protein
remodeler p97 and the fate of the removed helicase. To conduct this
research, the Student will use a cell-free system that recapitulates a whole
round of DNA replication in vitro and thus is invaluable for biochemical studies
of eukaryotic DNA replication and DNA repair processes.
References
Polyubiquitylation drives replisome disassembly at the termination of DNA replication.
Moreno SP, Bailey R, Campion N, Herron S, Gambus A.
Science. 2014 Oct 24;346(6208):477-81. doi: 10.1126/science.1253585.
PMID: 25342805 [PubMed - in process]
I will supervise the Student myself on a day to day basis and teach the
student all the techniques required. I will make sure that the Student
understands the project in depth, that her / his lab book is kept up to date
and that we discuss the progress of the project on the regular basis. Finally, I
plan to ask the student to prepare a number of presentations about different
aspects of the project to ensure that he / she gathers all required literature
background knowledge over the duration of the project rather than leaving it
till the end..
The student role.
I expect the Student to become proficient in all the techniques he / she will
need to use and to be able to carry on the experiments by him / herself after
the initial training. All of the work carried out by the Student for the purpose
of this project is laboratory based (wet science).
All of the research conducted by the Student will be novel and should result in
creating data that will be used for future grant applications and publications.
The Student will be a co-author of any publication resulting from this project
29
Lead Supervisor:
Paloma Garcia
Contact Email:
Telephone:
[email protected]
Co Supervisor:
Laila Cancian
Project Title:
DNA damage studies in Haemopoietic stem cells
(HSC) and progenitor cells with Mybl2
haploinsufficiency
Department:
Immunity and Infection, Institute of Biomedical Research
Yes (module 1)
Is the Project Cancer related? YES
Project Outline
Many blood disorders are associated with aging. More than 62% of people diagnosed with
myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN) and leukemia are over
60 years old. It has been proposed that DNA mutations accumulate during the life of an
individual as a consequence of failure to correct errors introduced in the genome during the
cell replicative process, leading to either activation of oncogenes or silencing of tumor
suppressors that operate as driving factors in the initiation of the disease (1,2).
Deletion of the long arm of chromosome 20 is observed in a 5-10% of patients suffering
haematological disorders related to ageing (MDS, MPN and leukaemias). The minimum
common deleted region published so far contains 9 genes (3). One of these genes is the
transcription factor MYBL2 (B-Myb) (3).
We generated a mouse model that expresses half the normal dose of MYBL2, ( MYBL2 )
paralleling MYBL2 haploinsufficiency in human del20q situation (4). We have demonstrated
that during ageing, mimicking what happens in humans, these mice developed
haematological disorders: 50% MPN and 50% MDS disorders (5).
Our hypothesis is that MYBL2 role in the maintenance of genome integrity is compromised
during ageing leading to the accumulation and selection of somatic mutations which drive the
development of blood disorders such as MDS and MPNs.
In this project we would like to get some insight into the molecular mechanism by
which MYBL2 haploinsufficiency leads to an increase in somatic mutations.
Specifically, we will study the DNA repair mechanisms in MYBL2 cells (HSCs and progenitor
cells).
This project will imply to work with mouse models and allow the student to learn the isolation
of bone marrow cells, flow cytometry, confocal microscopy, tissue culture, proliferation assays
and molecular biology techniques such as western blot and Taqman quantitative PCR.
References
1.-Kenyon J ,Gerson SL. 2007. The role of DNA damage repair in aging of adult stem cells. Nucleic Acids
Res 35: 7557-65.
2.- Maslov AY ,Vijg J. 2009. Genome instability, cancer and aging. Biochim Biophys Acta 1790: 963-9
3.- Bench AJ, Nacheva EP, Hood TL, Holden JL, French L, Swanton S, Champion KM, Li J, Whittaker P,
Stavrides G et al: Chromosome 20 deletions in myeloid malignancies: reduction of the common
deleted region, generation of a PAC/BAC contig and identification of candidate genes. UK
30
Cancer Cytogenetics Group (UKCCG). Oncogene 2000, 19(34):3902-3913.
4.- García, P., Berlanga, O., Watson, R., and Frampton J. (2005) Generation of a onditional allele of the
B-Myb gene. Genesis, 43: 189-95.
5.- Clarke, M.L., Dumon, S., Ward, C., Jager, R., Freeman, S., Dawood, B., Sheriff, L., Lorvellec, M.,
Kralovics, R., Frampton, J and García, P. MybL2 haploinsufficiency increases susceptibility to
age-related haemopoietic neoplasia. Leukaemia, 2013, 27: 661–670.
6- Mohrin, M., Bourke E, Alexander D, Warr MR, Barry-Holson K, Le Beau MM, Morrison CG, Passegué E.
Hematopoietic stem cell quiescence promotes error-prone DNA repair and mutagenesis. Cell
Stem Cell. (2010) 6;7(2):174-85.
Dr Laila Cancian will be the daily supervisor. Every time that student requires further
supervision or Dr Cancian is not available, Dr Garcia will be supervising. Dr Garcia’s
lab is shared with Prof Frampton, and together held around 8 postdoctoral
researchers who will be also able to help and provide the right environment for the
student to learn.
The student will also attend regular group meetings (every 15 days) on a round table
format to discuss progress/problems and to come up with solutions/ alternative
approaches.
The student will also benefit from the expertise of other researchers within the field
by attending group meetings with DNA repair groups (fortnightly) and floor meetings
with genome biology groups (weekly).
The student role.
The student will be studying whether haemopoietic stem cells and progenitor cells
expressing half the normal dose of Mybl2 (also known as B-Myb) have a greater
susceptibility to ionising radiation or other DNA damaging agents, compared to wildtype cells, in terms of cell proliferation/differentiation and ability of the cell to repair
the DNA lesions.
The student will then investigate the molecular mechanisms responsible for this
addressing the following questions: are specific DNA damage repair pathways
defective in Mybl2 haploinsufficient cells? Is Mybl2 integral part of the cell DNA
damage and repair machinery? Is Mybl2 regulating the expression of DNA damage
response genes or of cell proliferation/apoptosis genes?
The student will perform experiments, collect data, analyse it and present it. The
student will keep good record of the experimental data in his/her laboratory journal
and will also participate in the daily house-keeping of the lab. The student will attend
lab meetings and present and discuss his/her own work.
31
Lead Supervisor:
Contact Email:
Telephone:
Roger Grand
[email protected]
0121 414 2805
Co Supervisor:
Grant Stewart
Project Title:
Characterisation of novel DNA damage
response proteins
Cancer Sciences
Department:
No
Project Outline
The cellular genome is subject to continuous insult resulting in the occurrence of tens of thousands
of lesions in the DNA per cell per day. To counteract this, a complex series of pathways,
collectively known as the DNA damage response (DDR), has evolved and this is able to detect and,
if possible, repair the DNA. The inability to correct this damage is a major underlying cause of
cancers as well as other diseases. These repair pathways are based on the activities of three
related kinases-Ataxia Telangiectasia mutated (ATM), ATM and Rad3-related (ATR) and DNA
dependent protein kinase (DNA-PK). Following detection of DNA damage, one or more of the
kinases are activated leading to phosphorylation of multiple downstream targets and repair of the
lesion if the damage is not too great. The DDR pathways comprise a large number of components,
with, simplistically, specific proteins involved in homologous recombination, non-homologous end
joining and single strand break repair amongst others. We have recently identified an important
novel DDR protein, termed DIAD (Degraded and Induced by ADenovirus), which is involved in the
response to replicative stress and base modifications but not double strand breaks. We have also
shown by mass spectrometry that DIAD binds to the CNOT complex which has transcriptional
regulatory and deadenylation properties and has been well characterized in yeast but to a much
lesser extent in mammals. The aim of this project is to characterize this association in more detail
and determine to what extent DIAD is unique in its DDR role or whether the CNOT complex plays a
significant part in the DDR. The student will determine: 1, whether knock down of CNOT proteins
(using siRNAs) has the same deleterious effect on the DDR as loss of DIAD; 2, whether DIAD and
CNOT proteins locate to sites of DNA repair and 3, the role of CNOT proteins in cell cycle arrest
following DNA damage. Techniques include cell culture, immunofluorescence microscopy and
biochemical methods such as gel electrophoresis and western blotting.
References
1.The causes and consequences of genetic heterogeneity in cancer evolution.Burrell RA,
McGranahan N, Bartek J, Swanton C. Nature. 2013; 501:338-45.
2.More than just a focus: The chromatin response to DNA damage and its role in genome
integrity maintenance.Lukas J, Lukas C, Bartek J. Nat Cell Biol. 2011; 13:1161-9.
3.The DNA-damage response in human biology and disease. Jackson SP, Bartek J.Nature.
2009; 461:1071-8.
4.. The Ccr4--not complex. Collart MA, Panasenko OO. Gene. 2012 ; 492: 42-53
I spend most of my time in the lab and so will be able to supervise the student
personally. Also other group members will be working in the lab and so will be able
to help in supervision.
The student role.
32
The student will be expected to carry out the experiments outlined above. Towards
the end of the project it is hoped that they will contribute ideas of
alternative/additional approaches.
33
Lead Supervisor:
Dr Melissa Grant
Contact Email:
Telephone:
[email protected]
01214665520 or 01214142652
Co Supervisor:
Dr Gosia Wiench & Prof Iain Chapple
Project Title:
What is the role of epigenetic modifications in the
decrease of glutathione in periodontitis?
Department:
School of Dentistry
No
Project Outline
Glutathione (GSH) is a major antioxidant involved in maintaining redox balance.
Previously we have measured glutathione in gingival crevicular fluid (GCF), a serum
exudate and tissue transudate flowing from between the teeth and adjacent gingival
tissue (gums). In periodontitis, severe gingival inflammation initiated by the plaque
biofilm and exacerbated by an aberrant inflammatory and oxidative stress response,
GCF GSH is decreased compared to healthy donors. Remarkably upon successful
periodontal therapy, which reduces clinical measures of inflammation, the GSH level
in GCF does not rebound to healthy levels (Grant et al 2010). We would like to
understand if there are epigenetic mechanisms involved in this suppression. In
periodontal disease a number of studies have been published recently investigating
epigenetic modifications of inflammatory genes (eg TNFA (Zhang et al 2013); IFNG
(Zhang et al 2010); PTGS2 (Zhang et al 2010); hBD2 and CCL20 (Yin & Chung
2011), indicating this is an area of research expanding rapidly.
GSH is produced by a series of enzymatic steps which are controlled by the
production of the rate limiting enzyme glutamate cysteine ligase (GCL). This enzyme
is under transcriptional control by transcription factor Nrf2, which binds to the
antioxidant response element (ARE). We have already established in vitro (H400 oral
epithelial cell line) that we can induce GSH synthesis with Nrf2 agonist curcumin.
Additionally we have shown that this agonist inhibits HDAC activity and can inhibit
bacterial activation of the NFkB pathway (unpublished).
Histone modification is a multifaceted phenomenon controlling gene expression.
Mono-, di- and tri-methylation at lysine (K) residues has been associated with
transcriptional silencing and activation depending on which amino acid residue is
involved. Recent evidence has shown under oxidative stress conditions Histone3
lysine 4 dimethylation (H3K4me2) at the GCL gene (gclc) was increased and
H3K4me3 and H3K4me1 were decreased, but these remained in place after
alleviation of oxidative stress (Mishra et al 2014). Another study has shown the
increases of H4R3me2 and H2R17me2 in oxidative stress induced ARE activation
(Huang et al 2013).
In this project we propose to use chromatin precipitation (ChIP) experiments to
further our understanding of the control of GSH production in periodontitis. Initially
our cell culture model, using oral epithelial cell line H400, will be used to refine skills
34
and techniques in ChIP and to explore the dynamics of gclc control under model
conditions, using periodontopathogens such as Porphyromonas gingivalis and
Fusobacterium nucleatum, and induction of Nrf2 with known agonists such as
curcumin, with which we have copious experience. Later studies will utilise patient
tissue and potentially ChIP-Seq experiments for non-presumptive analysis of wider
Nrf2 targets.
References
Grant et al JOURNAL OR CLINICAL PERIODONTOLOGY 2010 37:17-23
Huang et al FASEB J 2013 27: 3763-74
Mishra et al FREE RADICAL BIOLOGY AND MEDICINE 2014 75:129-39
Yin & Chung Mucosal Immunity 2011 4: 409-19
Zhang et al J Periodontol 2013 84: 1606-16
Zhang et al J Clin Perio 2010 37:953-61
Zhang et al J Dent Res 2010 89:133-7
We will plan weekly structured meetings with the student and additionally will offer
an open door policy for discussion of progress. Our laboratories have many active
PhD students (7) and technicians (5) whom the student can ask initial questions dayto-day.
The student role.
The student will be responsible for cell culture and carrying out of experiments. We
will plan experiments together and discuss results prior to furthering the project.
They will develop ChIP techniques under supervisory guidance; explore the back
ground to the project through the literature; and will produce all documents required
for completion and submission of the project.
35
Lead Supervisor:
Dr. Paul Harrison
Contact Email:
Telephone:
[email protected]
Co Supervisor:
Professor Steve Watson
Project Title:
Measurement of circulating preplatelets in health
and disease
Department:
Immunity & Infection
No
Project Outline
The terminal differentiation mechanism of thrombopoiesis (platelet formation) has
recently been elucidated. A new population of large platelet-like structures (i.e.
preplatelets)undergoes final maturation into classical platelets within the circulating
blood via a remarkable division process involving barbell formation unique to this
anucleated cell. Measurement of the percentage and absolute levels of these
precursors may therefore not only provide an accurate estimation of platelet turnover
but provide mechanistic insights into the pathology of macrothrombocytopenia where
the terminal differentiation process may also be impaired resulting in the production
of large platelets. Although measurement of immature platelets by flow cytometry is
an established clinical tool the assays are poorly standardized and tend to be utilised
within specialised research laboratories. Routine measurement of immature platelets
is also possible by using an automated full blood counter that measures the IPF
(immature platelet fraction) using fluorescent flow cytometry principles. However,
although this method has been shown to have clinical utility it is still unclear whether
the measurement is an accurate reflection of the true circulating level of immature
platelets. A significant obstacle in these assays has been the widespread use of EDTA
anticoagulated whole blood which is the standard blood sample used for full blood
counters and IPF measurement. Indeed it has been recently demonstrated that
EDTA causes sphering of the newly identified barbell intermediates and therefore
prevents their accurate identification on blood films or by full blood counters. In this
project we therefore propose to set up and evaluate a number of new methods for
accurately quantitating the number of these newly identified platelet intermediates in
normal and pathological blood samples using alternative anticoagulants to EDTA to
inhibit sphering. A comparison of various anticoagulants will be undertaken and
immature platelets measured using fluorescent flow cytometry, Imagestream flow
cytometry (to facilitate imaging), Sysmex Xn full blood counting (IPF measurement
as a reference method) and image analysis of platelets in whiole blood or platelet
rich plasma (on blood films or poly-lysine coated coverslides that have been
fluorescently labelled using specific anti-platelet antibodies (e.g. anti-CD41 or CD61).
To facilitate the clear identification of the intermediate preplatelets samples will also
be fluorescently labelled for -tubulin. The percentage and absolute counts of the
preplatelets will be established in normal samples and then applied to the study of
platelet kinetics in a variety of clinical situations where platelet production is either
impaired or increased. Samples from patients with various forms of
macrothrombocytopenia will also be studied to determine whether there is a failure
36
in terminal maturation in some of these defects. We anticipate that this study will
result in the development of a new method of accurate immature platelet
enumeration with obvious clinical potential.
References
1) M.S.C. Robinson, I.J. Mackie, K. Khair, R. Liesner, A.H. Goodall, G.F. Savidge, S.J.
Machin, P. Harrison (1998) Flow cytometric analysis of reticulated platelets:
evidence for a large proportion of non-specific labelling of dense granules by
fluorescent dyes. British Journal of Haematology, 100, 351-357
2) JN. Thon, H. Macleod, AJ Begonja, J Zhu, K.C. Chen, A.Mogilner, J.H.Hartwig, J.E.
Italiano (2012) Microtubule and cortical forces determine platelet size during
vascular platelet production. Nature Communications, 138, 1-9.
Paul Harrison will supervise the student on a day to day basis as he is an established
expert on platelet counting, immature platelets and flow cytometry. The Sysmex Xn
analyser is located in his laboratory. Regular personal and laboratory meetings will
also be held with Steve Watson and the Birmingham platelet group to discuss project
progress and for the student to present his results to a critical audience. Fluorescent
microscopy and image analysis will be performed in collaboration with Steve Thomas
in the same group who is an expert on high resolution fluorescent microscopy.
The student role.
The student will be fully trained how to perform the repertoire of techniques required
for the success of the project including :- 1) preparation and processing of blood
samples (2) fluorescent labelling of platelets (3) Blood and PRP film preparation on
slides (4) Operation of the Sysmex Xn full blood counter (5) Flow cytometry using a
variety of instruments and the ImageStream system (6) Fluorescent microscopy and
image analysis. The student will record all results with a laboratory book and
regularly present the data to the supervisors. The project thesis write up will be
monitored so that this is deliverable by the completion date. The student will be
encouraged to present and write up his work in the form of seminars and
abstracts/papers.
37
Lead Supervisor:
Dr. Maarten Hoogenkamp
Contact Email:
Telephone:
[email protected]
Co Supervisor:
Dr. Vesna Stanulovic
Project Title:
Determining the effect of LMO2 overexpression
within the myeloid lineage
Department:
Cancer Sciences
No
Project Outline
LMO2 is a transcriptional regulator that is an essential partner of several
transcription factors and together they have a fundamental role in
haematopoietic, vascular and cardiac development.
Overexpression of LMO2 in T-cell progenitors has been shown to cause T-cell
acute lymphoblastic leukaemia. A significant proportion of acute myeloid
leukaemia (AML) has LMO2 overexpression but its contribution to the disease
state is not known.
We have generated LMO2 knockdown and overexpression models in mouse
embryonic stem cells and in a myeloid progenitor cell line that upon induction
differentiates into macrophages. Impaired LMO2 expression in myeloid
progenitors generated large cells with an endomytotic phenotype, implying
LMO2 function in chromosomal segregation.
This project will start by measuring LMO2 expression before and after the
knockdown induction at the mRNA (qPCR) and protein (Western blotting)
level, and assessing the cellular morphology and LMO2 localisation by
confocal microscopy. We will then establish the effects of high LMO2
expression, as observed in AML, on macrophage differentiation by monitoring
morphology, detection of surface markers (flow cytometry), and measuring
the expression of genes (qPCR) known to be involved in macrophage
development. The same experiments will be performed after abolishing LMO2,
mimicking potential LMO2-directed treatment.
References
Hoogenkamp, M. et al. (2009) Blood 114, 299-309
Matthews, J.M. et al. (2013) Nat. Rev. Cancer 13, 111-122
Both listed supervisors spend a significant proportion of their time at the
bench in the laboratory, are skilled in the techniques mentioned above, and
38
have prior experience in supervision of staff and students. This ensures that
supervision and support is available on a daily basis throughout the eight
months project. Every technique will initially be performed together with a
supervisor, hopefully resulting in more independence later into the project.
In addition there will be a weekly meeting to overview and discuss the
progress and next steps, or problems that have been encountered. A weekly
journal club will be held in which current literature on this and related topics
will be discussed.
The student role.
The student is expected to be motivated and to actively participate in the
research group. The student will be primarily responsible for performing the
experiments and interpreting the results of the above project, albeit in close
association with the supervisors.
The student should be motivated to learn the different techniques, which are
established protocols in the lab. We will work towards the student becoming
more independent over the eight months in both the practical aspects of the
project as well as in the ability to interpret the obtained data.
39
Lead Supervisor:
Contact Email:
Telephone:
[email protected]
Co Supervisor:
Project Title:
Identification of DNA binding partners of LMO
proteins in the myeloid lineage
Department:
Cancer Sciences
No
Project Outline
The four highly homologous LIM only (LMO1-4) proteins are all implicated in
cancerogenesis. They are part of DNA binding transcription factor complexes,
although they do not bind the DNA themselves. LMO2 and LMO4 are expressed in
the myeloid lineage of the blood and LMO2 is found to be overexpressed in a large
proportion of acute myeloid leukaemia (AML). Both LMO2 and LMO4 need to bind to
their protein partner LIM domain binding 1 (Ldb1) for their function within the
nucleus. The questions we want to address within this project are whether LMO2 and
LMO4 each form distinct DNA binding complexes and whether they need to compete
for Ldb1 to do this.
To answer these questions we will perform antibody pull-down assays, followed by
Western blotting and mass spectrometry. We will initially perform these using
antibodies recognising LMO2, LMO4, and Ldb1, which will give us insight in the
overlap between these complexes and which DNA binding components are present.
The information from these experiments will be used to perform further pull-down
assays for the newly identified proteins and chromatin immunoprecipitation to
identify where the proteins bind the DNA and which genes they regulate.
References
Meier, N. et al. (2006) Development 133, 4913-4923
Matthews, J.M. et al. (2013) Nat. Rev. Cancer 13, 111-122
40
will be discussed.
The student role.
41
Lead Supervisor:
Contact Email:
Telephone:
[email protected]
Co Supervisor:
Project Title:
The role of LMO proteins in neuroblastoma
Department:
Cancer Sciences
No
Project Outline
Neuroblastoma is one of the major types of cancers affecting young children
and arises from cells of the sympathetic nervous system. Recently it was
shown through a genome wide association study that LMO1 is involved in
neuroblastoma. LMO1 is a protein that does not bind DNA directly, but is a
component of particular DNA binding complexes, and its oncogenic potential
has already been observed in the onset of T cell leukaemia. In
neuroblastoma, higher expression levels of LMO1 were shown to lead to
enhanced proliferation rates. Another LMO family member, i.e. LMO3, has
already been implicated in neuroblastoma for a longer time. For both proteins
their elevated expression corresponds to poor prognosis and more advanced
disease.
Within this project we want to check which LMO family members are
expressed within the same cells, using a number of cell lines. We will perform
pull down assays to see which proteins associate with LMO1, and set up a
lentiviral system to knockdown LMO proteins within these cells. Combination
knock-down of two LMOs may work better than only LMO1 to stop cells from
growing or induce apoptosis. This would potentially be of therapeutic
relevance because the LMOs are structurally very similar. This means a drug
could be designed which interacts with a vital part of the LMO protein that is
structurally the same between the members, thereby targeting more than
one, functionally distinct, components of the cancerous phenotype.
References
Wang K. et al. (2011) Nature 469, 216-220
Ferronha et al. (2013) J. Neurosc. 33, 2773-2783
Aoyama et al. (2005) Cancer Res. 65, 4587-4597
42
will be discussed.
The student role.
43
Lead Supervisor:
Dr Marie-Christine Jones
Contact Email:
Telephone:
[email protected]
48188
Co Supervisor:
n/a
Project Title:
Overcoming platinum resistance in lung cancer by
targeting cancer stem cells with combinations of
chemotherapy with natural plant extracts.
Pharmacy and Therapeutics
Department:
No
Project Outline
Platinum drugs, alone or in combination, remain first line agents against non-small
cell lung cancer. However, development of drug resistance may limit the efficacy of
these agents over time. The exact mechanisms leading to platinum resistance are
not fully understood but increasing evidence supports a role for cancer stem cells
(CSCs). CSCs are thought to be mostly chemoresistant as they exploit different
pathways to avoid the damage induced by anticancer drugs.
Due to the ability of nanoparticles to bypass some of these resistance mechanisms, it
can be expected that encapsulation of chemotherapeutic agents in nanoparticles
could prevent the development of drug resistance and the proliferation of CSC. In
parallel, natural plant extracts, including curcumin (the curry spice) have been shown
to target CSCs.
The objectives of this project are two-fold. Firstly, the aim is to determine the
fraction of CSC in a common non-small lung cancer cell line and establish how that
population changes following treatment with platinum drugs. Secondly, nanoparticles
formulation of platinum-derivatives alone or combined with curcumin (turmeric
extract; curry) or resveratrol (red wine) can prevent the enrichment of CSCs exposed
to platinum drugs, overcome resistance and improve cytotoxicity.
References
[1] Barr MP, Gray SG, Hoffmann AC, Hilger RA, Thomale J, O’Flaherty JD, et al.
Generation and Characterisation of Cisplatin-Resistant Non-Small Cell Lung Cancer
Cell Lines Displaying a Stem-Like Signature. PLoS ONE. 2013;8:e54193.
[2] Alison MR, Lim SM, Nicholson LJ. Cancer stem cells: problems for therapy? J.
Pathol. 2011;223:147-61.
[3] Gottschling S, Schnabel PA, Herth FJ, Herpel E. Are we missing the target?
Cancer stem cells and drug resistance in non-small cell lung cancer. Cancer
Genomics Proteomics. 2012;9:275-86.
[4] Li Y, Zhang T. Targeting cancer stem cells by curcumin and clinical applications.
Cancer Lett. 2014;346:197-205.
[5] Sun TM, Wang YC, Wang F, Du JZ, Mao CQ, Sun CY, et al. Cancer stem cell
44
therapy using doxorubicin conjugated to gold nanoparticles via hydrazone bonds.
Biomaterials. 2014;35:836-45.
Active support will be provided at every step starting from experimental design to
data analysis, as required, by
1)
2)
3)
4)
5)
6)
7)
8)
9)
Providing adequate facilities for the work to be conducted
Clearly defining the objectives of the project
Identifying supervision needs
Providing sufficient background literature to put the project in context
Providing support in experimental design
Providing technical support as required during the experiments
Providing guidelines for data analysis and report writing
Arranging regular meetings to follow progress
Making sure any arising issues are addressed in a timely fashion
The student role.
The student is expected to conduct the cell culture experiments, analyse, interpret
and discuss the data generated. The student will be trained in using the different
techniques required for completion of the project. The student is also expected to
provide updates on project progression and participate actively in planning and
designing experiments.
45
Lead Supervisor:
Nick Jones
Contact Email:
Telephone:
[email protected]
43923
Co Supervisor:
David Withers
Project Title:
Characterisation of innate lymphoid cell
repopulation in a mouse model of bone-marrow
transplantation
MRC Centre for Immune Regulation
Department:
No
Project Outline
Graft versus host disease (GVHD) is a common side-effect following allogeneic
haematopoietic stem cell (HSC) transplantation and is a major cause of mortality and
morbidity amongst the 30,000 recipients with haematological malignancies
transplanted annually, worldwide. GVHD is mediated by donor T cells within the HSC
transplant however the same cells maximise the graft versus tumour effect of the
treatment. Therefore, medications to treat GVHD can increase susceptibility to
tumour relapse (in addition to opportunistic infections).
It has been recently shown that the presence of activated, donor-derived innate
lymphoid cells (ILC) and the presence of ILC3 (a sub-type of ILC expressing RORin the peripheral blood correlated with decreased incidence of GVHD in patients
receiving an allogeniec HSC transplant (Munneke, 2014). Furthermore, Hannash et al
found that recipient ILC3 produce IL-22 which limits GVHD in a mouse model of
GVHD (Hanash, 2012). Although both studies highlighted the potential of ILC3 to
control GVHD it remains unclear as to the kinetics of ILC3 repopulation after
transplantation and the relative importance of donor versus recipient ILC3.
The proposed project will evaluate the reconstitution/proliferation and activation of
the ILC compartment (with a focus on ILC3) following lethal irradiation and ILC3-GFP
bone-marrow transplantion in mice. Using ILC3 knockout bone-marrow we will
determine the impact of loss of donor ILC3 on alloreactive T cell responses and the
induction of acute GVHD.
The student will study a model of bone-marrow transplantation where residual ILC populations and recipient
repopulation by donor ILCs can be followed after bone-marrow transplantation. To this end, T cell depleted (TCD)
Ror -GFP (where ILC3 will express GFP) bone-marrow will be injected into lethally-irradiated C57BL/6xBALB/c F1
(CB6F1; H2d/b) recipients. Recipients will be analysed at multiple time-points following bone-marrow transplantation
and lymphoid and non-lymphoid tissues analysed for the proportion/number/phenotype/activation status and
localisation of ILCs by polychromatic flow cytometry and multi-colour confocal microscopy.
Mice that have received bone-marrow transplantation will be compared to non-irradiated wild-type control mice.
In order to assess the impact of donor-derived ILC3 populations on the T cell
response to the recipient (that results in GVHD), irradiated F1 recipient mice will
receive TCD bone-marrow from Ror -knockout mice (unable to generate ILC3)
together with GVHD-inducing CFSE+CD45.1+ TEa T cells (TCR-transgenic CD4+ T cells that recognise the
recipient MHC+peptide). The response of TEa T cells will be followed by flow cytometry and immunohistochemistry
both in lymphoid tissues and GVHD target tissues (gut, skin and liver) and the kinetics of disease followed.
46
Overall, these studies will provide vital information regarding the re-population of
ILCs in bone-marrow transplant recipients and will determine whether donor ILC3
seeding alters the subsequent T cell response to the recipient and induction of
GVHD.
References
McKenzie, A.N., H. Spits, and G. Eberl. 2014. Innate lymphoid cells in inflammation
and immunity. Immunity 41:366-374.
Munneke, J.M., A.T. Bjorklund, J.M. Mjosberg, K. Garming-Legert, J.H. Bernink, B.
Blom, C. Huisman, M.H. van Oers, H. Spits, K.J. Malmberg, and M.D. Hazenberg.
2014. Activated innate lymphoid cells are associated with a reduced susceptibility to
graft-versus-host disease. Blood 124:812-821
Hanash, A.M., J.A. Dudakov, G. Hua, M.H. O'Connor, L.F. Young, N.V. Singer, M.L.
West, R.R. Jenq, A.M. Holland, L.W. Kappel, A. Ghosh, J.J. Tsai, U.K. Rao, N.L. Yim,
O.M. Smith, E. Velardi, E.B. Hawryluk, G.F. Murphy, C. Liu, L.A. Fouser, R. Kolesnick,
B.R. Blazar, and M.R. van den Brink. 2012. Interleukin-22 protects intestinal stem
cells from immune-mediated tissue damage and regulates sensitivity to graft versus
host disease. Immunity 37:339-35
Initially I will meet with the student on a weekly basis with a joint
Jones/Withers lab meeting once a month to discuss student progress and
ideas pertaining to this project. In addition, the student will be directly
supervised on a day to day basis by an experienced post-doctoral researcher
(Kyoko Nakamura) as well as PhD students from both Jones and Withers
laboratories. We will review this arrangement every two months and alter
according to progress and required input from Dr Withers or myself.
The student role.
The student will be based within the MRC Centre for Immune Regulation in
the IBR. Importantly, the IBR houses over 300 scientists working on an array
of different immune processes, creating a hub of technological expertise and
an interactive research environment. The student will have access to and
training in key technologies within the College’s Core Technology Hub
including advanced confocal microscopy, flow cytometry, cell sorting and a
range of molecular techniques to support this project. The student will work
with mouse tissue but will not perform in vivo experiments directly. In vivo
experiments will be done in collaboration with Claire Dempsey who is
undertaking a PhD employing the aforementioned mouse model of GVHD.
The student will be expected to design, perform, analyse and interpret their
own experiments with the help of experts in the Jones and Withers group. All
necessary techniques have been established in either the Jones or Withers
group and the student will be taught by researchers well versed in such
techniques. Training on specialised equipment will be carried out either by
laboratory members or by dedicated members of staff as appropriate.
47
The student will participate in a weekly departmental meeting in addition to
supervisory 1 to 1 or lab meetings. The student will also be encouraged to
attend postgraduate PDR activities such as seminars (such as John Squire,
Happy Hour and CIIC) and journal clubs.
It is also expected that the student will produce a first-rate project report
where data is presented in either graph or photomicrograph form, the correct
statistical evaluation of data has been used, the data has been robustly
analysed without over-interpretation and that the data is discussed in the
context of the wider literature. It is expected that the report is consistent with
publication standards.
48
Lead Supervisor:
Dr Nils P Krone
Contact Email:
Telephone:
[email protected]
0121 414 2540
Co Supervisor:
Mr Graham Fews
[email protected]
Project Title:
Deciphering novel genetic variations associated with
disorders of sex development
Department:
Centre for Endocrinology, Diabetes and Metabolism
School for Clinical and Experimental Medicine
No
Project Outline
Disorders of Sex Development (DSD) are a heterogenous group of conditions.
Currently only approximately 40% of patients with DSD have a molecular genetic
diagnosis following classic diagnostic pathways. Patients typically present either in
the newborn period with atypical genitalia which may prevent immidiate gender
assignment; or during adolescence where atypical sex development may become
apparent. Current genetic testing strategies include chromosome analysis to establish
genetic sex, specific gene mutation testing as directed by biochemical profiles and
microarray analysis to detect genomic imbalances. The West Midlands Regional
Genetics Laboratory has recently validated a next generation sequencing based gene
panel to sequence 35 genes known to be causative of various DSD and identified
novel mutations and clarified the diagnosis in atypical cases, thus providing
personalised care. This includes gender assignment, stratified hormonal therapy and
information on potential future risk for gonadal malignancies. This current diagnostic
approach has already identified genetic variations in multiple genes in about 25% of
patients with DSD. Whilst our strategy for genetic testing in DSD has already
increased the effectiveness of genetic testing and provides an increased diagnosis, a
significant proportion (about 30-40%) of patients will remain without a confirmed
molecular basis for the underlying condition. Thus, the genetic service has developed
novel tests to analyse genes involved in DSD by clinical exome sequencing to screen
for mutations in genes known to be associated with sex development.
It will be vital to correlate the genetic findings with the detailed clinical phenotype
and model the functional consenquences of unknown gene variants to fully
understand the clinical conquences.
Thus this translational project has three main tasks:
1.
To assists the deep phenotyping of patients with DSD to collect
standardised clinical data.
2.
To analyse molecular genetic data generated by next generation
sequening.
3.
To perform funtional in vitro analysis of novel genetic variants
identified in patients with DSD
Task 1: Patients are ongoing recruited from the multidiciplinary Disorder of Sex
Development clinic (Clinical Lead Dr Krone) at Birmingham Children's Hospital (BCH).
Phenotypic data will be collected into the clinical data base at BCH and core data will
be also entered into the international I-DSD registry. The detailed examination will
included the exact phenotypic charaterisation of the external genitalia, examination
for additional dysmorphism and other clinical problem. In addition, data of
examinations under anaesthesia (EUA), ultrasounds, MRI and histological data will be
49
collected. Blood sampling for molecular genetic analysis is part of the standard care
pathway for these patients. DNA samples will be collected via phlebothomy and send
to the West Midlands Regional Genetics Laboratory for genetic analysis using NGS
approaches.
Task 2: Patient DNA will be stored and prepared for running on the Illumina
TruSight One Sequencing Panel and run on an Illumina HiSeq NGS system in the the
West Midlands Regional Genetics Laboratory (Mr G Fews). The resulting sequencing
data will be processed and analysed for variation against the genomic reference
sequence to ascertain mutations considered to be pathogenic and clinically
significant to the DSD. These mutations will then be confirmed by Sanger
sequencing.
Task 3: The genetic findings will be discussed at multidiplinary meetings and
pathogenic mutations will be analysed in vitro by functional expression analysis, to
characterise multiple hits within pathways involved in sex development. Mutations
will be introduced in respective cDNAs by in vitro mutagenesis. These will be
transfected into mammalian cell culture models to assess the in vitro protein
properties of respective. If transcription factors are involved, we will assess in vitro
transactivation potential towards respective receptors; in case of steroidogenic
enzymes the capacity to converst steroid hormones will be assessed. These studies
can be complemented by recreating mutations in vivo and study their effects in
zebrafish models.
This project is vital for the development of novel clinical pathways in rare genetic
disease as it will promote our understanding regading the clinical consequences of
genetic variants associated with disorders of sex development.
References
Kolesinska Z, Ahmed SF, Niedziela M, Bryce J, Molinska-Glura M, Rodie M, Jiang J,
Sinnott RO, Hughes IA, Darendeliler F, Hiort O, van der Zwan Y, Cools M, Guran T,
Holterhus PM, Bertelloni S, Lisa L, Arlt W, Krone N, Ellaithi M, Balsamo A, Mazen I,
Nordenstrom A, Lachlan K, Alkhawari M, Chatelain P, Weintrob N. Changes over time
in sex assignment for disorders of sex development. Pediatrics. 2014
Sep;134(3):e710-5.
Bashamboo A, McElreavey K. Gene mutations associated with anomalies of human
gonad formation. Sex Dev. 2013;7(1-3):126-46
Ahmed SF, Achermann JC, Arlt W, Balen AH, Conway G, Edwards ZL, Elford S,
Hughes IA, Izatt L, Krone N, Miles HL, O'Toole S, Perry L, Sanders C, Simmonds M,
Wallace AM, Watt A, Willis D. UK guidance on the initial evaluation of an infant or an
adolescent with a suspected disorder of sex development. Clin Endocrinol (Oxf).
2011 Jul;75(1):12-26
The proposed project is well embedded into ongoing research to model and
understand novel genetic variations in genes associated with disorders of sex
development. These projects are funded by European charities (IFCAH-ESPE) and
the EU fp7 framework program. Overarching structured supervision will formally take
place at least once weekly during lab meetings and on an informal basis when
required with the supervisors. To guarantee the maximum scientific outcome
(presentations, publications) and the best possible work experience (acquiring
scientific skills and broad cutting edge methods) the student will be able to get all
required support during daily interaction with postdocs working on related projects in
the lab.
The student role.
50
Throughout the project, the student will grow into the role with support of our
research teams to perform experiments with an increasing level of independence.
Together with the student, we will develop experimental outlines to enforce a
successful outcome of the proposed studies.
The project has three key elements embarking on a translational research
philosophy. The student will be able to attend multidisciplinary team clinics for
Disorders of sex development at Birmingham Children’s Hospital (Clinical Lead, Dr
Krone) and the student will be involved in the systematic collection of phenotypic
data. In addition, the student will be able to take part in the data analysis by next
generation sequencing in the Clinical Genetics Units (Principal Geneticist, Mr Fews)
and learn cutting edge methods of genetic analysis. Mutations of unknown relevance
will be modelled in vitro in Dr Krone’s lab at the Centre of Endocrinology, Diabetes
and Metabolism (CEDAM) across the road from the genetics units.
Over the course of the project, the student will acquire a multitude of generally
applicable lab methods including PCR, cloning, plasmid DNA (MiniPrep, MidiPrep) and
RNA preparation, transformation, transfection, cell culture techniques fluorescence
microscopy, mRNA synthesis and purification. These methods will equip the student
with the required skill mix to gain independence during the daily work. This will also
provide the vital basis for a potential career in academic medicine. In addition, the
student will acquire basic skill in cutting edge technologies such a steroid
metabolome analysis by LC-MS/MS. We see the student as an integral member of
our research teams and will provide them with state-of-the-art experience and
knowledge to master projects and in translational medicine.
51
Lead Supervisor:
Dr Nils P Krone
Contact Email:
Telephone:
[email protected]
0121 414 2540
Co Supervisor:
Dr Grareth G Lavery
[email protected]
Project Title:
The role of adrenodoxin in redox regulation of
steroid hormone biosynthesis
Department:
Centre for Endocrinology, Diabetes and Metabolism
School for Clinical and Experimental Medicine
Not necessary, but preferred
Project Outline
Background: Steroid hormones are key regulators of sex development,
homeostasis, and metabolism. Of the Cytochrome P450 (CYP) enzymes, CYP type-1
enzymes are active in the mitochondrion relying on electron transfer from
adrenodoxin (ADX) and adrenodoxin reductase (ADR). CYP type-2 enzymes are
localised to the endoplasmic reticulum and depend on P450 oxidoreductase as
electron donor. ADX/ADR dependent reactions account for more than half the
synthesis steps in steroid hormone production. Variations in mitochondrial redox
regulation of steroidogenesis are hypothesised to account for phenotypic differences
in common disease such as hypertension and modulators of phenotypic expression in
inborn errors of steroidogenesis. However, redox regulation of mitochondrial
steroidogenesis has not been studied in vivo, and in contrast to microsomal
steroidogenesis, no human mutations have been described in the mitochondrial cofactors ADX and ADR.
This project will elucidate the critical mechanisms regulating in vivo mitochondrial
steroidogenesis vitally important for understanding of range health and disease.
Activation of such pathways are likely to increase reactive oxygen species and apear
to be an attractive target in the treatment of hormone depedent cancers.
Hypothesis: Adrenodoxin control of CYP type-1 enzymes in the mitochondria is
central to the control of in vivo steroidogenesis
Experimental Design and Methods: We have recently created a global Adxdeletion and heterozygous Adx-deletion allele mice are readily available and will be
used to breed a global Adx knock-out mouse.
We will breed litters to study if the mutation has an impact on pregnancy or
development according to Mendelian inheritance. An impairment of pregnancy is
unlikely as progesterone, required to maintain pregnancy, is produced by the corpus
luteumand not by the placenta like in humans from mid-pregnancy. Adrenodoxin
expression will be assessed by Western Blot verifying adrenodoxin deficiency. As a
next step, thorough macroscopic analysis of size and growth of pups, organ size and
weight with focus on the adrenal and gonad will be conducted. The phenotype of
adrenocortical insufficiency might vary similar to effects observed in the deletion
model of sterodoidogenic acute regulatory protein (star) deletion mouse. Histological
methods will use H&E and oil red staining as well as immunohistochemistry for
adrenodoxin, adrenodoxin reductase, cytochrome P450 enzymes and transcription
factors involved in regulation of expression of steroidogenic enzymes. We will assess
the functional consequences of altered electron provision towards mitochondrial
steroidogenesis by analysing plasma and urine by LC/MSMS and GC/MS, which are a
well established methods in our laboratory and their key regulatory hormones
52
(ACTH, gonadotrophins).
Finally, we will conduct ex vivo whole organ cultures of the adrenals and gonads.
Employing such an approach will allow us to dissect the differential effects of an
adrenodoxin deletion on steroidogenic pathways by incubation with defined steroid
precursors specific for different CYP type 1 conversion reactions. These studies will
allow insight into the differential impairment of CYP type 1 enzymes (cyp11a1,
cyp11b1, cyp11b2) in an intact organ system.
Outcome: This project will generate unique data on the physiological role of
adrenodoxin. The work is well embedded into current research of both supervisors
will set the basis for further work to generate adrenal and gonadal specific Adxdeletion models. It will provide a postgraduate student with a huge skill set in animal
research and research publications.
References
Miller WL, Auchus RJ 2011 The molecular biology, biochemistry, and physiology of
human steroidogenesis and its disorders. Endocr Rev 32:81-151
Krone N, Arlt W 2009 Genetics of congenital adrenal hyperplasia. Best practice &
research 23:181-192
Parajes S, Kamrath C, Rose IT, Taylor AE, Mooij CF, Dhir V, Grötzinger J, Arlt W,
Krone N 2011 A novel entity of clinically isolated adrenal insufficiency caused by a
partially inactivating mutation of the gene encoding for P450 side chain cleavage
enzyme (CYP11A1). J Clin Endocrinol Metab 96:E1798-1806
Lisurek M, Bernhardt R 2004 Modulation of aldosterone and cortisol synthesis on the
molecular level. Mol Cell Endocrinol 215:149-159
The proposed project is well embedded into ongoing research to model and
understand steroidogenesis and steroid hormone metabolism. These projects are
funded by European charities (IFCAH-ESPE), the EU fp7 framework program and the
Wellcome Trust. Overarching structured supervision will formally take place at least
once weekly during lab meetings and on an informal basis when required with the
supervisors. To guarantee the maximum scientific outcome (presentations,
publications) and the best possible work experience (acquiring scientific skills and
broad cutting edge methods) the student will be able to get all required support
during daily interaction with postdocs working on related projects in the lab.
The student role.
Throughout the project, the student will grow into the role with support of our
research teams to perform experiments with an increasing level of independence.
Together with the student, we will develop experimental outlines to enforce a
successful outcome of the proposed studies.
Over the course of the project, the student will acquire a multitude of generally
applicable lab methods including PCR, cloning, plasmid DNA (MiniPrep, MidiPrep) and
RNA preparation, transformation, transfection, cell culture techniques bright fied and
fluorescence microscopy, mRNA synthesis and purification. These methods will equip
the student with the required skill mix to gain independence during the daily work.
This will also provide the vital basis for a potential career in academic medicine. In
addition, the student will acquire basic skill in cutting edge technologies such a
steroid metabolome analysis by LC-MS/MS. We see the student as an integral
member of our research teams and will provide them with state-of-the-art
experience and knowledge to master projects and in translational medicine.
53
Lead Supervisor:
Dr Patricia F. Lalor
Contact Email:
Telephone:
[email protected]
x46967
Co Supervisor:
Professor Stefan Hubscher
Project Title:
Prognostic and functional significance of hepatic
expression of L-FABP and varL-FABP
Department:
Is the Project Cancer related? - Yes
Project Outline
Liver fatty acid binding protein (L-FABP or FABP-1) is expressed in the pancreas,
small intestine, kidney and the liver. It is involved in intracellular transport
and chaperone functions for long chain fatty acids, and maintains appropriate
cytosolic concentrations of fatty acid. Cytoplasmic expression is reportedly
decreased in individuals with moderate to severe NASH [1] compared to
those with simple steatosis, whilst other studies suggest an increase in
expression in individuals with diabetes and metabolic syndrome. A recent
study has shown that polymorphisms of FABP1 make an individual more
susceptible to NAFLD [2] and also to developing type II diabetes[3], and we
have shown that it is possible to detect both variant and native protein in the
livers of individuals with NASH using novel MS technology[4]. Recent
evidence also suggests that l-FABP is a valuable tool for staging hepatic
adenomas[5] and colorectal neoplasms[6] but to date little data exists for
expression of variant forms during malignancy. Therefore this project will use
histochemical and molecular techniques to investigate the expression of
native and variant L-FABP in hepatic disease.
References
1.
2.
3.
4.
5.
6.
Charlton, M., et al., Hepatology., 2009. 49(4): p. 1375-84.
Peng, X.E., et al., Gene., 2012. 500(1): p. 54-8. Epub 2012 Mar 20.
Mansego, M.L., et al., PLoS One., 2012. 7(3): p. e31853. Epub 2012
Mar 2.
Sarsby, J., et al., J Am Soc Mass Spectrom, 2014. 25(11): p. 1953-61.
van Aalten, S.M., et al., J Hepatol, 2011. 55(1): p. 120-5.
Lawrie, L.C., et al., Br J Cancer, 2004. 90(10): p. 1955-60.
We have chosen to combine the technical expertise of experienced clinical and academic
supervisors to ensure the student is exposed to the maximal number of transferable research
skills and has access to appropriate clinical samples. All supervisors have a proven track
record in supervision of both undergraduate and postgraduate students, and prior experience
supervising successful Intercalation projects. For example one of Dr Lalor’s students has been
awarded a FALK/Core award for his research project (2014 – Ding Yang) Appropriate training
in research techniques and data analysis and presentation will be supplied. We will have
weekly meetings with supervisors and interactions with other members of the research
groups, who will be available day to day for advice and tuition will be encouraged.
The student role.
54
The student will be expected to perform experiments and conduct data analysis.
They will present data to the supervisory team and wider research groups and be
expected to assimilate available published literature under guidance from
supervisors. They will be trained and expected to work using good laboratory
practice under local standard operating procedures and be mindful of ethical use of
human tissue specimens. They will be working in large research teams and thus
would be expected to interact with colleagues and use local facilities in a respectful
manner.
55
Lead Supervisor:
Dr Patricia F. Lalor
Contact Email:
Telephone:
[email protected]
x46967
Co Supervisor:
Dr Tariq Iqbal
Project Title:
The consequences of mucosal platelet activation in
IBD
Department:
Liver research Laboratory, I+I
No
Is the Project Cancer related? - no
Project Outline
Platelets are traditionally considered as having a primary role in coagulation,
however studies from our group and others confirm that platelets can, under some
circumstances bind using integrins to endothelial cells where they can support leukocyte
recruitment to the vessel wall[1, 2]. They have beneficial roles in haemostasis [3],
pathogen clearance during sepsis[4], production of serotonin to assist wound healing in
multiple organs. Inflammatory bowel disease is associated with increased platelet
activation and risk of thromboembolism [5], and patients commonly have elevated
counts of small platelets [6, 7]with an activated phenotype[8, 9] and high levels of
CD40L expression. This is thought to contribute to elevated levels of circulating CD40L
found in patients and aggregation of platelets in gastric mucosal tissue[7]. The
importance of platelets to disease pathogenesis is illustrated by studies showing that
platelet-derived miRNA biomarkers are identified in screens of inflammatory bowel
disease susceptibility genes[10]. Importantly use of Clopidogrel to block platelet function
has shown promise for reduction of disease burden in animal models of colitis and
Crohns disease[11] and retrospective analysis of patients suggests reduced disease
activity in patients treated with anti-platelet agents[12].
Currently existing drugs which target the recruitment and activation of platelets
in the bowel would have promise in the treatment of IBD. However there is a risk of
bleeds and possible disturbance of normal mucosal anatomy in patients taking aspirin
and other antiplatelet agents[13] and thus whist of definite potential[14], this explains
the reluctance to adopt this therapeutic strategy. Importantly the mechanisms of platelet
activation in diseased bowel are not well characterized and nor are the mechanisms by
which the platelets bind to the colonic
vasculature. Additionally no studies in humans have demonstrated their contribution to
bowel inflammation. Identification of key anti-adhesive pathways would allow targeting
of anti-platelet therapies to the bowel, thereby minimizing compromise of coagulation.
We have demonstrated the efficiacy of this strategy I previos studies of the hepatic
microenvironment[2] In this study we will use functional assays and
immunocytochemical techniques to report the regulation of platelet activation and
adhesion within the bowel.
References
1.
2.
3.
Lalor, P. and G.B. Nash, Br.J.Haematol., 1995. 89(4): p. 725-732.
Lalor, P.F., et al., American journal of physiology. Gastrointestinal and liver
physiology, 2013. 304(5): p. G469-78.
Pereboom, I.T., T. Lisman, and R.J. Porte, Liver transplantation : 2008. 14(7): p.
923-31.
56
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Ma, A.C. and P. Kubes, Journal of thrombosis and haemostasis : JTH, 2008. 6(3): p.
415-20.
Webberley, M.J., M.T. Hart, and V. Melikian, Gut, 1993. 34(2): p. 247-51.
Larsen, T.B., et al., Pathophysiology of haemostasis and thrombosis, 2002. 32(2): p.
92-6.
Collins, C.E., et al., European Journal of Gastroenterology & Hepatology, 1997.
9(12): p. 1213-7.
Collins, C.E., et al., Gastroenterology, 1994. 106(4): p. 840-5.
Danese, S., et al., Gut, 2003. 52(10): p. 1435-41.
Duttagupta, R., et al., PloS one, 2012. 7(2): p. e31241.
Patel, S.H., M.A. Rachchh, and P.D. Jadav, Indian journal of pharmacology, 2012.
44(6): p. 744-8.
Vinod, J., et al., Journal of clinical gastroenterology, 2012. 46(6): p. 527-9.
Taha, A.S., et al., Alimentary pharmacology & therapeutics, 2006. 23(4): p. 489-95.
Pitchford, S.C., British journal of pharmacology, 2007. 152(7): p. 987-1002.
We have chosen to combine the technical expertise of experienced clinical and
academic supervisors to ensure the student is exposed to the maximal number of
transferable research skills and has access to appropriate clinical samples. All
supervisors have a proven track record in supervision of both undergraduate and
postgraduate students, and prior experience supervising successful Intercalation
projects. For example one of Dr Lalor’s students has been awarded a FALK/Core
award for his research project (2014 – Ding Yang) Appropriate training in research
techniques and data analysis and presentation will be supplied. We will have weekly
meetings with supervisors and interactions with other members of the research
groups, who will be available day to day for advice and tuition will be encouraged.
The student role.
The student will be expected to perform experiments, collect tissue samples from
clinic and conduct data analysis. They will present data to the supervisory team and
wider research groups and be expected to assimilate available published literature
under guidance from supervisors. They will be trained and expected to work using
good laboratory practice under local standard operating procedures and be mindful
of ethical use of human tissue specimens.
57
Lead Supervisor:
Dr Steven Lee
Contact Email:
Telephone:
[email protected] (tel 0121-414-2803)
Co Supervisor:
Prof Roy Bicknell
Project Title:
Targeting the tumour vasculature with genetically
engineered T cells.
Department:
No
Project Outline
Compared to normal tissue, angiogenesis in tumours is deregulated and/or aberrant,
resulting in a structurally and functionally abnormal vasculature. Targeting unique
features of the tumour vasculature to compromise blood flow in tumour tissue should
therefore provide therapeutic benefit. Anti-angiogenic monoclonal antibodies or small
molecules that target these tumour endothelial markers appears to have limited
curative potential, possibly because of their cytostatic action and the redundancy of
angiogenic pathways. In principle, cytotoxic strategies should be more effective
because they could prevent formation of new vessels and destroy existing tumour
vasculature.
T lymphocytes are self-replicating effectors that can persist for years and display
potent and specific cytotoxic activity. Recent clinical studies infusing cytotoxic T
lymphocytes (CTLs) specific for antigens expressed on malignant cells have
demonstrated remarkable efficacy in treating metastatic melanoma1. However,
extending this therapy to other cancers is limited by a lack of appropriate tumour
antigens. Targeting T cells to tumour endothelial markers offers an alternative
approach that in animal models has been shown to inhibit tumour growth and
prolong host survival2,3.
Melanoma Cell Adhesion Molecule (MCAM) was originally identified as an antigen on
metastatic melanoma cells4 and is thought to play a role in cell adhesion. More
recently, following an extensive analysis of renal cell carcinoma tissues, we have
found that MCAM is also a tumour endothelial marker, highly expressed on the
vasculature of renal cell carcinoma but absent or poorly expressed in normal tissues
(Fig. 1) (Wragg & Bicknell, unpublished data).
Fig. 1 MCAM is highly expressed on blood
vessels within renal cell carcinoma tissue
(indicated by brown staining) but not in
adjacent normal tissue.
Normal
tissue
Tumour
tissue
58
To date naturally occurring T cell responses to MCAM have not been described, but it
is possible to engineer T cells with a defined specificity by transducing genes
encoding “chimeric antigen receptors” (CARs). CARs combine the specificity of
antibodies with the cytotoxic and immunomodulatory functions of T cells and operate
in an MHC-unrestricted manner (reviewed5). Typically, CARs consist of a single chain
variable fragment (scFv) from a specific antibody linked to intracellular T cell
signalling domains (Fig. 2).
The anti-tumour effects of CAR-expressing T cells have been demonstrated in preclinical models6 and more recently with some dramatic results in clinical trials7-9.
Using the ETH-2 human antibody phage library10 we plan to isolate antibodies
specific for human MCAM, and then to generate CAR constructs that will target T
cells to recognise and destroy the tumour vasculature.
Aims: Generation and characterisation of MCAM-specific CARs to assess their
therapeutic potential for cancer.
The project will focus on the following areas:
1. Using our phage antibody library, we will use recombinant human MCAM to
isolate antibodies specific for this molecule.
2. Using molecular techniques, single chain variable fragment (scFv) genes that
encode these MCAM-specific antibodies will be cloned into an existing
retroviral expression plasmid designed to generate the CAR construct.
3. Human T cells will be transduced in vitro using these retroviruses to stably
express the CAR on the cell surface. Expression of the CAR will be explored
using flow cytometry.
4. To explore the specificity and function of these CAR-expressing T cells they
will be tested for their ability to proliferate and release cytokines in response
59
to purified MCAM and their ability to kill MCAM-expressing target cells.
5. If in vitro data look promising and if time permits, preliminary studies will be
conducted in vivo, using mouse tumour models to explore the safety and
efficacy of this approach.
This project is based on a joint study between two labs in the medical school,
combining the expertise of Dr Steve Lee (T cell therapy including engineering T cells
to express CARs) and Prof Roy Bicknell (Angiogenesis).
References
1. Rosenberg, SA et al. Clin Cancer Res 17, 4550-4557, (2011).
2. Chinnasamy, D et al. J Clin Invest 120, 3953-3968, (2010).
3. Niederman, TM et al. Proc Natl Acad Sci U S A 99, 7009-7014, (2002).
4. Lehmann, JM et al. Cancer Res 47, 841-845, (1987).
5. Sadelain, M et al. Cancer Discov 3, 388-398, (2013).
6. Brentjens, RJ et al. Nat Med 9, 279-286, (2003).
7. Maude, SL et al. N Engl J Med 371, 1507-1517, (2014).
8. Kalos, M et al. Sci Transl Med 3, 95ra73, (2011).
9. Brentjens, RJ et al. Sci Transl Med 5, 177ra138, (2013).
10. http://www.pharma.ethz.ch/institute_groups/biomacromolecules/protocols/eth
The student will be fully supported through scheduled weekly meetings with the
primary supervisor to discuss experiments and any concerns the student may have.
The student will work closely with the primary supervisor and have regular (almost
daily) access to both him and members of the Lee and Bicknell labs. Day to day the
student will work alongside other members of the Lee lab who have extensive
experience of this type of work.
The student role.
During the project, the student will spend most of their time engaged in laboratorybased research. Under the supervision of Dr Lee and following a period of training,
they will design, conduct and interpret the results of experiments. They will also
have time to read around the subject area, to understand the background to the
project and to keep up to date with recent developments. The student will take an
active role in weekly lab meetings/journal clubs in which they will have a chance to
discuss their own data and critique the work of others.
60
Lead Supervisor:
Dr Felicity de Cogan
Contact Email:
Telephone:
0121 4144859
Co Supervisor:
Prof Ann Logan
Project Title:
Self-cleaning surfaces: Preventing the spread of
hospital infection
Department:
School of Clinical and Experimental Medicine
No
Project Outline
Nosocomial (i.e. hospital acquired) infections affect 1 in 11 hospital patients at any
one time and cost the NHS approximately £1 billion year1. The most common way of
spreading infection is through contaminated surfaces such as beds, taps and door
handles. While hospital infection controls such as hand washing, personal protective
equipment and patient isolation can reduce the risk of infection, these approaches
are inadequate. Our work has developed a simple, cheap and durable protein coating
for different surfaces including metals, which prevents bacterial colonisation, using
titanium (material most commonly used in orthopaedic implants) as a base material.
The work carried out in this project will move this work away from orthopaedic
implants and towards general hospital surfaces, such as door handles, medical
equipment and surgical devices. The student will carry out synthetic biology
techniques for protein synthesis. They will carry out surface attachment of the active
proteins onto the target surfaces and then use microbiology assays to test the
efficacy of the coating and identify and specify targeted bacteria and carrying out a
randomised control trial of the surfaces in the Medical School infrastructure to test
the anti-microbial efficacy of the surfaces in a ‘real’ environment.
References
1.
UK National Audit Office ‘The management and control of hospital acquired
infection in acute NHS trusts in England, 17, February, 2000.
The student will be trained and supervised on a daily basis by Dr Felicity de Cogan.
The student will attend weekly group meetings to report back on the project to the
entire group and obtain feedback and ideas. The student will also have regular
progress/supervisory meetings with Professor Logan (Head of Section) and Dr Mark
Webber (Bioscience Collaborator) about their work.
The student role.
61
The student will carry out the day to day experimental work on the project. The
student will gain familiarisation with terminology and literature and existing
healthcare challenges in the field of antimicrobials and antibacterial resistance. They
will learn and exploit novel methods of antimicrobial surface attachment. They will
learn basic microbiology techniques and learn how to isolate and process pathogen
samples and to identify and track bacterial species. The student will gain experience
in carrying out a randomised controlled study and in blinded analysis.
62
Lead Supervisor:
Christian Ludwig
Contact Email:
Telephone:
[email protected]
Co Supervisor:
Jay Nath, Surgical Research Fellow & PhD Student.
Project Title:
Unlocking the metabolism of the kidney prior to
transplantation.
Department:
School of Cancer Sciences with crossover with School of
Immunity & Infection.
No
Project Outline
Our research vision is to optimise the metabolic support during the machine
perfusion of kidneys for transplantation to safely preserve an organ for 72 hours prior
to transplantation.
The development of hypothermic machine perfusion (HMP) has already
demonstrated clinical benefit and is associated with reduced rates of Delayed Graft
Function and improved graft survival in machine perfused kidneys compared to those
preserved in traditional Static Cold Storage (1-2).
There is increasing evidence that substantial metabolic activity occurs during HMP
and may have a protective effect. However the metabolic activity in the ex-vivo,
hypoxic, hypothermic environment provided by HMP is poorly understood. Initial
work from our group, using 1D 1H-NMR spectroscopy has identified a panel of 28
key metabolites within the kidney perfusate. We have found that the metabolomic
profile from perfusates of Immediate Graft Function kidneys differs from that of
Delayed Graft Function. These changes are apparent as little as 45 minutes after
perfusion commences (3,4).
A greater understanding of the active metabolic pathways within the HMP kidney
may allow a target for metabolic manipulation during perfusion. There is some
evidence that broad metabolic support with cell culture like perfusate improves the
viability of damaged porcine kidneys (5).
We intend to perform further studies utilising 2D NMR techniques incorporating
labelled metabolites into perfusion fluid to determine their metabolic fate.
13
C
The student will join an active research team, investigating an exciting topic within
renal transplantation. This study will facilitate a more complete understanding of the
metabolic processes within a machine perfused kidney using novel 13C technology
with the intention of developing a new metabolically supportive perfsion fluid. There
is no similar published work in this field and would be an exciting opportunity for an
aspiring academic clinician. This would be particularly suited to a canditate with an
interest in surgery or transplantation.
References
63
1. Moers C, Pirenne J, Paul A, Ploeg RJ, Machine Preservation Trial Study G (2012)
Machine perfusion or cold storage in deceased-donor kidney transplantation.
N Engl J Med 366: 770-771.
2. Lodhi SA, Lamb KE, Uddin I, Meier-Kriesche HU (2012) Pulsatile pump decreases
risk of delayed graft function in kidneys donated after cardiac death. Am J
Transplant 12: 2774-2780.
3. Guy AJ, Nath J, Cobbold M, Ludwig C, Tennant DA, et al. (2014)
Metabolomic Analysis of Perfusate During Hypothermic Machine
Perfusion of Human Cadaveric Kidneys. Transplantation Sep 12.
PMID: 25222017
4. Nath J, Guy A, Smith TB, Hodson J, Cobbold M, et al. (2014)
Metabolomic perfusate analysis during kidney machine perfusion:
the pig provides an appropriate model for human studies. PLOS One
(publication date due 12 Dec).
5. Brasile L, Stubenitsky BM, Haisch CE, Kon M, Kootstra G (2005) Repair of
damaged organs in vitro. Am J Transplant 5: 300-306.
The student will work under the close supervision of the current final year PhD
student who will oversee day-to-day training and will provide surgical training and
full supervision during any animal kidney perfusion experiments. As well as the daily
interactions with the research team, the student will be expected to attend regular
departmental ‘lab meetings’, ensuring exposure to a range of disciplines. We will
provide in house training on the fundamental concepts of NMR analysis and scientific
manuscript preparation.
The student role.
The student will be given appropriate training and be expected to assist during pig
kidney perfusion experiments, including visits to the abattoir with on-site perfusion.
They will be given the surgical training for this and all experiment will be overseen by
our surgical research fellow. They will be given training to and expected to perform
basic laboratory tasks including perfusion fluid preparation, Cell extraction, sample
preparation and Cell culture. A significant element of the project work relies on NMR
analysis and the student will be trained and expected to process and interpret NMR
spectra – (both 1D and various types of 2D NMR experiments).
Our research group has been expanding our publication output and has produced 2
high impact papers over the past 6 months (ref 3 & 4). The student would be
expected to contribute towards further manuscript preparation and we would expect
them to attain a publication as a result of their BMedSc.
64
Lead Supervisor:
Dr Yuk Ting Ma (Senior Clinical Lecturer)
Contact Email:
Telephone:
[email protected]
Co Supervisor:
Dr Sarah Leonard (Post-Doctoral Fellow)
Project Title:
The use of a genome-wide haploid genetic screen to
identify the critical genes that govern response to
sorafenib in the treatment of hepatocellular
carcinoma
School of Cancer Sciences, University of Birmingham
Department:
No
Project Outline
Loss-of-function genetic screens in model organisms have helped to elucidate many
biological processes, but such large scale gene disruption has not been possible in
human cells due to their diploid genome. Recently, a genome-wide loss-of-function
screening method has been developed in a human haploid cell line (Carette 2009). In
this approach, insertional mutagenesis was used to generate null alleles in a cancer
cell line haploid for all chromosomes except chromosome 8 (KBM7). Haploid
screening has recently been used to identify inter alia the critical mediator of
tunicamycin toxicity, the host receptor for Clostridium difficile toxin, and the host
factors important for Chlamydia trachmomatis infection (Reiling 2011,
Papatheodorou 2011, Rosmarin 2012). This approach avoids the potential off-target
effects and incomplete knockdowns of a siRNA screen, and has the advantage of
causing only a limited number of disrupted genes per cell, which can be readily
identified by deep sequencing. This haploid loss-of-function genetic screening
method thus provides an exciting and novel strategy for identifying the critical genes
that govern response to novel drugs or drug combinations.
Hepatocellular carcinoma (HCC) is a leading cause of cancer death globally and its
incidence is increasing in the West, with the increasing burden of chronic liver
disease. Until recently, systemic treatment options for advanced disease were
limited. However, randomised clinical trials have demonstrated that the multikinase
inhibitor, sorafenib, prolongs survival in appropriately selected patients (Llovet 2008)
and this drug has become the standard for patients with advanced HCC. However
the effects of sorafenib are modest (2.8 months improvement in median survival), is
associated with significant toxicity and is expensive (approx. £3000 per month per
patient). Restricting the use of sorafenib to patients who will respond will improve its
clinical effectiveness and thus cost-effectiveness, and will also spare unnecessary
toxicity in patients who will not respond. However, there is currently no predictive
biomarker of response.
We have exposed gene-trapped mutagenized KBM7 cells to sorafenib and surviving
(resistant) clones have been expanded, harvested and genomic DNA extracted. The
mutated genes are currently being identified by mapping of the insertion sites using
high throughput sequencing.
References
65
Carette JE, Guimaraes CP, Varadarajan M, et al. Haploid genetic screens in human cells
identify host factors used by pathogens. Science 2009; 326: 1231-1235.
Llovet JM, Ricci S, Mazzaferro V et al. Sorafenib in advanced hepatocellular carcinoma. N Engl
J Med 2008; 359: 378-390.
Papatheodorou P, Carette JE, Bell GW, et al. Lipolysis-stimulated lipoprotein receptor (LSR) is
the host receptor for the binary toxin Clostridium difficile transferase (CDT). Proc Natl Acad
Sci U S A 2011; 108: 16422-16427.
Reiling JH, Clish CB, Carette JE, et al. A haploid genetic screen identifies the major facilitator
domain containing 2A (MFSD2A) transporter as a key mediator in the response to
tunicamycin. Proc Natl Acad Sci U S A 2011; 108: 11756-11765.
Rosmarin DM, Carette JE, Olive AJ, et al. Attachment of Chlamydia trachomatis L2 to host
cells requires sulfation. Proc Natl Acad Sci U S A 2012; 109: 10059-10064.
The lead supervisor, Dr Ma, will meet with the student weekly. Dr Leonard will
supervise the student in the laboratory on a daily basis.
The student role.
In this project, the student will perform the validation experiments on selected
candidate genes identified from the haploid genetic screen described above.
To confirm that the gene trap insertion of the affected locus is responsible for the
observed resistance to sorafenib in mutagenized KBM7 cells, individual resistant
clones carrying the defined gene-trap insertion will be isolated. Absence of gene
expression will be confirmed using immunoblotting, and the wild-type cDNA
sequence of the gene of interest will then be ectopically expressed in these null-cells
to assess if drug sensitivity can be restored. Candidate genes will then be examined
for their conserved function in hepatocellular carcinoma cells by first determining if
expression level correlates with cell line sorafenib sensitivity, and second, by
knocking down or over-expressing the gene in sensitive and resistant HCC cell lines,
respectively.
66
Lead Supervisor:
Konstantinos Manolopoulos
Contact Email:
Telephone:
[email protected]
4147525
Co Supervisor:
Gareth Lavery
Project Title:
Understanding adipose tissue dysfunction in obesity
and type 2 diabetes
Department:
CEM-CEDAM
No
Project Outline
Obesity is the leading cause of insulin resistance and type 2 diabetes. Adipose tissue
dysfunction has been proposed as being central in the pathophysiology of obesityrelated complications. However, the exact mechanisms remain unclear. Furthermore,
there is a big variation in the response to weight-loss promoting therapies in patients
with type 2 diabetes, suggesting intrinsic differences in adipose tissue function
between individuals. The aims of this project are two-fold: Firstly, to analyse a
clinical database of obese diabetes patients receiving treatments known to promote
weight loss (GLP-1 agonists, DPP-4 antagonists, SGLT-2 antagonists), and establish
predictors of weight loss responses. Secondly, to characterise adipose tissue function
in various degrees of obesity in a lab-based project. For this, adipose tissue biopsies
obtained from a range of patients with obesity will be assessed in terms of gene and
protein expression profiles, histomorphology and in vitro metabolic function. Lab
methods will involve cell culture, RNA extraction and qPCR techniques, Western blots
and in vitro assays of glucose and lipid metabolism. By linking the in vitro findings
with the clinical data this project will help deepending our understanding of adipose
tissue dysfunction in obesity and type 2 diabetes.
References
Shulman GI , Ectopic fat in insulin resistance, dyslipidemia, and cardiometabolic
disease.
N Engl J Med. 2014 Sep 18;371(12):1131-41.
KN Manolopoulos, F Karpe, Frayn KN, Gluteofemoral body fat as a determinant of
metabolic health. Int J Obes (Lond). 2010 Jun;34(6):949-59
Initial period of focused training in data analysis methodology and in vitro methods.
Open door policy for day-to-day queries and support with data analysis and lab work.
Weekly meetings to assess progress and provide support.
The student role.
Following an initial training period you would be expected to carry out the database
analysis and apply statistical regression models to identify predictors of weight loss.
In the lab, you would be responsible for your own cell cultures, and in vitro
experiments in adipocytes derived from the patient biopsies. While you are expected
to work independently, ample training and support will be provided.
67
Lead Supervisor:
Dr Helen McGettrick
Contact Email:
Telephone:
[email protected]
0121 414 4043
Co Supervisor:
Dr Andrew Filer
Project Title:
Synovial fibroblasts release microvesicles with different
bioactivity in acute and chronic inflammation: Role in
regulating the inflammatory infiltrate?
Department:
No
Project Outline
Rheumatoid arthritis (RA) is a prototype inflammatory disease in which synovial
fibroblasts induce joint damage and maintain the persistence of the inflammatory
episode [1]. Multiple epigenetic changes drive the acquisition of a pathogenic
phenotype in rheumatoid synovial fibroblasts that underpins these aberrant
behaviours. A window of opportunity exists during the first 3 months of RA when the
disease is still evolving and more responsive to current treatment strategies [2,3].
This raises intriguing questions about the development of the fibroblast phenotype
during the earliest stages of persistent arthritis.
One way fibroblasts moderate the inflammatory infiltrate is by “talking” to
neighbouring vascular endothelium. We have previously shown that rheumatoid
synovial fibroblasts activate endothelium to inappropriately recruit leukocytes in vitro
[4]. Using the Birmingham Early Arthritis Cohort (BEACON) we have recently shown
that fibroblasts from acutely resolving arthritis are immunosuppressive, inhibiting
lymphocyte adhesion to cytokine-treated endothelium in an IL-6 and TGFb
dependent manner. Interestingly, this effect was lost in very early RA, such that
fibroblasts no longer suppressed recruitment but rather usurped the action of IL-6
and TGFb to support increased lymphocyte infiltration [5].
One hypothesis is that synovial fibroblasts release membrane microvesicles (MV >
1µm) that bind to other cells, such as endothelial cells, and deliver signalling
molecules or change the cell's adhesive surface [6,7]. Indeed increased levels of
circulating MV in RA have been shown to be related to disease activity [8]. We now
wish to characterise the MV derived from resolving and very early RA fibroblasts and
assess their role in modulating endothelial and leukocyte behaviour. Identifying key
fibroblast derived mediators responsible for the switch from resolving to persistent
lymphocyte recruitment will enable the development of novel therapeutic agents that
target fibroblasts to restore normal patterns of lymphocyte entry and exit.
References
1. Filer A. The fibroblast as a therapeutic target in rheumatoid arthritis. Current
Opinion in Pharmacology 2013;13:413-9.
2. Cush JJ. Early rheumatoid arthritis -- is there a window of opportunity? J
Rheumatol Suppl 2007 Nov;80:1-7.
3. Raza K et al. Early rheumatoid arthritis is characterized by a distinct and transient
68
synovial fluid cytokine profile of T cell and stromal cell origin. Arthritis Res Ther
2005;7(4):R784-R795
4. McGettrick HM et al. Fibroblasts from different sites may promote or inhibit
recruitment of flowing lymphocytes by endothelial cells. Eur J Immunol 39, 113-125,
2009.
5. McGettrick, HM et al. Fibroblasts lose their immunosuppressive ability early in the
development of Rheumatoid Arthritis: Effects on lymphocyte recruitment. Annals of
Rheumatic Diseases, 2014, 73s1:A19 doi: 10.1136/annrheumdis-2013-205124.43.
6. Loyer X et al. Microvesicles as cell-cell messengers in cardiovascular diseases. Circ
Res 2014;114:345-353.
7. Barteneva NS et al. Circulating microparticles: square the circle. BioMed Central
Review 2013, 14;23.
8. Sellam J et al. Increased levels of circulating microparticles in primary Sjogren’s
syndrome, systemic lupus erythematosus and rheumatoid arthritis and relation with
disease activity. Arthritis Res Ther 2009, 11:R156.
The student will be part of the Leukocyte Trafficking Group
(www.birmingham.ac.uk/leukocyte-trafficking ~20 members ) based in the IBR
West Extension, and the Rheumatology Research Group (~40 members) located
within University Hospital Birmingham. In addition, the supervisors collaborate with
Dr Paul Harrison, a world leading expert on MV biology and their analysis, based in
the Centre for Translational Inflammation Research. Collectively, these have created
a strong mentoring environment and well established training system which will
support the student throughout their studies. The student will attend 3 separate
weekly meetings dedicated to (i) stromal cell biology, (ii) leukocyte trafficking and
(iii) Rheumatology. Supervisors, or existing lab members, have extensive experience
in the specialist techniques, such as 3D multi-cellular flow-based adhesion assays,
required for the project. Moreover, this project will run alongside research being
undertaken as part of Dr Helen McGettrick’s Arthritis Research UK Career
Development Fellowship.
The student role.
The student will test the hypothesis that the phenotype and function of MV release
from synovial fibroblasts are different in acute and chronic inflammation.
The main aims of the project will be to:
1. Characterise MV released from synovial fibroblasts isolated from patients
attending BEACON.
2. Analyse the ability of fibroblast derived MV to bind to endothelial cells.
3. Using flow based adhesion assays, examine the bioactivity of fibroblast
derived MV (i.e. the ability of fibroblast derived MV to effect endothelial
recruitment of leukocytes).
Key practical skills developed will include cell culture (aseptic technique), quantitative
PCR, flow cytometry, confocal microscopy, flow-based adhesion assays.
69
Lead Supervisor:
Prof. Jane McKeating
Contact Email:
Telephone:
Co Supervisors:
Dr. Alan Zhuang
Project Title:
Schools:
Circadian rhythms and HIV infection.
Project Outline
The immune system is modulated by a wide variety of environmental influences and
recent studies highlight a role for circadian rhythms to regulate host innate and adaptive
immune responses via the expression of “clock genes”1,2. Macrophages show circadian
responses to pathogen-associated challenges via clock genes such as Rev-erbA that
regulate expression of cytokines IL6, CXCL6, CXCL11 and chemokine CCL23,4. Much of the
published literature is based on animal models and there is a need to investigate the
influence of circadian rhythms on innate immunity and susceptibility to infection in
humans. Recent studies reporting that T cell activation in human immunodeficiency virus
(HIV) infection associates with cortisol levels5 and that HIV-encoded Tat protein alters
circadian activity6 suggest a role for clock genes in the viral lifecycle.
Hypothesis: The cellular response to HIV infection is regulated by oscillations in clock
gene expression.
We will evaluate this hypothesis with the following objectives:
1. Does T cell circadian cycle associate with permissivity to support HIV replication?
Experiments will utilize reporter HIV strains engineered to express GFP or
luciferase, enabling rapid quantitation of viral replication.
2. Do changes in viral replication correlate with clock gene expression?
3. Does modulation of clock gene expression affect HIV infectivity or cytokine
responses?
These studies will increase our understanding of host pathways that regulate HIV
replication and will influence HIV vaccine trial design.
References
1. Curtis et al (2014). Circadian Clock protein and immunity. Immunity 40: 178-86.
2. Gibbs et al (2014). An epithelial circadian clock controls pulmonary inflammation and
glucocorticoid action. Nat Med 20: 919-26.
3. Keller et al (2009). A circadian clock in macrophage controls inflammatory immune
responses. Proc Natl Acad Sci USA 106:21407-21412.
4. Gibbs et al (2012) The nuclear receptor Rev-ErbA mediates circadian regulation of innate
immunity through selective regulation of inflammatory cytokines. PNAS USA 109:582-587.
5. Patterson et al (2013). Cortisol patterns are associated with T cell activation in HIV.
PLOS ONE 8: e63429.
6. Wang, T. et al. (2014). HIV Tat protein affects circadian rhythmicity by interfering with
the circadian system. HIV Med 15: 565-70.
The student will be supervised by Prof McKeating and Dr Zhuang, providing constant
advice and supervision during the course of the project. The student will have weekly 1-1
70
meetings with the supervisors to monitor progress, troubleshoot technical problems and
provide advice and encouragement.
The student role.
This project offers the potential for training in key aspects of medical research including
molecular virology and cell biology. The student would work alongside a team of
scientists, working in novel areas of research with key relevance to medically important
pathogens. The student will be trained to work with ACDP2 pathogens as well as a critical
working knowledge of techniques such as tissue culture, virus replication, Western blot,
transfections and immuno-fluroescence. Within the course of the placement it is expected
the student would develop as an independent researcher able to design and conduct their
own experiments; with the aim to acquire sufficient quality and novelty to merit
publication. In addition the student would be expected to play an active part in the group
including journal clubs and lab meetings.
71
Lead Supervisor:
Jane McKeating
Contact Email:
Telephone:
Co Supervisors:
Dalan Bailey
Project Title:
Optimising lentiviral pseudotypes to study Ebola
virus tropism.
Schools:
Project Outline
Ebola virus (EBOV) is a member of the Filoviridae family that causes severe
hemorrhagic fever with up to 90% mortality. Unsurprisingly EBOV is classified as a
bio-safety level 4 pathogen, limiting studies to designated laboratories with
appropriate containment facilities. Lentiviral pseudotypes expressing foreign viral
encoded glycoproteins have been used to study internalization pathways of many
pathogenic human viruses including HIV and SARS under general laboratory
conditions. Pseudotypic viruses undergo a single round of infection and expression of
reporter proteins provides a quantitative assessment of glycoprotein-dependent
particle entry. Recent reports demonstrate the infectivity of lentiviral pseudotypes
expressing the EBOV glycoprotein GP1,2, validating this approach for studying viral
tropism.
EBOV GP1,2 regulates virus entry into cells and is a major virulence factor implicated
in
pathogenesis,
including
cytopathicity,
endothelial
dysfunction
and
immune suppression (1,2). GP1,2 expression is regulated by an RNA editing
mechanism where full-length GP1,2 mRNA is produced by slippage of the viral
polymerase at an editing site (3,4). Approximately 20% of transcripts are produced
in this way with the remaining transcripts containing a premature stop codon which
encodes a soluble truncated glycoprotein (sGP). A recent study reported that high
levels of GP1,2 reduced the infectivity of lentiviral pseudotypes by undefined
mechanisms (5). Previous studies highlight a role for GP1,2 in evading host
immune responses leading us to suggest that the relative expression of GP1,2 and
sGP regulate virus production, infectivity and evasion of innate and adaptive host
immune responses.
In this project we will modify the region of EBOV at which editing occurs to alter the
rate of GP1,2 and sGP production and assess the impact on lentiviral pseudotype
production and infectivity. These studies will optimise the production of EBOV
pseudoparticles to study cellular tropism.
References
1. Mohamadzadeh M, et al. 2007. How Ebola and Marburg viruses battle the immune
system. Nat Rev Immunol 7:556-567.
2. Yang ZY, et al. 2000. Identification of the Ebola virus glycoprotein as the main
viral determinant of vascular cell cytotoxicity and injury. Nature Medicine 6:886-889.
3. Sanchez A, et al. 1996. The virion glycoproteins of Ebola viruses are encoded in
two reading frames and are expressed through transcriptional editing. PNAS USA
93:3602-3607.
72
4. Volchkov VE, et al. 1995. GP mRNA of Ebola virus is edited by the Ebola virus
polymerase and by T7 and vaccinia virus polymerases. Virology 214:421-430.
5. Mohan GS, et al. 2014. Less is more: Ebola surface glycoprotein expression levels
regulate virus production and infectivity. J Virology, in press.
The student will be co-supervised by members of the McKeating and Bailey groups.
Our joint expertise in viral entry and cell-cell fusion mechanisms will ensure the
prospective student is supervised to a high level, both technically and theoretically.
The student will have regular (weekly) 1-1 meetings with the supervisors to monitor
progress, troubleshoot technical problems and provide advice and encouragement.
The student role.
This project offers the potential for training in key aspects of medical research
including molecular virology and cell biology. The student would work alongside a
team of scientists, working in novel areas of research with key relevance to medically
important pathogens. The student will be trained to work with ACDP2 pathogens as
well as a critical working knowledge of techniques such as tissue culture, virus
replication, Western blot, transfections and immuno-fluroescence. Within the course
of the placement it is expected the student would develop as an independent
researcher able to design and conduct their own experiments; with the aim to
acquire sufficient quality and novelty to merit publication. In addition the student
would be expected to play an active part in the group including journal clubs and lab
meetings.
73
Lead Supervisor:
Sally Roberts
Contact Email:
Telephone:
Co Supervisor:
[email protected]
Project Title:
The role of hypoxia inducible factors in the human
papilloma virus lifecycle and pathogenesis.
Schools:
Cancer Sciences and Immunity and Infection
Jane McKeating
No
Project Outline
Human papilloma virus (HPV) infections are associated with the development of over
300,000 epithelial cancers each year. Of the 275,000 women who die from cervical
cancer, 80% occur in low-economic countries where the incidence of HPV-associated
cancers is increasing. HPV prophylactic vaccines are not widely available in these
countries and offer no therapeutic cure to individuals who have developed cancer.
Thus, there is an urgent need for more affordable and easily administered
anti-viral and therapeutic interventions. To do this we need a better
understanding of the HPV life cycle and of mechanisms associated with HPV
pathogenesis.
HPV has evolved numerous strategies to hijack the host cell machinery to establish
and maintain infection, including the stabilization of hypoxia inducible transcription
factors (HIF)1. HIFs regulate cellular metabolism, angiogenesis, proliferation and
migration, enabling a cell to respond to a low oxygen or hypoxic environment2. HPV
and HIF-1α have been reported to synergistically promote cancer lesions in
transgenic mice3. Furthermore, increased HIF expression correlates with a poor
prognosis of patients with cervical cancer lesions.
We will investigate whether: (1) HPV promotes HIF transcriptional activity at
different stages of the HPV life cycle; (2) HPV stabilizes HIF in anogenital and
oropharyngeal culture systems and (3) inhibiting HIF activity impacts on HPV
genome replication and protein expression.
This project will increase our
understanding of the role HIFs play in the HPV lifecycle and whether they provide a
new therapeutic target.
References
1. Tang, et al. Overexpression of human papilloma type 16 oncoproteins
enhances HIF protein accumulation and VEGF expression in human cervical
carcinoma cells. (2007). Clin Cancer Research 13: 2568-2576.
2. Wilson, et al. Hypoxia inducible factors in liver disease and hepatocellular
carcinoma: Current understanding and future directions. (2014). J Hepatol, in
press.
3. Lu, et al. HIF-1 facilitates cervical cancer progression in HPV 16 transgenic
mice. (2007). Am J Pathol 171: 667-681.
The student will be co-supervised by members of the Roberts and McKeating groups.
As such there will be constant advice and supervision during the course of the
project from group leaders, their postdocs and PhD students. Our joint expertise in
HPV biology (Roberts) and HIF biology (McKeating) will ensure the prospective
74
student is supervised to a high level, both technically and theoretically. The student
will have regular (weekly) 1-1 meetings with the supervisors to monitor progress,
troubleshoot technical problems and provide advice and encouragement.
The student role.
This project offers the potential for training in key aspects of medical research
including molecular virology and cell biology. The student would work alongside a
team of scientists, working in novel areas of research with key relevance to medically
important pathogens. The student will be trained to work with ACDP2 pathogens as
well as a critical working knowledge of techniques such as tissue culture, virus
replication, Western blot, transfections and immuno-fluroescence. Within the course
of the placement it is expected the student would develop as an independent
researcher able to design and conduct their own experiments; with the aim to
acquire sufficient quality and novelty to merit publication. In addition the student
would be expected to play an active part in the group including journal clubs and lab
meetings.
75
Lead Supervisor:
Dr Mike Milward
Contact Email:
Telephone:
[email protected]
0121 466 5132
Co Supervisor:
Prof Paul Cooper, Dr Will Palin
Project Title:
Novel non-antibiotic-based light irradiation
approaches for decontamination
Department:
School of Dentistry
NO
NO
Project Outline
Increasing bacterial antibiotic resistance is causing major concern within the
community and for healthcare professions and ultimately the World Health
Organisation has identified antibiotic resistance as a major threat to public health.
The Assistant Director-General for Health Security has stated that “Without urgent,
coordinated action by many stakeholders, the world is headed for a post-antibiotic
era, in which common infections and minor injuries which have been treatable for
decades can once again kill”. It is therefore of major importance that alongside
development of new types of antibiotics that other modalities to treat bacterial
infections are developed.
Photobiomodulation or Low level light therapy (LLLT) utilises low power (<500mW)
light emitting diodes (LEDs) or lasers which have a direct biological effect. Varying
the wavelength and dose (irradiance x time) of light delivered has been shown to
provide a range of beneficial biological actions including increased healing, reduced
inflammation and antibacterial effects. Addition of a photosensitizer can also be used
to enhance the antimicrobial action of light – this approach is termed photodynamic
therapy (PDT). A number of natural plant based extracts as well as synthetic
nanoparticles have been shown to exhibit antimicrobial action when exposed to
certain light wavelengths and irradiation conditions. Notably in general it is the
generation of reactive oxygen species following the light activation of these
intermediaries which results in the bactericidal activity.
This project aims to investigate the potential for LLLT and PDT to inhibit bacterial
growth and viability. Studies will investigate both direct action of light (LLLT) and via
use of photosensitizing agents, such as natural plant-based extracts and synthetic
nanoparticles (PDT). The ultimate aim is to develop a non-toxic, non-antibiotic based
approach for decontaminating infected wounds throughout the body as well as
related healthcare devices and equipment.
The study will comprise 3 core research areas:
1. Light measurement:
The initial objective is to accurately characterise the light sources to be used
in this study. Research into LLLT often fails to appropriately and/or accurately
characterise and report the light energy that cells receive, which undermines
76
the findings reported. Therefore initial studies will underpin the downstream
understanding of the light delivered for use in LLLT & PDT using established
methods in the research unit.
2. Bacterial Culture and Antibacterial assay:
A range of Gram positive & negative bacteria associated with wound
infections will be grown using in both broth and biofilm cultures. The growth
characteristics will be determined using turbidity and colony counting assays.
Data generated will identify optimal conditions for use in subsequent
antibacterial assays.:
Studies will subsequently determine the effect on bacterial cell growth and
viability of (i) varying light irradiances/doses and wavelengths, and (ii) the
use of a range of novel photosensitizing agents.
3. Cytotoxicity analysis:
Light irradiation conditions identified as having antibacterial activity by direct
action or via the photosensitizing agent intermediary will be investigated with
regards to their application safety in eukaryotic cells in vitro. Standard
metabolic and genotoxic assays, eg MTT and DNA comet, will be applied in
this aspect of the work.
It is envisaged that many irradiation parameters will be investigated as part
of this work using a bespoke high-throughput light delivery device in order to
determine the optimal wavelength and dose for decontamination. Combined
this data will be used to underpin the development of a new therapeutic
device for the disinfection of wounds as well as healthcare contaminated
devices and equipment.
References
1. Ultraviolet Radiation in Wound Care: Sterilization and Stimulation.
Gupta A, Avci P, Dai T, Huang YY, Hamblin MR. Adv Wound Care (New
Rochelle). 2013 Oct;2(8):422-437.
2. Novel strategies to fight Candida species infection. Rodrigues ME, Silva S,
Azeredo J, Henriques M. Crit Rev Microbiol. 2014 Nov 10:1-13.
3. Ultra-low power laser stimulation impairs the adhesion of Staphylococcus
aureus to primary human cells, and interferes with the expression of
staphylococcal pathogenic factors. Petruzzelli S, Congiu A, Gallamini M,
Pompei R. New Microbiol. 2014 Apr;37(2):193-9. Epub 2014 Apr 1.
4. Antimicrobial photodynamic therapy using a diode laser with a potential new
photosensitizer, indocyanine green-loaded nanospheres, may be effective for
the clearance of Porphyromonas gingivalis. Nagahara A, Mitani A, Fukuda M,
Yamamoto H, Tahara K, Morita I, Ting CC, Watanabe T, Fujimura T, Osawa
K, Sato S, Takahashi S, Iwamura Y, Kuroyanagi T, Kawashima Y, Noguchi T.
J Periodontal Res. 2013 Oct;48(5):591-9.
5. Flow cytometric assessment of Streptococcus mutans viability after exposure
to blue light-activated curcumin. Manoil D, Filieri A, Gameiro C, Lange N,
Schrenzel J, Wataha JC, Bouillaguet S. Photodiagnosis Photodyn Ther. 2014
Sep;11(3):372-9.
77
All the techniques proposed are well established within our research group, with staff
and associated laboratory personnel experienced in undertaking these experiments.
In addition we have a dedicated team of laboratory technicians who support students
in initial laboratory induction/training and also provide the day-to-day bench support
for experiments. Any student undertaking this project will have the necessary
resource & support required to successfully complete their project. Regular meetings,
likely weekly initially, will be organised to discuss progress and to provide guidance
and further ad hoc meetings will occur. The supervisors have extensive experience in
both undergraduate and postgraduate supervision.
The student role.
For students to get the most out of this project they will need to work diligently and
show commitment to developing the necessary technical skills to undertake this
study. Initially this will involve a thorough literature review to familiarise themselves
with the underpinning published literature and attending training in the laboratory
work. The student is always encouraged to discuss any project related issues with
the supervisors who operate an ‘open door’ approach in their support.
78
Lead Supervisor:
Professor Tim Mitchell
Contact Email:
Telephone:
[email protected] Tel: 46779
Personal assistant : [email protected]
Co Supervisor:
Dr Andrea Mitchell
[email protected]
Project Title:
Evaluation of statins to reduce inflammation using in
vitro models of pneumococcal pneumonia and
meningitis
Institute of Microbiology and Infection, School of Immunity
and Infection, Biosciences Building
Department:
Project Outline
Streptococcus pneumoniae (the pneumococcus) is part of the normal bacterial flora
of humans but can also cause serious life threatening diseases such as
pneumonia and meningiitis. We have shown that a pore forming toxin
(pneumolysin) plays a major role in the pathology of these diseases. This
toxin uses membrane cholesterol as its receptor. This project will therefore
investigate whether the use of statins (which block cholesterol synthesis in
mammalian cells) can reduce the effect of the toxin in disease pathology. In
order to do this you will test the effect of statins (simvastatin) on the ability
of the pneumococcus to induce pathology in cell culture models of blood
brain barrier (human brain endothelial cells) and lung (alveolar basal
epithelial cells). You will use several clinical isolates of the pneumococcus that
differ in their ability to produce active toxin as well as bacterial mutants in
which the gene for the toxin has been deleted or altered. The readouts will
be measurement of baterial attachment and toxicity to the cell culture
models. The effect of statins on cells will be monitored by measuring levels of
cholesterol in the cell. The amount of inflammation induced by the bacteria or
isolated toxin in the presence or absence of statins will be quantified by
measuring cytokine profiles. Level of apoptosis will also be measured using
standard assays.
References
Mitchell, T.J. The pathogenesis of streptococcal infections: from tooth decay to
meningitis. Nat. Rev. Microbiol. 2003. 1 (3) 219-30.
Boyd, A.R., Hinojosa, C.A., Rodriguez, P.J., Orihuela, C.J. Impact of oral simvastatin
therapy on acute lung injury in mice during pneumococcal pneumonia. BMC
Microbiology 2012, 12; 73.
Day to day routine laboratory supervision(how to use equipment, grow cell lines etc)
will be provided by the laboratory manager. Day to day scientific supervision will be
provided by the named second supervisor or one of the post-doctoral fellows in the
79
laboratory. The student will have weekly meetings with Professor Mitchell to discuss
project progress and plans. The student will also attend and present at the weekly
laboratory meetings so that he/she is also aware of the other research projects
running in the laboratory and what scientific techniques can be learned.
The student role.
The student will initially be given one-to-one supervision but will then be expected to
gain independence in experimental design and take ownership of the project. The
student will be expected to present data at regular group meetings and to interact
with collaborators. Ideally the student will be expected to produce a publication at
the end of the project.
80
Lead Supervisor:
Dr Neil Morgan
Contact Email:
Telephone:
[email protected]
0121 414 6820
Co Supervisor:
Mr Ben Johson
Project Title:
Molecular Genetic Investigation
Platelet-based Bleeding Disorders
Department:
Clinical and Experimental Medicine
of
Inherited
Yes or
No
Project Outline
Background
Platelets play an important role in normal haemostasis to prevent excess blood flow
following vascular injury and can be regulated by aggregation, adhesion, secretion or
procoagulant activities. Platelet bleeding disorders can present with variable
penetrance ranging from mild to severe bleeding. For example Von Willebrand
disease is the most common inherited bleeding disorder in which the mild forms are
highly under diagnosed. Therefore there are a large number of patients with
unclassified platelet bleeding disorders which underlies the need for comprehensive
molecular diagnostic tools which will increase the capacity for early and rapid
identification of these disorders. The recent advent of whole exome sequencing and
next generation technologies has greatly enhanced the probability and speed of
identifying mutations and hence causative genes in such conditions. Using this
approach we have recently identified novel genes (ANKRD18A and SLFN14) in
affected patients with inherited forms of thrombocytopenia (low platelet count)
which causes severe bleeding.
We have established a cohort of over 400 patients with platelet-based bleeding
disorders. In this project, you will be trained in the use of next generation
sequencing to identify genetic mutations in a small subgroup of these with a clear
phenotype, and perform corresponding biochemical studies to verify the defect.
Techniques to be used in the project
Gene identification studies (genetic mapping, bioinformatic analysis, second
generation sequencing), functional analysis of mutant gene products (protein
expression analysis, Western blotting, cell localisation studies, analysis of
downstream target genes).
References
81
1. Daly ME, Leo VC, Lowe GC, Watson SP, Morgan NV (2014) What is the role of
genetic testing in the investigation of patients with suspected platelet function
disorders? Br J Haematol, 165, 193-203.
2. Stockley J*, Morgan NV*, Bem D, Lowe GC, Lordkipanidzé M, Dawood B,
Simpson MA, Macfarlane K, Horner K, Leo VC, Talks K, Motwani J, Wilde JT,
Collins PW, Makris M, Watson SP, Daly ME: UK Genotyping and Phenotyping of
Platelets Study Group (2013) Enrichment of FLI1 and RUNX1 mutations in
families with excessive bleeding and platelet dense granule secretion defects.
Blood, 122, 4090-3. * equal contribution
3. Watson SP, Lowe GC, Lordkipanidzé M, Morgan NV; GAPP consortium (2013)
Genotyping and phenotyping of platelet function disorders. J Thromb Haemost 11
(Suppl 1), 351-63.
From a day to day basis both myself and Mr Johnson will be directly involved in the
laboratory supervision of the student.
The student role.
The student will perform the techniques mentioned above as well as playing a role in
defining the clinical phenotype of the inherited platelet-based bleeding disorders.
The immediate focus of the planned project will be the identification of new
causative genes using the powerful technique of whole exome sequencing. Further
investigation of the role of the identified disease-causing mutations/genes will be
performed. The precise techniques to be applied will depend on what type of gene is
identified and what is already known, as well as the availability of relevant patient
material such as cryopreserved peripheral blood mononuclear cells, disease tissue
and/or cell lines. Functional analysis of human mutated genes could include cellular
transfection and localisation by immunocytochemistry/flow cytometry, and in situ
mutagenesis and expression of mutant proteins. Real-time PCR and transcriptional
array analysis may be required to investigate expression of the wild type gene or, in
the case of a transcription factor, to investigate the effect of mutations on regulatory
activity.
The project is highly likely to yield novel genes for these platelet-based bleeding
disorders leading to a publication.
82
Lead Supervisor:
Professor Paul Moss
Contact Email:
[email protected]
Telephone:
Co Supervisors:
Miss Louise Hosie
Dr Jianmin Zuo
Project Title:
‘HLA-C restricted’ T cell immunotherapy for the
prevention of cytomegalovirus disease after stem
cell transplantation
Department:
School Of Cancer Sciences
No
Project Outline
Previous clinical trials within our group have demonstrated the successful treatment
of cytomegalovirus (CMV) disease within transplant (HSCT) patients involving the
transfer of CMV-specific CD8+ T cells isolated from seropositive donors1. Despite
these encouraging results, this therapy becomes difficult to apply to seronegative
patients that have either undetectable or rare CMV-specific CD8+ T-cells to isolate2.
In order to overcome this problem, an alternative approach is to transfer TCR
specificity to transgenic, i.e. transfer known TCR specificities to primary T cells.
Currently, adoptive therapy with HCMV-specific CD8+ T-cells TCR restricted through
HLA-A2 alleles are successful; however this allele is only present in ~30% of the UK
Caucasian population. Studies in our laboratory have recently identified a novel
immunodominant HLA-Cw*0702 restricted CD8+ T-cell response which stimulates
very strong T cell immune responses in healthy people (Hosie et al, unpublished
data). This HLA-C allele is expressed at a much higher percentage within the human
population, reaching a prevalence of 41%.
We plan to generate HLA-C restricted CMV-specific CD8+ T cells using TCR transfer
technology. These cells can then be used to treat CMV disease, especially for the
CMV negative immunosuppressed HSCT recipient.
83
Experimental Plan:1.
CD8+ T cell clones have been generated and will be purified with
MHC class I tetramers. RNA will then be extracted and the TCR gene will be
cloned, sequenced and subcloned into retrovirus expression vector.
2.
The retrovirus expressing the TCR will then be produced through
transfection and introduced into primary CD8+ T cells. The expression of the
TCR will be confirmed by Flow Cytometry after staining with the HLA-peptide
tetramer.
3.
The TCR transfected T cell will be assessed for their ability to kill
human cells which are infected with cytomegalovirus in the laboratory. This a
prelude towards using these reagents in cellular immunotherapy protocols.
The student will join one of the largest translational research units within the
University. The group has successfully trained over 20 B.Med.Sci (Clinical Sciences)
students in recent years.
References
1
Paul Moss, and Alan Rickinson, 'Cellular Immunotherapy for Viral Infection after Hsc
Transplantation', Nat Rev Immunol, 5 (2005), 9-20.
2
Andrea Schub, Ingrid G. Schuster, Wolfgang Hammerschmidt, and Andreas Moosmann,
'Cmv-Specific Tcr-Transgenic T Cells for Immunotherapy', The Journal of
Immunology, 183 (2009), 6819-30.
Miss Louise Hosie and Dr Zuo will be guiding the student about the project and
meeting regularly for discussion. Miss Hosie will help the student to master the labbased technologies and also perform the day to day direct supervision.
The student role.
1. In the lab, the student will carry out the lab work, such as PBMC separation,
FACS staining and in vitro function assay of CD8+ T cells.
2. The student will attend the internal and external seminars of the department,
will attend the group lab meetings and present their data, also will attend the
journal club and discussion relevant paper to broad their scientific knowledge.
84
Lead Supervisor:
Dr Ye Htun Oo
Contact Email:
Telephone:
[email protected]
MRC Clinician Scientist & Honorary Consultant Hepatologist
Room 536, 5th Floor, IBR; Centre for Liver Research& NIHR BRU
University of Birmingham & UHB NHS Foundation Trust
Wolfson Drive, B15 2TH, Edgbaston, Birmingham, UK
Co Supervisor:
Mr Thomas Pinkney
Dr Tariq Iqbal
Project Title:
Human hepatic innate lymphoid cells can be
controlled by regulatory T cells
Department:
Project Outline
Background
Innate lymphoid cells (ILCs) are emerging as crucial effectors of innate immunity and
tissue remodeling. ILCs are instrumental in immunity to invading microbes and also
in mucosa immunity. ILC had been classified into Type1-3 depending on their surface
markers, transcription factors and cytokine profile. The role of innate lymphoid cells
in liver diseases in unexplored especially its regulation by regulatory T cells. Our
recent preliminary data suggested the ILC are present in human inflammatory liver
disease and inflammatory bowel diseases.
Aim of the project
To investigate the phenotypic of ILC subsets in human inflammatory liver disease
and inflammatory bowel diseases.
Project outline
To investigate the ILC subsets, frequency in different human liver and bowel
tissues by immunohistochemistry and confocal microscopy.
Liver infiltrating ILC cells phenotype by analysing surface markers (including
lineage markers, CD127, CD161. We then analyse its homing receptors,
functional receptors, effector cytokines and transcription factors in explanted
human liver tissues from diseased and normal livers/ hepatic resection. Same
experiments will be performed with inflammatory bowel tissue.
If time permits, we will co-culture ILC with regulatory T cells and assess its
regulation by suppression of proliferation of ILC
Benefits
This will be the first study to investigate the role of ILC cells subsets in human liver
tissue and inflammatory bowel diseases. We could apply this knowledge of
85
immunopathogenesis in the translational medicine to target specific ILC cells subsets
to prevent inflammatory bowel diseases, cirrhosis, liver failure in future.
Student will have a opportunity to sit in and learn liver diseases in autoimmune and
viral hepatitis clinics and learn translational medicine and liver clinical trials.
Student will have an opportunity to present the work in national and European
meeting in results are novel.
References
Human type 1 innate lymphoid cells accumulate in inflamed mucosal tissues
Jochem H Bernink1,10, Charlotte P Peters1,2,10, Marius Munneke3,4, Anje A te
Velde1, Sybren L Meijer5, Kees Weijer4, Hulda S Hreggvidsdottir1,6, Sigrid E
Heinsbroek1, Nicolas Legrand4,9, Christianne J Buskens7, Willem A Bemelman7,
Jenny M Mjösberg1,8 & Hergen Spits Nature immunology
We have chosen to combine the technical expertise of three supervisors to ensure
the student is exposed to the maximal number of transferable research skills. Dr Ye
Htun Oo is dedicated to 80% of his time in the laboratory, which is well equipped
and set up for this type of study. Dr Iqbal and Mr Pinkey are both internationally
renowned for their work in inflammatory bowel diseases.
The Centre for Liver Research is part of the MRC Centre for Immune regulation and
includes a large number of scientists working on liver immunology so the student will
be working in a stimulating and supportive environment providing the student with
exposure to research staff at all stages of their research careers. Appropriate training
in research techniques and data analysis and presentation will be provided. We have
weekly meetings with supervisors and interactions with other members of the
research groups, who will be available day to day for advice and tuition.
The student role.
The student will be expected to perform experiments and conduct data analysis.
They will present data to the supervisors and team and wider research groups and
be expected to assimilate available published literature under guidance from the
supervisors. They will be trained and expected to work using good laboratory
practice under local standard operating procedures and be mindful of ethical use of
human tissue specimens. They will be working in a large research teams and thus
would be expected to interact with colleagues and use local facilities in a respectful
manner.
They could have an opportunity to sit in and observe in dedicated autoimmune
hepatitis and viral hepatitis clinics to correlate laboratory bench work findings to
pathogenesis and investigation of hepatic inflammation and autoimmunity. This
would provide the student with an opportunity to link basic science to the patient.
86
Lead Supervisor:
Dr. Jo Parish
Contact Email:
Telephone:
[email protected]
x58151
Co Supervisors:
Dr. Gosia Wiench, Dr. Sally Roberts
Project Title:
Genome-wide analysis of CTCF binding distribution
and HPV-induced reorganisation.
Department:
Cancer Sciences
Project Outline
Human papillomaviruses (HPVs) are the cause of benign and malignant lesions of the
cutaneous and mucosal surfaces of the skin. High-risk HPV types (e.g. 16, 18, 31)
are the cause of cervical cancer and many other anogenital carcinomas and cancers
of the head and neck. To maintain persistent infection, papillomaviruses target
several host cell pathways to alter cell cycle control and cellular gene expression
creating a favourable environment for viral gene expression and genome replication1.
Work in the Parish laboratory has shown that the host cell DNA binding factor CTCF
associates with the genomes of low- and high-risk HPV types. The normal cellular
function of CTCF is to regulate gene expression in multiple ways; CTCF binds to
defined regions of the host cell genome and is required for maintaining epigenetic
boundaries, gene enhancer blocking and regulating gene splicing and genetic
imprinting. CTCF is able to contribute to these important mechanisms of host cell
gene regulation by binding to DNA and either creating loop structures among cisregulatory elements or by physically blocking the path of the transcription
machinery2.
We have shown that CTCF recruitment to the HPV genome is important in the
regulation of HPV gene regulation and splicing. We also have good evidence that
HPV infection alters the normal cellular functions of CTCF leading to the hypothesis
that recruitment of CTCF to the HPV genome alters CTCF distribution within
the host cell genome and causes aberrant expression of host cell genes.
To address this hypothesis, the specific aims of the project are as follows:
1. Determine the distribution of CTCF in the cellular genome before
and after HPV infection. The genome-wide distribution of CTCF will be
compared in primary human foreskin keratinocytes (HFKs) before and after
transfection with HPV16 or HPV18 genomes. Chromatin immunoprecipitation
followed by next generation sequencing (ChIP-Seq) will be used to determine
the differences in CTCF distribution in this isogenic primary cell culture
87
system.
2. Determine whether HPV infection alters CTCF-dependent expression of
candidate host genes. An alteration in CTCF binding at specific genomic loci
could dramatically alter the activity of regional promoters and expression of
imprinted genes. Regions that are identified to differentially bind CTCF following
establishment of persistent HPV episomes will be analysed. The expression of
genes within close proximity to these regions will be assessed by quantitative
real-time PCR and western blot.
Completion of these aims will provide important insight as to how an oncogenic
virus manipulates host cell gene expression to enhance productivity of infection.
Furthermore, identification of genes that are aberrantly expressed following HPV
infection could enhance our understanding of how HPV causes cellular
transformation and highlight possible biomarkers that could be used for early
detection of HPV infection.
References
1. Doorbar, J. (2005) The papillomavirus life cycle. J. Clin. Virol. 32(suppl):S7-15.
2. Phillips, J.E. and Corces V.G. (2009) CTCF: Master weaver of the genome. Cell. 137:11941211.
The student will meet with the supervisors regularly to discuss progress and short
and long-term plans. In addition, the student will attend and present at weekly lab
meetings. Each week a member of the group presents their most recent
experimental data, which is then discussed in an informal and supportive manner.
We also have frequent journal club style meetings in which students and postdocs
present and critique a recent publication. These meetings provide useful discussion
points and an opportunity for feedback on presentation and critical analysis skills.
In addition, the student will be informally supervised within the laboratory by an
experienced postdoctoral research assistant. This provides a firm support network for
students in the laboratory that can be adapted to an individual’s specific needs.
The student role.
The student will be trained in all necessary techniques and will therefore perform the
laboratory work required to deliver the project goals. Importantly, to ensure
ownership of the project, the student will be given every opportunity to contribute
intellectually to the direction of the project, through discussion with the supervisors
during weekly review meetings and during laboratory group meetings. To aid this
intellectual input and to facilitate writing the thesis, the student will be expected to
review the literature underpinning the project and to keep abreast of the current
literature enabling the development and direction of the project and to ensure it
remains cutting edge.
88
Lead Supervisor:
Dr Eva Petermann
Contact Email:
Telephone:
[email protected]
41 49165
Co Supervisor:
Dr Yavor Hadzhiev
Project Title:
DNA replication stress in the developing embryo
Department:
Cancer Sciences (Petermann)
Clinical and Experimental Medicine (Hadzhiev)
Yes
Project Outline
DNA replication is the fundamental process by which all dividing cells copy their
chromosomes. If progression of DNA replication forks is impaired, this can be a
significant source of DNA damage, which in turn causes genomic instability or cell
death. This is known as replication stress (1).
A substantial number of gene that regulate DNA replication in human cells have been
discovered (e.g. ATR, CHK1), and human patients with germ line mutations in these
genes have been identified (2,3). Mutations in “DNA replication” genes cause
autosomal recessive disorders including Seckel syndrome, Meier-Gorlin syndrome,
microcephalic osteodysplastic primordial dwarfism, and others (4).
Patients with these syndromes experience developmental defects, particularly
proportional dwarfism and microcephaly. This suggests that replication stress during
embryonic development may prevent organismal growth, especially that of the brain.
This observation is fascinating because it suggests that control of DNA replication
may be one of the fundamental mechanisms regulating human growth and
development.
However the roles of replication stress during development are not understood.
Evidence that proper DNA replication underpins development is still lacking as data
from in cell culture stduies have been inconclusive. What is really needed are animal
models of the developing embryo, where conditions are very different from cells in
culture (4).
The aim of this project is to investigate whether deficiency in the gene mutated in
ATR-Seckecl syndrome (ATR) causes replication stress and developmental defects in
animal models of embryo development.
Zebrafish embryos will be used as model system that allows us to study molecular
processes including DNA replication as well as growth and development in vivo.
You will use embryos that have a mutation in the ATR gene, and also treat wild type
embryos with drugs that inhibit ATR or cause replication stress directly (e.g.
hydroxyurea). You will then measure replication stress using DNA fibre analysis and
DNA damage assays (5) and also monitor developmental defects and apoptosis,
particularly in the brain. The main techniques to be used will be immunological and
histological staining methods, microscopy, and Western blotting.
This project will help us to understand how replication stress in the embryo is
89
connected to human growth disorders. Deciphering the molecular mechanisms
underlying such disorders will be important for the development of treatments for
growth defects. The project will be ideal for those who would like to gain experience
in working with animal models and molecular biology (the molecular investigation of
DNA and proteins).
References
1. Zeman, M.K. & Cimprich, K.A. Causes and consequences of replication stress. Nature Cell Biology 16,
2-9 (2014).
2. Petermann, E. & Caldecott, K.W. Evidence that the ATR/Chk1 pathway maintains normal replication
fork progression during unperturbed S phase. Cell Cycle 5, 2203-9 (2006).
3. O'Driscoll, M., Ruiz-Perez, V.L., Woods, C.G., Jeggo, P.A. & Goodship, J.A. A splicing mutation
affecting expression of ataxia-telangiectasia and Rad3-related protein (ATR) results in Seckel syndrome.
Nat Genet 33, 497-501 (2003).
4. Klingseisen, A., & Jackson, A. P. Mechanisms and pathways of growth failure in primordial dwarfism.
Genes & Development, 25, 2011–2024 (2011).
5. Jones, R. M., Kotsantis, P., Stewart, G. S., Groth, P., & Petermann, E. BRCA2 and RAD51 Promote
Double-Strand Break Formation and Cell Death in Response to Gemcitabine. Molecular Cancer
Therapeutics, 13, 2412–2421 (2014).
The student will be supervised by Lead Supervisor Dr Petermann and Co-Supervisor
Dr Hadzhiev, a postdoctoral researcher in the group of Dr Ferenc Muller. Day-to-day
supervision will be ensured by Dr Petermann, who will introduce the student to the
project, hold weekly meetings with the student and operate an open-door policy for
the rest of the week. The student will further be supervised on a day-to-day basis by
members of the Petermann lab, who have expertise in the scientific background and
all molecular techniques used in this project. Dr Hadzhiev and members of the Muller
lab will advise the student if and when needed with aspects relating to work with
Zebrafish embryos.
The Petermann lab holds weekly meetings were results are presented to the group
and feedback obtained. The student will be encouraged to work on drafting the final
thesis over the course of the project and regularly submit drafts to the Lead
Supervisor to obtain feedback.
Our group is part of a larger cluster of research groups with extensive expertise in
human genetic disorders, DNA replication and DNA damage who hold weekly
meetings and will provide further opportunities of support for the student. All
previous project students in the Petermann lab have obtained first degrees on their
projects.
The student role.
The student will be expected to familiarise themselves with the background and
purpose of the project and the key literature in the field before and during the
course of the project. With the help of the supervisor and group members, she/he
will obtain a personal licence for Zebrafish and learn central laboratory methods in
Zebrafish embryo culture and molecular biology of the DNA damage response. With
support from the lab, the student will then apply the learnt methods to new
experiments, and analyse and interpret the data obtained. She/he will keep a
constant record of experiments conducted and results obtained, and regularly
present the work to the group in lab meetings. The student is expected to develop
increasing autonomy during the course of the project, take ownership of the project
as much as possible, and be able to write a small thesis at the end.
The student should spend all of the allocated time working on the project. The
90
student will be expected to work responsibly as part of a team, honour the rules of
the lab, and immediately report any problems encountered to the supervisor.
91
Lead Supervisor:
Laura JV Piddock
Contact Email:
Telephone:
[email protected]
0121 414 6966
www.antimicrobialagentsresearchgroup.com
Mark Webber
Co Supervisor:
Project Title:
From farm to fork: evaluating the dangers of antibiotic
resistance in the food chain
Department:
No
Is the Project Cancer related? No (Infection)
Project Outline
Bacteria resistant to antibiotics are an ever increasing threat to human health with
pathogens for which no effective treatment remains being observed in hospitals
around the world. Antibiotic resistance is a natural phenomenon and resistance
genes and resistant strains can spread between environments and hosts. A number
of pathogenic bacteria are common commensals or contaminants of food and the
food chain is a significant reservoir of human infection. The use of antibiotics in
farming has been implicated in the selection of resistant mutants which can then
infect humans. Of particular concern is the emergence of resistance in animals to
antibiotics used in humans as ‘last resort’ drugs for example colistin and
carbapenems. Resistance to both these agents has been seen in bacteria from food
animals.
The aim of this project is to determine the level of resistance to critical human
antibiotics in isolates of bacteria from food animals. Resistant isolates will be further
analysed to identify the specific resistance genes and their immediate genetic
context (i.e. are they on mobile elements which can be transferred between bacterial
strains). Identification of antibiotic resistance genes in bacteria from animals will
inform antibiotic use in animals, identify the emergence and prevalence of novel
resistance mechanisms and ultimately help prevent the selection of mutant strains
which pose a risk to human health.
The project will contain a mix of clinical microbiology methods, antibiotic
susceptibility testing, molecular biology, bacterial whole genome sequencing and
bioinformatics.
1.
2.
3.
References
Blair JM, Webber MA, Baylay AJ, Ogbolu DO, Piddock, LJ. Molecular
mechanisms of antibiotic resistance. Nat Rev Micro. 2015 In Press.
Cottell JL, Saw HT, Webber MA, Piddock LJ. Functional genomics to identify
the factors contributing to successful persistence and global spread of an
antibiotic resistance plasmid. BMC Microbiol. 2014 Jun 24;14:168. doi:
10.1186/1471-2180-14-168.
Jiang HX, Song L, Liu J, Zhang XH, Ren YN, Zhang WH, Zhang JY, Liu YH,
Webber MA, Ogbolu DO, Zeng ZL, Piddock LJ. Multiple transmissible genes
encoding fluoroquinolone and third-generation cephalosporin resistance co-
92
located in non-typhoidal Salmonella isolated from food-producing animals in
China.
Int
J Antimicrob
Agents.
2014 Mar;43(3):242-7. doi:
10.1016/j.ijantimicag.2013.12.005. Epub 2013 Dec 17.
Students will meet weekly with Prof Piddock and bi-weekly with both Prof Piddock
and Dr Webber. They will be supervised daily by members of the antimicrobials
research group which currently contains 15 full time members
(www.antimicrobialagentsresearchgroup.com) ensuring appropriate cover will be
available for student supervision. Piddock has successfully supervised numerous
intercalating medical student projects to completion.
The student role.
The student will be responsible for performing and analysing experiments under the
direction of Prof Piddock and Dr Webber and will be treated within the laboratory as
any other member of the research team. Daily supervision will be by Dr Vito Ricci, a
post-doctoral research fellow. This will ensure appropriate cover will be available for
student supervision. Piddock and Webber have successfully supervised eight
intercalating students in the last six years. The student will have responsibility for
investigating the background of the project and developing a good awareness of the
context and aims of the project
93
Lead Supervisor:
Dr Myriam Chimen
Contact Email:
Telephone:
[email protected]
01214144042
Co Supervisor:
Prof. Ed Rainger
Project Title:
Effect of
pathway
Department:
ageing
on
the
adiponectin/PEPITEM
No
Project Outline
We recently identified a novel pathway that regulates the recruitment
of T-lymphocytes across inflamed endothelium. This anti-inflammatory
pathway involves adiponectin, an anti-inflammatory cytokine produced by the
adipose tissue [1], known to modulate leukocyte recruitment in vivo [2]. In
our laboratory, we have shown that adiponectin inhibits T-lymphocyte
migration across inflamed endothelium. Adiponectin does not directly target
T-lymphocytes; rather it stimulates B-lymphocytes to secrete a novel
endogenous peptide (PEPITEM) that in turn modulates T-lymphocyte
recruitment (Figure 1). This novel regulatory peptide stimulates the release
of sphingosine-1-phosphate (S1P) by the endothelium that in turn modulates
T-lymphocyte recruitment. Interestingly, the adiponectin/PEPITEM pathway is
altered in autoimmune and chronic inflammatory diseases such as type 1
diabetes and rheumatoid arthritis (RA) and is therefore an exciting potential
therapeutic target. Interestingly, our preliminary data indicate that the
adiponectin/PEPITEM pathway is altered in the elderly which implies that
there is a natural process of senescence in this pathway that could contribute
to the risk of developing diseases such as RA.
This project will overall aim to determine the effets of age on the
adiponectin/PEPITEM pathway.
1
Lymphocyte
rolling
2
β1/β2-integrin
activation
4
3
6
Lymphocyte Lymphocyte spreading
arrest
and migration
8
Adiponectin
5
PEPITEM
T-lymphocyte
AdipoR1/2
CXCR3
9
DP2
S1PR1/4
T
7
B-lymphocyte
Spsn2
CXCL9-11)
Endothelial Cell
COX
PGD2
AA
S1P
SPHK1
B
PEPITEM
SPHK1 receptor
Endothelial cell
New steps in the regulation
of Lymphocyte migration
Figure
1:
Schematic representation of the endogenous B-cell
94
mediated regulation of T-cell migration during inflammation.
PEPITEM secretion from B-cells. PEPITEM signalling through an unknown
receptor on the endothelium results in S1P production, which subsequently
inhibits T-cell migration. (AdipoR1/2: adiponectin receptors 1 and 2; S1P:
sphingosine-1-phosphate; S1P1/4: S1P receptors 1 and 4).
Plan of investigation:
Aim 1: Quantification of Adiponectin receptors in healthy ageing cohorts.
We have measured the expression of both adiponectin receptors (AdipoR1
and AdipoR2) on healthy controls from a small cohort of subjects and found a
negative correlation between both receptors expression and age. Our aim in
this study is to quantify the expression of both receptors on defined age
cohorts of 20-30 years old, 30 to 40 years old, 40 to 50 years old and 60 plus.
In addition, the subjects will be grouped according to gender and ethnicity.
The expression of both adiponectin receptors will be measured by flow
cytometry and real-time-PCR following well-established protocols in our
laboratory.
Aim 2: Measure the effests on adiponectin and PEPITEM on lymphocyte
transmigration in the healthy ageing cohort.
In our T1D and RA study, the levels of adiponectin receptors on Blymphocytes negatively correlates with the inhibitory effects of adiponectin on
peripheral blood lymphocyte transmigration. In this project we would like to
assess the response of lymphocyte to adiponectin in a functionnal
transmigration assay and correlate this with age and levels of adiponectin
receptors on B-lymphocytes.
References
1. Kadowaki T et al. (2006) Adiponectin and adiponectin receptors in insulin
resistance, diabetes, and the metabolic syndrome. J Clin Invest. 116:17841792.
2. Ouedraogo R et al. (2007) Adiponectin deficiency increases leukocyteendothelium interactions via upregulation of endothelial cell adhesion
molecules in vivo. J Clin Invest. 117:1718-1726.
The student will join the well-established collaborative research group of Prof.
Rainger within the Institute for Biomedical Research. The student will
collaborate with the group of Prof. Janel Lord in order to obtain the different
age cohort samples. Within these groups, the student will attend meetings on
a weekly basis with the core of people working on the PEPITEM project.
95
These meetings will allow informal and supportive discussion of the student’s
progress and establishment of short and long term plans. The student will be
supervised in the laboratory on a daily basis by an experienced post-doctoral
fellow who conducted her PhD on PEPITEM (Dr Myriam Chimen).
The student role.
The student will learn a variety of useful and cutting-edge techniques
including immune cell isolation and culture, culture of endothelial cells, flow
cytometry, real-time PCR.The student will be involved in in-vitro work with
human tissue only. The student will be required to read around the subject,
present and participate in lab meetings and will be in a supportive laboratory
atmosphere.
The student will be responsible for analysing the data and keep detailed
record of the work accomplished. Upon initial training, the student will be able
to gain independence in the lab, contribute to experimental design and
present the data in an appropriate manner. The student will have many
opportunities to interact with lab members who will kindly offer support at a
daily basis.
96
Lead Supervisor:
Sally Roberts
Contact Email:
Telephone:
0121 414 7459
Co Supervisor:
Joanna Parish
Project Title:
Mapping viral transcription profiles in life cycle
models of oncogenic human papillomaviruses
Department:
Cancer Sciences
Project Outline
Oncogenic human papillomaviruses (HPV) infect the squamous epithelia lining the
anogenital and oropharyngeal tracts and these infections are at risk of progressing to
cancer. The introduction of prophylactic vaccines to protect against infection with
the most prevalent HPV genotypes HPV16 and 18 is effective at reducing high-grade
cervical premalignant disease. However, the bulk of cervical disease occurs in
countries where it is difficult to introduce an expensive and cold-dependent vaccine.
Furthermore, the incidence of HPV-induced oropharyngeal cancers are increasing at
an alarming rate, primarily in men who are not offered the vaccine. Thus, this
cancer-causing virus remains a significant threat to the human health and there is an
urgent demand for cheap anti-viral/therapeutic interventions.
The HPV life cycle is intimately linked to the differentiation of the host cell - the
keratinocyte, such that new progeny are only produced in the most differentiated
cells. This is achieved through a strict coordination of viral gene expression. We
have modelled the life cycle of HPV16 and HPV18 in anogenital and oropharyngeal
human keratinocytes. Upon growth of the HPV genome-containing cells in three
dimensional organotypic raft culture we observe signficant differences in the
execution of the life cycle in cells derived from different body sites. We hypothezie
that these variations explain the different pathogenicities of HPV infection at these
two sites.
To gain insight into HPV infection at different body sites, the project aims to
investigate the transcription profiles of HPV16 and HPV18 in anogenital and
oropharyngeal keratinocytes. The project will use RT-PCR in combination with laser
capture microdissection to define the spatial and temporal profiles of HPV transcripts
in organotypic raft cultures. It is anticipated that this approach will identify major
differences in the HPV16 and 18 life cycles in different target tissues which will help
explain the natural history of cancer development in different epithelia.
References
Doorbar et al., 2012 The biology and life-cycle of human papillomaviruses. Vaccine
30S F55-70.
The student will be embedded in the groups of Parish and Roberts and will benefit
from the joint expertise of these two groups in HPV molecular and cell biology. The
97
student will meet weekly with the two supervisors to discuss progress and future
direction. They will also participate in the combined weekly group meetings where
the student will have the opportunity to present their data to an interested and
knowledgeable group and where they will benefit from constructive feedback.
The student role.
The student will be exposed to HPV molecular and cell biology. They will be trained
in all necessary techniques including primary epithelial cell culture, organotypic raft
culture, RNA purification, RT-PCR and laser capture microdissection and will
therefore perform the laboratory work required to deliver the project goals.
Importantly, to ensure ownership of the project, the student will be given every
opportunity to contribute intellectually to the direction of the project. To aid this
intellectual input and to facilitate writing the thesis, the student will be expected to
review the literature underpinning the project, enabling the development and
direction of the project and ensuring it remains cutting edge.
98
Lead Supervisor:
Dr Claire Shannon-Lowe
Contact Email:
Telephone:
[email protected]
Co Supervisor:
Dr Doug Ward
Project Title:
Identification of critical B cell and epithelial cell
factors involved in entry of Epstein Barr virus.
Department:
Cancer Sciences
No
Yes
Project Outline
Epstein Barr virus (EBV) is a human herpesviruses which infects approximately 90%
of the human population and maintains a lifelong latent infection in the infected host.
Importantly, EBV was also the first human tumour virus to be identified, and is
associated with several cancers from different cell lineages including B-, T- and
natural killer lymphocytes and epithelail cells.
During the natural life cycle of the virus, EBV infects B lymphocytes and epithelial
cells, and thanks to recombinant virus technology we have been able to identify
which viral proteins are absolutely essential for infection of these two cell types. This
is of critical importance when designing a vaccine. Entry into B lymphocytes requires
the specific interaction between the viral glycoproteins gp350 and gp42 with the B
cell CD21 and HLA class II respectively. In contrast, gp350 and gp42 are not
essential for viral entry into epithelial cells. This is alternately mediated by the viral
gH/gL interaction with various epithelial cell integrins (1-3). The interaction partner
of one essential viral glycoprotein however remains elusive.
Glycoprotein B is the viral protein which initiates the fusion between the viral
envelope and both the B cell and epithelial cell plasma membranes, enabling delivery
of the viral capsid inside the target cell (4). Recombinant viruses with the gB gene
deleted cannot infect either cell type.
In this project, the student will investigate which proteins present on the B cell and
epithelial cell surface interact with the viral glycoprotein gB. The student will use
both recombinant wild-type and gB-deleted viruses as well as purified multimerised
gB to interact with the cell surface. We will use co-immunoprecipitation and
proteomics to identify potential binding partners and immunoassays to confirm the
protein-protein interactions. The student will be trained in various research
techniques including primary cell culture, virological techniques, co-IP and Western
blotting.
1.
2.
3.
4.
References
Shannon-Lowe et al., 2006. PNAS, 103(18):7065-70.
Shannon-Lowe and Rowe, 2014. Curr Opin Virol, 78-84.
Shannon-Lowe and Rowe, 2011. PLoS Pathogens, 7(5):e1001338.
Neuhierl et al., 2009. J Virol, 83(9):4616-23.
99
This project will be supervised by Dr Claire Shannon-Lowe who will teach the student
the techniques necessary for the project. Dr Shannon-Lowe will be available to meet
on a daily basis to plan and discuss work. Dr Shannon-Lowe has expertise in the
entry of EBV into B cells and epithelial cells and associated virological techniques. Dr
Ward is an expert in proteomics and associated techniques and will be available to
meet and give advice when necessary.
The student role.
The student will work alongside Dr Shannon-Lowe, Dr Ward and members of the B
cell group. The project will provide the student with training in basic laboratory
techniques which will not only provide a good basic understanding of how techniques
are performed, but will enable the student to design and appropriately control
experiments and correctly interpret the results. Laboratory techniques involved in
this project include isolation and purification of lymphocyte subsets, cell culture,
production of recombinant viruses, proteomics, co-immunoprecipitation and Western
blotting. The student will also have the opportunity to discuss their work regularly at
group meetings and will attend group journal clubs, seminars by members of Cancer
Sciences and by visiting international researchers.
100
Lead Supervisor:
Dr Adnan Sharif, Consultant Nephrologist.
Contact Email:
Telephone:
[email protected]
Co Supervisor:
Andrew Ready, Consultant Transplant Surgeon.
Jay Nath, Surgical Research Fellow & PhD Student.
Project Title:
Analysis of modifiable and non modifiable risk
factors to predict renal transplant outcome.
Department:
Department of renal transplantation, University Hospital
Birmingham NHS Foundation Trust.
No
Project Outline
Renal transplantation remains the optimal treatment for patients with end stage renal
failure conferring survival and quality of life advantages (1). Such are the benefits of
renal transplantation that a greater number of patients are now offered this
treatment, including those with complex medical problems as even in high risk
patients survival is superior to remaining on dialysis.
There has been a considerable change to both the donor and recipient transplant
population over the past decade. Many these are reflective of the change in general
population trends such as increasing age and BMI. However further immunological
complexity has been observed with a greater proportion of highly sensitised patients,
often from multiple previous failed transplants.
Despite the ever-growing demand, transplantation is limited by the paucity of
available organs and great efforts have been made to expand the donor pool over
the past decade (2). These include utilisation of more high risk extended criteria
donors, Donation after Circulatory Death kidneys, living kidney donation,
anatomically complex organs and transplantation across ABO blood groups.
Encouragingly, despite the increasing complexity of both donor and recipient factors,
the outcome following kidney transplantation continues to improve with current 1
year graft and patient survival rates at over 90% (3).
Most of the identified risk factors to predict transplant outcome (type of donor,
immunological compatibility) has been derived from the American dataset (UNOS).
However many of these population based findings are historical and have not been
validated on a large second population.
Through NHSBT (NHS Blood and Transplant) we have research permission to access
the national renal transplant database containing over 22,000 renal transplant
patients between 2001-2014. To our knowledge this is the largest and most complete
dataset other than the UNOS registry, containing comprehensive donor and
transplant information as well as both patient and graft survival information.
The aim of this project would be analyse this dataset to identify risk factors that
govern renal transplant outcome. We are particularly interested in the outcome
amongst South Asian transplant recipients as this has been an underpublished area
of research. Results and risk factors amongst high risk groups such as ABO
101
bloodgroup incompatible (ABOi) transplants, Multiorgan transplant recipients (eg
combined liver kidney) and those with multiple previous failed transplants are also of
great interest.
The student will be based in the QE hospital Birmingham as part of an active clinical
research team. They will be performing an interesting and highly productive role in
analyzing a large national dataset. This project would be particularly suited to an
aspiring academic clinician, ideally with an aptitude and interest in statistics.
This position is heavily orientated towards publication and we would expect at least
two publications from any BMedSc student, one of which should be as first author.
Access to such a dataset in combination with the clinical and statistical knowledge
within the trust make this an exciting and potentially highly lucrative opportunity.
References
1. Wolfe RA, Ashby VB, Milford EL, Ojo AO, Ettenger RE, Agodoa LY, et al.
Comparison of mortality in all patients on dialysis, patients on dialysis
awaiting transplantation, and recipients of first cadaveric transplant. N Engl J
Med 1999;341:1725-30.
2. Nagaraja P, Roberts GW, Stephens M, Horvath S, Kaposztas Z, et al. (2014)
Impact of Expanded Criteria Variables on Outcomes of Kidney Transplantation
from Donors After Cardiac Death. Transplantation.
3. http://www.organdonation.nhs.uk/statistics/transplant_activity_report/current
_activity_reports/ukt/survival_rates.pdf
The student will receive a departmental induction and set clear objectives by the
supervisors. Initial work will include a literature review to familiarise the student with
current evidence in renal transplantation. They will work under the close supervision
of the current final year PhD student who will oversee day to day training. Statistical
guidance will be provided by the clinical team and the trust statistician (Dr Hodson).
The student will be handling large amounts of patient sensitive data and will be given
training in data protection. We will provide in house training on scientific writing and
manuscript preparation.
The student role.
The student will be expected to act professionally and maintain ethical standards to
protect patient sensitive data. In time they will be expected to become familiar
statistical concepts and with the SPSS statistical programme.
We would expect the student to be highly motivated and aim towards producing high
quality pieces of work leading to publication. This project represents a significant
opportunity for the aspiring academic. The student will be expected to attend and
present their findings at the in house renal audit meetings.
102
Lead Supervisor:
Shishir Shetty
Contact Email:
Telephone:
[email protected]
07879691053
Co Supervisor:
Fedor Berditchevski, Christopher Weston
Project Title:
The role of CD151 in leucocyte recruitment to
the human liver and hepatocellular cancer
Department:
Centre for Liver Research, Cancer Sciences
No
Project Outline
Liver disease is dramatically increasing in the UK and globally. New medical
therapies are urgently required to reduce the burden on liver transplantation.
Inflammatory liver diseases are caused by a variety of factors but they all
progress through a common pathway. This involves leucocyte recruitment
from the circulation into liver tissue which drives tissue injury, fibrosis and can
progress to cirrhosis. Leucocyte recruitment to the liver occurs within the low
shear channels of the hepatic sinusoids where leucocytes interact with
specialized sinusoidal endothelial cells (1).
These channels are a unique
environment for recruitment, they lack conventional adhesion molecules such
as selectins and recruitment is mediated by atypical adhesion molecules such
as vascular adhesion protein-1 (VAP-1) and the common lymphatic and
vascular endothelial receptor- 1 (CLEVER-1) (2,3,4). Recently, using confocal
microscopy, we have also demonstrated that leucocyte transmigration across
human sinusoidal endothelial cells (HSEC) occurs via the conventional
paracellular route and a novel transcellular route (3) Figure 1.
Figure 1: (A) Confocal imaging of fluorescently stained lymphocytes transmigrating
across monolayers of HSEC with demonstration of
(B) paracellular and (C)
transcellular migration.
Intercellular interactions play a fundamental role during transendothelial
migration. Assembly and reorganisation of cell-cell junctions are controlled at
multiple levels and require tight co-ordination of various signalling pathways.
The regulators of this process in the liver sinusoids are poorly understood and
identifying key players in this process could lead to new treatments for
inflammatory liver disease and liver cancer.
Tetraspanin CD151 is a member of the family of four-transmembrane proteins
(5). At the cellular level CD151 is known to interact with and regulates function
103
of laminin-binding integrins (adhesion, migration and invasion) (6). Along with
several other tetraspanins CD151 was found at the cell-cell junctions and
described as a regulator of homotypic intercellular interactions in epithelial and
endothelial cells.
In preliminary work we discovered that depletion of tetraspanin
CD151 altered the cellular distribution and expression levels of ZO-1,
a key structural component of intercellular junctions in epithelial and
endothelial cells.
We have also demonstrated that CD151 is
expressed in diseased human liver tissue and in primary human liver
sinusoidal endothelial cells (Figure 2).
Figure 2: (A) Lysates from control (CD151+) and CD151-depleted cells (CD151-) were
probed with specific antibodies. Re-expression of the wild type protein but not CD151
mutant which is unable to bind integrins in CD151- cells (CD151rec and CD151QRD
cells, respectively), restored the cellular level of ZO-1.
(B) CD151 expression in
inflamed human liver tissue by immunohistochemistry and (C,D) ZO-1 and CD151
expression by immunofluorescence in isolated human HSEC.
Aims
1) Study the expression of CD151 in normal and chronically inflamed
human liver tissue and primary liver cancer. Use dual colour
immunohistochemistry to confirm the cell specific expression of CD151.
2) Study the regulation of CD151 expression in primary human liver
endothelial cells by using cytokine stimulation, co-culture with other
liver cell populations and mediators of fibrosis and hypoxia.
3) The functional role of CD151 in leucocyte trafficking to the liver using in
vitro flow adhesion assays with a combination of phase contrast and
confocal microscopy and lentiviral knockdown of CD151 in primary
human liver endothelial cells and tumour endothelial cells.
References
104
1)
2)
3)
4)
5)
6)
Shetty S, Toxicology Dec 30;254(3):136-46. (2008)
Lalor P. J Immunol 169(2), 983-92 (2002)
Shetty S. J. Immunol 186(7), 4147-4155 (2011)
Shetty S Hepatology 56(4):1521-31 (2012)
Tsujino, K. Am. J. Respir. Crit Care Med. 186, 170-180 (2012)
Detchokul, S Jun 3 epub.Br. J. Pharmacol. (2013)
This project involves three supervisors, Dr Shetty and Dr Weston are based in the
centre of liver research in the medical school. They will provide guidance and
supervision for studying liver tissue and isolating liver cells, furthermore they can
directly supervise in the use of flow adhesion assays and confocal microscopy. Dr
Berditchevski is based in Cancer Sciences and he will be able to supervise the student
in transfection techniques and protein analysis. All three supervisors will liaise
regularly to ensure that the student is gaining adequate training and guidance to
complete the project.
The student role.
The student will be mainly based in the centre for liver research where they will be
working both on liver tissue sections and isolated liver cells which are available
through the liver transplant programme at the Queen Elizabeth Hospital Birmingham.
All the protocols and techniques are established in the department- and the student
will initially be taught these techniques followed by focussing on the molecule CD151.
The role of CD151 in liver disease is poorly understood and there is limited literature
on this subject. Using established techniques to study the role of CD151 will allow the
student to gather novel data for publication. The student will also spend a part of
their time in cancer studies with Dr Berditchevski. Dr Berditchevski is an expert on
CD151 and has several important reagents and techniques which the student can use
to study CD151 regulation and trafficking. The experiments in cancer studies will
complement and support the work performed in the centre for liver research.
105
Lead Supervisor:
Attila Sik
Contact Email:
Telephone:
[email protected]
46018
Co Supervisor:
Ferenc Muller
[email protected]
Project Title:
Development of high throughput
electrocardiogram recording of zebrafish
embryos for drug screening
Department:
Yes
Project Outline
Effective chemical compound screening is of paramount importance for
safe cardiac drug development. A difficult issue relates to the risk of serious
adverse events that may only be discovered at a late stage in the adoption of
new drug, such as idiosyncratic events or toxic effects that are difficult to
identify and predict from preclinical development programmes. The vast
majority of drug candidates fail during various test phases because of side or
toxic effects. Using mammals for preliminary screening is expensive, slow and
requires enormous numbers of animals. Alternatively, zebrafish embryos can
be used because the electro cardiogram (ECG) is similar to mammals, a
minimal amount of chemicals are necessary for drug testing, and embryo
production is fast and inexpensive. Video-recording of zebrafish embryos’
heart activity is currently used for drug screening, but unlike ECG recording it
lacks the temporal and dynamic resolution necessary for cardiac cycle
component analysis. By combining expertise from electrical engineering,
genetics, physiology and neuroscience, we are developing a method to
perform high throughput recording from the heart of ~100 zebrafish
embryos. Multichannel electrodes will register ECG signals under control
conditions and after drug treatment for automatic analysis. The high
throughput screening of drugs will potentially become an essential tool in
cardiovascular research in the near future.
References
S.S.Dhillon, E.Doro, I.Magyary, S.Eggington, A.Sík and F.Müller (2013)
Optimisation of embryonic and larval ECG measurement in zebrafish for
quantifying the effect of QT prolonging drugs Plos One, 8(4):e60552
The student have access to both supervisors in a daily basis. Focused lab meeting is
held every week to discuss the project where everyone involved in the project
participate.
The student role.
106
The student will perform single and multichannel recording from zebrafish
heart, actively involved in electrode design, analyse data and test drugs for
side and toxic effects.
107
Lead Supervisor:
Alex Sinclair
Head of the CSF Disorders Research Group, Neurobiology,
Contact Email:
Telephone:
[email protected]
Co Supervisor:
Wiebke Arlt
Professor of Medicine, Head, Centre for Endocrinology,
Diabetes and Metabolism (CEDAM)
Project Title:
Characterising a novel relationship between idiopathic
intracranial hypertension and polycystic ovarian syndrome
Department:
Neurobiology & Centre for Endocrinology, Diabetes and
Metabolism College of Medical and Dental Sciences
Yes or
No
Project Outline
Idiopathic intracranial hypertension (IIH) is a condition of unknown aetiology
characterised by elevated intracranial pressure (ICP) (Mollan, Markey et al.
2014). The condition causes chronic disabling headaches and papilloedema
which results in visual loss in up to 25% of cases. Of particular interest is that
the condition occurs almost exclusively (>90%) in obese
women of
childbearing age (Corbett, Savino et al. 1982). Intracranial pressure
regulation is dependent on the balance between CSF production at the
choroid plexus and drainage at the arachnoid granulation tissue but the
underlying pathogenesis leading to disordered ICP in IIH is not known.
A number of studies have highlighted the increased incidence of polycystic
ovarian syndrome (PCOS) amongst IIH patients (39 – 57% of patients with
IIH having PCOS, whilst the background risk of PCOS in the general
population is 7-15% (Glueck, Iyengar et al. 2003, Tsilchorozidou, Overton et
al. 2004, Glueck, Aregawi et al. 2005). IIH and polycystic ovarian syndrome
(PCOS) share number phenotypic characteristics including female gender,
obesity, and often hirsutism. PCOS is a heterogeneous condition, comprising
clinical and biochemical hyperandrogenism, oligo/amenorrhoea and metabolic
syndrome (Tsilchorozidou, Overton et al. 2004). 5-alphα reductase functions
as a cortisol inactivating enzyme and it also plays a key role in activating
testosterone into 5-alphα dihydrotestosterone (Bruchovsky and Wilson 1968).
A significant body of evidence is accumulating suggesting dysregulation of 5αlpha reductase activity in PCOS: Increased 5-αlpha reductase activity has
been demonstrated in urinary metabolites in PCOS (Stewart, Shackleton et al.
1990, Chin, Shackleton et al. 2000, Fassnacht, Schlenz et al. 2003,
Tsilchorozidou, Honour et al. 2003, Vassiliadi, Barber et al. 2009)
We suggest that the strong relationship between IIH and obese female
patients strongly suggests that a neuroendocrine dysfunction, potentially
108
through dysregulation of androgen pathways, may play an important
aetiological role.
Hypothesis: Androgen dysregulation is involved in the aetiology of IIH
AIM:
1. Characterisation of the key elements of the androgen signalling
pathway in the choroid plexus.
2. Evaluate the effects of exogenous androgens (including androgen
metabolites and regulating enzymes) on cerebrospinal fluid secretion
(CSF) using a primary choroid plexus in vitro model.
3. Characterise the serum androgens and urinary androgen metabolites in
IIH patients and compared to obese controls and PCOS patients.
Methods:
1. Key elements of the androgen signalling pathway will be examined in
the choroid plexus from brain sections, choroid plexus explants and
primary choroid plexus epithelial cells (human and porcine) (Haselbach,
Wegener et al. 2001): gene expression will be investigated by qPCR
will be used to assess the expression and further protein localisation
will be conducted by immunohistochemistry.
2. The effects of exogenous androgens on cerebrospinal fluid secretion
(CSF) will be examined on a functional primary porcine choroid plexus
in vitro model currently established in house: CSF secretion will be
assessed using: 1) a fluorescent dextran assay; 2), by measuring
Na+,K+-ATPase activity and 3) by Real Time PCR. Briefly, the
fluorescent dextran will be added to both the apical and basolateral
chambers and the change in the concentration of dextran in each
chamber will be measured over a period of time. Na+,K+,ATPase
activity will be assessed by measuring the amount of inorganic
phosphate produced by the cells.
3. Using previously collected samples from IIH and control subjects,
serum androgens will be evaluated by tandem mass spectrometry and
24-hour urine androgen excretion by gas chromatography/mass
spectrometry. Data will be analysed and critically evaluated.(O'Reilly,
Taylor et al. 2014)
Key References
109
Bruchovsky, N. and J. D. Wilson (1968). "The conversion of testosterone to 5alpha-androstan-17-beta-ol-3-one by rat prostate in vivo and in vitro." J Biol
Chem 243(8): 2012-2021.
Chin, D., C. Shackleton, V. K. Prasad, B. Kohn, R. David, J. ImperatoMcGinley, H. Cohen, D. J. McMahon and S. E. Oberfield (2000). "Increased
5alpha-reductase and normal 11beta-hydroxysteroid dehydrogenase
metabolism of C19 and C21 steroids in a young population with polycystic
ovarian syndrome." J Pediatr Endocrinol Metab 13(3): 253-259.
Corbett, J. J., P. J. Savino, H. S. Thompson, T. Kansu, N. J. Schatz, L. S. Orr
and D. Hopson (1982). "Visual loss in pseudotumor cerebri. Follow-up of 57
patients from five to 41 years and a profile of 14 patients with permanent
severe visual loss." Arch Neurol 39(8): 461-474.
Fassnacht, M., N. Schlenz, S. B. Schneider, S. A. Wudy, B. Allolio and W. Arlt
(2003). "Beyond adrenal and ovarian androgen generation: Increased
peripheral 5 alpha-reductase activity in women with polycystic ovary
syndrome." J Clin Endocrinol Metab 88(6): 2760-2766.
Glueck, C. J., D. Aregawi, N. Goldenberg, K. C. Golnik, L. Sieve and P. Wang
(2005). "Idiopathic intracranial hypertension, polycystic-ovary syndrome, and
thrombophilia." J Lab Clin Med 145(2): 72-82.
Glueck, C. J., S. Iyengar, N. Goldenberg, L. S. Smith and P. Wang (2003).
"Idiopathic intracranial hypertension: associations with coagulation disorders
and polycystic-ovary syndrome." J Lab Clin Med 142(1): 35-45.
Haselbach, M., J. Wegener, S. Decker, C. Engelbertz and H. J. Galla (2001).
"Porcine Choroid plexus epithelial cells in culture: regulation of barrier
properties and transport processes." Microsc Res Tech 52(1): 137-152.
Mollan, S. P., K. A. Markey, J. D. Benzimra, A. Jacks, T. D. Matthews, M. A.
Burdon and A. J. Sinclair (2014). "A practical approach to, diagnosis,
assessment and management of idiopathic intracranial hypertension." Pract
Neurol 14(6): 380-390.
O'Reilly, M. W., A. E. Taylor, N. J. Crabtree, B. A. Hughes, F. Capper, R. K.
Crowley, P. M. Stewart, J. W. Tomlinson and W. Arlt (2014).
"Hyperandrogenemia predicts metabolic phenotype in polycystic ovary
syndrome: the utility of serum androstenedione." J Clin Endocrinol Metab
99(3): 1027-1036.
Stewart, P. M., C. H. Shackleton, G. H. Beastall and C. R. Edwards (1990). "5
alpha-reductase activity in polycystic ovary syndrome." Lancet 335(8687):
431-433.
Tsilchorozidou, T., J. W. Honour and G. S. Conway (2003). "Altered cortisol
metabolism in polycystic ovary syndrome: insulin enhances 5alpha-reduction
but not the elevated adrenal steroid production rates." J Clin Endocrinol
Metab 88(12): 5907-5913.
Tsilchorozidou, T., C. Overton and G. S. Conway (2004). "The
pathophysiology of polycystic ovary syndrome." Clin Endocrinol (Oxf) 60(1):
1-17.
Vassiliadi, D. A., T. M. Barber, B. A. Hughes, M. I. McCarthy, J. A. Wass, S.
Franks, P. Nightingale, J. W. Tomlinson, W. Arlt and P. M. Stewart (2009).
"Increased 5 alpha-reductase activity and adrenocortical drive in women with
polycystic ovary syndrome." J Clin Endocrinol Metab 94(9): 3558-3566.
110
Our research groups are translational groups made up of clinicians and
scientists.
Dr Alex Sinclair (Principal Supervisor) has a research portfolio of clinical and
translational studies applied to IIH and ICP. She heads the Cerebrospinal
Disorders Research Group which has developed specialised in vitro and in vivo
models to investigate choroid plexus function and
neuroendocrine
dysregulation. This lab also has a number of translational laboratory studies
running at this time.
The student will work with the CSF disorders research group and will be
supported by weekly supervisor meetings. Additionally, day to day support
and coaching will come from the other members of the CSF research group
(post doc, PhD student, senior lecturer and Clinical Research Fellow). The
project will fit into the portfolio of studies (both basic science and clinical)
conducted by the group. The student will benefit from state of the art
equipment, excellent training and a supportive environment.
The student role.
The student will be part of the CSF Disorders Research Group and will
contribute to discussions and present their work in the weekly lab meetings.
As part of the team the student will be able to interact on a daily basis with
other members of the group and will have the opportunity of learning how
medical knowledge is acquired in the laboratory setting.
The student will learn a variety of molecular biology, immunohistochemical,
cell culture techniques. The student will also utilise clinical samples collected
from IIH patient (translational work from existing ongoing clinical studies).
Skills in data interpretation, statistical analysis and writing will also be
acquired. If successful, the work will likely lead to a peer reviewed publication
and opportunities to present the work at local and national conferences.
111
Lead Supervisor:
Dr Zania Stamataki
Contact Email:
Telephone:
[email protected] 01214146967
Co Supervisor:
Dr Patricia Lalor
Project Title:
T cell entosis in the liver: the impact of
inflammation.
Department:
Yes or
No
Is the Project Cancer related? Potentially.
Project Outline
Entosis is the process whereby one cell invades another, and can remain internalised
in its own vesicle in the host cell’s cytoplasm for long periods of time. The
internalised cell may die or it may be released unharmed, and little is known
regarding the molecules guiding these decisions. We recently discovered that T cells
internalise into hepatocytes in the liver via entosis. This may have implications for
the role of T cells in the regulation of liver immunity, and it is therefore a hot topic in
immunology as well as cell biology. The successful candidate will learn how to coculture human hepatocyte and lymphocyte cell lines, label them with fluorescent
markers and measure internalised and released cells by flow cytometry.
Lymphocyte entosis will be performed in the presence of a series of
proinflammatory cytokines, anti-inflammatory mediators and drugs
relevant in liver inflammation to help identify the conditions of lymphocyte
release from hepatocytes. The techniques used have already been established in
our lab.
References
Overholtzer, M. and J.S. Brugge, The cell biology of cell-in-cell
structures. Nat Rev Mol Cell Biol, 2008. 9(10): p. 796-809.
2.
Overholtzer, M., et al., A nonapoptotic cell death process, entosis, that
occurs by cell-in-cell invasion. Cell, 2007. 131(5): p. 966-79.
3.
Benseler, V., et al., Hepatocyte entry leads to degradation of
autoreactive CD8 T cells. Proc Natl Acad Sci U S A, 2011. 108(40): p. 1673540.
1.
The successful candidate will work directly with the primary supervisor (Dr Zania
Stamataki) who is an early career research scientist that spends a lot of her time in
the laboratory. The student will work closely with Zania and PhD students with an
interest in T cell entosis and T cell biology. The project will take part in the liver labs,
a vibrant research environment where the student will have the opportunity to
interact with postgraduate students (MRes, MD and PhD), postdoctoral scientists and
clinical and non-clinical researchers. Weekly lab meetings will provide the opportunity
to liaise with Prof. David Adams and other members of the lab to have input in the
project, and will be also a valuable forum for the student to hear about the progress
of other projects in our lab.
The student role.
112
The successful candidate will join a highly productive research lab with expertise in
basic/translational and clinical research. Key research interests in the Liver Labs
involve ongoing projects in immunology, hepatology and virology so there is plenty
of opportunity for the student to sample multiple research areas. During their time in
the lab, the student will be trained in laboratory techniques relevant to the project
and learn how to perform experiments in a quality-controlled manner. Beyond
research excellence, the intellectual contribution of the student to this project will be
strongly encouraged. The student will learn to evaluate research publications and
interpret results, analyse experiments and form hypotheses with the aim to foster
the ability to place their research into “the bigger picture” in the field. On a daily
basis, the student will plan and perform experiments, analyse results and discuss
their findings in brainstorming sessions with other scientists. As the project
progresses, experimental data may be put together for presentation in scientific
conferences and as part of scientific publications.
113
Lead Supervisor:
Professor David Thickett
Contact Email:
Telephone:
[email protected] or [email protected]
Co Supervisor:
Dr. Aaron Scott
Project Title:
The effect of e-cigarette smoking on macrophage
function in acute inflammation
Department:
Department of Clinical Respiratory Sciences
Centre for Translational Inflammation Research
No
Project Outline
Macrophages play a critical role in sepsis, lung injury and inflammation. They
are derived from peripheral blood monocytes and the bone marrow and are
recruited to sites of tissue injury and infection forming the first line of defense
towards pathogen and particulate exposure, acting to induce and perpetuate
innate and adaptive immune responses [1]
E-cigarettes (ECIG) have recently become popular as a substitute for and as a
tool in smoking cessation. As these systems deliver nicotine without
combusting tobacco these vapour devices undoubtedly reduce the exposure
to carcinogens from other agents in cigarette smoke. However the negative
impact of these devices remains to be fully explored. Murine studies
examining
asthmatic
airway
inflammation
(AI)
and
airway
hyperresponsiveness (AHR) have shown e-cigarette (ECIG) cartridge fluid can
increase inflammatory cell infiltration into the airways and increased
inflammatory cytokine production [2]
Recent work examining gene expression in human bronchial epithelial cells
has shown similarities in gene expression when cells are exposed to ECIG
fluid when compared to cigarette smoke-conditioned media[3]. In agreement
with the murine studies, high levels of nicotine from ECIG-conditioned media
has also been shown to induce a proliferative, invasive phenotype in human
bronchial epithelial cells in an air-liquid interface model [3].
Many practitioners actively recommend these nicotine delivery systems,
especially to those at risk of developing respiratory problems and yet
relatively little is known about the effect on normal biology and importantly
the effects in acute inflammation and resolution. This project therefore aims
to investigate the effects of smoking and ECIG fluid on the function of
monocytes and macrophages from both healthy volunteers, patients
undergoing lung resection surgery and patients suffering from ALI/ARDS.
HYPOTHESIS
Smoking and ECIGs supress macrophage function and therefore cause
increase in post-operative complications and poor outcomes from ARDS.
AIMS:
1] To investigate the effect of ECIG-fluid on phagocytosis and inflammatory
114
response to stimulation in monocyte derived macrophages (from healthy
volunteers) and primary human alveolar macrophages
2] To investigate the mechanism of action of smoking versus non-smoking on
macrophages from patients undergoing lung resection surgery and in those
with ARDS to determine if this correlates to surgical complication rates and
ARDS outcomes.
References
1. Herold S, Mayer K, Lohmeyer J: Acute lung injury: how macrophages
orchestrate resolution of inflammation and tissue repair. Front
Immunol 2011, 2:65.
2. Lim HB, Kim SH: Inhallation of e-Cigarette Cartridge Solution
Aggravates Allergen-induced Airway Inflammation and Hyperresponsiveness in Mice. Toxicol Res. 2014 Mar;30(1):13-8.
3. Stacy J. Park, Tonya C. Walser, Catalina Perdomo, Teresa Wang, Paul C.
Pagano, Elvira L. Liclican, Kostyantyn Krysan, Jill E. Larsen, John D. Minna,
Marc E. Lenburg, Avrum Spira, Steven M. Dubinett. The effect of ecigarette exposure on airway epithelial cell gene expression and
transformation. [abstract]. In: Proceedings of the AACR-IASLC Joint
Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; Clin Cancer
Res 2014;20.
Both the supervisors are located in the new Centre for Translational Inflammation
Research in the University Labs in the new QEHB. Weekly supervisor meetings will be
arranged to ensure student progress and to plan the evolution of the project. The
student will attend respiratory research in progress meetings weekly.
Considerable expertise exists within the respiratory research group in the techniques
necessary to make this project successful and have extensive experience of BMedSci
student supervision with a good track record with 1st class degree results as well as
outputs with publications and abstracts presented at national and international
meetings. Our most recent student has not only won the best BMedSci project award
but also a British Thoracic Society Medical Student Prize and is an ambassador award
to promote the Clinical Intercalated BMedSci degree.
Patient samples are available for the primary alveolar macrophage work in
collaboration with Mr Babu Naidu, thoracic surgeon and Sepsis/ARDS blood samples
from other trials ongoing within the group and in collaboration with Dr David Thickett
and Dr Elizabeth Sapey. Regular updates of progress with these collaborators will
ensure detailed multidisciplinary support for the successful student.
The student role.
The student will learn the cell isolation and culture techniques to obtain monocyte
derived macrophages from healthy and sepsis/ARDS blood, and primary alveolar
macrophages from lung resection tissue samples and bronchoalveolar lavage fluid.
The student will perform stimulation experiments and perform phagocytosis assays
and measure markers of inflammation by ELISA and phenotype the macrophages by
flow cytometry.
115
Lead Supervisor:
Dr. Kai-Michael Toellner
Contact Email:
Telephone:
Email: [email protected]
Telephone: +44 (0)121 415 8687
Co Supervisor:
Laura Garcia Ibanez
Project Title:
Migration Events from the Germinal Centre during
Vaccine Responses
Department:
School of Immunity and Infection
No, can be acquired
Project Outline
Vaccination results in the production of high affinity protective antibody. The
differentiation of high affinity memory B cells and plasma cells occurs in germinal
centres (GC) in secondary lymphoid organs. Affinity maturation involves migration
between the GC’s dark and light zones, and also migration of effector cells out of the
GC.
The signals that indicate to a B cell that it is time to differentiate and leave the GC
are far from being fully understood. Chemokines, small secreted proteins that
regulate leukocyte migration, are prime candidates to regulate this exit. It has been
shown that chemokines have key roles in B cell differentiation and migration.
We will evaluate the effect of the deficiency of chemokine receptor CCR7 on the exit
of plasma cells and memory B cells from the spleen to other organs such as lymph
nodes, blood and bone marrow. CCR7 binds to chemokines CCL19 and CCL21,
produced by stromal cells in the vicinity of GCs. Mouse models deficient for CCR7 on
B cells are available.
Preliminary data shows that CCR7-deficient antigen-specific B cells form different
memory B cell populations than CCR7-sufficient antigen-specific B cells but the
relevance of these changes in memory B cell populations is unclear. The response of
these memory B cells to subsequent antigen encounters may be affected and needs
to be studied.
The project will help to understand migration and differentiation of effector cells,
especially of the different subsets of memory B cells, which may lead to new ways of
manipulating antibody producing cells in vaccine responses.
References
Allen, C. D., T. Okada, J. G. Cyster (2007). “Germinal-center organization and cellular
dynamics.” Immunity 27(2): 190-202.
Comerford, I., Y. Harata-Lee, M. D. Bunting, C. Gregor, E. E. Kara, S. R. McColl
(2013). “A myriad of functions and complex regulation of the CCR7/CCL19/CCL21
chemokine axis in the adaptative immune system.” Cytokine Grow Factor Rev 24(3):
269-283.
Zhang, Y., M. Meyer-Hermann, L. A. George,M.T. Figge, M. Khan, M. Goodall, S. P.
Young, A. Reynolds, F. Falciani, A. Waisman, C. A. Notley, M.R. Ehrenstein, M.
Kosko-Vilbois, K. M.Toellner (2013). “Germinal center B cells govern their own fate
116
via antibody feedback.”J Exp Med 11;201(3): 457-464
All the experiments will be planned in regular meetings between student, PhD
student co-supervising and main supervisor ensuring the logical progress of the
project.
Experiments performed in the lab will be directly supervised and guided by PhD
student, who is expert in all techniques and procedures involved, and works on a
closely related project with similar questions covering other chemokine receptors.
Weekly meetings will be set up to ensure the satisfactory progress of the project and
to deal with any difficulties that may have arisen.
Further, our research group has weekly lab meetings, where problems are put in
common with all the members of the lab and weekly journal clubs, where the most
recent and relevant papers in the field are discussed.
The student role.
The student will receive full training in the following techniques: murine tissue
preparation (spleen, lymph nodes, bone marrow and blood), flow cytometry,
immunohistochemistry, light and fluorescence microscopy, ELISA and data analysis.
The student will be directly involved in lab meetings, having the opportunity to
discuss their work with the rest of the group for feedback and possible new ideas, in
order to train in scientific discussion and project planning.
Also, he will participate in journal clubs, giving him the possibility to improve
presentation skills in a dissented environment.
Procedures on animals can be carried out by PhD student, and a Home Office license
to perform procedures on animals can be acquired during the course of the project.
117
Lead Supervisor:
Dr Zania Stamataki
Contact Email:
Telephone:
[email protected] 01214146967
Co Supervisor:
Dr Gary Reynolds
Project Title:
Deciphering the B cell compartment in human liver
disease.
Department:
Yes or
No
Project Outline
The liver is the largest internal organ in the human body and performs ~500
important functions. Blood traffics continuously through this organ, yet little is known
about its contribution to immunity. B cells are important lymphocytes as they
perform essential immune functions that protect against pathogens via i) antibody
production, ii) antigen presentation to T cells, iii) cytokine expression, and others.
This project is designed to investigate the distribution of B cell subsets in
human explanted livers from patients with end stage liver disease.
Outcomes from this work will help identify B cell differences between chronic liver
diseases of viral origin, autoimmune aetiology or other forms of liver injury.
We are based at the Institute for Biomedical Research, adjacent to the New Queen
Elisabeth Hospital and the University of Birmingham Medical School. Our group
benefits from access to well-characterised patient cohorts that allow unique insights
into liver disease.
The successful candidate will be part of a multidisciplinary environment of biologists,
immunologists, virologists and clinicians that work together to extend our knowledge
about liver immunity, in health and in inflammation.
References
Microanatomy of the liver immune system. Nemeth E, Baird AW, O'Farrelly C. Semin
Immunopathol. 2009 Sep;31(3):333-43.
Human B cell subsets. Jackson SM, Wilson PC, James JA, Capra JD. Adv Immunol.
2008;98:151-224.
The successful candidate will work directly with the primary supervisor (Dr Zania
Stamataki) who is an early career research scientist that spends a lot of her time in
the laboratory. The student will work closely with Sudha Purswani, a final year PhD
student that has an interest in B cell biology. The project will take part in the liver
labs, a vibrant research environment where the student will have the opportunity to
interact with postgraduate students (MRes, MD and PhD), postdoctoral scientists and
clinical and non-clinical researchers. Weekly lab meetings will provide the opportunity
to liaise with Prof. David Adams and other members of the lab to have input in the
project, and will be also a valuable forum for the student to hear about the progress
118
of other projects in our lab.
The student role.
The successful candidate will join a highly productive research lab with expertise in
basic/translational and clinical research. Key research interests in the Liver Labs
involve ongoing projects in immunology, hepatology and virology so there is plenty
of opportunity for the student to sample multiple research areas. During their time in
the lab, the student will be trained in laboratory techniques relevant to the project
and learn how to perform experiments in a quality-controlled manner. Beyond
research excellence, the intellectual contribution of the student to this project will be
strongly encouraged. The student will learn to evaluate research publications and
interpret results, analyse experiments and form hypotheses with the aim to foster
the ability to place their research into “the bigger picture” in the field. On a daily
basis, the student will plan and perform experiments, analyse results and discuss
their findings in brainstorming sessions with other scientists. As the project
progresses, experimental data may be put together for presentation in scientific
conferences and as part of scientific publications.
119
Lead Supervisor:
Richard Tuxworth
Contact Email:
Telephone:
[email protected]
40476
Co Supervisor:
Tim Barrett
Project Title:
Lysosomal storage disorders: genetic models of
neurodegeneration.
Department:
Clinical and Experimental Medicine
No
Project Outline
Lysosomes are low pH organelles central to the regulation of growth, autophagy and
catabolism. Dysfunction of lysosomes is thought to contribute to most or all
neurodegenerative diseases but its relative importance is difficult to ascertain in the
common late-onset neurodegenerative disorders which are complex and multifactorial. The lysosomal storage disorders (LSDs) are a group of approximately 50
inherited diseases that result in lysosomal failure in cells. Early-onset neuronal
pathology occurs in many of the LSDs, including neurodegeneration. Since each
disease is caused by a single gene defect, similar mutations can be generated in
genetically tractable animals such as mice and fruit flies to use as simple models to
study the cell biology and synaptic biology of neurodegeneration. This project will
use a combination of fruit fly genetics, cell culture, biochemistry and cell biology to
ask what is special about the lysosomes of neurons that makes them particularly
vulnerable in the LSDs.
References
Kollmann K, Uusi-Rauva K, Scifo E, Tyynelä J, Jalanko A, Braulke T. 2013
Cell biology and function of neuronal ceroid lipofuscinosis-related proteins.
http://www.ncbi.nlm.nih.gov/pubmed/23402926
Hindle S, Hebbar S, Sweeney ST. 2011
Invertebrate models of lysosomal storage disease: what have we learned so far?
http://www.ncbi.nlm.nih.gov/pubmed/22038288
Richard Tuxworth will supervise full-time.
The student role.
The student will generate novels mutations, markers and cell biological probes in
cells and flies to examine how complexes of lysosomal membrane proteins differ as
the environment changes. The student will learn a broad range of molecular, cell
biological and genetic techniques.
120
Lead Supervisor:
Dr Graham Wallace
Contact Email:
Telephone:
[email protected] 0121 371 3255
Co Supervisor:
Miss Saaeha Rauz
Project Title:
Does endogenous cortisol induce miRNA
Department:
Ophthalmology,
No
Project Outline
The eye is regarded as an immune privileged site, due to effective barriers and an
immunosupprssive microenvironment. We have shown that ocular barrier cells can
convert inactive cortisone to active cortisol. Related to this we have previously shown
that treatment of human corneal fibroblasts with dexamethsone, a synthetic
glucocoticoid, induced the expression of several miRNA. These are small RNA
molecules that bind to mRNA to control translation. Among the miRNA identified
included two associated with fibrosis. This is of interest as topical treatment with
dexamethasone can caused raised intraocular pressure that may be associated with
fibrosis. In this project we wish to determine whether endogenous cortisol production
mediates the same changes in miRNA expression. Primary corneal cells will be
prepared from tissue rims, following transplant. Cells will be treated with
cortisone and miRNA expression will be assessed by a fluorometric assay. Target
mRNAs will be identified by analysing on-line databases and candidate genes
expression will be assessed by PCR. The potential for stress responses to
alterprotein expression in corenal cells is intriguing and potentially important in
controlling site-threatening inflammatory disease.
References
Susarla R, Liu L, Walker EA, Bujalska IJ, Alsalem J, Williams GP, Sreekantam S,
Taylor AE, Tallouzi M, Southworth HS, Murray PI, Wallace GR, Rauz S. Cortisol
biosynthesis in the human ocular surface innate immune response. PLoS One. 2014 Apr
15;9(4):e94913
Liu L, Walker EA, Kissane S, Khan I, Murray PI, Rauz S, Wallace GR.
Gene expression and miR profiles of human corneal fibroblasts in response to
dexamethasone. Invest Ophthalmol Vis Sci. 2011 Sep 21;52(10):7282How are you planning to ensure adequate supervision?
The student will be supervised on a daily basis by Dr Wallace in the Centre for
Translational Inflammation laboratories in the QE Hospital. Weekly meeting with both
supervisors will take place every Wednesday morning. Miss Rauz will supervise the
tissue collection at the Birmingham and Midland Eye centre, City Hospital.
The student role.
121




The student will prepare the corneal cells from tissue rims and grow under
tissues culture conditions.
Test for miRNA using a commercial fluorometric kit.
Analyse miRNA databases for potential targets
PCR and protein analysis (ELIsA) of potential targets
122
Lead Supervisor:
Dr Doug Ward
Contact Email:
Telephone:
[email protected]
01214149528
Co Supervisor:
Dr Rik Bryan and Prof Jean-Baptiste Cazier
Project Title:
Correlating the genomic landscape of bladder cancer
with clinical outcome.
Department:
No
Project Outline
Bladder cancer is the 5th most common cancer in Western societies, responsible for
10,000 new cases and 5,000 deaths annually in the UK. Patient management and
treatment have not changed significantly for decades and the prognosis is dismal for
patients with muscle-invasive disease.
75% of new cases of bladder cancer are non-muscle invasive and the majority of
these are papillary tumours with activating mutations in the FGFR3–PIK3CA pathway.
Most cases of muscle invasive disease carry mutations inactivating tumour
suppressors. Within both invasive and non-invasive disease there is considerable
heterogeneity in gene mutation, gene expression and clinical behaviour. Next
generation sequencing data is now available for several hundred bladder tumours
painting a complex picture with more than 30 genes mutated at statistically
significant frequency in these patients and on average several hundred genes are
mutated in each tumour.
We have collected tumour tissue and matched whole blood from 1000 patients with
bladder cancer and well collated outcome data. We are generating exome and RNASeq data on a large number of these tumours with a focus on particularly difficult to
manage clinical sub-groups. The aim of this project is to analyse these datasets in
the context of outcome to develop prognostic and predictive biomarker signatures.
References
Whole-genome sequencing of bladder cancers reveals somatic CDKN1A mutations
and clinicopathological associations with mutation burden. Cazier JB, Rao SR,
McLean CM, Walker AL, Wright BJ, Jaeger EE, Kartsonaki C, Marsden L, Yau C,
Camps C, Kaisaki P; Oxford-Illumina WGS500 Consortium, Taylor J, Catto JW,
Tomlinson IP, Kiltie AE, Hamdy FC.
Nat Commun. 2014 5 3756
Combined proteome and transcriptome analyses for the discovery of urinary
biomarkers for urothelial carcinoma. Shimwell NJ, Bryan RT, Wei W, James ND,
Cheng KK, Zeegers MP, Johnson PJ, Martin A, Ward DG. Br J Cancer. 2013 108
1854-61.
Daily office/laboratory supervision and fortnightly meetings with all supervisors.
123
The student role.
The student will perform bioinformatic analyses of next generation sequencing data
to aid in our understanding of how gene copy numbers, mutations and expression
levels influence phenotype in bladder cancer. This data will be used to develop
clinically relevant predictive models to aid in the development of precision medicine
in bladder cancer. If the student wishes the project may also contain a wet-lab
element using targeted re-sequencing, qPCR, Western blotting and possibly mass
spectrometry to confirm and extend the NGS data.
124
Lead Supervisor:
Professor Hisham Mehanna
Contact Email:
Telephone:
Co Supervisor:
[email protected]
0121 414 6547 (Mrs Gemma Jones, PA to Hisham
Mehanna)
Prof Jean-Baptiste Cazier
Project Title:
In Silico methylation analysis of head and neck
Department:
Cancer Sciences
cancer.
Will the project require a Home Office working with animals licence? NO
Project Outline
The Institute of Head and Neck Studies and Education (InHANSE), led by Professor
Hisham Mehanna, focuses on research into diseases of the head, neck and thyroid,
and on the education of health professionals in the field.
For head and neck cancer one of the most complex areas of management is the
choice of primary treatment (surgery vs chemo-therapy) and this often relies on
clinical factors (mainly clinical stage) and the therapeutic preference of the treating
centre. InHANSE are near completion of a clinical trial Predictr, this is a large
multicentred trial examining the differential expression of 10 biomarkers between
normal, pre-malignant and malignant tissue. The aim of this trial is to utilise the
biological characteristics of a tumour alongside clinical factors to more reliably predict
those cases that have a high risk of transformation. This will allow more aggressive
treatment of high risk cases, whilst sparing the majority of patients from potentially
disabling side effects.
Epigenetic modification of DNA has been increasingly recognized as performing an
important role in carcinogenesis. However, there are suprisingly few studies of
genome-wide methylation and its effects in head and neck cancer. In order to
improve the prognostic classifier obtained from the Predictr trial we are currently
investigating methylation status in head and neck cancers compared to normal
tissue. In collaboration with the National Cancer Institute of Brazil (INCA) we have
preliminary data showing differential methylation between normal, pre-malignant
and malignant tissue as well as HPV- and HPV+ disease. The aims of this project are
two fold:
1. Validate our in vitro methylation findings using in silico database searches
2. Use in silico database searches to find novel methylation gene signatures that
will predict tumours that are high risk.
References
 Mehanna H et al. Head and neck cancer--Part 2: Treatment and prognostic
factors. Bmj. 2010;341:c4690.
 Nejat Dalay. Role of DNA methylation in head and neck cancer. Clin Epigenet
(2011) 2:123-150.
 Beggs A et al. Whole-genome methylation of benign and malignant colorectal
tumours. J Pathol (2013)229:697-704.
125
 Cazier, J.B. et al. Whole-genome sequencing of bladder cancers reveals somatic
CDKN1A mutations and clinicopathological associations with mutation burden. Nat
Commun (2014) 5, 3756.
InHANSE is a team of over 15 researchers, which will include a bioinformatician by
January 2015. The student will be primarily supervised within InHANSE but will also
co-supervised by Professor Jean-Baptiste Cazier, the Director Designate of the Centre
for Computational Biology, with the involvement of Dr Andrew Beggs who runs a
bioinformatics “dry-lab” to search for novel therapeutic targets and stratification
markers in colorectal cancer.
The student will have 1 to 1 meetings 2-weekly with the InHANSE bioinformatician
and monthly meetings with Professor Mehanna and/or Professor Cazier.
The student role.
The student will learn to use and interrogate publicly available, online genomics and
methylation data repositories bases, and carry out analyses and meta-analysis on the
data, examining hypotheses developed from our pilot data. They will examine
potential associations and risk factors, as well undertake survival analyses. They will
liaise and collaborate with our colleagues in INCA Brazil to analyse the extension of
the pilot data.
126
Lead Supervisor:
Contact Email:
Telephone:
[email protected]
Mehanna)
Dr Rachel Watkins
Co Supervisor:
Project Title:
Accelerated discovery and development of novel
therapeutic agents for the treatment of head
and neck cancer patients
Department:
Cancer Sciences
Yes
Project Outline
In the last 30 years, there have been only 2 new anti-cancer drugs introduced
for the treatment of head and neck cancer. As a result, traditionally the
survival of patients with advanced head and neck cancer has been very poor,
with only a 50% survival rate. These survival rates have not increased
significantly in the past 30 years.
A key goal of Accelerated is to increase the speed with which these new
treatments are implemented into clinical practice, whilst reducing the costs
associated with identification, testing and development. The Accelerated
project provides a platform for the identification and validation of novel drugs
for the management of head and neck cancer through examining currently
approved, non-cancer, off-patent drugs for anti-cancer activity.
Potential therapeutic agents are taken through a series of in vitro cell-based
assays to determine their efficacy against head and neck cancer survival and
proliferation. These studies are carried out using a panel of cell lines and
primary cultures derived from donated patient tissues, and include
proliferation assays, migration assays, flow cytometry and 3D organotypic
cultures.
Following on from this in vivo xenograft mouse models will be used to confirm
the efficacy of the candidate agents in living organisms. Once targets have
been identified and confirmed we will explore their mechanism of action using
a range of molecular biology techniques.
References
 Mehanna H et al. Head and neck cancer--Part 2: Treatment and prognostic
factors. Bmj. 2010;341:c4690
 Fung C et al. Emerging drugs to treat squamous cell carcinomas of the head and
neck. Expert opinion on emerging drugs. 2010;15(3):355-73
 DiMasi JA et al. The price of innovation: new estimates of drug development
costs. Journal of health economics. 2003;22(2):151-85
127
 Bodmer M et al. Long-term metformin use is associated with decreased risk of
breast cancer. Diabetes care. 2010;33(6):1304-8.
 Wu Z et al. Quantitative chemical proteomics reveals new potential drug targets in
head and neck cancer. Molecular & cellular proteomics: MCP. 2011;10(12):M111
011635
The Accelerated platform was established in 2013. Currently there are 3
postdocs and 2 research technicians working full time on this project, in total
there will be 5 members of staff to help with Supervision. The translation
team have informal weekly meeting’s to discuss data and to plan the
following weeks experiments. These meeting’s are supported by fortnightly
meetings, in which data is presented and discussed in detail.
In addition the student has 1 to 1 meetings 2-weekly meetings with Dr
Watkins the team leader and monthly meetings with Prof Mehanna
The student role.
The student will participate in the delivery of the Accelerated platform. Using
a panel of characterised head and neck cancer cell lines they will screen
agents for efficacy against HNC survival and proliferation using high
throughput Alamar blue proliferation assays. They will test agents alone and
in combination with chemotherapy and radiotherapy to mimic current
treatment in head and neck. They will then validate any hits further with a
series of in vitro cell based assays such as clonogenic, scratch-wound
migration, apoptosis and cell cycle analysis. Those agents that show promise
in the in vitro models will then be tested in head and neck xenograft animal
models to verify their efficacy against head and neck cancer tumours in living
organisms.
128
Lead Supervisor:
Prof Ben Willcox
(http://ciic.org.uk/team-member/prof-ben-willcox/)
Contact Email:
Telephone:
0121 414 9533/07919167723
Co Supervisor:
Dr Carrie Willcox, Dr Fiyaz Mohammed
Project Title:
Exploring recognition and manipulation of stressinduced ligands by the NKG2D immunoreceptor.
Department:
No
Project Outline
The project focusses on NKG2D, a key activatory receptor expressed on immune
effector cells, that recognises a family of related MHC-like ligands that are
upregulated after cellular stress, such as infection or tumourigenesis. Ligand
recognition by NKG2D is important in cancer immunosurveillance, and might be
exploitable for cancer immunotherapy, however it is currently poorly understood.
This project builds on research by Prof Willcox’ and Prof Moss’ research groups, and
aims to:
(i) Assess which NKG2D ligands (NKG2DL) are the most potent activators of
immune effector cells.
(ii) Structure-based approach to defining a core recognition motif on NKG2DL
that is conserved across the family.
(iii) High-throughput screening approach to identify how to therapeutically
manipulate expression of the most bioligically potent NKG2DL.
References
Zuo-J, Willcox-CR, Mohammed-FM, Antoun-A, Malladi-R, Inman-C, Croudace-J,
Briggs-D, Willcox-BE, Moss-PA
ULBP6 polymorphism modulates NKG2D-mediated immunity by creating an ultrastable NKG2D receptor/ligand interaction
Nature Immunology in preparation
There are four excellent postdoctoral scientists who will play a role in supervising the
student. Dr Jianmin Zuo is an excellent cellular immunologist focusing on the role of
NKG2D in transplantation; Dr Carrie Willcox is an expert on cellular stress recognition
with outstanding molecular and cellular skills; Dr Martin Davey will assist Dr Zuo in
cellular assays. Dr Fiyaz Mohammed is a structural biologist with a strong interest in
immune receptor/ligand interactions.
The student role.
The student will gain core laboratory skills in a wide range of techniques, including
molecular skills in construct design and generation, molecular modelling, generation
of transfectants, and cellular activation assays; also, time permitting, in high
throughput screening approaches to identify chemotherapy drugs that upregulate
key NKG2D ligands.
129
Lead Supervisor:
Jayne Wilson
Co Supervisor:
Professor Keith Wheatley, Professor Pam Kearns
Project Title:
Systematic reviews of treatment strategies for
hepatoblastoma in children and young adults
Department:
CRCTU
Contact Email:
Telephone:
[email protected]
0121 414 3461
No
Is the project cancer related?
Yes
Discipline:
Cancer Studies
Histopathology
Endocrinology
Haematology
Infection

Immunology & Renal
Rheumatology / Orth
Evolutionary Med
Liver & GI Medicine
Project Outline
Hepatoblastoma is a very rare childhood cancer of unknown aetiology. Approximately
8 children a year are diagnosed in the UK. The main treatment is surgery but
chemotherapy is used to shrink the tumour prior to surgery or treat unresectable or
metastatic disease. Survival estimates are very good for patients with local disease
but decrease significantly in patients with advanced or metastatic disease.
The aim of this project is to identify trials and studies that have reported the efficacy
and effectiveness of therapeutic interventions for hepatoblastoma. We aim to focus
on chemotherapy and will look at single arm studies as well as controlled trials.
These systematic reviews will help inform clinical practice and contribute to defining
the pertinent clinical research questions to be addressed in future trials in order to
improve the treatment for these diseases.
References
References on why you need a systematic review in clinical trials
Clarke M, Hopewell S, Chalmers I. Clinical trials should begin and end with
systematic reviews of relevant evidence: 12 years and waiting. Lancet 2010;
376(9734):20-21.
Goudie AC, Sutton AJ, Jones DR, Donald A. Empirical assessment suggests that
existing evidence could be used more fully in designing randomized controlled trials.
J Clin Epidemiol 2010; 63(9):983-991.
Guides to undertaking a systematic review.
Systematic Reviews – CRD’s guidance for undertaking reviews in health care.
Centre for Reviews and Dissemination. University
130
of York 2009
ISBN: 978 1 900640 47 3
http://www.york.ac.uk/inst/crd/pdf/Systematic_Reviews.pdf
pragmatic guide to how to do SR
Cochrane Handbook for Systematic Reviews of Interventions.
Julian PT Higgins, Sally Green. Wiley
Blackwell 2008
ISBN: 978 0 470 69951 5
http://www.cochrane.org/training/cochrane-handbook
mainly deals with RCTs but does have info on other designs
Hepatoblastoma
Herzog CE, Adnrassy RJ, Eftekari F., Childhood Cancers: Hepatoblastoma. The
Oncologist 2000 5:445-453.
Pinkerton R, Shankar A, Matthay KK., Evidence-based pediatric oncology. Third
Edition. Wiley-Blackwell. 2013
Keith Wheatley is Professor of Medical Statistics at CRCTU and statistical lead for the
Children’s Cancer Trials Team (CCTT) and Jayne Wilson is a Senior Systematic
Reviewer at CRCT. Both have substantial experience and expertise in evidence
synthesis that will enable us to supervise and educate the student(s) appropriately.
Supervision will be in the form of regular meetings. The student will also have the
opportunity to attend training courses in systematic review methods.
The student role.
The student will learn through working on their systematic review and participation
in meetings to discuss the review.
The student will learn how to design and conduct a systematic review. The student
will
 Write the review protocol, learning about how to formulate an answerable
question.
 Systematically search for studies using electronic databases and reference
manager software.
 Data extract information and critically appraise the studies included in the
review. By the end of the review the student will learn about the different
statistics used in research papers and how to interpret them, they will also
learn about different trial designs and how to recognise whether a study has
been well conducted.
 Synthesize the data using statistical techniques such as meta-analysis.
 Write up findings in a scientific format, but also learn who to make findings
accessible to users of systematic reviews including patients and clinicians.
The student will also learn about how systematic reviews are used in clinical decision
making process’s, learn about the institutions who produce and use systematic
reviews such as NICE and the Cochrane Collaboration and learn how systematic
reviews are used in the development of clinical trials.
131
Lead Supervisor:
Jayne Wilson
Co Supervisor:
Dr Neil Steven, Mr Neil Sharma, Miss Lisha McClelland
Project Title:
Systematic review of management of sinonasal
melanoma
Department:
CRCTU
Contact Email:
Telephone:
[email protected]
0121 414 3461
No
Yes
Discipline:
Cancer Studies
Histopathology
Endocrinology
Haematology
Infection

Immunology & Renal
Rheumatology / Orth
Evolutionary Med
Liver & GI Medicine
Project Outline
Sinonasal melanoma is a rare malignancy with a poor prognosis, and its aggressive
nature is highlighted by the omission of stage I and II mucosal melanomas from the
UICC/AJCC TNM Classification of Malignant Tumours. Primary management is
surgical, and although the previously held view that these cancers were radioresistant has changed, there is no clear consensus regarding use of radiation or
chemotherapy. Adjuvant therapy has not led to increased prognosis, with high rates
of loco-regional recurrence and metastases and low 5 year survival.
The aim of this project is to work with a cross disciplinary team to develop clinical
guidelines for managing sino-nasal melanoma and explore the feasibility of clinical
trials investigating potential novel therapeutic strategies. The objectives are:
A. to undertake a systematic review of publications reporting surgical, radiation,
cytotoxic, immune and molecularly targeted therapeutic interventions for sinonasal
melanoma.
B. to carry out a review of the presentation, investigation (including molecular
phenotyping) and management of the sino-nasal melanoma at University Hospital
Birmingham.
References
References on why you need a systematic review in clinical trials
Clarke M, Hopewell S, Chalmers I. Clinical trials should begin and end with
systematic reviews of relevant evidence: 12 years and waiting. Lancet 2010;
376(9734):20-21.
Goudie AC, Sutton AJ, Jones DR, Donald A. Empirical assessment suggests that
existing evidence could be used more fully in designing randomized controlled trials.
132
J Clin Epidemiol 2010; 63(9):983-991.
Guides to undertaking a systematic review.
Systematic Reviews – CRD’s guidance for undertaking reviews in health care.
Centre for Reviews and Dissemination. University
of York 2009
ISBN: 978 1 900640 47 3
http://www.york.ac.uk/inst/crd/pdf/Systematic_Reviews.pdf
pragmatic guide to how to do SR
Cochrane Handbook for Systematic Reviews of Interventions.
Julian PT Higgins, Sally Green. Wiley
Blackwell 2008
ISBN: 978 0 470 69951 5
http://www.cochrane.org/training/cochrane-handbook
mainly deals with RCTs but does have info on other designs
Sinonasal melanoma
Gore MR, Zanation AM, Survival in Sinonasal Melanoma: A Meta-analysis. J Neurol
Surg B Skull Base. 2012 Jun;73(3):157-62
Roland NJ, Paleri V (eds). Head and Neck Cancer: Multidisciplinary Management
Guidelines. 4th edition. London: ENT UK; 2011
This project is led by a multidisciplinary panel of supervisors who will ensure a well
rounded approach to the project with supervision in the form of regular meetings
and electronic updates. The student will also have the opportunity for formal and
practical training in systematic review methods. Clinically, the student will attend
diverse clinics including rhinology and oncology and join the multidisciplinary team
discussing case management. The student will meet and speak to patients suffering
from sinonasal melanoma, giving them a unique insight into the patient perspective.
The supervisory team comprise:
Jayne Wilson is a Senior Systematic Reviewer at CRCT with substantial experience
and expertise in evidence synthesis that will ensure the student is supervised and
educated appropriately.
Neil Steven is a Consultant Oncologist and Deputy Clinical Director of the CRUK
Clinical Trials Unit with considerable experience in the use of cytotoxic, molecular
targeted and immune therapies for melanoma.
Neil Sharma is an NIHR Clinical Lecturer in ENT at the Institute of Head and Neck
Studies and Education, involved in clinical trials relating to head and neck cancers.
Lisha McClelland is a Consultant ENT/Sinonasal surgeon frequently treating patients
with this disease.
The student role.
133
The student will learn through working on their systematic review and participation
in meetings to discuss the review.
The student will learn how to design and conduct a systematic review. The student
will
 Write the review protocol, learning about how to formulate an answerable
question.
 Systematically search for studies using electronic databases and reference
manager software.
 Data extract information and critically appraise the studies included in the
review. By the end of the review the student will learn about the different
statistics used in research papers and how to interpret them, they will also
learn about different trial designs and how to recognise whether a study has
been well conducted.
 Synthesize the data using statistical techniques such as meta-analysis.
 Write up findings in a scientific format, but also learn who to make findings
accessible to users of systematic reviews including patients and clinicians.
The student will also learn about how systematic reviews are used in clinical decision
making process’s, learn about the institutions who produce and use systematic
reviews such as NICE and the Cochrane Collaboration and learn how systematic
reviews are used in the development of clinical trials.
The student will also learn how to extract and systematise diverse clinical,
radiological and pathological data from case records.
134
Lead Supervisor:
Dr Jonathan W Mueller (CEDAM)
Co Supervisor:
Dr Daniel A Tennant (Cancer Sciences)
Prof Wiebke Arlt (CEDAM)
Hypoxia as a regulator of steroid synthesis and sulfation
Project Title:
Department:
Contact Email:
Telephone:
CEM – Clinical and Experimental Medicine
CEDAM – Centre for Endocrinology, Diabetes and
Metabolism
[email protected]
x58819
Discipline:
No
Cancer Studies
Histopathology
Endocrinology
Haematology
Infection
X
Immunology & Renal
Rheumatology / Orth
Evolutionary Med
Liver & GI Medicine
Project Outline
During fetal development, the fetal zone of the adrenal gland produces large
amounts of DHEAS, the sulfate ester of the crucial androgen precursor
dehydroepiandrosterone (DHEA). DHEA sulfation in the adrenal requires two
enzymes, PAPS synthase 2 (PAPSS2), to produce the sulfate donor PAPS, and DHEA
sulfotransferase (SULT2A1, to attach the sulfate group to the DHEA molecule [1]). It
is one of the mysteries in endocrinology how this fetal zone of the adrenal quickly
changes after birth, significantly shrinking in size with a steep decline in circulating
DHEAS levels (Fig.1A). These dramatic physiologic changes have previously been
suggested to involve enhanced apoptosis [2], but the underlying mechanisms remain
unclear. A previous study has provided evidence that FZ involution occurs
irrespective of gestational age at birth (Fig.1.B+C), i.e. that parturition itself appears
to be the most important trigger for the initiation of rapid FZ involution [3].
Figure 1: A, Schematic representation of serum levels of the major adrenal androgen dehydroepiandrosterone-sulfate (DHEA-S) over the human lifetime. Normal DHEA-S levels in adults are as high
as up to 10 µM. B and C, adrenal volume decrease and drop in DHEA-S serum levels at birth coincide
irrespective of gestational age. B, mean ultrasonographic adrenal volume in cubic centimeters for 26–
135
28 wk gestation () and 29–32 wk gestation (). C, mean DHEA-S serum levels (micromoles per
liter) for 26–28 wk (), 29–32 wk (), and 33–35 wk gestation (). Shaded area, Normal adult
reference values. Figure A adapted from Ref. 7, Figures B and C from Ref. 3.
During fetal development, the adrenal is exposed to an oxygen tension of 3% (~22
mmHg). However, upon the onset of pulmonary function at birth, a significant
increase in adrenal oxygenation is observed, reaching around 12% (90 mmHg
(Fig.2). The human adrenal is an organ with very high vascularisation. Hence, one
can approximate its tissue oxygen tension to that of capillary arterioles. The
influence of the massive increase in oxygenation after birth on adrenal development
has never been investigated before. This is especially noteworthy, as adrenal
steroidogenesis strongly relies on oxygen [4]. Therefore in this PhD project we
propose the influence of hypoxia and changes in oxygen tension on adrenal steroid
synthesis and sulfation.
Figure 2: Oxygen concentration
in the fetal and adult adrenal
(adapted from Ref. 8).
Hypothesis
We hypothesise that changes in oxygen tension represent a hitherto
unknown modulator of steroid synthesis and sulfation and underlies the
early neonatal involution of the fetal zone of the human adrenal cortex.
References (supervisors)
1) Mueller JW, Shafqat N. FEBS J. 2013 Jul;280(13):3050-7.
2) Spencer SJ, Mesiano S, Lee JY, Jaffe RB. J Clin Endocrinol Metab. 1999
Mar;84(3):1110-5.
3) Ben-David S, Zuckerman-Levin N, Epelman M, Shen-Orr Z, Levin M, Sujov P,
Hochberg Z. J Clin Endocrinol Metab. 2007 Jan;92(1):93-7.
4) Prasad R, Kowalczyk JC, Meimaridou E, Storr HL, Metherell LA. J Endocrinol. 2014
Jun;221(3):R63-73.
5) Wang T, Rainey WE. Mol Cell Endocrinol. 2012 Mar 31;351(1):58-65.
6) Junqueira LC, Carneiro J (Eds) Basic Histology 2002.
7) Miller and Auchus. Endocrine Rev, 2011, 32(1):81-151.
8) Wenger RH, AINS 2004, 39, S38-43.
136
The student will receive daily supervision by Dr Mueller, during the initial phase
working alongside him at the bench, including a thorough introduction to cell culture
techniques, transfection, fluorescence microscopy, SDS-PAGE, quantitative western
blotting, nucleic acid techniques and other basic methodology as well as steroid
analytics. The student will receive special training for cell culture work using the
hypoxia workstations in Dr Tennant’s lab. Previous lab experience is not a
prerequisite for this project, but scientific curiosity and enthusiasm for research are
very much welcome. As the lab skills of the student develop, he or she will soon be
able to carry out defined elements of the research project autonomously with daily
feedback.
In addition, the student will participate and present at the weekly Arlt group
meetings and discuss his/her work in detail with Prof Arlt, Dr Tennant and Dr Mueller
in bi-weekly research meetings of the steroid sulfation sub-group. Finally, active
participation in national or international endocrine conferences will be supported.
The student role.
The human cell line NCI-H295 derived from an adrenocortical carcinoma is a wellestablished model for adrenal function [5] with fetal characteristics. The student will
subject NCI-H295 cells to extended periods of 3% oxygen mimicking the fetal
oxygen tension. This will re-set their ‘normoxic’ set point, so that 3% becomes
normoxia for them, as it is in utero. This adaptation will be monitored through
regular sampling of the cell culture supernatant for analysis of steroid and steroid
sulfate secretion and other metabolic markers of oxygenation (lactate, alanine). He
or she will then model the change in oxygenation induced by birth by moving them
to 12% oxygen, effectively “giving birth in the petri dish”. Thereafter the student will
analyse the resulting changes in steroidogenesis (including expression changes of
the sulfation enzymes SULT2A1 and PAPSS2), and alterations in cell viability,
specifically through apoptosis.
Mass spectrometric analysis of steroid sulfation is primarily carried out by specialist
core facility staff, but the student will liaise with them and gain detailed insight into
the methodologies, their pitfalls and opportunities. Data analysis, literature research
and planning of further experimental procedures will be carried out by the student in
close collaboration with Dr Mueller.
137
Lead Supervisor:
Dr Stephen Young
Contact Email:
Telephone:
[email protected]
(0121) 371 3249
Co Supervisor:
Dr Jane Falconer
Project Title:
How does the genetic variant of PTPN22 regulate
macrophage metabolism and signalling?
Department:
Rheumatology Research group, Centre for translational
inflammation Research, QEH
No
Is the Project Cancer related? Possibly
Project Outline
A genetic variant of the protein tyrosine phosphatase PTPN22, in which arginine 620
is changed to a tryptophan (R620W), is the most widely distributed polymorphism
associated with a broad range of autoimmune disease. This includes rheumatoid
arthritis, lupus and type I diabetes. We have shown that neutrophils expressing this
variant are hyperactive and so may contribute to the inflammatory disease processes
in autoimmunity. Preliminary data shows that this also affects the function of
macrophages, and intriguingly also affects the metabolism of these cells.
We have shown that metabolic profiling using NMR spectroscopy of serum and urine
of rheumatoid arthritis patients can predict outcome early in disease and can also
predict responses to therapeutic intervention. The source of the metabolites in the
blood and urine may be partly from the inflamed synovium tissue in the joints but
also from activated immune cells both locally and systemically. Macrophages are
likely to contribute in a significant way to these metabolites and we hypothesise that
macrophages expressing the PTPN22 will have a signature metabolic profile which
will indicate significant differences in the function and may show how they contribute
to driving the chronic immune inflammation associated with rheumatoid arthritis.
References
1.
2.
3.
4.
Bayley R, Kite KA, McGettrick HM, Smith JP, Kitas GD, Buckley CD, Young SP. The
autoimmune-associated genetic variant PTPN22 R620W enhances neutrophil activation and
function in rheumatoid arthritis patients and healthy individuals. Ann Rheum Dis. 2014;epub
ahead.
Young SP, Kapoor SR, Viant MR, Byrne JJ, Filer A, Buckley CD, Kitas GD, Raza K. The impact of
inflammation on metabolomic profiles in patients with arthritis. Arthritis Rheum.
2013;65(8):2015-23.
Kapoor SR, Filer A, Fitzpatrick M, Fisher BA, Taylor PC, Buckley CD, McInnes IB, Raza K, Young
SP. Metabolic profiling predicts response to anti-TNFα therapy in patients with rheumatoid
arthritis. Arthritis Rheum. 2013;65(6):1448-56.
Fitzpatrick MA, Young SP. Metabolomics – A novel window into inflammatory disease. Swiss
Med Wkly. 2013;143:w13743.
Dr Young and Dr Falconer both actively work in the laboratory and would be
available for direct supervision of the student. Dr Young supervises two further
postdocs, two technicians and two graduate students working in this area all of
whom could contribute to day-to-day supervision. The rheumatology group as a
whole comprises around 50 people with two active research laboratories into which
the student will be fully integrated.
The student role.
138
In this project the student will differentiate blood monocytes into different types of
macrophage and characterise their metabolic profiles using NMR-based analysis. This
will be done with monocytes from individuals expressing wild-type and variant
PTPN22 to determine how this phosphatase influences metabolism. The effect of the
variant on signalling pathways regulated by PTPN22 will also be investigated using
the analysis of calcium signalling and western blotting for phosphorylated targets of
PTPN22.
139
Lead Supervisor:
Dr. Kai-Michael Toellner
Contact Email:
Telephone:
Email: [email protected]
Telephone: +44 (0)121 415 8687
Co Supervisor:
Laura Garcia Ibanez
Project Title:
Migration Events from the Germinal Centre during
Vaccine Responses
Department:
Yes or
No, can be acquired
Project Outline
Vaccination results in the production of high affinity protective antibody. The
differentiation of high affinity memory B cells and plasma cells occurs in germinal
centres (GC) in secondary lymphoid organs. Affinity maturation involves migration
between the GC’s dark and light zones, and also migration of effector cells out of the
GC.
The signals that indicate to a B cell that it is time to differentiate and leave the GC
are far from being fully understood. Chemokines, small secreted proteins that
regulate leukocyte migration, are prime candidates to regulate this exit. It has been
shown that chemokines have key roles in B cell differentiation and migration.
We will evaluate the effect of the deficiency of chemokine receptor CCR7 on the exit
of plasma cells and memory B cells from the spleen to other organs such as lymph
nodes, blood and bone marrow. CCR7 binds to chemokines CCL19 and CCL21,
produced by stromal cells in the vicinity of GCs. Mouse models deficient for CCR7 on
B cells are available.
Preliminary data shows that CCR7-deficient antigen-specific B cells form different
memory B cell populations than CCR7-sufficient antigen-specific B cells but the
relevance of these changes in memory B cell populations is unclear. The response of
these memory B cells to subsequent antigen encounters may be affected and needs
to be studied.
The project will help to understand migration and differentiation of effector cells,
especially of the different subsets of memory B cells, which may lead to new ways of
manipulating antibody producing cells in vaccine responses.
References
Allen, C. D., T. Okada, J. G. Cyster (2007). “Germinal-center organization and cellular
dynamics. ” Immunity 27(2): 190-202.
Comerford, I., Y. Harata-Lee, M. D. Bunting, C. Gregor, E. E. Kara, S. R. McColl
(2013). “A myriad of functions and complex regulation of the CCR7/CCL19/CCL21
chemokine axis in the adaptative immune system.” Cytokine Grow Factor Rev 24(3):
269-283.
Zhang, Y., M. Meyer-Hermann, L. A. George,M.T. Figge, M. Khan, M. Goodall, S. P.
Young, A. Reynolds, F. Falciani, A. Waisman, C. A. Notley, M.R. Ehrenstein, M.
Kosko-Vilbois, K. M.Toellner (2013). “Germinal center B cells govern their own fate
via antibody feedback.”J Exp Med 11;201(3): 457-464
140
All the experiments will be planned in regular meetings between student, PhD
student co-supervising and main supervisor ensuring the logical progress of the
project.
Experiments performed in the lab will be directly supervised and guided by PhD
student, who is expert in all techniques and procedures involved, and works on a
The student role.
Also, she/he will participate in journal clubs, giving her/him the possibility to improve
Procedures on animals can be carried out by the PhD student, and a Home Office
license to perform procedures on animals can be acquired during the course of the
project.
141
Lead Supervisor:
Kai-Michael Toellner
Contact Email:
Telephone:
[email protected]
Tel: 0121 415 8687
Co Supervisor:
Yang Zhang
Project Title:
The gut, immune responses and the effects of aging
Department:
Yes or
No
Project Outline
Immunosenescence is a well known phenomenon of old age. Our ability to produce
adequate responses to vaccination declines dramatically as we enter our 70s. How B
cells are affected by aging remains a topic of study. Different B cell subsets have
been shown to increase as we age (and are also associated with autoimmunity)
(1.2). Our lab examined the contributions of the splenic environment compared to
that of the intrinsic effects of aging on B cells. The data show that aged B cells
produced more antibody and longer lived germinal centres when transferred into
young hosts compared to young B cells. Further, the aged environment is greatly
repressive to the immune responses of young B cells. The histological staining
showed changes in B cell follicle structure in aged lymph nodes. Recently, a role for
fibroblastic reticular cells (FRC) in the control of B cell homeostasis was shown in
lymph nodes (3). Stromal cells also regulate production and migration of plasma
cells. We have identified FRCs are in intimate contact with plasma cell differentiating
from the germinal centre, and may regulate germinal centre derived plasma cell
production. We hypothesize that changes of stromal cells in old age affect B cell
homeostasis and immune responses after antigen challenge.
The project will study these stromal cells before and after antigen challenge in gut,
spleen, and other lymphoid tissues of young and aged mice, to investigate how
ageing affects the distribution of stromal cells and their interactions with B cells. The
project will involve immunohistochemistry, light and confocal microscopy and, time
allowing, RT-PCR.
References
1. Ademokun A et al, The ageing B cell population: composition and function,
Biogerontology, 2010 11: 125-137
2. Scholz JL et al, A comparative review of aging and B cell function in mice and
humans, Curr Opin Immunol, 2013, 25(4): 504-10
3. Cremasco V et al, B cell homeostasis and follicle confines are governed by
fibroblastic reticular cells. Nat Immunol, 2014 15(10): 973-81
All the experiments will be planned in regular meetings between student, postdoctor
co-supervising and main supervisor ensuring the logical progress of the project.
Experiments performed in the lab will be directly supervised and guided by the
postdoctor, who is expert in all techniques and procedures involved, and works on a
142
The student role.
Procedures on animals can be carried out by postdoctor, and a Home Office license
143
Lead Supervisor:
Kai-Michael Toellner
Contact Email:
Telephone:
[email protected]
Tel: 012 4158687
Co Supervisor:
Yang Zhang
Project Title:
Regulation of B cell responses specific for tumour
expressed antigens
Department:
No
Project Outline
More than 1/3 people in the UK will develop cancer, and more than 1/4 will die from
it. Consequently there is great need for safer and more effective therapies. There is
more and more evidence from clinical practice that antibodies are highly efficient
drugs for the treatment of cancer. We recently published a new vaccine design that
allows induction of high titres of antibody specific to tumour endothelium expressed
antigen Robo4 (1). By chemically linking autoantigen to a foreign carrier protein to
which pre-existing immunity is present, autoreactive B cells are able to recruit T cell
help from primed carrier-specific T helper cells. Our published method used Robo4 as
the tumour expressed autoantigen and chicken gamma globulin as the foreign
carrier. We recently also have shown that coupling another tumour endothelium
expressed antigen – CLEC14a – to the carrier tetanus toxoid (TT) results in antibody
response with similarly efficiency are induced that confer tumour growth inhibition.
CLEC14a, is highly present on the vasculature of many common human cancers
(including liver, oesophageal, pancreatic, breast, bladder, ovarian) but is
low/undetectable in healthy tissue (2). We hypothesize that target CLEC14a by using
this type of vaccine can induce antibodies which specifically react with CLEC14a, and
then damage tumour vessels and inhibit tumour growth without affecting normal,
non-tumour vessels.
The project will test the efficiency of the vaccine response when the tumour is
already established, long term antibody production, and also test whether antibody
response is inhibited by the presence of autoantigen. The project will involve
fluorescent staining, Flow cytometry, ELISA, and, time allowing, RT-PCR.
References
1. Zhuang, X., Ahmed, F., Zhang, Y., Ferguson, H. J., Steele, J. C., Steven, N.
M., Nagy, Z., Heath, V. L., Toellner, K.-M. and Bicknell, R. (2014) Robo4
vaccines induce antibodies that retard tumor growth Angiogenesis. Available
online
2. Mura, M., Swain, R. K., Zhuang, X., Vorschmitt, H., Reynolds, G., Durant, S.,
Beesley, J. F., Herbert, J. M., Sheldon, H., Andre, M., Sanderson, S., Glen, K.,
Luu, N. T., McGettrick, H. M., Antczak, P., Falciani, F., Nash, G. B., Nagy, Z. S.
and Bicknell, R. (2012) Identification and angiogenic role of the novel tumor
endothelial marker CLEC14A. Oncogene. 31, 293-305
144
All the experiments will be planned in regular meetings between student, postdoctor
co-supervising and main supervisor ensuring the logical progress of the project.
Experiments performed in the lab will be directly supervised and guided by the
postdoctor, who is expert in all techniques and procedures involved, and works on a
The student role.
Procedures on animals can be carried out by postdoctor, and a Home Office license
145
Lead Supervisor:
Dr Jianmin Zuo
Contact Email:
Telephone:
[email protected]
0121-4144473
Co Supervisor:
Dr Francesca Kinsella
Project Title:
Study of the role of NK Activation and Inhibition
Receptors in the Graft versus Leukaemia effect and
Graft versus Host Disease
School Of Cancer Sciences
Department:
No
Project Outline
Stem Cell transplantation (SCT) is used widely in the management of haematological
malignancies where its curative potential relies largely on the ‘graft versus leukaemia’
(GvL) effect that is mediated by the donor immune system. The mechanism of the
GvL effect is uncertain but is believed to be mediated by donor-derived T and NK
cells that recognise alloreactive proteins on the tumour target cell. As GvL is often
associated with the detrimental effect of ‘graft versus host disease’(GvHD), a key aim
within the SCT field is to selectively promote GvL whilst controlling GvHD.
Work in our group has recently provided support for a role of the activatory NK
receptor, NKG2D in GvL, by showing the impact of polymorphisms in its ligand ULBP6
on SCT outcomes. Recently, murine models have also provided evidence to suggest
a role for other activatory receptors in modulating GvHD.
We therefore propose to undertake a comprehensive study of NK cell activatory and
inhibitory receptors in the SCT patients, including the stem cell bags and PBMCs post
transplantation to assess their impact on GvL and GvHD.
1. NK cell activatory / inhibitory receptor expression before and after SCT will be
studied using multi-colour flow-cytometry. NK subset cytokine production will be
assessed ex vivo with intracellular cytokine staining, and their cytotoxic capacity
assessed in vitro by their ability to kill target cells.
2. We propose to study the cohort patients to compare the phenotype of expanding
T cells post-SCT and correlate with their NK phenotype. We will analyze the
proportions of memory and naïve CD8 T cell populations, their proliferation (Ki67
146
staining), and cytokine production after SCT. A correlation will be sought between
the CD8+ T cell phenotype, NK pheotype, and clinical outcome.
3. The Mixed Lymphocyte Reaction (MLR) is a functional assay which can measures
the proliferative response of T lymphocytes from one individual to lymphocytes from
another individual. It will be used to test the hypothesis of the regulation of T cell
activation by NK cells.
Importantly, by studying NK activation and inhibition receptors in the SCT will
potentially guide strategies for obtaining better GvL while minimizing GvHD in SCT by
manipulating NK population.
References
Antoun A, et al. (2012), 159(5), 589-598. The genotype of RAET1L (ULBP6), a ligand
for human NKG2D (KLRK1), markedly influences the clinical outcome of allogeneic
stem cell transplantation. British Journal of Haematology.
Ghadially H, et al. (2014), 7(6), 1809–1814. NK Cell Receptor NKp46 Regulates
Graft-versus-Host Disease. Cell Reports.
Dr Zuo and Dr Kinsella will be guiding the student about the project and meeting
regularly for discussion. Dr Zuo, Dr Kinsella and Mr Luke Maggs will help the student
to master the lab-based technologies and also perform the day to day direct
supervision.
The student role.
1. In the lab, the student will carry out the lab work, such as PBMC separation, FACS
staining, in vitro function assay (including NK cytotoxicity assay and Mixed
Lymphocyte Reaction).
2. The student will attend the internal and external seminars of the department, will
attend the group lab meetings and present their data, also will attend the journal
club and discussion relevant paper to broad their scientific knowledge.
147
Lead Supervisor:
Contact Email:
Telephone:
[email protected]
Mehanna)
Dr Paul Nankivell, University of Birmingham
Dr Mark Prince, Aston University
Co Supervisor:
Project Title:
Developing a surgical robot for oropharyngeal cancer
surgery.
Department:
Cancer Sciences
Will the project require a Home Office working with animals licence? NO
Project Outline
The Institute of Head and Neck Studies and Education (InHANSE), led by Professor
Hisham Mehanna, focuses on research into diseases of the head, neck and thyroid,
and on the education of health professionals in the field.
For head and neck cancer one of the most complex areas of management is the
choice of primary treatment (surgery vs chemoradiotherapy) and this often relies on
clinical factors (mainly clinical stage) and the therapeutic preference of the treating
centre.
Increasingly oropharyngeal cancer is being treated surgically with the advent of the
intuitive surgical robot. However, this robot was designed for abdominal surgery and
is not ideally suited for operating within the mouth and oropharynx. Our aim,
therefore is to develop a robotic platform that is designed specifically for operating
within the mouth and the oropharynx, especially the base of tongue and tonsil.
We have started working on designing the robot. This is being done in collaboration
with Dr Mark Prince from Aston University and Mr Chris Coulson from the Queen
Elizabeth Hospital. Both have had experience in working on surgical robotic
platforms in the head and neck.
InHANSE is a team of over 15 researchers. This includes a clinical lecturer with an
interest in the development of surgical devices – Paul Nankivell.
The student will be supervised in their work both by Professor Mehanna and Dr
Nankivell. Day to day supervision will be by Dr Nankivell, weekly updates to
Professor Mehanna at the InHANSE weekly team meeting and monthly supervisory
meetings with Professor Mehanna.
The student role.
148
The student will undertake the following.
1. A review of the literature and the competitive landscape, examining
approaches to operating in the oropharynx and existing robotic solutions.
2. Will carry out interviews with clinicians who have used the intuitive robot to
explore what the current impediments are and how to improve it if possible.
3. Carry out an evaluation of dimensions and volumes of the oropharynx and
mouth using 3D reconstructions of CT scans.
149
Lead Supervisor:
Mark Webber
Contact Email:
Telephone:
Co Supervisor:
[email protected]
0121 414 2859
www.antimicrobialagentsresearchgroup.com
Laura Piddock
Project Title:
Supercoiling and Superbugs
Department:
No
Is the Project Cancer related? No (Infection)
Project Outline
Antibiotic resistance is one of the great global health challenges of the 21st century
and a problem which is raidly worsening with the emergence and spread of
multidrug resistant pathogens and a dearth of new drugs in development. This has
raised the real scenario of infections with untreatable pathogens and threatens an
extraordinarily broad range of clinical activities where antibiotics are needed to both
treat infection and provide prophylactic cover to allow complex surgery etc.
Bacteria can employ a range of mechanisms to resist the actions of antibiotics,
recently we have discovered that mutations within DNA gyrase which confer
resistance to the powerful quinolone antibiotics by altering their target site also give
a low level of resistance to many other drugs which do not target this enzyme. DNA
gyrase controls how DNA is packaged (supercoiled) within the cell and changes in its
activity alter chromosome structure and as a result expression of many other genes.
We have found that gyrase mutants show up-regulation of stress response pathways
which provide broad antimicrobial protection which we hypothesise to be due to a
result of altered supercoiling.
In this project we aim to take some of the stress responsive genes implicated in the
antibiotic resistance phenotype and move them to new locations within the bacterial
genome. We will then measure their expression (in response to mutation of DNA
gyrase and in the presence and absence of quinolones) in order to determine
whether local DNA supercoiling is key to production of a protective stress response.
The project will contain a mixture of microbiology, molecular biology, antibiotic
sensitivity testing and cellular permeability assays.
4.
5.
6.
References
Blair JM, Webber MA, Baylay AJ, Ogbolu DO, Piddock, LJ. Molecular
mechanisms of antibiotic resistance. Nat Rev Micro. 2015 In press.
Redgrave LS, Sutton SB, Webber MA, Piddock LJ. Fluoroquinolone
resistance: mechanisms, impact on bacteria, and role in evolutionary
success. Trends Microbiol. 2014 Aug;22(8):438-445. PMID:24842194
Clinically relevant mutant DNA gyrase alters supercoiling, changes the
transcriptome and confers multi-drug resistance. Webber MA, Ricci V,
Whitehead RN, Patel M, Fookes M, Ivens A, Piddock LJ. mBio. 2013. Jul
150
23;4(4). doi:pii:e00273-13.
23882012.
10.1128/mBio.00273-13.
PubMed
PMID:
Students will meet weekly with Dr Webber and bi-weekly with both Dr Webber and
Prof Piddock. They will be supervised daily by Dr Webber or other members of the
antimicrobials research group which currently contains 15 full time members
(www.antimicrobialagentsresearchgroup.com) ensuring appropriate cover will be
available for student supervision. Webber has successfully supervised seven
intercalating students in the last six years.
The student role.
The student will be responsible for performing and analysing experiments under the
direction of Dr Webber and Prof Piddock and will be treated within the laboratory as
any other member of the research team. The student will have responsibility for
investigating the background of the project and developing a good awareness of the
context and aims of the project
151
Lead Supervisor:
Babu Naidu
Contact Email:
Telephone:
[email protected]
Co Supervisor:
Amy Kerr ( nee Bradley)
Project Title:
[email protected]
Department:
CEM
No
Is the Project Cancer related? yes
Project Outline
5700 patients a year in the UK undergo major surgery to remove part of their lungs;
primarily to cure cancer. A common post-operative complication is collapse or
infection in the remaining lung (15% of cases). These complications are linked to
increased risk of death, likelihood of admission into an intensive care unit and longer
hospital stay. (1)
We developed a comprehensive pulmonary rehabilitation programme (ROC- 2010)
of an out-patient based programme consisting of exercise training, self-management
education, nutritional and smoking cessation support and demonstrated in initial
results improvement in complication and hospital readmission rates though data. (2)
Lack of immediate access to pulmonary rehabilitation programmes across the
country has hampered spread. There is thus a need to develop a service that can be
delivered immediately at the convenience and in control of the patient.
A tailored at home smart device ‘app’ based lung rehabilitation programme for lung
surgery has already been developed but requires testing and refinement. (3) As with
outpatient based pulmonary rehabilitation it aims to improve patient “fitness” for
surgery, thus reducing post-surgical complications and enhancing recovery. This will
improve long term patient centric outcomes such as quality of life and freedom from
associated symptoms such as breathlessness.
References
1. Agostini P, Cieslik H, Bishay E, Kalkat MS, Rajesh PB, Steyn RS, Naidu B The
impact and risk factors for Post operative Pulmonary Complications following Lung
Resection in current UK practice – Are there any modifiable factors?” Thorax. 2010
Sep;65(9):815-8.
2. Bradley A, Marshall A, Stonehewer L, Reaper L, Parker K, Bevan-Smith E, Jordan
C, Gillies J, Agostini P, Bishay E, Kalkat M, Steyn R, Rajesh P, Dunn J, Naidu B.
Pulmonary rehabilitation programme for patients undergoing curative lung cancer
surgery. Eur J Cardiothorac Surg. 2013 Oct;44(4):e266-71
3.
Scan for demo
152
The student will form part of the team of three research nurses, scientist and 2
clinical research fellows based at Heartlands hospital . We have a regular team
meeting every two weeks followed by one to one sessions as required. The student
will thus benefit from working in a supportive team environment
The student role.
The student will have two roles
1. The ROC programme is ongoing and since the initial publication collected data
needs to be cleaned analysed written up and presented.
2. The ROC app has been developed but qualitative testing gathering feedback from
patients carers and health care professionals is required. This will be collected into a
report which will be disseminated at regional national and international meetings and
will serve to refine the app prior to wider testing
153
Lead Supervisor:
Dr Farhat Khanim
Contact Email:
Telephone:
[email protected]
Co Supervisor:
Prof Chris Bunce, Prof Mark Drayson
Project Title:
Department:
Can we identify microRNAs that are prognostic indicators
of response to BaP therapy in AML/MDS patients?
School of Biosciences and Immunity and Infection
No
Project Outline
3.3.4. Effect of BaP on microRNA (miRNA) and non-coding RNA (ncRNA) expression
MiRNAs are a class of small noncoding RNA (∼22 nucleotides) that regulate gene expression
by triggering message RNA (mRNA) degradation or inhibiting protein translation. Almost
1,000 miRNAs have been identified in the human genome and are thought to regulate 30%
of the transcriptome. Not only have some miRNAs been demonstrated to have a role in the
development of some types of haematological cancers, there is growing appreciation that
heterogeneity amongst AML and CLL, and responses to therapy may be regulated at the
level of miRNA activity. There are also direct links between miRNAs and the regulation of
core metabolism and lipid metabolism. For example mitochondrial glutaminase (GLS), a key
enzyme that converts glutamine to glutamate, which serves as a substrate in the TCA cycle
for the production of ATP is regulated by miR-23a and miR-23b. MiRNAs are surprisingly
stable and detectable in the circulation where they are proving to be a powerful diagnostic
and prognostic tool in several diseases including AML, B cell lymphomas and myeloma.
Our translational approach addresses acute myeloid leukaemia (AML), chronic lymphocytic
leukaemia (CLL) and B- cell Non-Hodgkins lymphomas (B-NHL) including endemic Burkitt's
lymphoma (eBL). These haematological malignancies share a common need for innovative
therapies that display clinically significant anti-tumour activity without haematological
toxicity. Our work began in AML and has led us to trialling a drug redeployment approach
using combined Bezafibrate and medroxyprogesterone acetate (BaP). Our studies have also
identified that the same drug combination has potential against CLL and B-NHL.
It is therefore timely and necessary to investigate the role of miRNAs in mediating the antileukaemic effects of BaP and whether they can be used as prognostic indicators of response
to BaP.
We will perform microRNA microarrays on control vs BaP treated AML, CLL, and B-NHL cell
lines and primary samples. These data will be integrated using bioinformatics approaches
with responses to BaP treatment as measured using a range of cell-based and flow
cytometric assays.
Where targets of identified miRNAs are known we will investigate their modulated
expression in response to treatments and their expression between responders and nonresponders (immunoblotting/QRTPCR). Where targets are unknown we will use lentiviral
transgenic, proteomics and array approaches to identify proteins and mRNAs targets.
References
154
Esquela-Kerscher, A. and F.J. Slack, Oncomirs - microRNAs with a role in cancer. Nat
Rev Cancer, 2006. 6(4): p. 259-69.
Hayden, R.E., et al., Treatment of primary CLL cells with bezafibrate and
medroxyprogesterone acetate induces apoptosis and represses the pro-proliferative
signal of CD40-ligand, in part through increased 15dDelta12,14,PGJ2. Leukemia :
official journal of the Leukemia Society of America, Leukemia Research Fund, U.K,
2009. 23(2): p. 292-304.
Khanim, F.L., et al., Combined bezafibrate and medroxyprogesterone acetate:
Potential novel therapy for acute myeloid leukaemia. PLoS ONE, 2009. In Press.
Murray, J.A., et al., Combined bezafibrate and medroxyprogesterone acetate have
efficacy without haematological toxicity in elderly and relapsed acute myeloid
leukaemia (AML). British journal of haematology, 2010. 149(1): p. 65-9
The student will be co-supervised by Dr Farhat Khanim, Prof Chris Bunce and Prof
Mark Drayson. We will have regular meetings to discuss progress and to address
any developments in the project. Daily laboratory supervision will be undertaken by
Dr Farhat Khanim. Some of the techniques used in the project are undertaken in the
Clinical Immunology Service run by Prof Mark Drayson. This will give the student a
very useful insight into clinical laboratory techniques to compliment the cell and
molecular techniques they will learn in the research laboratory.
The student role.
Clinical researchers are absolutely essential for successful translation of research
findings into the clinic. Hence it is important that intercalating students have a
positive research project experience and leave with the motivation and enthusiasm to
continue with research. Hence, the student will work within the supportive network
of a larger group of post-docs and students, will be encouraged to interact with them,
and will be treated as a member of the research team. The project is organised such
that the student will be very closely supervised during the first few weeks and
semester. Once the student is confident in the lab and with the laboratory protocols,
the students are given more independence to organise their own time and design the
experiments. The student will be encouraged to read papers and develop their own
ideas and hypotheses about the work and design their own experiments. They will
also be encouraged to attend national and international meetings to present their
work if a relevant meeting/conference comes up. This supervisory approach has
been extremely successful with our previous students. Blair Merrick won a poster
prize at the International Myeloma Workshop in Paris 2011 and Hannah Giles
presented the work at a national student conference 2010 and won third prize for
best oral presentation. Suzanne Raffles and Lauren Ferretti both won prizes and
came top of the year for their intercalation projects (2012, 2013, respectively). Both
Suzanne and Lauren also won prizes for best oral presentation on their projects at
the International Doctors Research Symposium. Lauren also presented her work at
a meeting in Vienna in 2013. Matt Fenton won best intercalation project prize, the
Frank Kerr prize, and second place for the Claire Ripley award in 2014.
Our goal is to give the students real experience of research and the importance of
the interface between the lab and the clinic.
155
Lead Supervisor:
Dr Farhat Khanim
Contact Email:
Telephone:
[email protected]
Co Supervisor:
Project Title:
New use of old drugs in the treatment of myeloma;
investigation of mechanisms of action against the malignant
clone
Department:
No
Project Outline
Rationale
Myeloma is a cancer of bone marrow plasma cells that impairs normal haemopoiesis
and antibody production, destroys the skeleton and by secretion of M-protein causes
renal failure. Treatment with high dose cytotoxic drugs and stem cell rescue
improves survival. More recently biological therapies including thalidomide and its
analogues and proteasome inhibitors have provided further improvement in survival
but at a pharmaceutical cost of up to £50,000 per patient per year. Cures remain
elusive and the disease kills 3,000 people per year in the UK.
Translational research in Birmingham identified new molecular targets for a
combination of two old drugs (progesterone and bezafibrate). As a result the drug
combination is being tested as treatment for adult acute leukaemia and childhood
Burkitt’s lymphoma. This strategy of redeployment of old drugs has been
investigated by previous intercalating students who have identified a combination
(VaN) of an anti-epileptic (valproate) and an anti-helminthic (niclosamide) with
activities against myeloma cell lines. This very successful project has already resulted
in 1 publication and a second manuscript is under preparation with all intercalating
students as co-authors.
The proposed project for 2015/2016 is a continuation of this work. We will be
investigating the effect of VaN combination therapy on primary myeloma tumour
cells both in vitro and also using a mouse model in collaboration with a group in
Sheffield. The mouse model will be used to study the effect of the drugs on myeloma
induced lytic bone lesions, cytokine secretion and disease progression. A previous
student identified that VaN modifies the “acetylome” (acetylated proteins) in
myeloma cells. We will continue these studies looking at the effect of VaN therapy on
the acetylome of myeloma cells using a combination of molecular, cellular and
biochemical approaches. The ultimate aim is to generate enough data to initiate
phase II clinical trials of VaN therapy in myeloma.
References
Khanim, F.L., et al., Redeployment-based drug screening identifies the anti-helminthic
niclosamide as anti-myeloma therapy that also reduces free light chain production. Blood
cancer journal, 2011. 1(10): p. 21.
156
The student role.
157
Lead Supervisor:
Dr Farhat Khanim
Contact Email:
Telephone:
[email protected]
Co Supervisor:
Project Title:
Can bacterial NDK proteins regulate human haemopoiesis
and leukaemic progression?
Department:
No
Is the Project Cancer related? YES, Infection related also
Project Outline
Rationale
We have shown that the human nucleotide diphosphate kinase (NDK) protein, NM23-H1, is
able to promote survival and induce proliferation of human haemopoietic stem cells, both
normal and leukaemic (Lilly et al, 2011, Manuscript in preparation). NDKs are highly
evolutionarily conserved and are also expressed by bacteria. Based on our previous data
with human NDK (NM23-H1) and the fact that bacterial infection is a common clinical
feature of leukaemia’s, also myelodysplastic syndromes (MDS), and is often the cause of
mortality in these patients, we hypothesised that bacterial NDK proteins generated during
infection may promote progression of the leukaemia or MDS. In collaboration with Mark
Webber (IMI), we have cloned NDK ORFs from 2 gram-positive and 2 gram-negative bacteria
and produced recombinant HIS-tagged protein. Although we need to treat more samples,
preliminary data shows clearly that the bacterial NDK proteins are able to induce similar
effects on human umbilical blood derived haemopoietic stem cells as recombinant human
NDK (NM23-H1). This work has major implications for how bacterial infections are managed
in leukaemia/MDS patients but also raises very interesting questions about the influence of
bacteria on the evolution of the haemopoietic system and haemopoietic stem cells.
The proposed project for 2015/2016 is a continuation of this work. We will be investigating
the effect of human and bacterial NDK proteins on adult normal donor derived
haemopoietic stem cells (HSCs) and leukaemic cells. We will look at the effect on cell
proliferation as measured using cellular and flow cytometry based assays. Luminex analysis
will be used to dissect the influence of human and bacterial NDK proteins on cytokine and
growth factor secretion. We will use a combination of molecular and proteomics strategies
to identify the receptor for bacterial NDKs on HSCs and leukaemia cells. We will also
investigate the possibility of generating antibodies specific for the bacterial NDKs so that we
can develop an ELISA that can be used on patient sera to monitor bacterial NDK levels in
MDS and leukaemia patients.
References
Lilly A.J., Khanim F.L., Hayden R.E. et al. Nm23-h1 indirectly promotes the survival
of acute myeloid leukemia blast cells by binding to more mature components of the
leukemic clone. Cancer research 71, 1177-1186, doi:10.1158/0008-5472.CAN-101704
Niitsu N., Okabe-Kado J., Okamoto M. et al. Serum nm23-H1 protein as a prognostic
factor in aggressive non-Hodgkin lymphoma. Blood 97, 1202-1210 (2001)
Chopra P., Singh A., Koul A. et al. Cytotoxic activity of nucleoside diphosphate
kinase secreted from Mycobacterium tuberculosis. Eur J Biochem 270, 625-634,
158
doi:3402 [pii] (2003).
The student role.
159

Available Projects - University of Birmingham

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