http://www.dspace.cam.ac.uk:80/handle/1810/221766 Mon, 20 May 2013 16:20:39 GMT 2013-05-20T16:20:39Z http://www.dspace.cam.ac.uk:80/handle/1810/241721 Title: Fusion genes in breast cancer Authors: Batty, Elizabeth Abstract: Fusion genes caused by chromosomal rearrangements are a common and important feature in haematological malignancies, but have until recently been seen as unimportant in epithelial cancers. The discovery of recurrent fusion genes in prostate and lung cancer suggests that fusion genes may play an important role in epithelial carcinogenesis, and that they have been previously under-reported due to the difficulties of cytogenetic analysis of solid tumours. In particular, breast cancers often have complex, highly rearranged karyotypes which have proved difficult to analyse using classical cytogenetic techniques. The aim of this project was to search for fusion genes in breast cancer by using high-resolution mapping of chromosome rearrangements in breast cancer cell lines. Mapping the chromosome rearrangements was initially done using high-resolution DNA microarrays and fluorescence in- situ hybridisation, but moved to high-throughput sequencing as it became available. Interesting candidate genes identified from the mapped chromosome rearrangements were investigated on a larger set of cell lines and primary tumours. The complete karyotypes of two breast cancer cell lines were constructed using a combination of microarrays, fluorescence microscopy, and high-throughput sequencing. A number of potential fusion genes were identified in these two cell lines. Although no expressed fusion genes were found, the complete karyotypes gave insight into the number and mechanisms of chromosome rearrangement in breast cancer, and identified interesting candidate genes which may be of importance in tumourigenesis. Two genes which were fused in other breast cancer cell lines, BCAS3 and ODZ4, were disrupted by chromosome rearrangements and identified as interesting candidate genes in tumorigenesis. A bioinformatic pipeline to process high-throughput sequencing data was set up and validated, and shown to more accurately predict fusion genes than other methods, and can be used to investigate further cell lines and tumours for recurrent fusion genes. The pipeline was used to analyse data from 3 other breast cancer cell lines and predict chromosomal rearrangements and fusion genes, several of which were found to be expressed. Of the fusions predicted in the cell line ZR-75-30, 7 expressed fusion genes were identified, and may have functional significance in breast cancer. Tue, 07 Feb 2012 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/241721 2012-02-07T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/241516 Title: Glycoprotein M and ESCRT in herpes simplex virus type 1 assembly Authors: Ren, Yudan Abstract: Herpes simplex virus type 1 (HSV-1) has a large linear double-stranded DNA genome in an icosahedral capsid shell, a cell-derived lipid envelope and a proteinaceous tegument layer. There are over fifty viral proteins and many host proteins identified in HSV-1 virions. The final formation of mature virus particles requires the membrane wrapping of tegumented capsids in the cytoplasm, a process termed secondary envelopment. This process involves the coordination of numerous viral and cellular proteins and results in double-membrane structures with enveloped virions contained within cellular vesicles. Mature viruses are then released through the fusion of these virion-containing vesicles and plasma membranes. This thesis describes investigation into the functions of viral glycoprotein M (gM) and the cellular Endosomal Sorting Complexes Required for Transport (ESCRT) in secondary envelopment. Firstly, it has been reported that gH/L can be efficiently internalised and targeted to the TGN by the co-expression of gM in transfection assays. In order to examine the role of gM in guiding the localisation of viral proteins in infected cells, a HSV-1 gM deletion virus (∆gM), and its revertant virus were constructed. The major phenotype demonstrated was that the absence of gM caused the internalisation of cell surface gH/L to be inhibited and higher levels of gH/L to be observed on the cell surface. Further, lower levels of gH/L were detected in purified ∆gM virions, which was in agreement with the delayed entry kinetics, smaller plaque sizes and greater replication deficits at low multiplicity of infection observed in ∆gM infected cells. Over all the results presented in this thesis demonstrate that in infected cells the efficient incorporation of gH/L into virions relies on the function of gM in HSV-1. Secondly, during HSV-1 secondary envelopment the budding and scission of the viral envelope from the host membrane share topological similarities with the formation of intraluminal vesicle in multivesicular bodies, retrovirus budding, and abscission at the end of cytokinesis, processes that require the cellular ESCRT machinery. There are four multiprotein ESCRT complexes and many associated proteins involved in their regulation. It has been previously shown that the ESCRT-III complex and a functional ATPase VPS4 are required for HSV-1 secondary envelopment, but different from the strategy utilised by HIV-1, the recruitment of ESCRT during HSV-1 infection is independent of TSG101 and/or ALIX. Data presented in this thesis demonstrate that CHMP4A/B/C proteins of the ESCRT-III complex are specifically crucial for HSV-1 secondary envelopment. Simultaneous depletion of CHMP4A/B/C proteins significantly inhibited HSV-1 replication. Ultrastructure analysis revealed that there were virtually no extracellular virions in CHMP4A/B/C depleted samples while more free capsids were observed in the cytoplasm, although the nuclear capsids and primary envelopment events appeared to be normal. In order to identify interactions between HSV-1 and ESCRT proteins, 22 HSV-1 tegument proteins were cloned and tested against a panel of ESCRT and ESCRT-associated proteins in yeast two-hydrid assays. Analysis of positive hits from yeast two-hybrid interaction screens using GST pull-down, co-immunoprecipitation and protein co-localisation assays have validated interactions of pUL47 with CC2D1A/1B, CIN85, CHMP6 and ALIX, pUL46 and pUL49 with CC2D1A/1B and CIN85, and pUL16 with CC2D1A/1B. Furthermore, the newly identified ESCRT associated proteins CC2D1A and CC2D1B have been detected in purified virions. The role of the identified ESCRT proteins in HSV-1 replication has been investigated using siRNA depletion. Unfortunately siRNA depletions of the various ESCRT candidates individually or in combinations did not show any significant effect on HSV-1 replication. Overall these data suggest that unlike HIV and other retroviruses, HSV-1 has evolved multiple parallel pathways to hijack the ESCRT machinery to facilitate its replication, particularly, through the interactions that lead directly to the recruitment of CHMP4A/B/C proteins. Disruption of some of these pathways did not prevent HSV-1 replication in tissue culture, suggesting any one potential pathway is sufficient for ESCRT recruitment to sites of HSV-1 assembly. Tue, 10 Jan 2012 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/241516 2012-01-10T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/239397 Title: The structure and evolution of breast cancer genomes Authors: Newman, Scott Abstract: Chromosome changes in the haematological malignancies, lymphomas and sarcomas are known to be important events in the evolution of these tumours as they can, for example, form fusion oncogenes or disrupt tumour suppressor genes. The recently described recurrent fusion genes in prostate and lung cancer proved to be iconic examples as they indicated that important gene fusions are found in the common epithelial cancers also. Breast cancers often display extensive structural and numerical chromosome aberration and have among the most complex karyotyes of all cancers. Genome rearrangements are potentially an important source of mutation in breast cancer but little is known about how they might contribute to this disease. My first aim was to carry out a structural survey of breast cancer cell line genomes in order to find genes that were disrupted by chromosome aberrations in “typical” breast cancers. I investigated three breast cancer cell lines, HCC1187, VP229 and VP267 using data from array painting, SNP6 array CGH, molecular cytogenetics and massively parallel paired end sequencing. I then used these structural genomic maps to predict fusion transcripts and demonstrated expression of five fusion transcripts in HCC1187, three in VP229 and four in VP267. Even though chromosome aberrations disrupt and fuse many genes in individual breast cancers, a major unknown is the relative importance and timing of genome rearrangements compared to sequence-level mutation. For example, chromosome instability might arise early and be essential to tumour suppressor loss and fusion gene formation or be a late event contributing little to cancer development. To address this question, I considered the evolution of these highly rearranged breast cancer karyotypes. The VP229 and VP267 cell lines were derived from the same patient before and after therapy-resistant relapse, so any chromosome aberration found in both cell lines was probably found in the common in vivo ancestor of the two cell lines. A large majority of structural variants detected by massively parallel paired end sequencing, including three fusion transcripts, were found in both cell lines, and therefore, in the common ancestor. This probably means that the bulk of genome rearrangement pre-dated the relapse. For HCC1187, I classified most of its mutations as earlier or later according to whether they occurred before or after a landmark event in the evolution of the genome - endoreduplication (duplication of its entire genome). Genome rearrangements and sequence-level mutations were fairly evenly divided between earlier and later, implying that genetic instability was relatively constant throughout the evolution of the tumour. Surprisingly, the great majority of inactivating mutations and expressed gene fusions happened earlier. The non-random timing of these events suggests many were selected. Mon, 11 Jul 2011 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/239397 2011-07-11T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/238398 Title: Molecular Pathogenesis of MALT lymphoma Authors: Hamoudi, Rifat A Abstract: Mucosa associated lymphoid tissue (MALT) lymphoma is characterized by t(11;18)(q21;q21)/API2-MALT1, t(1;14)(p22;q32)/BCL10-IGH and t(14;18)(q32;q21)/IGH-MALT1, which commonly activate the NF-κB pathway. Gastric MALT lymphomas harbouring such translocation do not respond to Helicobacter pylori eradication, while those without translocation can be cured by antibiotics. To understand the molecular mechanism of MALT lymphoma with and without chromosome translocation, 24 cases (15 translocation-positive and 9 translocation-negative) of MALT lymphomas together with 7 follicular lymphomas and 7 mantle cell lymphomas were analysed by Affymetrix gene expression microarray platform. Unsupervised clustering showed that cases of MALT lymphoma were clustered as a single branch. However, within the MALT lymphoma group, translocation-positive cases were intermingled with translocation-negative cases. Gene set enrichment analysis (GSEA) of the NF-κB target genes and 4394 additional gene sets covering various cellular pathways, biological processes and molecular functions showed that translocation-positive MALT lymphomas were characterized by an enhanced expression of NF-κB target genes, particularly TLR6, CCR2, CD69 and BCL2, while translocation-negative cases were featured by active inflammatory and immune responses, such as IL8, CD86, CD28 and ICOS. Separate analyses of the genes differentially expressed between translocation-positive and negative cases and measurement of gene ontology term in these differentially expressed genes by hypergeometric test reinforced the above findings by GSEA. The differential expression of these NF-κB target genes between MALT lymphoma with and without translocation was confirmed by quantitative RT-PCR and immunohistochemistry or Western blot. Expression of TLR6, in the presence of TLR2, enhanced both API2-MALT1 and BCL10 mediated NF-κB activation in vitro. In addition, there was cooperation between expression of BCL10, MALT1 or API2-MALT1, and stimulation of the antigen receptor or CD40 or TLR in NF-κB activation as shown by both reporter assay and IκBα degradation. Interestingly, expression of BCL10 but not API2-MALT1 and MALT1, in the presence of LPS stimulation, also triggered IκBβ degradation, suggesting activation of different NF-κB dimers between these oncogenic products. Study by co-immunoprecipitation showed that BCL10 directly interacts with MALT1. Sub-cellular localisation experiments in BJAB B-cells, showed that BCL10 localisation was affected by MALT1. When BCL10 was over-expressed, the protein was predominantly expressed in the nuclei, but when MALT1 was over-expressed, BCL10 was mainly localised in the cytoplasm. When both BCL10 and MALT1 were over-expressed, BCL10 was expressed in the cytoplasm in the early hours when the protein level was low, but in both the cytoplasm and nuclei after 9 hours when the protein level was high. Over-expression of API2-MALT1 did not shown any apparent effect on BCL10 sub-cellular localisation in vitro. Finally, comparison of MALT lymphoma expression microarray with other lymphomas showed lactoferrin to be highly expressed in MALT lymphoma. This was confirmed by qRT-PCR, showing lactoferrin to be significantly over-expressed in MALT lymphoma compared to FL and MCL. Thus lactoferrin may be a potential marker for MALT lymphoma. Description: The original dissertation included a number or articles which were excluded from the digital file for copyright reasons. This is a list of the articles:; 1) Hamoudi RA, Appert A, Ye H, Ruskone-Fourmestraux A, Streubel B, Chott A, Raderer M, Gong L, Wlodarska I, De Wolf-Peeters C, MacLennan KA, de Leval L, Isaacson PG, & Du MQ. Differential expression of NF-kappaB target genes in MALT lymphoma with and without chromosome translocation: insights into molecular mechanism. Leukemia. 2010 Aug;24(8):1487-1497 http://www.ncbi.nlm.nih.gov/pubmed/20520640; 2) Ye H, Gong L, Liu H, Hamoudi RA, Shirali S, Ho L, Chott A, Streubel B, Siebert R, Gesk S, Martin-Subero JI, Radford JA, Banerjee S, Nicholson AG, Ranaldi R, Remstein ED, Gao Z, Zheng J, Isaacson PG, Dogan A & Du MQ. MALT lymphoma with t(14;18)(q32;q21)/IGH-MALT1 is characterized by strong cytoplasmic MALT1 and BCL10 expression. J Pathol. 2005 Feb;205(3):293-301. http://www.ncbi.nlm.nih.gov/pubmed/15682443; 3) Liu H, Hamoudi RA, Ye H, Ruskone-Fourmestraux A, Dogan A, Isaacson PG & Du MQ. t(11;18)(q21;q21) of mucosa-associated lymphoid tissue lymphoma results from illegitimate non-homologous end joining following double strand breaks. Br J Haematol. 2004 May;125(3):318-329. http://www.ncbi.nlm.nih.gov/pubmed/15086412; 4) Liu H, Ye H, Ruskone-Fourmestraux A, De Jong D, Pileri S, Thiede C, Lavergne A, Boot H, Caletti G, Wündisch T, Molina T, Taal BG, Elena S, Thomas T, Zinzani PL, Neubauer A, Stolte M, Hamoudi RA, Dogan A, Isaacson PG & Du MQ. T(11;18) is a marker for all stage gastric MALT lymphomas that will not respond to H. pylori eradication. Gastroenterology. 2002 May;122(5):1286-1294. http://www.ncbi.nlm.nih.gov/pubmed?term=11984515; 5) Liu H, Ye H, Dogan A, Ranaldi R, Hamoudi RA, Bearzi I, Isaacson PG & Du MQ. T(11;18)(q21;q21) is associated with advanced mucosa-associated lymphoid tissue lymphoma that expresses nuclear BCL10. Blood. 2001 Aug 15;98(4):1182-1187. http://www.ncbi.nlm.nih.gov/pubmed?term=11493468; 6) Liu H, Ruskon-Fourmestraux A, Lavergne-Slove A, Ye H, Molina T, Bouhnik Y, Hamoudi RA, Diss TC, Dogan A, Megraud F, Rambaud JC, Du MQ & Isaacson PG. Resistance of t(11;18) positive gastric mucosa-associated lymphoid tissue lymphoma to Helicobacter pylori eradication therapy. Lancet. 2001 Jan 6;357(9249):39-40. http://www.ncbi.nlm.nih.gov/pubmed?term=11197361 Fri, 01 Jan 2010 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/238398 2010-01-01T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/237703 Title: Investigating the molecular mechanisms of the metabolic syndrome Authors: Morris, Tiffany J Abstract: This thesis aims to highlight molecular mechanisms that have been altered by prenatal undernutrition and may be involved in the metabolic syndrome. Two sepa- rate studies were conducted both using a rat model developed through manipulation of the maternal diet to provoke the key features of the metabolic syndrome in adult o spring. Microarray technology was used to detect changes in gene expression in tar- get tissues between o spring of control (normally fed, AD) and undernourished (UN) mothers to obtain a broader picture of the cellular functions and genetic pathways that may be implicated in the metabolic syndrome. The rst study compared gene expression di erences in liver, skeletal muscle, and white adipose tissue between 55 day old male o spring of AD and UN mothers. No signi cant changes were found in muscle or adipose tissue; however, the di erences in the liver suggested the UN animals had been metabolically programmed to favour fat as an energy source. To investigate whether DNA methylation might be responsible for the observed transcriptional changes, pooled liver samples from the rst study were used with the McrBC restriction enzyme assay to determine full, partial, incomplete, or no methylation between AD and UN. Two di erentially expressed genes (Zfand2a and Mapk4) showed methylation changes. The same liver samples were hybridised to a miRNA array. Two miRNAs showed a nearly 2-fold upregulation in the UN livers. Both were found to be either directly or indirectly associated with the metabolic syndrome. MiR-335 has been shown to be upregulated in the livers of obese/diabetic mice. By association with miR-27a, miR-451 might be involved in aspects of lipid metabolism in adipose tissue. A second study used microarray to analyse the liver tissues of day 170 female o - spring of the same rat model with additional insults (neonatal leptin treatment and post-weaning high-fat (HF) diet). Leptin has been shown to reverse the programming e ects of the restricted maternal diet and this study aimed to highlight mechanisms that could be involved in this reversal. The results revealed the importance of the in- teraction between treatments. Signi cant gene expression changes were only present when two or more treatments were combined. This study revealed signi cantly, dif- ferentially expressed genes involved in immune function, regulation of the circadian rhythm, and metabolism. These ndings provide a number of interesting genes and pathways for further studies and also highlight the need to conduct a thorough study in multiple tissues at di erent time-points to pinpoint the window of developmental plasticity. Tue, 16 Nov 2010 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/237703 2010-11-16T00:00:00Z