Cardiovascular disease (CVD) is a major cause of morbidity and mortality in the U.S. and worldwide. Atherosclerosis, the buildup of plaque in the arteries, is a common cause of CVD. For many years, research in atherosclerosis was focused on lipid metabolism and the accumulation of low-density lipoprotein in the arteries. While this research set public health guidelines for lipid management, lipid concentration was not the only factor influencing atherosclerosis and CVD events. Many scientists, as far back as the 1850’s recognized the role of inflammation in the progression of atherosclerotic disease. The continuous low levels of immune activation in the body contribute to atherosclerosis. Research in animal models and epidemiologic studies have shown the involvement of both the innate and the adaptive immune systems in plaque development and to elucidate the roles of monocytes and T cells. In addition to animal studies and epidemiologic research, CVD and atherosclerotic research has extended to genetic analysis in the search for associations with risk factors and outcomes. The first chapter is a review of the literature studying the immune system’s involvement in atherosclerosis. Beginning with an examination of the impact of CVD and atherosclerosis, the basic pathophysiology, and the involvement of the innate and adaptive immune systems through animal models and epidemiology. Some of the significant cohort studies in CVD and genome wide association studies are also discussed. Chapter 2 examines the associations of soluble interleukin 2 receptor alpha (sIL-2Rα) with clinical events in the Cardiovascular Health Study and genetic variants. Interleukin 2 (IL-2) and its receptor regulate both tolerance and immunity, IL-2 induces the proliferation and differentiation of T cells, part of the adaptive immune system. The results showed an association between sIL-2Rα and CVD events. The genome-wide association study found 52 variants to be significantly associated with sIL-2Rα in European Americans. Chapter 3 assesses the involvement of the innate immune system in atherosclerosis through the associations of soluble CD163 (sCD163). CD163 is a marker of macrophage activation, specifically associated with M2 macrophages. In CHS, sCD163 levels were analyzed for associations with cardiovascular events and genetic variants. sCD163 was found to be associated with CVD risk factors and with cardiovascular events. In a genome-wide association study six variants in European Americans and three variants in African Americans were found to be significant. Chapter 4 summarizes the results and discusses some bench to bedside translational science already seen in atherosclerosis treatment and prevention. Continued investigation of markers of T-cell and monocyte differentiation in animal models and cohort studies may lead to opportunities for the prevention of atherosclerosis and/or treatment through an increased understanding of the biology and genetics of the innate and adaptive immune.

The sweetpotato whitefly Bemisia tabaci is a highly destructive agricultural and ornamental crop pest. It damages host plants through both phloem feeding and vectoring plant pathogens. Introductions of B. tabaci are difficult to quarantine and eradicate because of its high reproductive rates, broad host plant range, and insecticide resistance. A total of 791 Gb of raw DNA sequence from whole genome shotgun sequencing, and 13 BAC pooling libraries were generated by Illumina sequencing using different combinations of mate-pair and pair-end libraries. Assembly gave a final genome with a scaffold N50 of 437 kb, and a total length of 658 Mb. Annotation of repetitive elements and coding regions resulted in 265.0 Mb TEs (40.3%) and 20 786 protein-coding genes with putative gene family expansions, respectively. Phylogenetic analysis based on orthologs across 14 arthropod taxa suggested that MED/Q is clustered into a hemipteran clade containing A. pisum and is a sister lineage to a clade containing both R. prolixus and N. lugens. Genome completeness, as estimated using the CEGMA and Benchmarking Universal Single-Copy Orthologs pipelines, reached 96% and 79%. These MED/Q genomic resources lay a foundation for future 'pan-genomic' comparisons of invasive vs. noninvasive, invasive vs. invasive, and native vs. exotic Bemisia, which, in return, will open up new avenues of investigation into whitefly biology, evolution, and management.

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During the evolution of multicellularity from unicellular ancestors, cells transition from being evolutionary individuals to components of more complex, multicellular evolutionary individuals. The volvocine green algae provide a powerful model system for understanding the genetic and morphological changes that underlie and are caused by the evolution of multicellularity. This dissertation concerns the role of cell cycle regulation, cellular differentiation, and sexual traits during the evolution of multicellularity. While some of these are shown to be causally important in the origins of multicellularity (Appendix B), others are driven by the evolution of multicellularity (Appendix D). We provide a review of recent mathematical models on the evolution of multicellularity, which are found to focus heavily on the later, subsequent stages of the evolution of multicellular complexity. We found that many of these models assume multicellular ancestors and instead evolve cellular differentiation, bringing attention to a gap in our understanding of the events in the initial stages of the evolution of multicellularity. We show that a focus on the early stages of the evolution of multicellularity reveals a powerful and critical role for regulation of the cell cycle at the origins of multicellularity (Appendix B). We further find that the genetic basis for cellular differentiation evolved sometime after the evolution of cell cycle regulation. We find that while the genetic basis for cellular differentiation evolved after cell cycle regulation, it also evolved earlier than previously predicted in the volvocine green algae, suggesting an important role in undifferentiated species (Appendix C). Lastly, having elucidated the origins and evolution of multicellularity, we find that multicellularity causes the evolution of sexual traits including anisogamy, internal fertilization, and subsequently sexual dimorphism (Appendix D). This work emphasizes the important role that multicellularity plays in driving the evolution of sexual diversity seen across the eukaryotic tree and well as informs critical hypotheses on the evolution of anisogamous sex, among the most challenging problems in evolutionary theory.

Small Cell Lung Cancer (SCLC) is a very aggressive neuroendocrine tumour of the lung, which demonstrates a 5 year survival of only 10% for extensive stage disease (20-30% for limited stage), with only modest improvement over the last few decades. Identification of new molecular diagnostic and therapeutic targets is thus imperative. Previous efforts in identifying molecular changes in SCLC by gene expression profiling using microarrays have facilitated disease classification but yielded very limited information on SCLC biology. Previous DNA studies have been successful in identifying several loci important to SCLC. However the low resolution of conventional chromosomal Comparative Genomic Hybridization (CGH) has limited the findings to large chromosomal regions with only a few specific candidate genes discovered to date. Thus, to further understand the biological behaviour of SCLC, better methods for studying the genomic alterations in SCLC are necessary. This thesis highlights the development of array CGH technology for the high resolution dissection of aneuploidy in cancer genomes and the application of this new technology to the study of SCLC. I present the development of the first whole genome CGH array which offered unprecedented resolution in the profiling of cancer genomes allowing fine mapping of genes in a single experiment. Through application of DNA based analysis in conjunction with integrated expression analysis and comparison of SCLC to less aggressive non-small cell lung tumours I have identified novel patterns of pathway disruption specific to SCLC. This included alteration to Wnt pathway members and striking patterns of cell cycle activation through predominantly downstream disruption of signalling pathways including direct activation of the E2F transcription factors, which are normally repressed by the Rb gene. Analysis of targets of the E2F/Rb pathway identified EZH2 as being specifically hyper-activated in SCLC, compared to NSCLC. EZH2 is a polycomb group gene involved in the control of many cellular functions including targeted DNA methylation and escape from senescence in hematopoietic stem cells. Taken together these results suggest that in SCLC, downstream disruption may replace multiple upstream alterations leading to activation independent of a specific mitogenic pathway, and that EZH2 represents a potentially important therapeutic target. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Baculoviruses have been used as biocontrol agents to control insect pests in agriculture since the 1970s. Out of the fifteen virus families known to infect insects, baculoviruses offer the greatest potential as insect biopesticides, due to their high host specificity which makes them extremely safe to humans, other vertebrates, plants and non-target microorganisms. They comprise of two genera: nucleopolyhedroviruses (NPVs) and granuloviruses (GVs). The South African isolate of Cryptophlebia leucotreta granulovirus (CrleGV-SA) which is infectious for the false codling moth (FCM), Thaumatotibia leucotreta, (Meyrick) (Lepidoptera: Tortricidae), has been successfully developed into two commercial biopesticides; Cryptogran® and Cryptex®, for the control of FCM in citrus crops. The current method of enumeration used for CrleGV-SA virus particles in routine experiments during the production of the GV as biopesticides, is dark field microscopy. However, due to the small size of GVs (300-500 nm in length), the technique is not easy to perform on these viruses, and no systemic comparison has been made of potential alternative methods. Therefore, the main objective of this study was to develop a quantitative enumeration method for CrleGV-SA occlusion bodies (OBs) which is accurate, reliable, and feasible, and compare the developed methods of enumeration to the current method. Purified and semi-purified CrleGV-SA viral stocks were prepared for enumeration studies using spectrophotometry, dark field microscopy, scanning electron microscopy (SEM) and real time qPCR. Spectrophotometry was found to be an unreliable method for enumeration of GVs in the production, standardisation, and quality control of biopesticides. Dark field microscopy and SEM were found to be accurate, and statistically comparable (p = 0.064) enumeration techniques. qPCR is currently being optimised for the enumeration of GVs. This technique was demonstrated to generate accurate standard curves for absolute quantification of virus particles for pure and semi-pure virus preparations. qPCR offers the greatest potential as an accurate enumeration method because it is not affected by contamination with non-biological contaminating debris, nor by other biological material due to the specificity of PCR primers. Further work is required to fully develop qPCR as an enumeration method for GVs. However, dark field microscopy has been successfully validated as an enumeration method. SEM, which has a high resolution compared to light microscopy, has an added advantage over dark field microscopy, which is to distinguish virus particles in semi-pure viral stock preparations during counting. Therefore, SEM currently provides the most unambiguous and feasible enumeration method for GVs in both purified and semi-purified virus samples.

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 inVP267. 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.

Lysine acetylation is a post-translational modification (PTM) studied extensively in the context of histone proteins as a regulator of chromatin dynamics. Recent proteomic studies have revealed that as much as 10% of prokaryotic and mammalian proteins undergo lysine acetylation, and as such, the study of its biological consequences is rapidly expanding to include virtually all cellular processes. Unravelling the complex regulatory network governed by lysine acetylation will require an in depth knowledge of the lysine acetyltransferase enzymes that mediate catalysis, and moreover the development of methods that can identify enzyme-substrate relationships in vivo. This is complex task and will be aided significantly through the use of model organisms and systems biology approaches. The work presented in this thesis explores the function of the highly conserved NuA4 lysine acetyltransferase enzyme complex in the model organism Saccharomyces cerevisiae using systems biology approaches. By exploiting genetic screening tools available to the budding yeast model, I have systematically assessed the cellular roles of NuA4, thereby identifying novel cellular processes impacted by the function of the complex, such as vesicle-mediated transport and the stress response, and moreover identified specific pathways and proteins that are impacted by NuA4 KAT activity, including cytokinesis through the regulation of septin protein dynamics. Moreover, I have developed a mass spectrometry-based technique to identify NuA4-dependent acetylation sites amongst proteins that physically interact with NuA4 in vivo. Together this work demonstrates the diversity of processes impacted by NuA4 function in vivo and moreover highlights the utility of global screening techniques to characterize KAT function.

Despite the considerable expansion of knowledge in the development of pancreatic cancer, there has been little progress made in facilitating an early diagnosis of this disease and predicting an accurate response to treatment. We aim to translate this knowledge to clinical practice by using a prospective database of precursor cystic lesions in pancreas cancer, assessing the use of over-expressed genes in pancreatic juice as a surrogate marker of these pancreas cancer and finally, downstream of these changes at the genetic level, use metabolomic techniques to look for biomarkers in pancreas cancer in serum. In the first study, we investigate the natural history of pancreatic cystic neoplasms, specifically IPMNs, using a prospectively collected database to examine the profiles and outcomes of main duct IPMN, branch duct IPMN and cystic lesions measuring less than 3 cm in size. A total of 99 patients with suspected pancreatic cystic tumours were enrolled over 3 years. Median follow-up was 24 months (range 0 – 124). Cystic tumours comprised of 13 MD-IPMN, 40 BD-IPMN, 11 MCN and 8 adenocarcinomas among others. The complete cohort showed an overall risk of adenocarcinoma of 8%. Main duct IPMN showed a cumulative risk of 46% with evidence of progression of disease in a further 23%. The associated mortality in MD-IPMN was related to the underlying adenocarcinoma and was 38% in our group. The incidence of adenocarcinoma in branch duct IPMN was 11% with disease progression seen 13.8%. Evidence of extra-pancreatic malignancies was seen in 37.7% of patients with IPMN. In the second study, we explore the feasibility of gene expression profiling from RNA isolated from matched pancreatic juice and tumour tissue in patients with pancreatic cancer and pancreatic cystic tumours. RNA was isolated and Poly(A) PCR was used to globally amplify the RNA. RT-PCR was used to measure expression levels of 18 genes common to both pancreas cancer and pancreatic cystic tumours. Spearman’s rank correlation test was used to examine the relationship of gene expression between pancreatic juice and tissue. One gene out of eighteen, MSLN (p<0.008), showed significant correlation in the expression levels between paired pancreatic juice and tissue samples in pancreas cancer. In the cystic tumour group, only one gene MMP-7 (p<0.01), showed a significant correlation between paired juice and tissue samples. When the whole cohort was analysed for the false discovery rate, these genes did not exhibit statistically significant correlation between the samples. RNA analysis of pancreatic juice is feasible using the Poly(A) cDNA technique and correlation of gene expression is shown to exist, albeit with low sensitivity, indicating its potential use in clinical practice with small tissue and juice samples. In the final study, we performed a literature review on the use of metabolomics in pancreas cancer. We performed metabolic profiling of serum samples from selected cancer patients and noncancerous controls using UPHLC-MS to generate and compare the metabolic profiles in serum samples from a cohort of patients with pancreas cancer, ampullary cancer and endocrine cancer. Thirty nine serum samples (including 19 pancreatic cancers, 9 ampullary cancers and 5 endocrine cancers) and 21 matched HUSERMET controls were analysed using Ultra high performance liquid chromatography mass spectrometry (UHPLC-MS) in both positive and negative ESI modes. The output was generated as a data matrix of mass spectral features with related accurate m/z and retention time pairs. The data was then signal corrected and individual peaks were normalised and the resultant spectra were compared against a metabolite reference library and analysed using univariate and multivariate statistical tests. We found a disparity in the metabolite peaks between the cases and controls on PCA that did not permit the accurate interpretation of the data in the case study set compared to the control set. No obvious reason other than metabolite degradation during storage could account for this difference. PC-DFA analysis of metabolite peaks between pancreas cancer, ampullary cancer and endocrine cancer showed significant difference between endocrine cancers and the other two groups. Significant ESI positive metabolites included those involved in lipid pathways and metabolites involved in glucose metabolism. There is encouraging scope for studies using prospective controls to identify and develop metabolic biomarkers in pancreas cancer.

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