Over the past two decades, high-throughput (HTP) technologies such as microarrays and mass spectrometry have fundamentally changed the landscape of aging and disease biology. They have revealed novel molecular markers of aging, disease state, and drug response. Some have been translated into the clinic as tools for early disease diagnosis, prognosis, and individualized treatment and response monitoring. Despite these successes, many challenges remain: HTP platforms are often noisy and suffer from false positives and false negatives; optimal analysis and successful validation require complex workflows; and the underlying biology of aging and disease is heterogeneous and complex. Methods from integrative computational biology can help diminish these challenges by creating new analytical methods and software tools that leverage the large and diverse quantity of publicly available HTP data. In this thesis I report on four projects that develop and apply strategies from integrative computational biology to identify improved biomarkers and therapeutics for aging and disease. In Chapter 2, I proposed a new network analysis method to identify gene expression biomarkers of aging, and applied it to study the pathway-level effects of aging and infer the functions of poorly-characterized longevity genes. In Chapter 4, I adapted gene-level HTP chemogenomic data to study drug response at the systems level; I connected drugs to pathways, phenotypes and networks, and built the NetwoRx web portal to make these data publicly available. And in Chapters 3 and 5, I developed a novel meta-analysis pipeline to identify new drugs that mimic the beneficial gene expression changes seen with calorie restriction (Chapter 3), or that reverse the pathological gene changes associated with lung cancer (Chapter 5). The projects described in this thesis will help provide a systems-level understanding of the causes and consequences of aging and disease, as well as new tools for diagnosis (biomarkers) and treatment (therapeutics).

Metastasis initiation is a complex process encompassing numerous steps. To identify molecular alterations associated with early and late stages of metastasis, we used high throughput screening techniques. Early events in metastasis were investigated by differential proteomic analysis of lymph node-negative and positive breast cancer samples. Two candidate biomarkers (DCN and HSP90B1) were discovered and further validated through tissue microarray analyses. To examine late events in metastasis formation, we prospectively evaluated genomic differences between disseminated tumour cells in bone marrow, and metastatic tumour cells obtained from computed tomography guided biopsies of bone metastases. Results indicate that specific subsets of genes are required for breast cancer cells to initiate bone metastases. Discovery of proteomic and genomic alterations specifically associated with metastases may yield biomarkers capable of stratifying patients into different risk categories. Proteins and genes identified in this work may form the foundation of a biomarker panel for metastatic risk assessment.

Magister Scientiae - MSc / Breast cancer (BC) is the most common cancer in women worldwide, and is the second most common cancer in the world, responsible for more than 500 000 deaths annually. Estimates are that 1 in 8 women will develop BC in their lifetime. In South Africa, BC in women affects about 16.6 % of the population and could see a 78 % increase in cases by 2030. The failure of conventional diagnostic tools to detect BC from an early onset has revealed the need for diagnostic tools that would enable early diagnosis of BC. The current diagnostic tools include breast self-examination, mammography magnetic resonance imaging, ultrasonography and serum biomarkers; BRACA1, BRACA2, HER2. These conventional methods lack sensitivity, specificity and positive predictive value, and some of these diagnostic tools may be expensive and quite invasive. Therefore, novel diagnostic tools such as microRNAs which address the short comings of current methods are required for early diagnosis as well as BC management. MicroRNAs are a class of non-coding RNA molecules, which are important in RNA stability and gene expression. Various methodologies have been employed to identify novel microRNAs for diagnostics such as bioinformatics, also referred to as in silico analysis. The aim of this study is to identify novel microRNAs that can potentially detect BC at its earliest stage.

Evidence from international comparisons and migrant studies suggest that environmental factors, such as a Western diet, may be important in prostate cancer development, possibly through effects on hormone and growth factor secretion and metabolism. However, despite considerable research, convincing associations between diet and risk for prostate cancer have not been established. Random and systematic measurement error in dietary assessment using traditional survey methods may contribute to inconsistent findings, particularly as they may not capture adequately specific nutritional constituents of the diet that may be associated with risk, such as fatty acids or vitamins. Validated biomarkers of nutritional factors and hormonal activity, as used in this thesis, provide more precise, objective and integrated measures of exposure, with the capacity to clarify potential mechanisms in the causal pathway of prostate cancer development. Nutritional and hormonal biomarkers investigated for their potential role in the development of prostate cancer include: folate and vitamin B₁₂, which are essential for DNA methylation, repair and synthesis; phytanic acid, obtained predominantly from ruminant fat intake and associated with an enzyme (α-Methylacyl-Coenzyme A Racemase (AMACR)) that is consistently over-expressed in prostate cancer tissue; and insulin-like growth factor (IGF-I), a growth factor influenced by diet and involved in the regulation of cell proliferation, differentiation, and apoptosis. All work presented in this thesis is from the European Prospective Investigation into Cancer and Nutrition (EPIC) study of 500,000 European men and women, using prospectively collected diet and lifestyle data and biological samples. The large number of prostate cancer cases diagnosed during long-term follow-up of EPIC participants enabled investigation of heterogeneity in risk for prostate cancer by time from recruitment to diagnosis (of particular importance for a disease with a long pre-clinical phase) and cancer characteristics such as disease grade and stage. Plasma phytanic acid concentration was highly correlated with dietary intake of fat from dairy products (r = 0.46) and beef (r = 0.30); capturing differences between countries in consumption of fat from these foods. Although phytanic acid is a useful biomarker of ruminant fat consumption, there was little evidence to support the hypothesis that the association between dairy products and prostate cancer risk (as suggested by previous work in EPIC and other studies) is mediated by phytanic acid (OR for doubling in concentration 1.05; 95%CI 0.91 – 1.21; P _trend = 0.53). There was strong evidence for an association between higher circulating IGF-I concentration and risk for prostate cancer (OR for highest versus lowest fourth 1.69; 95% CI: 1.35, 2.13; P _trend = 0.0002). Furthermore, the positive association observed among men diagnosed with advanced stage disease and among men diagnosed more than seven years after blood collection, supports the hypothesis that high IGF-I concentration is associated with clinically significant prostate cancer many years before diagnosis. There was no evidence of an association between prostate cancer risk and dietary folate or vitamin B₁₂ intake, or between circulating levels of folate (OR for doubling in concentration 1.05; 95%CI 0.95 – 1.15; <en>P _trend = 0.33) or vitamin B₁₂ (1.05; 95%CI 0.92 – 1.21; P _trend = 0.47) and only limited evidence for an increased risk associated with elevated vitamin B₁₂ in a meta-analysis of six prospective studies, that included the present study. All of these analyses were based on a blood sample taken at one point in time, with the assumption that this reflects the ‘true’ underlying concentration over the long-term. The poor to modest reliability estimates (intra-class correlation coefficients ranging from 0.18 to 0.48) for circulating concentrations of folate, IGF-I, phytanic acid and vitamin B₁₂ taken in samples approximately six years apart in a sub-sample of participants from EPIC Oxford, show that estimates of usual concentrations based on a single blood measurement weaken the ability to detect associations with disease risk. Where small effect sizes are anticipated, this may bias associations toward the null. In conclusion, there is convincing evidence that IGF-I is an important and potentially modifiable risk factor for prostate cancer many years before diagnosis. However, there is little evidence for an association between biomarkers of folate, vitamin B₁₂ and phytanic acid concentrations and risk for prostate cancer. Future studies should, where possible, incorporate multiple blood samples taken several years apart to better characterise long term relationships between biomarkers of nutritional and hormonal exposure and disease risk and pool individual participant data from multiple prospective studies to strengthen the power to detect modest associations.

Background Breast cancer is the most commonly diagnosed cancer in women worldwide. The identification of breast cancer molecular biomarkers would provide a more accurate assessment of individual disease risks and prognosis. Exosomes, small extracellular vesicles, have been shown to contribute to various aspects of cancer development and progression. Within the last decade, the content of exosomes has been increasingly explored as a new source of potential biomarker molecules for early disease detection. Methods Exosomal proteomes of MDA-MB-231, a metastatic breast cancer cell line, and MCF-10A, a non-cancerous epithelial breast cell line, were compared. Proteomic analysis was conducted using nano-liquid chromatography coupled to tandem mass spectrometry. The expression of proteins in MDA-MB-231cells was analyzed using label-free protein quantification methods. For the selection of potential biomarkers, the following criteria were used: (i) proteins must be unique to MDA-MB-231 cells when compared to MCF-10A cells, ii) localized on the membrane, (iii) abundant in breast cancer and (iii) are reported to increase in expression as the disease progresses. The presence of selected proteins on exosomes was verified using flow cytometry methods. Results In total, 1,107 exosomal proteins were identified in both cell lines, 726 of which were unique to the MDA-MB-231 breast cancer cell line. The biomarker selection process identified three exosomal proteins (glucose transporter 1, glypican 1, and “disintegrin and metalloproteinase domain-containing protein 10”) as potential breast cancer biomarkers. The presence of these three proteins was validated using flow cytometry methods. The proteomics dataset was also rich in other interesting breast cancer proteins, such as 16 metastasis-associated proteins and two kinases. Conclusion We demonstrate that breast cancer exosomes are a rich source of protein biomarkers that may be beneficial for diagnosis and prognosis.

Interstitial lung disease is one of the main causes of mortality in Systemic Sclerosis. The course of the disease is clinically variable where patients can suffer from a range of stable disease to rapid progressive clinical deterioration. Therefore, it is important to identify biomarkers that can predict the clinical course of patients in order to provide early treatment. We evaluated 1129 proteins utilizing novel high-throughput SOMAlogic proteomic technology from the serum of 13 LSSc, 13 progressive ILD and 11 stable ILD patients. Calpain-1 was significantly elevated in progressive ILD patients (median 15129 RFU, 11091-24561) compared to LSSc patients (12759, 9904-15498, p=0.0015) and stable ILD patients (11876, 10271-14249, p=0.0005). Coagulation Factor V was significantly lower in the progressive ILD patients (7161 RFU, 2140-8296) compared to LSSc patients (10311 RFU, 6396-12260, p=0.001) and stable ILD patients (9646 RFU, 6510-11941, p=0.0016). The combination of Coagulation Factor V and Calpain-1 produced an area under the curve of 0.97 (95% CI, 0.921-0.99), sensitivity of 99% and specificity of 91% for the identification of progressive ILD. We have identified a combination of proteins that show potential to be prognostic biomarkers for ILD in SSc. / 2016-12-31T00:00:00Z

This study was carried out primarily to evaluate the cryo-tolerance of equine semen from four stallions through assessing the spermatozoa motion characteristics with Computer-Assisted Sperm Analysis (CASA). Four stallions were collected during the breeding season (summer). For each ejaculate, fresh and cryopreserved samples were taken for sperm motility characteristics evaluation. Data analysis demonstrated that sperm cells of stallions were significantly affected by (P<0.05) cryodamage. Stallion (A) was cryotolerant, and was classified as a good freezer, whereas stallion (D) was not and classified as a poor freezer regardless of the concentration of sperm. In addition, a concentration of 0.4 x 109 sperm cells/ml had higher percentages of rapid sperm and velocity parameters (P<0.05) compared to 0.8 x 109 sperm/ml. Further research is necessary to identify potential biomarkers for good and poor freezer stallions.

Indiana University-Purdue University Indianapolis (IUPUI) / Large-scale genome-wide association studies for Alzheimer’s disease (AD) have identified more than 20 risk loci and several pathways including lipid metabolism. Lipids are fundamental to cellular structure and organization, where they compose biological bilayer membranes surrounding the cell. In their structural role, lipids provide a scaffold for cell signaling, such as neurotransmission. There is a large body of evidence linking lipids and AD, yet the relationship between AD pathogenesis and lipid dyshomeostasis is not well understood. Here, we performed manual PubMed searches to identify the most studied lipid classes and risk genes in AD. We discussed pathological alterations of the key lipids and their potential contribution to the recent NIA-AA “A/T/N” framework. We also summarized what is known between the key lipids and etiological hypotheses of AD. Finally, we characterized relationship of the key lipids with AD genomic risk factors to identify possible downstream mechanisms of lipid dysfunction in AD. There is a large body of evidence linking lipids and AD, yet the relationship between AD pathogenesis and lipid dyshomeostasis is not well understood. In particular, we investigated the association between triglyceride (TG) species and AD. The overall goal was to test the hypothesis that TGs would associate with AD endophenotypes, based on their fatty acid composition. Diagnostic groups (cognitively normal older adults (CN), mild cognitive impairment (MCI), and AD) differed on two principal components extracted from 84 serum TG levels. Fish oil-type and olive oil-type TGs were significantly lower in MCI and AD compared to CN. Next, association analysis of TG principal components with “A/T/N/V” (amyloid-β, tau, neurodegeneration, and cerebrovascular) biomarkers for AD showed that the fish oil-type and olive oil-type TGs were also significantly associated with atrophy on MRI. Finally, a mixed model regression analysis investigated the association between baseline TGs and longitudinal changes of AD endophenotypes to show that olive oil-type TGs predicted changes in AD brain atrophy. Our results indicate that a specific subcategory of TGs is associated with an early prodromal stage of cognitive impairment and early-stage biomarkers for AD, providing the foundation for future therapeutic development related to TG metabolism. / 2023-05-05

Breast cancer (BC) is the second most commonly occurring malignant disease in women and one of the leading causes of cancer-related death worldwide, globally accounting for almost half-a-million deaths per year. In Canada, BC is the second leading cause of death in women preceded only by lung cancer. Invasion and metastasis are the most common causes of mortality in patients with BC. Studies show that extracellular vesicles (EVs) play an important role in immune system evasion, invasion and metastasis. Studies have shown a significant elevation of EVs in the serum of cancer patients compared to healthy subjects. Furthermore, elevated secretion of EVs has been correlated with cancer malignancy. Therefore, it has been suggested that EVs may be an important non-invasive diagnostic and prognostic tool for cancer. Herein our in vitro studies show that ER-α is secreted via EVs from MCF-7 cells. Furthermore, our mass spectrometry (MS)-based proteomic study showed that the proteomic profile of EVs from the plasma of BC patients differs from that of healthy subjects. In addition, we have also shown that vesicular abundance of proteins associated with tumour malignancy, such as tissue factor (TF), plasminogen activator inhibitor (PAI-1), a disintegrin and metalloproteinase 12 (ADAM12) and β-Catenin is different between primary tumour and metastatic disease. / Thesis / Master of Science (MSc)

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