No description available.

abstract: Given the process of tumorigenesis, biological signaling pathways have become of interest in the field of oncology. Many of the regulatory mechanisms that are altered in cancer are directly related to signal transduction and cellular communication. Thus, identifying signaling pathways that have become deregulated may provide useful information to better understanding altered regulatory mechanisms within cancer. Many methods that have been created to measure the distinct activity of signaling pathways have relied strictly upon transcription profiles. With advancements in comparative genomic hybridization techniques, copy number data has become extremely useful in providing valuable information pertaining to the genomic landscape of cancer. The purpose of this thesis is to develop a methodology that incorporates both gene expression and copy number data to identify signaling pathways that have become deregulated in cancer. The central idea is that copy number data may significantly assist in identifying signaling pathway deregulation by justifying the aberrant activity being measured in gene expression profiles. This method was then applied to four different subtypes of breast cancer resulting in the identification of signaling pathways associated with distinct functionalities for each of the breast cancer subtypes. / Dissertation/Thesis / M.S. Computer Science 2011

Suicidal behavior represents the most severe, yet inherently preventable, outcome of psychiatric disease. Despite tremendous efforts to improve the awareness and treatment of psychiatric illness, suicidal behavior rates have been on the rise. The greatest challenge to confronting this crisis is the effective identification and treatment of those at risk for suicide. This challenge has been difficult to address due, in part, to the lack of a clear biological basis for suicidal behavior. Toward addressing this knowledge gap, evidence has been identified of a significant heritable component to suicidal behavior. Subsequent genetic research efforts have focused on the examination of common sites of genetic variation within candidate genes and throughout the genome. These efforts have identified many potentially important risk loci, but the majority of the risk expected to arise from genetic variation remains unexplained by current data. The primary objective of this dissertation was to examine the contribution of largely unexplored rare and potentially damaging genetic variation within suicidal behavior. To do this, targeted next-generation sequencing approaches were employed within a cohort of individuals diagnosed with bipolar disorder, a group particularly enriched for suicidal behavior. Sequence data was generated that examined essentially all protein-coding regions of the human genome (“exome”), with expanded sequencing around and within candidate genes hypothesized to play a role in suicidal behavior risk. The secondary objective of this dissertation focused on the assessment of rare variation within bipolar disorder through sequenced pedigrees and followup in a large collaborative bipolar disorder versus normal control sequencing dataset. These objectives were addressed through the thoughtful application of diverse and complimentary methods. These methods were selected to investigate individual variants, genes, and biological pathways. This approach offered examinations of the potential impact of rare genetic variation within focused regions and across complex biological process pathways that could be disrupted through damaging variation in many different genes. The presented efforts represent the largest examinations of rare functional variation with suicidal behavior and bipolar disorder performed, to date. No individual variant or gene survived correction for multiple testing for either phenotype. These results are consistent with other initial sequencing efforts in complex psychiatric phenotypes, offering conclusions that larger samples will likely be required to identify significant associations for single variants and genes. Within pathway analyses, however, we identified a significant enrichment of rare damaging variation that segregated within bipolar disorder pedigrees in genes that have been implicated in de novo studies of autism. This finding was further replicated within three large case/control sequencing samples, providing support to emerging evidence of a potential overlap of risk loci for autism and bipolar disorder. Many additional results approached significance that bear further consideration. These results offer potential candidate genes and pathways that could be utilized in future sequencing efforts for suicidal behavior and bipolar disorder. In addition, highly valuable resources in the form of datasets strongly enriched for novel rare loci were produced that can significantly contribute to ongoing efforts to investigate bipolar disorder and suicidal behavior. These data can be used in combination with other emerging datasets to generate more powerful meta- and mega-analyses to confidently identify risk loci for both phenotypes.

No description available.

This work helps to determine the extent of immune system involvement in the adaptive response to subacute ruminal acidosis (SARA) in three parts. The first (Chapter 2) uses non-lactating cows to study specific changes in inflammatory protein expression in which SARA is created. The second (Chapter 3), uses the same model as Chapter 2. However, in this case, lactating cows are used to help establish the time course for adaptation to acidosis. The third part (Chapter 4) delineates the genomic changes that occur in the rumen epithelium when a therapeutic intervention is introduced using exogenous supplemental butyrate. In the first experiment, the expression of the extracellular matrix (ECM) proteins type IV collagen and laminin β1 decreased, and the monocarboxylate transporter MCT1, increased during the acidotic challenge. Nuclear factor of activated T-cells, NFATc2, and tumour necrosis factor alpha (TNF-α) decreased while interleukin-1 beta (IL-1β) increased during the experimental treatment period. Chapter 3 measured lipopolysaccharide (LPS) and its carrier, LPS binding protein, LBP, which were found to be elevated due to SARA. Moreover, NFATc2 was reduced during this period. Exogenous butyrate resulted in increased plasma LBP, plasma beta hydroxyl butyrate (BHBA), and ruminal butyrate. Milk parameters (total protein and fat) were unaffected by treatment, as were rumen LPS, acetate, valerate, isovalerate, and isobutyrate. Moreover, exogenous butyrate increased gene transcription of genes involved in non-specific host defences (NHSD) such as mucin, and remodelling (RM), such as matrix metallopeptidase 16 (MMP16), and decreased the transcription of genes of the immune response (IR), such as nuclear factor kappa B2 (NFκB2). Together, these three experiments have demonstrated that although wound healing is mediated by the immune system in more severe models of epithelial damage, our model of SARA did not involve full-thickness, penetrating lesions and hence did not involve the systemic immune system to such a degree than was previously thought. In addition, we were able to demonstrate that the addition of butyrate to this model of grain-induced acidosis was beneficial, as it decreased the local inflammatory response and helped the epithelium adapt to its harsher environment. / Agriculture and Agri-Food Canada, the National Sciences and Engineering Council of Canada (NSERC), the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA), the Canadian Dairy Commission (CDC), and the Dairy Farmers of Ontario (DFO).

Résumé : L’adénocarcinome colorectal est parmi les plus importants cancers au Canada en terme de mortalité et morbidité. Cependant, nous n’en connaissons encore que peu, entres autres sur les voies cellulaires importantes et les protéines présentant un potentiel comme biomarqueur. Cette étude fut divisée en deux sous-projets. Sous-projet A. Il n’y a présentement aucun biomarqueur permettant de prédire la réponse à la radiothérapie comme modalité de traitement pour le cancer colorectal. Le but de ce sous-projet était de mettre au point les méthodes permettant d’effectuer une étude prospective ou rétrospective par spectrométrie de masse sur la réponse à la radiothérapie en utilisant des échantillons de tissu de patient. Des échantillons de tissu de souris et de tissu humains anonymisés ont été utilisés pour évaluer la faisabilité d’une telle étude. Différentes techniques d’extraction protéique ont été évaluées. Les extraits totaux et fractionnements subcellulaires de tissu frais ont permis une analyse appropriée des protéines cellulaires. Il en était de même pour l’extraction totale de tissus fixés. Cependant, les protéines extraites suite à microdissection au laser de tissu fixé étaient inadéquates et en nombre insuffisant. Sous-projet B. Afin d’investiguer l’importance de fonctions, voies ou protéines dans différents types de cancer colorectaux, neuf lignées cellulaires de cancer colorectal et de côlon normal ont été fractionnées en quatre compartiments subcellulaires et analysées par spectrométrie de masse. Aucun groupe de recherche n’avait analysé jusqu’à présent plus de cinq lignées et plus d’un compartiment subcellulaire à la fois. Les résultats montraient que certaines voies canoniques et fonctions cellulaires étaient de haute importance dans plusieurs des lignées analysées, dont la voie de signalisation par eIF2. De plus, les régulateurs de transcription TP53, MYC et TGFB1, pouvant être responsables des caractéristiques cellulaires observées, ont été identifiés. En conclusion, ce projet nous a permis d’améliorer nos connaissances sur les caractéristiques moléculaires d’importance dans le cancer colorectal et de mettre au point des techniques qui pourraient permettre la découverte de nouveaux biomarqueurs. / Abstract : Colorectal adenocarcinoma is one of the most important cancers in Canada in terms of mortality and morbidity. However, we still know very little on its molecular features. This study was divided into two sub-projects. Sub-project A. At this time, no biomarker has the capacity of predicting a patient’s response to radiotherapy, which is a commonly used treatment of colorectal cancer. The goal of this section was to develop the methods to conduct a prospective or retrospective mass spectrometry study on the patient response to radiotherapy, through the use of human tissues. Mouse tissues and tissues of an anonymous patient were obtained in order to evaluate the feasibility of such a study. Different protein extraction techniques were evaluated. Total lysates and subcellular fractionations of fresh tissues allowed for a successful analysis of the samples. The same was true of total lysates of fixed tissues. However, proteins extracted from cells isolated through laser capture microdissection were insufficient in numbers and their types were inconsistent with the expected results. Sub-project B. In order to study the importance of proteins and cellular functions or pathways in different types of colorectal cancers. nine cell lines originating from colorectal carcinoma and from normal colon were fractionated according to four subcellular compartments and analysed through mass spectrometry. Until now, no research group had analysed, in a single study more than 5 cell lines as well as more than one subcellular compartment at once. Some cellular functions and canonical pathways were shown to be of high importance in many of the studied cell lines, such as the signalling through eIF2 pathway. Furthermore, the transcription regulators TP53, MYC and TGFB1were identified as potentially responsible for the observed proteomic characteristics. In conclusion, this study allowed for a better understanding of important molecular caracteristics of colorectal cancer and allowed for the optimization of techniques that may serve in the discovery of new biomarkers relative to the use of radiotherapy as a treatment.

<p><a>Prostate cancer is the second leading cause of cancer death among men in the United States. The androgen receptor (AR) antagonist enzalutamide is an FDA-approved drug for treatment of patients with late-stage prostate cancer and is currently under clinical study for early-stage prostate cancer treatment.</a> After a short positive response period to enzalutamide, tumors will develop drug resistance. In these studies, we uncovered that NOTCH signaling and DNA methylation are a deregulated in enzalutamide-resistant cells. <i>NOTCH2</i> and<i> c-MYC </i><a>gene expression<i> </i>positively correlated with <i>AR </i>expression in samples from patients with hormone refractory disease in which AR expression levels correspond to those typically observed in enzalutamide-resistance</a>. The expression of Notch signaling components was upregulated in enzalutamide-resistant cells suggesting the activation of the pathway. Inhibition of this pathway <i>in vitro</i> and <i>in vivo</i> promoted an increase in the sensitivity to enzalutamide with an impact on AR expression. On the other hand, DNMT activity and DNMT3B expression were upregulated in resistant lines. Enzalutamide induced the expression of DNMT3A and DNMT3B in prostate cancer cells with a potential role for p53 and pRB in this process. The overexpression of DNMT3B3, a DNMT3B variant, promoted an enzalutamide-resistant phenotype in C4-2 cells. DNA methylation inhibition, using low-concentration decitabine, and <i>DNMT3B</i> knockdown induced a re-sensitization of resistant prostate cancer cells and tumors to enzalutamide. Decitabine treatment in enzalutamide-resistant induced a decrease in the expression of AR-V7 and changes in genes from the apoptosis, DNA repair and mRNA splicing pathways. Decitabine plus enzalutamide treatment of 22RV1 xenografts induced a decrease in tumor weight, KI-67 and AR-V7 expression and an increase in Cleaved-Caspase3 levels. All the above suggest that Notch signaling and DNA methylation pathways are deregulated after enzalutamide resistance onset, and targeting these pathways restores the sensitivity to enzalutamide.<b><u></u></b></p>

No description available.

Cardiovascular disease (CVD) is the leading cause of death worldwide, and is projected to remain so for at least the next decade. Ever since its discovery in the urine and blood of children with inborn errors of metabolism, homocysteine (Hcy) at elevated plasma concentrations has been associated with CVD clinically and epidemiologically. Observational studies and meta-analyses have noted that changes in plasma Hcy by 5&mu;M increase the odds ratio of developing coronary artery disease by 1.6-1.8 among other CVD. Clinical trials aimed at reducing plasma Hcy for benefit against development of subsequent cardiovascular events have had unconvincing results, but have moreover failed to address the mechanisms by which Hcy contributes to CVD. Recommendations from national agencies like the American Heart Association and the United States Preventive Services Task Force emphasize primordial prevention as a way to combat CVD. Reducing plasma Hcy as secondary and primary interventions does not fulfill this recommendation. In order to best understand the role of Hcy in CVD, an investigation into its mechanisms of action must be undertaken before measures of primordial prevention can be devised. Numerous experimental studies in the literature identify vascular endothelium as a target for the pathological effects of Hcy. Endothelial injury and impairment are contributory processes to atherosclerosis, and Hcy has been demonstrated to inhibit endothelial cell (EC) growth and proliferation through mechanisms involving cell cycle arrest, oxidative stress, and programmed cell death in vitro. Animal models have also confirmed that high levels of Hcy accelerate atherosclerotic plaque development and lead to impairment of vascular reendothelialization following injury. Hcy has been shown to have the opposite effect in vascular smooth muscle cells (SMC), causing their proliferation and again contributing to atherosclerosis. The cell-type specificity of Hcy remains to be understood, and among the aims of this research was to further characterize the effects of Hcy in EC. The overarching goal was discovery in order to direct future investigations of Hcy-mediated pathology. To begin, the first investigation considered the transcriptional and regulatory milieu in EC following exposure to Hcy. High-throughput screening using microarrays determined the effect of Hcy on 26,890 mRNA and 1,801 miRNA. Two different in vitro models of hyperhomocysteinemia (HHcy) were considered in this analysis. The first used a high dose of 500µ Hcy to mimic plasma concentrations of patients wherein the transsulfuration pathway of Hcy metabolism is impaired as in inborn cystathionine-ß-synthase deficiency. The other set of conditions used 50µ Hcy in the presence of adenosine to approximate impairment of the remethylation pathway of Hcy metabolism wherein s-adenosylhomocysteine accumulates, thus inhibiting s-adenosylmethionine formation and methylation reactions. These distinctions are important because most clinical trials do not distinguish between causes of HHcy, thereby ignoring the specific derangements underlying HHcy. mRNA and miRNA expression changes for both sets of treatment conditions identified CVD as a common network of Hcy-mediated pathology in EC. Moreover, methylation-specific conditions identified cell cycle modulation as a major contributory mechanism for this pathology, which agrees with recent findings in the literature. Analysis of significant mRNA changes and significant miRNA changes independently identified roles for Hcy in CVD and cell cycle regulation, thereby suggesting that miRNA may mediate the effects of Hcy in addition to gene expression changes alone. To investigate the role of Hcy in the cell cycle further, the next set of investigations considered the effect of Hcy under conditions approximating impaired remethylation in early cell cycle events. Previous studies have demonstrated that Hcy inhibits cyclin A transcription in EC via demethylation of its promoter. Conversely, Hcy induces cyclin A expression in SMC, again making the case for a cell type-specific mechanism in EC. Preceding cyclin A transcription and activation, canonical events in the early cell cycle include D-type cyclin activation, retinoblastoma protein (pRB) phosphorylation, and transcription factor E2F1 activation. In a series of in vitro experiments on EC, it was seen that Hcy inhibits expression of cyclin D2 and cyclin D3, but not cyclin D1. Next, pRB phosphorylation was seen to be decreased following treatment with Hcy. This also led to decreased E2F1 expression. However, this series of events could be reversed with E2F1 supplementation, allowing the cell cycle to proceed. As Hcy exerts a number of its effects via regulation of gene transcription, a final series of investigations aimed to predict potential targets of Hcy by examining patterns of transcription factor binding among known targets of Hcy regulation. Gene promoters of Hcy-modulated genes were analyzed in order to determine common transcription factors that potentially control their regulation. The locations of CpG-rich regions in promoters were identified to determine which regions would be most susceptible to regulation by DNA methylation. Next, high-throughput next-generation sequencing (NGS) and bisulfite NGS was performed for DNA from EC treated with Hcy in order to determine methylation changes after Hcy treatment. A number of potential transcription factors and their binding sites were identified as potential mediators of Hcy-mediated gene regulation. Taken together, these investigations represent an exploration of Hcy-mediated pathology in CVD, by focusing upon novel regulatory mechanisms in EC. Objective high-throughput arrays identified roles for Hcy in CVD and cell cycle pathways regulated by miRNA and gene expression, which were confirmed experimentally in vitro. These observations led to an investigation and identification of common transcription factors that potentially regulate Hcy-altered gene expression. This framework may be used to guide future investigations into the complex pathological network mediating the effects of Hcy in CVD. First, identification of a role for miRNA in mediating the effects of Hcy represents a novel regulatory mechanism, heretofore largely unexplored. Next, expanding the role of Hcy in EC cell cycle regulation to identify upstream mediators greatly adds to the published literature. Finally, noting that these changes center upon transcriptional and post-transcriptional regulation gives import to developing methods to characterize promoter and transcription factor regulation. The investigations presented herein and their results provide evidence that the future of Hcy research is vibrant, relevant, and not nearly surfeit. / Pharmacology

No description available.