Cells can mediate intercellular communication by the secretion and uptake of microvesicles, nano-sized membranous particles that carry signaling molecules, antigens, lipids, mRNA and miRNA between cells. The biological function of these vesicles is dependent upon their composition and cellular origin which is regulated by mechanisms that are not well understood. Based on their molecular content, microvesicles may play a role in immune regulation, cancer progression, the spread of infectious agents and numerous other important normal and pathogenic processes. The proteomic content of microvesicles from diverse sources has been intensely studied. In contrast, little is known about their glycomic content. The glycosylation pattern of a protein or lipid plays a key role in determining its functional properties in several ways. Glycans can determine the trafficking of a protein to particular regions of the cell as well as the protein’s half life. In addition, the glycan-dervied oligomerization of glycolipids and glycoproteins is a known mechanism for the activation of receptors and recognition of ligands on the surface of the cell. Glycomic analysis may thus provide valuable insights into microvesicle function. I utilized lectin microarray technology to compare the glycosylation patterns of microvesicles derived from a variety of biological sources. When compared to cellular membranes, microvesicles were enriched in high mannose, polylactosamine, α2-6 sialic acid, and complex N-linked glycans but exclude terminal blood group A and B antigens. The polylactosamine signature in microvesicles from different cell lines derives from distinct glycoprotein cohorts. After treatment of Sk-Mel-5 cells with lactose to inhibit lectin-glycan interactions, secretion of microvesicle resident proteins was severely reduced. Taken together, this work provides evidence for a role of glycosylation in microvesicle-directed protein sorting. / text

The development of wireless DNA microelectrochemical microarray sensors is described. The operational principles of these sensors are based on bipolar electrochemistry. Bipolar electrodes are used to fabricate the wireless microarrays in this work. The systems are configured so that DNA sensing is carried out at the cathodic end of a bipolar electrode (BPE) and the result of the sensing experiment is reported at the anodic end of the BPE. There are two types of reporting platforms developed in this study. The first type relies on the emission of electrogenerated chemiluminescence (ECL). The system is configured so that ECL is emitted at the anodic end of the BPE when the target DNA is hybridized to the capture probe DNA immobilized on the cathodic end of the BPE. However, when there is no hybridization reaction occurs, there is no ECL emission on the electrode surface. The second type of reporting platform developed is based on silver electrodissolution at the anodic end of a BPE. When a reduction reaction occurs at the cathodic end of a BPE, it triggers oxidation and dissolution of silver deposited at the anodic end of the BPE. The loss of silver can easily be detected by the naked eye. This detection principle is used for DNA detection: when the target DNA is hybridized to capture probe DNA on the BPE, the BPE becomes shorter. However, if target DNA does not hybridize to the electrode surface, the length of the BPE remains the same. The BPE microarrays described in this work eliminate the need for complicated microfabrication procedures and instrumentation. For example, as many as 1000 BPEs can be simultaneously controlled using just two driving electrodes and a simple power supply. To fully utilize BPE microarrays for specific sensing tasks, a method based on robotic spotting was developed to modify the cathodic end of each BPE in the array. Because each BPE in a microarray is individually addressable, this development allows each BPE to perform a particular sensing operation. / text

The goal of this dissertation was to evaluate genomic regulation during bovine mammary epithelial cell (BMEC) growth and differentiation. To accomplish this goal, a collagen gel cell culture system was developed that was capable of mimicking the prepartum stages of epithelial development and differentiation. In addition, a 4,600-cDNA bovine microarray was developed in order to profile gene expression. Analysis of BMEC in collagen cultures using various lactogenic conditions highlighted the critical importance of both hormonal and structural signals. The objective of the first study utilizing the microarray was to evaluate the contribution of the two prominent lactogenic factors in vitro, 1) prolactin and 2) gel release. Collectively, lactogenic stimulation appears to turn off genes associated with structural progression and morphogenesis, and turn on genes involved in alveolar MEC differentiation such as cell polarization, milk protein synthesis and ER/Golgi transport. The objective of the second study utilizing these resources was to evaluate the direct effects of thermal stress on BMEC growth and development. The structural response to thermal stress was characterized by morphogenic inhibition and dramatic regression of the ductal branches. Microarray analysis revealed an overall up-regulation of genes associated with stress response, DNA repair, protein degradation and cell death. In contrast, genes associated with cellular and MEC-specific biosynthesis, metabolism, and morphogenesis, were generally down-regulated. Subsequent to the analysis of BMEC differentiation was a targeted effort focusing on two small molecules hypothesized to be involved in regulating the BMEC secretory response: serotonin and prostaglandin E2. A pilot study suggested that serotonin is produced by bovine MEC and a model was proposed that describes serotonin's role as a feedback inhibitor during milk synthesis and secretion. A second pilot study demonstrated that PGE2 had a consistently positive influence on lumen diameter of alveolar structures in vitro. Overall, this dissertation provides new resources for studying bovine functional genomics, particularly within the mammary gland, and it provides a strong foundation for understanding genomic regulation of mammary epithelial structure and function. Furthermore, it establishes potential roles for local regulation of milk production by serotonin and PGE2.

The type II tumour suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. To investigate the putative role of ING3 in melanoma development, we examined the expression of ING3 in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas with tissue microarray and immunohistochemistry. Overall ING3 was reduced in metastatic melanomas compared with dyslastic nevi and primary melanomas. Reduced nuclear ING3 staining also correlated with melanoma progression, increased cytoplasmic ING3 level, tumour location at sun-exposed sites, and a poorer disease-specific 5-year survival of patients with primary melanoma. Multivariate analysis revealed that nuclear ING3 staining can independently predict patient outcome in primary melanomas. In melanoma cells, ING3 expression was rapidly induced by UV irradiation. Using stable clones of melanoma cells overexpressing ING3, we showed that ING3 significantly promoted UV-induced apoptosis. Unlike its homologues ING1b and ING2, ING3-enhanced apoptosis upon UV irradiation was independent of functional p53. Furthermore, ING3 did not affect the expression of mitochondrial proteins but increased the cleavage of Bid and caspases. Moreover, ING3 upregulated Fas expression and ING3-mediated apoptosis was blocked by inhibiting caspase-8 or Fas activation. Knockdown of ING3 expression decreased UV-induced apoptosis remarkably, suggesting that ING3 plays a crucial role in cellular response to UV radiation. To explore how ING3 is deregulated in advanced melanomas, we examined ING3 expression in metastatic melanoma cells and found that ING3 was downregulated due to a rapid protein turnover in these cells. Further studies demonstrated that ING3 undergoes degradation via the ubiquitin-proteasome pathway. We also demonstrate that ING3 interacts with the SCF (Skp1/Cul1/Roc1/Skp2) E3 ligase complex. Knockdown of Cul1 or Skp2 significantly stabilized ING3 in melanoma cells. In addition, lysine residue 96 is essential for ING3 ubiquitination as its mutation to arginine completely abrogated ING3 turnover and enhanced ING3-stimulatd apoptosis upon UV irradiation. Taken together, ING3 is deregulated in melanomas as a result of both nucleus-to-cytoplasm shift and rapid degradation. The level of ING3 in the nucleus may be an important marker for human melanoma progression and prognosis. Restoration of ING3 expression significantly sensitizes melanoma cells to UV radiation through the activation of Fas/caspase-8 pathway.

The analysis of microarray data is a challenging task because of the large dimensionality and small sample size involved. Although a few methods are available to address the problem of small sample size, they are not sufficiently successful in dealing with microarray data from extremely small (~<20) sample sizes. We propose a method to incorporate information from diverse sources to analyze the microarray data so as to improve the predictability of significant genes. A transformed data set, including statistical parameters, literature mining and gene ontology data, is evaluated. We performed classification experiments to identify potential allergy-related genes. Feature selection is used to identify the effect of features on classifier behaviour. An exploratory and domain knowledge analysis was performed on noisy real-life allergy data, and a subset of genes was selected as positive and negative class. A new set of transformed variables, depending on the mean and standard deviation statistics of the data distribution and other data sources, was identified. Significant allergy- and immune-related genes from the microarray data were selected. Experiments showed that classification predictability of significant genes can be improved. Important features from the transformed variable set were also identified.

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).

In vitro gut models provide several advantages over in vivo models for the study of the human gut microbiota. However, because communities developed in these models are simplified simulations of the in vivo environment it is necessary to characterize the reproducibility, repeatability and stability of cultured communities. We also need to broadly define the differences between in vitro consortia and the communities from which they are derived. In this study we characterized and validated a twin-vessel (independent, identical) single-stage chemostat model of the human distal gut. Samples were analyzed using a molecular fingerprinting technique (Denaturing Gradient Gel Electrophoresis) to compare and monitor changes in the overall structure of the communities while a phylogenetic microarray (Human Intestinal Tract Chip) was used to obtain phylogenetic information. We found that twin-vessels inoculated with feces developed and maintained diverse communities that reached stable compositions by at most 36 days post-inoculation. Communities were enriched in Bacteroidetes but not Clostridium cluster XIVa, Bacilli or other Firmicutes relative to the fecal inocula. Vessels were very reproducible when inoculated with identical fecal inocula, less similar when inoculated with consecutive fecal donations from the same donor, and maintained donor-specific identities when inoculated with feces from different donors. Norepinephrine exposure (undefined perturbation) did not appear to have a substantial effect on the structure of chemostat communities, while clindamycin treatment (defined perturbation) caused large changes in the structure of chemostat communities. Packed-column biofilm reactors incorporated a simulated mucosal environment into our chemostat system, allowing us to simultaneously culture biologically relevant planktonic and biofilm communities that were complex, reproducible, and distinct. Defined communities were comparable to fecal communities at the phylum/class-level but established stable compositions more rapidly. While it was difficult to assess the persistence of synthetic stool in a healthy fecal chemostat community (+/- antibiotic perturbation), mixing communities from two donors resulted in a mixed community that more closely resembled one donor over the other. Although future experimentation is required, the results presented here show our twin-vessel single-stage chemostat model represents a valid simulation of the human distal gut environment and can support complex, representative microbial communities ideal for experimental manipulation. / Canadian Institutes of Health Research; Ontario Ministry of Agriculture, Food and Rural Affairs; Ontario Ministry of Research and Innovation; Canada Foundation for Innovation; Ontario Ministry of Training, Colleges and Universities

<p>Autism is a pervasive developmental disorder (PDD) that&rsquo / s incidence is approximately 1 in 158. It is four times more prevalent in males than females and is believed to be caused by both genetic and environmental factors. Research indicates that several genes are involved in autism and it is believed that these genes act together to produce autism. Many genes implicated in this disorder are involved with brain structure formation and brain functioning. Studies have identified the reelin (RELN) gene as necessary for proper formation of brain, which indicates that RELN abnormalities could contribute to the aetiology of several neurogenetic diseases such as schizophrenia, bipolar and autism. The aims of the study were (i) to genotype two SNPs (exonic rs3622691 and intronic rs736707) in the RELN gene using Taqman&reg / SNP Genotyping assays to detect association with autism in three distinct South African (SA) ethnic groups (Black, Caucasian and Mixed), and (ii) to detect candidate genes that are over and under-expressed in the samples taken from a SA Caucasian autistic group and compare those with samples taken from a healthy Caucasian group using cDNA microarray. The Taqman&reg / study indicated significant association for the intronic SNP, rs736707, with a p-value of 0.0009 in the total SA group. More so, the Mixed group displayed the highest significance amongst the ethnic groups, with a p-value of 0.00014. The microarray study yielded 21 genes with 95% significance in the Caucasian sample group. Most genes were hypothetical proteins and formed part of the FAM90A family. The LOC83459 showed the highest level of expression in the autistic samples, while the BTNL8 gene was shown to be highly suppressed in the control samples.</p>

No abstract

Rat exon microarrays were utilized to detect changes in mRNA expression and alternative splicing in the liver, duodenum, jejunum, and ileum of the lactating rat when compared to age-matched virgin controls. Analysis of data at the level of gene expression revealed differential expression of genes involved in cholesterol biosynthesis in each tissue examined, suggesting increased Sterol Response Element Binding Protein activity. We also detected decreased mRNA from components of the T-cell signaling pathway in the jejunum and ileum. We characterized expression of solute carrier and adenosine triphosphate binding cassette proteins. In addition to characterizing genes by pathway, we have also grouped genes based on their pattern of expression to identify important genes. Amongst genes upregulated in all tissues was Slc39a4, which is a critical transporter in the absorption of zinc in enterocytes. Alternative splicing analysis detected a substantial amount of alternative splicing in the ileum compared to other tissues. In addition, in the liver Abcg8, a protein that functions as a heterodimer to export cholesterol in the bile, shows differential splicing in the liver, but not in other tissues. We also detected differential expression of Ugt1a6 in the liver based on usage of an alternative first exon, which is consistent with altered protein levels observed previously. Differential splicing also appears to occur in Ace2 in the ileum, which could have consequences on the renin-angiotensin pathway.