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A mild traumatic brain injury (mTBI), commonly referred to as a concussion, is when the brain experiences an abrupt acceleration and/or deceleration that sends shock waves through the brain tissue, upsetting its structure and function. A mTBI is a heterogeneous condition with acute and chronic outcomes for patients. The chronic form of mTBI can lead to a wide range of neurological, behavioral, and cognitive symptoms. Critically, this injury is not defined by a simple process or pathophysiological event but rather biomechanical and neurological brain damage that can trigger highly complex physiological cascades. These further lead to a wide range of cellular, molecular, and functional changes that alter genes and associated metabolites. These changes, if specifically characterized, could be used to predict a patient’s outcome and recovery timeline. Recently, genetic studies showed that specific genotypes could increase an individual’s risk of more severe injury and impaired recovery following mTBI. Consequently, an improved understanding of gene alteration and genetic changes is necessary to develop personalized diagnostic approaches which can guide the design of novel treatments. The current study proposes utilizing bioinformatic tools, biological networks, and databases to identify potential genes and microRNAs associated with the mTBI in order to aid the early diagnosis of mTBI and track recovery for individual patients. With bioinformatic techniques, we were able to identify and compare genetic and epigenetic data associated with mTBI, as well as understand the various aspects of molecular changes after brain injury. Ultimately, we analyzed and cataloged the biological pathways and networks associated with this injury. A critical search of online bioinformatics databases was performed to determine interactions between mTBI-related genes, and relevant molecular processes. The major finding was that APOE, S100B, GFAP, BDNF, AQP4, COMT, MBP, UCHL1, DRD2, ASIC1, and CACNA1A genes were significantly associated with mTBI outcome. Those genes are primarily involved in different neurological tasks and neurological pathways such as neuron projection regeneration, regulation of neuronal synaptic plasticity, cognition, memory function, neuronal cell death and the dopaminergic pathway. This study predicted specific miRNAs linked to mTBI outcomes and candidate genes (hsa-miR-204-5p, hsa-miR-16-5p, hsa-miR-10a-5p, has-miR-218-5p, has-miR-34a-5p), and RNA-seq analysis on the GSE123336 data revealed that one miRNA found (hsa-miR-10a-5p) matched our predictions related to mTBI outcomes. Pathway analysis revealed that the predicted miRNA targets were mainly engaged in nervous system signaling, neuron projection and cell differentiation. These findings may contribute to developing diagnostic procedures and treatments for mTBI patients who are still experiencing symptoms, but validation of these genetic markers for mTBI assessment requires patient participation and correlation with advanced personalized MRI methods that show concussion related changes. / Thesis / Master of Applied Science (MASc) / Traumatic brain injury (TBI) is a highly prevalent neurological injury affecting millions of individuals globally. Mild TBI (mTBI), sometimes called concussion, makes up over 85% of TBI cases. A mTBI is a heterogeneous condition with acute and chronic outcomes for patients and involves complex cascades of cellular and molecular events that can lead to functional changes in genes and associated metabolites. In recent genetic studies, it has been shown that certain genotypes are associated with a higher risk of experiencing a more serious injury and a slower recovery after mTBI. These genes can be utilized as crucial biomarkers to predict how long it will take for a person to recover from a concussion. The purpose of this study was to find potential biomarkers that could help in the early detection of mTBI and the monitoring of individual patients’ recovery. It was hypothesized that genes and miRNAs (and their associated proteins) involved in neuronal body, axonal and myelin integrity and regeneration would be identified as important markers of severity.

MicroRNAs (miRNAs) function as key regulators in immune responses and cancer development. In the contexts of infection with oncogenic viruses, miRNAs are engaged in viral persistence, latency establishment and maintenance, and oncogenesis. In this review, we summarize the potential roles and mechanisms of viral and cellular miRNAs in the host-pathogen interactions during infection with selected tumor viruses and HIV, which include (i) repressing viral replication and facilitating latency establishment by targeting viral transcripts, (ii) evading innate and adaptive immune responses via toll-like receptors, RIG-I-like receptors, T-cell receptor, and B-cell receptor pathways by targeting signaling molecules such as TRAF6, IRAK1, IKKε, and MyD88, as well as downstream targets including regulatory cytokines such as tumor necrosis factor α, interferon γ, interleukin 10, and transforming growth factor β, (iii) antagonizing intrinsic and extrinsic apoptosis pathways by targeting pro-apoptotic or anti-apoptotic gene transcripts such as the Bcl-2 family and caspase-3, (iv) modulating cell proliferation and survival through regulation of the Wnt, PI3K/Akt, Erk/MAPK, and Jak/STAT signaling pathways, as well as the signaling pathways triggered by viral oncoproteins such as Epstein-Barr Virus LMP1, by targeting Wnt-inhibiting factor 1, SHIP, pTEN, and SOCSs, and (v) regulating cell cycle progression by targeting cell cycle inhibitors such as p21/WAF1 and p27/KIP1. Further elucidation of the interaction between miRNAs and these key biological events will facilitate our understanding of the pathogenesis of viral latency and oncogenesis and may lead to the identification of miRNAs as novel targets for developing new therapeutic or preventive interventions.

Rainbow trout are an important salmonid species whose poor utilization of dietary carbohydrates spurred research investigating molecular and physiological components of its glucoregulation. Among the environmental factors described to exert robust changes in glucose metabolism in rainbow trout, nutrition and social stress are among the most studied: Diets exceeding 20% of carbohydrates and chronic social stress induce hyperglycemia in adult and juvenile rainbow trout, respectively. Common to both responses is a contribution of hepatic de novo gluconeogenesis, which has been described to evade repression in response to high dietary carbohydrate content and to be stimulated in subordinate rainbow trout. Compared to previous studies investigating the regulation of hepatic gluconeogenesis at the molecular level, the recent publication of the annotated rainbow trout genome has opened novel possibilities to investigate paralogue-specific and posttranscriptional regulation of gluconeogenesis. In this thesis, I identify and describe the regulation of the novel phosphoenolpyruvate carboxykinase paralogue pck2b in rainbow trout and identify specific miRNA candidates predicted to contribute to gene paralogue-specific regulation of gluconeogenesis in nutritional and social contexts using small RNA next generation sequencing, real-time RT-PCR and in silico target prediction approaches. In nutritional and social status experiments, in silico predicted targets of differentially expressed hepatic miRNAs are enriched for gluconeogenesis regulation, suggesting a posttranscriptional component in regulating gluconeogenic transcript abundance. Differentially expressed hepatic miRNAs in both experiments comprise evolutionarily conserved and teleost-specific miRNAs, and are indicative of both environmental factor-specific and common regulation of gluconeogenesis transcripts in rainbow trout liver. Together this work provides novel comparative insight into hepatic miRNA-dependent glucoregulation and identifies several specific candidate miRNAs for future functional validation in hepatic glucoregulation in rainbow trout.

Sex differences are widespread in both mouse and human liver, and are associated with sex differences in drug metabolism and liver pathophysiology. The secretory patterns of growth hormone (GH) is one of the major drivers of liver sex specificity, where intermittent and continuous secretion in male and female respectively lead to sex bias in the expression of more than 1000 genes in mouse liver, via a complex interplay of GH-responsive transcription factors and epigenetic mechanisms. This thesis explores three themes of molecular control in the regulation of liver sex differences: microRNAs, DNA methylation, and translational control. Studies herein identified two microRNAs, miR-1948-5p and miR-802-5p, whose expression is sex biased and regulated by GH and the transcription factor STAT5b. Small RNA sequencing confirmed the sex specificity of these two microRNAs and identified an additional 18 sex-biased microRNAs. Computational and experimental characterization of miR-1948-5p and miR-802-5p confirmed their authenticity. In vivo inhibition of these microRNAs by locked nucleic acids indicated that miR-1948-5p and miR-802-5p played a functional role in repressing female-biased genes and male-biased genes, respectively. This thesis also investigated the impact of GH and STAT5b on liver DNA methylation profiles. Reduced representation bisulfite sequencing was performed on liver tissues from four mouse models that perturbed the GH and STAT5b axis. In the wildtype liver, sex biased demethylation was positively associated with sex biased chromatin opening and gene expression. Global hypermethylation was observed in livers of mice with lit/lit mutation resulting in GH deficiency or with hepatocyte-specific deletion of the STAT5ab locus. Strikingly, these hypermethylated loci were enriched for enhancer elements and STAT5b binding sites found in wild-type mouse liver. Hypophysectomy followed by GH replacement mouse models identified differentially methylated regions that were sex-biased and rapidly methylated and demethylated in response to GH stimulation. Finally, we used ribosome profiling to validate sex-biased protein translation and identify mechanisms of translational control. In sum, this body of work provides novel insights and broadens our understanding of the diverse molecular mechanisms underlying sexual dimorphism in the liver. / 2020-10-08T00:00:00Z

Mosquitoes are notorious vectors for multiple diseases like malaria, yellow fever and dengue fever. To manipulate gene expression in mosquito and spread desired genes among natural population for vector control, a thorough understanding of mosquito development and gene regulation is critical. Early embryogenesis is a rapid, complex yet crucial process in the very beginning of development. Previous research in other species indicated genes transcribed that early evolved fast and played essential roles. The study of mosquito early zygotic genes (EZGs) would offer unique insights into mosquito gene evolution as well as potential targets for mosquito control. In this study, I identified 61 pure EZGs (pEZGs) in mosquito Aedes aegypti. These pEZGs were enriched in architectures adapting to the rapid embryonic cell cycles and were over represented by domains or functions related to maternal zygotic transition. Phylogenetic analysis showed that pEZGs originated mainly from duplication, retrotransposition and de novo emergence. The comparison of pEZGs in Ae. aegypti with those in Drosophila revealed an interesting evolutionary paradox where the early zygotic genes turned over fast but the regulatory motif was conserved in two species. Curiously, the motif binding protein in Drosophila (zelda) seemed unable to initiate the earliest zygotic transcription in Ae. aegypti due to late temporal expression. The regulatory motif (VBRGGTA) found in Ae. aegypti pEZGs was shown necessary and sufficient for driving early zygotic gene expression by transient reporter assays and one motif-bearing promoter was tested with success in driving gene expression as early as 2-4h after egg laying in transgenic Ae. aegypti. This was the first characterized promoter with early zygotic but no maternal expression in Ae. aegypti that can be used for future genetic studies and mosquito control strategies. As important gene regulators, miRNAs also play essential roles in early embryogenesis. The genome-wide predictions and systematic analysis of miRNAs in Ae. aegypti and Anopheles stephensi were conducted in this study. The first miRNA profiling in mosquito across all developmental stages was also performed to provide basis for future functional study. Several lineage-specific miRNAs were found highly expressed in embryos, indicating their special roles in the embryogenesis of mosquitoes. / Ph. D.

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with rising global incidence. Merkel cell polyomavirus (MCV) was discovered in 2008 in 80% of MCC samples and since then a causal link between MCV and the majority of MCC cases has been established. microRNAs (miRNA, miR) are a family of small non-coding RNAs which play a key role in post-transcriptional regulation of gene expression and are considered significant players in disease and development in many species. Whilst the focus of MCV research has thus far been on the oncogenic MCV early proteins, large tumour (LT) and small tumour (sT) antigens, there is a knowledge gap regarding MCV miRNA and its functional significance in MCV pathogenesis. Given the emerging importance of viral miRNAs in virus-host interaction and pathogenesis, the aim of this doctoral research project was to investigate alterations in host cell transcripts induced by MCV miRNA and determine any functional significance these might have on virus-host cell interaction. RNA sequencing (RNA-Seq) in the presence and absence of MCV miRNA uncovered a multitude of downregulated cellular transcripts. Gene ontology analysis revealed that MCV miRNA targets transcripts associated with multiple cellular processes, however, regulation of immune response was overrepresented in our datasets. Validation of RNA-Seq data using MCV miRNA mimics and a synthetic, fully replicative MCV genome (MCVSyn) confirmed RNA-Seq data at mRNA and protein expression level for several targets, including the cytokine stimulating gene, SP100, and the neutrophil stimulator chemokine, CXCL8. Moreover, dual luciferase assays revealed that SP100 and MAPK10 (a member of mitogen-activated protein kinases (MAPK) family which is involved in regulation of CXCL8 expression) are directly and specifically targeted and downregulated by MCV miRNA. The MCV miRNA-dependent dysregulation of CXCL8 secretion is associated with impaired neutrophil migration, suggesting that the virus miRNA may be implicated in evasion of the host immune response.

MicroRNAs (miRNAs) are short non-coding RNAs that function in post-transcriptional gene silencing and mRNA regulation. Although the number of nucleotides of miRNAs ranges from 17 to 27, they are mostly made up of 22 nucleotides. The expression of miRNAs changes significantly in cancer, causing protein alterations in cancer cells by preventing some genes from being translated into proteins. In this research, a structural analysis of 587 miRNAs that are differentially expressed in myeloid cancer was carried out. Length distribution studies revealed a mean and median of 22 nucleotides, with an average of 21.69 and a variance of 1.65. We performed nucleotide analysis for each position where Uracil was the most observed nucleotide and Adenine the least observed one with 27.8% and 22.6%, respectively. There was a higher frequency of Adenine at the beginning of the sequences when compared to Uracil, which was more frequent at the end of miRNA sequences. The purine content of each implicated miRNA was also assessed. A novel motif analysis script was written to detect the most frequent 3–7 nucleotide (3–7n) long motifs in the miRNA dataset. We detected CUG (42%) as the most frequent 3n motif, CUGC (15%) as a 4n motif, AGUGC (6%) as a 5n motif, AAGUGC (4%) as a 6n motif, and UUUAGAG (4%) as a 7n motif. Thus, in the second part of our study, we further characterized the motifs by analyzing whether these motifs align at certain consensus sequences in our miRNA dataset, whether certain motifs target the same genes, and whether these motifs are conserved within other species. This thorough structural study of miRNA sequences provides a novel strategy to study the implications of miRNAs in health and disease. A better understanding of miRNA structure is crucial to developing therapeutic settings.

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