<p>Coronaviruses are the largest known RNA viruses and infect a wide range of hosts. Human coronaviruses traditionally have been known to be the cause of the common cold and have been vastly understudied due to low morbidity and mortality. The emergence of SARS-CoV and MERS-CoV has altered the landscape of coronavirus research and proven the deadly capabilities of human coronaviruses. With two recent zoonotic events, it is increasingly important to understand the molecular biology of human coronaviruses. The coronavirus nucleocapsid protein is an essential structural protein that complexes with the viral genome. Though nucleocapsid formation is the protein’s major role, it has also been found to have other functions and effects during infection. The following research aimed to examine how the human coronavirus nucleocapsid protein affects the innate immune response <em>in vitro</em>. Modulation of the type I interferon response by the nucleocapsid was first investigated and the nucleocapsids were shown to have the ability to block interferon signalling. Additionally, the nucleocapsid protein was found to cause a dysregulation of transcription factor NFKB1. We propose a novel mechanism of this NFKB1 negative regulation interference. Taken together, we have further characterized the significant role of the coronavirus nucleocapsid protein in innate immune evasion.</p> / Doctor of Philosophy (Medical Science)

Hepatitis C virus (HCV) infection is common among injection drug users (IDUs). There is accumulating evidence that circulating microRNAs (miRNAs) are related to HCV infection and disease progression. The present study was undertaken to determine the in vivo impact of heroin use on HCV infection and HCV-related circulating miRNA expression. Using the blood specimens from four groups of study subjects (HCV-infected individuals, heroin users with/without HCV infection, and healthy volunteers), we found that HCV- infected heroin users had significantly higher viral load than HCV-infected non-heroin users (p=0.0004). Measurement of HCV-related circulating miRNAs in plasma showed that miRs-122, 141, 29a, 29b, and 29c were significantly increased in the heroin users with HCV infection, whereas miR-351, an HCV inhibitory miRNA, was significantly decreased in heroin users as compared to control subjects. Further investigation identified a negative correlation between the plasma levels of miR-29 family members and severity of HCV infection based on aspartate aminotransferase to platelet ratio index (APRI). Heroin use and/or HCV infection also dysregulated a panel of plasma miRNAs. Taken together, these data for the first time revealed in vivo evidence that heroin use and/or HCV infection alter circulating miRNAs, which provides a novel mechanism for the impaired innate anti-HCV immunity among IDUs. Recent studies revealed that extracellular miRNAs were able to incorporate into cell-derived exosomes as a method of cell-to-cell interaction. Exosomes are a class of cell-released small vesicles that mediate intercellular communication by delivering functional factors to recipient cells. During HCV infection, the interaction between liver resident macrophages and hepatocytes is important for host defense and viral elimination, triggered by innate immune activation, especially Toll like receptors (TLR). In our study, we explored the role of macrophage-derived exosomes in the transmission of innate immune responses against HCV infection in hepatocytes, and the involvement of exosomal miRNAs in transferring the anti-HCV activities. We reported that upon TLR3 activation, macrophages shed exosomes that were able to attenuate HCV-JFH1 infection in Huh7 cells. We further demonstrated that exosomes from poly I:C treated macrophages were internalized by Huh7 cells, which induced the intercellular anti-HCV responses (type I interferon, interferon stimulated genes, etc.) and thus drastically inhibited HCV infection in Huh7 cells. Moreover, using an in vitro macrophage and Huh7 cell co-culture model, we also found exosomes mediated HCV suppression in Huh7 cells after TLR3 activation. The presence of exosome inhibitor in co-culture compromised the anti-HCV activity by TLR3-activated macrophages. Interestingly, the miRNA-29 family, which was reported to suppress HCV infection, was significantly increased in the macrophage exosomes after TLR3 activation. The inhibition of miRNA-29 partially compromised the anti-HCV activity of TLR3-activated macrophages, indicating the potential involvement of exosomal miRNAs in the transmission of anti-HCV activity from macrophages to Huh7 cells through exosomes. In conclusion, this study proposed an antiviral mechanism of TLR3 activation that involves the intercellular communication between immune cells and hepatic parenchymal cells via exosomes, and exosomal miRNAs. This discovery sheds light on exploiting the therapeutic potential of new drugs against HCV infection. / Pathology

Research has shown that our gut microbiota confers many beneficial functions, including aiding the development of the immune system, metabolism, modulating stress reactivity and behaviour. The diverse population of the gut microbiota has been shown to be heterogeneous between individuals, with host genetic factors emerging as a contributor to gut microbiota composition. Recent work suggests that microRNA may act as a mediator of communication between the host and resident gut microbiota. Here we explore host genetic differences in gut microbiota composition and fecal miRNA profiles in two inbred mouse strains BALB/C and C57BL/6, in relation to gastrointestinal homeostasis. Furthermore, we evaluate the role of host genetics in response to perturbation of the gut microbiota using broad-spectrum antibiotic treatment. Distinct differences in the gut microbiota composition evaluated by fecal 16s rRNA gene sequencing between BALB/c and C57BL/6 mice were found with notable significant differences in genera Prevotella, Alistipes, Akkermansia and Ruminococcus. Significant host genetic differences were also observed in fecal miRNA profiles evaluated using the nCounter Nanostring platform. A BLASTn analysis was used to identify conserved fecal miRNA target regions in bacterial metagenomes, which identified numerous bacterial gene targets. Of those miRNA targets that were conserved in our dataset, 14 significant correlations were found between fecal miRNA and predicted taxa relative abundance. Treatment with broad-spectrum antibiotics for a period of 2 weeks resulted in BALB/c mice exhibiting a decrease in barrier permeability while C56BL/6 barrier permeability remained unchanged, demonstrating a host-specific physiological response to antibiotics at the gastrointestinal barrier. Differential response to antibiotics was also observed in the expression of barrier regulating genes in both host strains. Individual taxa were found to respond differentially by host strain, with Parabacteroides and Bacteroides associating with changes in barrier function. Together these findings suggest that host genetics play a role in determining the host-microbe relationship in both healthy homeostatic conditions and altered microbial conditions. / Thesis / Master of Science (MSc)

Yes / Skin development is governed by complex programs of gene activation and silencing, including microRNA-dependent modulation of gene expression. Here, we show that miR-214 regulates skin morphogenesis and hair follicle (HF) cycling by targeting β-catenin, a key component of the Wnt signaling pathway. miR-214 exhibits differential expression patterns in the skin epithelium, and its inducible overexpression in keratinocytes inhibited proliferation, which resulted in formation of fewer HFs with decreased hair bulb size and thinner hair production. The inhibitory effects of miR-214 on HF development and cycling were associated with altered activities of multiple signaling pathways, including decreased expression of key Wnt signaling mediators β-catenin and Lef-1, and were rescued by treatment with pharmacological Wnt activators. Finally, we identify β-catenin as one of the conserved miR-214 targets in keratinocytes. These data provide an important foundation for further analyses of miR-214 as a key regulator of Wnt pathway activity and stem cell functions during normal tissue homeostasis, regeneration, and aging.

The MEF2 transcription factor is a central regulator of skeletal and cardiac muscle development. Recently, we showed that MEF2A regulates skeletal muscle regeneration through direct regulation of the Gtl2-Dio3 microRNA mega-cluster. In addition to their expression in skeletal muscle, temporal expression analysis of selected Gtl2-Dio3 microRNAs revealed high enrichment in cardiac muscle. Therefore, I investigated the role of selected microRNAs from the Gtl2-Dio3 noncoding RNA locus in the heart. First, I found that Gtl2-Dio3 microRNAs are expressed at higher levels in perinatal hearts compared to adult, suggesting they function in cardiac maturation shortly after birth. I also demonstrated that these microRNAs are dependent on MEF2A in the perinatal heart and in neonatal cardiomyocytes. To determine the specific role in cardiac muscle, I overexpressed selected microRNA mimics in neonatal rat ventricular myocytes (NRVMs). My results showed that miR-410 and miR-495 stimulate cell cycle re-entry and proliferation of terminally differentiated NRVMs. Subsequent target prediction analyses revealed a number of candidate target genes known to function in the cell cycle and/or in cardiac muscle. One of these was Cited2, a cofactor required for proper cardiac development. Subsequently, I showed that Cited2 is a direct target of these miRNAs and that siRNA knockdown of Cited2 in NRVMs resulted in robust cardiomyocyte proliferation. This phenotype was associated with reduced expression of Cdkn1c/p57/Kip2, a cell cycle inhibitor, and increased expression of Vegfa, a growth factor with proliferation-promoting effects. Given the exciting possibility of manipulating the expression of these microRNAs to repair the damaged heart by stimulating cardiomyocyte proliferation, I then investigated whether they were regulated in cardiac disease and function in pathological signaling. Toward this end, I examined expression of miR-410, miR-495, miR-433, as well as the Gtl2 lncRNA in various cardiomyopathies. Interestingly, the microRNAs and lncRNA were dynamically regulated in mouse models of cardiac disease including myocardial infarction and chronic angiotensin II stimulation. Furthermore, I showed for the first time that the Gtl2 lncRNA and miRNAs are differentially regulated in myocardial infarction, indicating that the complex regulation of the Gtl2-Dio3 noncoding RNA locus may be important for response to cardiac injury. Lastly, I showed that inhibiting select Gtl2-Dio3 microRNAs in pathological signaling attenuated cardiomyocyte hypertrophy in vitro. Therefore, differential targeting of the Gtl2-Dio3 noncoding RNAs could provide new therapeutic strategies to control the response of the heart to cardiac diseases with diverse etiologies.

No / Bone morphogenetic proteins (BMPs) play essential roles in the control of skin development, postnatal tissue remodelling and tumorigenesis. To explore whether some of the effects of BMP signalling are mediated by microRNAs, we performed genome-wide microRNA (miRNA) screening in primary mouse keratinocytes after BMP4 treatment. Microarray analysis revealed substantial BMP4-dependent changes in the expression of distinct miRNAs, including miR-21. Real-time PCR confirmed that BMP4 dramatically inhibits miR-21 expression in the keratinocytes. Consistently, significantly increased levels of miR-21 were observed in transgenic mice overexpressing the BMP antagonist noggin under control of the K14 promoter (K14-noggin). By in situ hybridization, miR-21 expression was observed in the epidermis and hair follicle epithelium in normal mouse skin. In K14-noggin skin, miR-21 was prominently expressed in the epidermis, as well as in the peripheral portion of trichofolliculoma-like hair follicle-derived tumours that contain proliferating and poorly differentiated cells. By transfecting keratinocytes with a miR-21 mimic, we identified the existence of two groups of the BMP target genes, which are differentially regulated by miR-21. These included selected BMP-dependent tumour-suppressor genes (Pten, Pdcd4, Timp3 and Tpm1) negatively regulated by miR-21, as well as miR-21-independent Id1, Id2, Id3 and Msx2 that predominantly mediate the effects of BMPs on cell differentiation. In primary keratinocytes and HaCaT cells, miR-21 prevented the inhibitory effects of BMP4 on cell proliferation and migration. Thus, our study establishes a novel mechanism for the regulation of BMP-induced effects in the skin and suggests miRNAs are important modulators of the effects of growth factor signalling pathways on skin development and tumorigenesis.

Yes / Malignant pleural mesothelioma (MPM) is associated with an extremely poor prognosis, and most patients initially are or rapidly become unresponsive to platinum-based chemotherapy. MicroRNA-31 (miR-31) is encoded on a genomic fragile site, 9p21.3, which is reportedly lost in many MPM tumors. Based on previous findings in a variety of other cancers, we hypothesized that miR-31 alters chemosensitivity and that miR-31 reconstitution may influence sensitivity to chemotherapeutics in MPM. Reintroduction of miR-31 into miR-31 null NCI-H2452 cells significantly enhanced clonogenic resistance to cisplatin and carboplatin. Although miR-31 re-expression increased chemoresistance, paradoxically, a higher relative intracellular accumulation of platinum was detected. This was coupled to a significantly decreased intranuclear concentration of platinum. Linked with a downregulation of OCT1, a bipotential transcriptional regulator with multiple miR-31 target binding sites, we subsequently identified an indirect miR-31-mediated upregulation of ABCB9, a transporter associated with drug accumulation in lysosomes, and increased uptake of platinum to lysosomes. However, when overexpressed directly, ABCB9 promoted cellular chemosensitivity, suggesting that miR-31 promotes chemoresistance largely via an ABCB9-independent mechanism. Overall, our data suggest that miR-31 loss from MPM tumors promotes chemosensitivity and may be prognostically beneficial in the context of therapeutic sensitivity.

Much research has been directed toward understanding the roles of essential components in the cell, such as proteins, microRNAs, and genes. This dissertation focuses on two interesting problems in bioinformatics research: microRNA-target prediction and the identification of conserved protein complexes across species. We define the two problems and develop novel approaches for solving them. MicroRNAs are short non-coding RNAs that mediate gene expression. The goal is to predict microRNA targets. Existing methods rely on sequence features to predict targets. These features are neither sufficient nor necessary to identify functional target sites and ignore the cellular conditions in which microRNA and mRNA interact. We developed MicroTarget to predict microRNA-mRNA interactions using heterogeneous data sources. MicroTarget uses expression data to learn candidate target set for each microRNA. Then, sequence data is used to provide evidence of direct interactions and ranking the predicted targets. The predicted targets overlap with many of the experimentally validated ones. The results indicate that using expression data helps in predicting microRNA targets accurately. Protein complexes conserved across species specify processes that are core to cell machinery. Methods that have been devised to identify conserved complexes are severely limited by noise in PPI data. Behind PPIs, there are domains interacting physically to perform the necessary functions. Therefore, employing domains and domain interactions gives a better view of the protein interactions and functions. We developed novel strategy for local network alignment, DONA. DONA maps proteins into their domains and uses DDIs to improve the network alignment. We developed novel strategy for constructing an alignment graph and then uses this graph to discover the conserved sub-networks. DONA shows better performance in terms of the overlap with known protein complexes with higher precision and recall rates than existing methods. The result shows better semantic similarity computed with respect to both the biological process and the molecular function of the aligned sub-networks. / Ph. D. / Much research has been directed toward understanding the roles of essential components in the cell, such as proteins, microRNAs, and genes. The processes within the cell include a mixture of small molecules. It is of great interest to utilize different information sources to discover the interactions among these molecules. This dissertation focuses on two interesting problems: microRNA-target prediction and the identification of conserved protein complexes across species. We define the two problems and develop novel approaches for solving them. MicroRNAs are a recently discovered class of non-coding RNAs. They play key roles in the regulation of gene expression of as much as 30% of all mammalian protein encoding genes. MicroRNAs regulation activity has been implicated in a number of diseases including cancer, heart disease and neurological diseases. We developed MicroTarget to predict microRNAgene interactions using heterogeneous data sources. The predicted target genes overlap with many of the experimentally validated ones. Proteins carry out their tasks in the cell by interacting with each other. Protein complexes conserved among species specify the cell core processes. We identify conserved complexes by constructing an alignment graph leveraging on the conservation of PPIs between species through domain conservation and domain-domain interactions (DDI) in addition to PPI networks. Better integration of domain conservation and interactions in our developed conserved protein complexes identification system helps biologists benefit from verified data to predict more reliable similarity relationships among species. All the test data sets and source code for this dissertation are available at: https://bioinformatics.cs.vt.edu/∼htorkey/Software.

Die Multiple Sklerose (MS) ist die häufigste chronisch-entzündliche Erkrankung des zentralen Nervensystems und die häufigste Ursache erworbener Behinderung im jungen Erwachsenenalter. Histopathologisches Kennzeichen der MS sind fokale Läsionen, die durch Demyelinisierung, Entzündungsinfiltrat, gliale Narbenbildung, Astrozytose, Oligodendrozytenschädigung und unterschiedlich stark ausgeprägte Axondegeneration gekennzeichnet sind. Die Läsionen können im gesamten ZNS auftreten. Bei der Pathogenese der MS geht man von einem multifaktoriellen Geschehen aus. Nach dem derzeitigen Wissenstand ist die MS eine T-Zell-vermittelte Autoimmunerkrankung gegen bestimmte Bestandteile des zentralen Nervensystems, bei der zusätzlich genetische, epidemiologische und Umweltfaktoren eine Rolle spielen. MicroRNAs (miRNAs) haben wichtige Funktionen in der Genregulation und sind in viele physiologische Zellprozesse involviert. Man geht daher davon aus, dass miRNAs auch eine wichtige Rolle in der Pathogenese der MS spielen. Sowohl an MS-Patienten als auch im Tiermodell werden diesbezügliche Untersuchungen durchgeführt. Das wichtigste Tiermodell der MS ist die experimentelle autoimmune Enzephalomyelitis (EAE). In der vorliegenden Arbeit wurde die miRNA-Regulation ausgewählter miRNAs in EAE-Läsionen von C57/BL6 Mäusen (MOG35-55-induziert) und Weißbüschelaffen (MOG1-125-induziert) im Gegensatz zu Kontrollgewebe ermittelt und mit der bekannten miRNA-Regulation in aktiven humanen MS-Läsionen verglichen. Aus den EAE-Rückenmarksläsionen der Mäuse und den makrosezierten EAE-Hirnläsionen der Weißbüschelaffen wurde zunächst die RNA extrahiert. Im Anschluss erfolgte die Umschreibung in eine cDNA und dann eine miRNA-Detektion und -Quantifizierung mit Hilfe einer qPCR. Es konnte gezeigt werden, dass die miRNA-Regulation in den EAE-Läsionen von Mäusen und Weißbüschelaffen mit der miRNA-Hoch- und -Herabregulation in den aktiven humanen MS-Läsionen vergleichbar ist. Die im Menschen konservierten und regulierten miRNAs miR-155, miR-142-3p, miRNA-146a, miR-146b und miR-21 waren auch in Maus und Weißbüschelaffe in ähnlicher Weise hochreguliert.

Die chronische Herzinsuffizienz ist ein schwerwiegendes progredientes Krankheitsbild, das sich neben Dyspnoe und abnehmender Leistungsfähigkeit in einer nachgewiesenen Verschlechterung der HDL-Funktion manifestiert. In zahlreichen Studien, in denen der Einfluss von körperlichem Training auf die Progredienz der chronischen Herzinsuffizienz untersucht wurde, korrelierte dauerhaftes Ausdauertraining mit einer Verbesserung der eNOS-Aktivität und damit der HDLFunktion in Gefäßen. Ein Regulationsmechanismus von Endothelzellen besteht in der Expression von angiogenen microRNAs, die über negative Regulation die Proteinexpression beeinflussen. Ziel dieser Studie ist es nun, einen möglichen Zusammenhang zwischen der HDL-Funktionalität und der microRNA-Expression in Endothelzellen zu prüfen und damit die Funktionsänderung von HDL bei Herzinsuffizienten auf molekularer Ebene nachzuweisen. Zudem soll eine Beeinflussung der HDL-Funktion durch körperliches Training geprüft werden. Dafür wurde das HDL von gesunden und herzinsuffizienten Probanden (NYHAIII-Stadium) vor und nach einem vier- bzw. zwölfwöchigen Trainingsprogramm aus dem Plasma isoliert. Anschließend erfolgte mit dem gewonnenen HDL die 24-stündige Inkubation von HAEC-Kulturen. Nach Isolation der microRNAs aus dem gewonnenen Zelllysat konnte die Menge ausgewählter proangiogener miRs über RT-PCR quantifiziert werden. Die molekularbiologische Analyse der Proben zeigte eine, im Vergleich zu den Kontrollzellen, signifikant verringerte Menge an miR-21, -126 und -222 in den, mit HDLNYHAIII-inkubierten, Endothelzellen. Die miR-Expression der Endothelzellen zeigte nach dem Trainingsprogramm eine Annäherung an das Expressionsniveau der Kontrollen. Aus der dargelegten Studie wird so ersichtlich, dass das HDL von Herzinsuffizienten die Expression von proangiogenen microRNAs in Endothelzellen hemmt, was scheinbar in Korrelation mit der Ausbildung von endothelialen Dysfunktionen bei Herzinsuffizienz steht. Zudem konnte gezeigt werden, dass körperliches Training mit einer verbesserten Endothelfunktion über die Erhöhung der miR-Expression in Endothelzellen einhergeht.