Human Cytomegalovirus Reprograms the Expression of Host Micro-RNAs whose Target Networks are Required for Viral Replication: A Dissertation

The parasitic nature of viruses requires that they adapt to their host environment in order to persist. Herpesviruses are among the largest and most genetically complex human viruses and they have evolved mechanisms that manipulate a variety of cellular pathways and processes required to replicate and persist within their hosts. Human cytomegalovirus (HCMV), a member of the β- herpesvirus sub-family, has the capacity to influence the expression of many host genes in an effort to create an optimal environment for infection. One mechanism utilized by HCMV to alter gene expression is the host RNA interference (RNAi) pathway. This is evidenced by a requirement of host factors to process viral micro-RNAs (miRNAs) and by the dynamic expression of host miRNAs during infection. The work presented in this dissertation demonstrates that productive HCMV infection reprograms host miRNA expression in order to positively influence infection. I was able to identify a cohort of infection-associated host miRNAs whose change in expression during infection was highly significant. Using the enhancer-promoter sequences of this panel of host miRNAs, I statistically enriched for the presence of functional transcription factor binding sites that regulated the expression of two highly conserved clusters of host miRNAs: miR132/212 and miR143/145. Given that inhibiting their infection-associated change in expression during infection was detrimental to viral replication, it suggests that HCMV mechanistically influences the expression of these miRNA clusters. In order to determine the functional relevance of these miRNAs, I assembled a cohort of potential miRNA target genes using gene expression profiles from primary fibroblasts. By statistically enriching for miRNA recognition elements (MRE) in the respective 3’-UTR sequences, I generated a miRNA target network that includes thousands of host genes. I evaluated the efficacy of our novel miRNA target prediction algorithm by confirming the functionality of enriched MREs present in the 3’-UTR of KRas and by confirming anecdotal miRNA targets from published studies. Gene ontology terms enriched from infection-associated host miRNA target networks suggest that the utility of host miRNAs may extend to multiple host pathways that are required for viral replication. The targeting of multiple miRNAs to shared genes increased the statistical likelihood of target site enrichment. I propose that identifying cooperative miRNA networks is essential to establishing the functional relevance of miRNAs in any context. By combining contextual data on the relative miRNA/mRNA abundance with statistical MRE enrichments, one will be able to more accurately characterize the biological role of miRNAs

An E2F1-Mediated DNA Damage Response Contributes to the Replication of Human Cytomegalovirus

DNA damage resulting from intrinsic or extrinsic sources activates DNA damage responses (DDRs) centered on protein kinase signaling cascades. The usual consequences of inducing DDRs include the activation of cell cycle checkpoints together with repair of the damaged DNA or induction of apoptosis. Many DNA viruses elicit host DDRs during infection and some viruses require the DDR for efficient replication. However, the mechanism by which DDRs are activated by viral infection is poorly understood. Human cytomegalovirus (HCMV) infection induces a DDR centered on the activation of ataxia telangiectasia mutated (ATM) protein kinase. Here we show that HCMV replication is compromised in cells with inactivated or depleted ATM and that ATM is essential for the host DDR early during infection. Likewise, a downstream target of ATM phosphorylation, H2AX, also contributes to viral replication. The ATM-dependent DDR is detected as discrete, nuclear γH2AX foci early in infection and can be activated by IE proteins. By 24 hpi, γH2AX is observed primarily in HCMV DNA replication compartments. We identified a role for the E2F1 transcription factor in mediating this DDR and viral replication. E2F1, but not E2F2 or E2F3, promotes the accumulation of γH2AX during HCMV infection or IE protein expression. Moreover, E2F1 expression, but not the expression of E2F2 or E2F3, is required for efficient HCMV replication. These results reveal a novel role for E2F1 in mediating an ATM-dependent DDR that contributes to viral replication. Given that E2F activity is often deregulated by infection with DNA viruses, these observations raise the possibility that an E2F1-mediated mechanism of DDR activation may be conserved among DNA viruses

A Novel Technique for the Expression and Purification of HIV-1 VIF Co-factor APOBEC3G

HIV-1 has evolved to protect itself against the body's innate viral defense mechanisms. One such example of this phenomenon is illustrated by the viral protein vif, which is expressed in the later stages of infection. Vif binds the host cell protein Apobec3G, a cytidine deaminase which is ectopically expressed in non-permissive cell lines to allow for reverse transcription of the HIV-1 viral genome. Apobec3G has the ability to prevent HIV-1 replication by mutating the cDNA, so the goal of this research was to develop a novel technique for the expression and purification of Apobec3G. An expression and purification technique was developed, and the purified Apobec3G protein was shown to have the capacity for binding viral Vif in vitro

Transgenic animals

The purpose of this IQP was to investigate the controversial new topic of transgenic animals, and determine the impact of this new technology on society. Through extensive medical, scientific and ethical research, this paper documents the impact of transgenic animals on society. Topics such as the description and construction, classification and examples, ethical issues, legal issues and the impact that this newly developed technology has made are detailed in the work. Illustrations, tables and direct quotes have been utilized to convey some of the more technical aspects of the topic. The authors conclude that with cautious oversight, transgenic animals can indeed be made with minimal or no animal suffering, which have enormous potential benefit to society

2008-2009 Design and Fabrication of a SAE Baja Race Vehicle

The objective of this project was to design and fabricate a racing vehicle for participation in SAE's Baja World Challenge. The vehicle was designed using mathematical and computer-aided modeling and simulation, resulting in a safe, high-performance vehicle for off-road competition, with a lightweight, high strength, and high durability. The vehicle was fabricated meticulously by the team, using WPI facilities, comprehensively satisfying both the design goals and manufacturing constraints, and will compete in June 2009