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Human Cytomegalovirus (HCMV), the prototypic β-herpesvirus, is the most common cause of congenital infections as well as morbidity and mortality in immunocompromised patients. The anti-HCMV drugs currently available have a number of drawbacks (i.e. detrimental side-effects and/or the appearance of drug resistant strains), which limit their clinical usefulness. Therefore, a better understanding of host-virus interactions is important to develop new, safe and effective ways to treat HCMV. HCMV has evolved various strategies to make the host cell more conducive for the replication process, many of these involve modulation of host signalling pathways through proteins or non-coding RNAs. The focus of this thesis is on the regulation of one class of non-coding RNA, microRNAs (miRNA) by HCMV as well as murine CMV (MCMV). miRNAs are short ~22 nucleotide RNA sequences, which negatively regulate the stability and translational efficiency of specific target messenger RNAs (mRNAs). It has been previously shown that three host-encoded miRNAs, miR-199-3p, miR-199-5p and miR-214, are down-regulated in both MCMV and HCMV infected cells. Despite the biological and genomic differences between the two viruses, this down-regulation occurs in both infections, suggesting a possible conserved antiviral role of the miRNAs in mouse and human cells. Consistent with this, miR-199-3p and miR-214 manifest antiviral properties against MCMV and HCMV when over-expressed in vitro. This thesis investigates two hypotheses: 1) CMV down-regulates the expression of these host miRNAs through a mechanism involving viral factors, 2) The down-regulation of miR-199-3p leads to the up-regulation of its targets and this influences the cell in a way that favours some aspect of the viral life cycle. The first part of this project examined the regulation of miR-199-3p, miR-199-5p, and miR-214, which derive from a single primary transcript (pri-miRNA). The down-regulation of all three miRNAs was found to occur at the transcriptional level by 4 hours post infection. The promoter of the miR-199a/214 cluster was therefore cloned into a reporter vector in order to interrogate the factors regulating transcription of pri-miRNA in infection; this was carried out in the murine model based on availability of reagents. The reduction in the pri-miRNA was found to correlate with a decrease in the transcriptional activity of miR-199a/214 promoter in infected cells. Further analysis revealed the presence of a sequence between -421 to -273 relative to the transcription start site (TSS) that was critical for promoter activity. This sequence contains a putative serum response element (SRE), which includes two binding sites for the SRF dimer (serum response factor) and a binding site for a molecule of TCF (ternary complex factor), ELK-1. Initial knock-down studies suggest that these transcription factors are required for basal activity but it remains unknown whether they are involved in the differential expression of miR-199a/214 observed during infection. Another binding site for the transcription factor TWIST-1 was found outside this region, which is known to regulate the miR-199a/214 cluster in other cell types. Western blot analysis showed reduced expression of TWIST-1 in cells infected with HCMV and MCMV infections, by 24 and 48 hours, respectively, suggesting a role of TWIST-1 in regulating miR-199a/214 cluster during these infections. This regulation seems to be dependent on viral gene expression, as a replication deficient viral mutant fails to repress the promoter function and subsequent pri-miRNA production. Taken together, these results suggest an active viral mechanism for transcriptional repression of the miR-199a/214 promoter. To understand the antiviral function of miR-199-3p, the second part of this thesis examined whether miR-199-3p regulates host signalling pathways important for CMV replication and/or the life cycle. A microarray analysis was carried out with samples from cells transfected with miR- 199-3p mimic versus inhibitor. This revealed 198 genes significantly down-regulated by the miRNA. From the 198 genes, Ingenuity pathway analysis (IPA) software identified several host pathways with a potential role in HCMV infection including: PI3K/AKT signalling, the ERK-MAPK cascade, and prostaglandin production. This thesis examined the role of miR-199-3p in regulating the PI3K/AKT pathway in HCMV infection. It was found that miR-199-3p modulates the phosphorylation of the central regulator of PI3K/AKT signalling, AKT. Transfection of miR-199-3p before the infection impedes the complete phosphorylation of AKT, which is known to be required for the immediate early viral gene expression and replication. This provides an explanation for the antiviral function of miR-199-3p, through its ability to modulate AKT phosphorylation. An open question, however, is how the natural down-regulation of miR-199-3p from 24 to 72 hours post infection naturally affects AKT phosphorylation. Several predicted targets of miR-199-3p, such as PIK3CB, ITGA3, and ITGA6 were shown to be up-regulated at these late time points, correlating with the miR-199-3p down-regulation. The interaction of miR-199-3p with target sites in the 3′UTRs of PIK3CB and ITGA3 was validated by luciferase reporter assays and western blotting and qRT-PCR results indicated that protein and mRNA levels of ITGA6 were regulated by miR-199-3p mimic transfection. However, the knock-down of these three targets did not result in a significant decrease of the viral growth, and thus cannot alone explain the antiviral function of miR-199-3p. Overall, this study suggests that the transcriptional repression of miR- 199a/214 is likely a strategy employed by CMV to support its own growth through attenuating the biological effect of miR-199-3p within the host cell.

The autophagy pathway is an essential component of the innate immune response, capable of rapidly targeting intracellular bacteria, which are subsequently degraded by lysosomal enzymes. Recent work has begun to elucidate the regulatory signalling for autophagy induction in response to pathogenic bacteria. However, the initial signalling regulating autophagy induction in response to the detection of pathogens remains largely unclear. Here we report that AMPK, an important upstream activator of the autophagy pathway, is rapidly stimulated upon detection of pathogenic bacteria, prior to bacterial invasion. Bacterial recognition is initially achieved through detection of outer membrane vesicles (OMVs). Additionally, we show that AMPK signalling relieves mTORC1-mediated repression of the autophagy pathway in response to Salmonella infection, positioning the cell for a rapid induction of autophagy. Surprisingly, we found that the activation of AMPK and inhibition of mTORC1 in response to extracellular Salmonella are not accompanied by an induction of bulk autophagy. However, upon Salmonella invasion AMPK signalling is required for efficient and selective targeting of bacteria-containing vesicles by the autophagy pathway through activation of pro-autophagic kinase complexes. Collectively, these results demonstrate a key role for AMPK signalling in coordinating the rapid autophagic response prior to invasion of pathogenic bacteria.

Thesis advisor: Hiroshi Nakazato / In addition to negotiation, nonverbal signaling plays a large part in diplomacy. One such nonverbal technique is diplomatic revocation, in which a sending state summons its ambassador from a receiving state. Such an act has strategic value and can be used to discourage politically reprehensible acts in the receiving state, or further delegitimize its leaders or government to the international community, especially when accompanied by other sanctions or a comprehensive political agenda. Other times, revocation is reactionary, as in the cases of recalling an ambassador for poor conduct or as a precautionary measure against dwindling security conditions in the host state. In consulting scholarly work on the nonverbal dynamics of diplomacy and using an original dataset of over 1,000 instances of diplomatic revocation, this thesis examines the efficacy of diplomatic sanctions and concludes that 53% of diplomatic revocations are not intended as politically persuasive tools. / Thesis (BA) — Boston College, 2017. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Arts and Sciences Honors Program. / Discipline: International Studies.

Behçet’s disease (BD) is a chronic, multi systemic, recurrent vasculitis disease of unknown aetiology. The clinical manifestations are composed of relapsing episodes of recurrent oral ulcers, uveitis, skin lesions and genital ulcers along with musculoskeletal and neurological involvement. Pro-inflammatory cytokines are a key feature of the disease but the triggers for their induction are not well understood and/or controversial. Many cytokines (including IFNγ, IL-12, IL-23, IL-10 and IL-6) activate the JAK-STAT signalling pathway which is negatively regulated by Suppressor of Cytokine Signalling (SOCS) proteins. Therefore, it was hypothesised that SOCS proteins may be dysregulated in BD. The expression of SOCS 1-3 mRNA and protein was studied in peripheral blood mononuclear cells (PBMCs), Neutrophils and buccal mucosal cells (BMC) of BD patients and compared with healthy controls (HC) and recurrent aphthous stomatitis (RAS) patients. SOCS 1 and 3 were significantly upregulated in PBMCs of BD patients compared with HC (p=0.0149; p=0.0007) and there were subtle differences between expression in relapsed and symptom free BD (quiet BD). SOCS1 and SOCS 3 also significantly upregulated in BMC from oral ulcers of BD compared with HC (both at p=0.0001). Cytokines were examined in serum, saliva and culture supernatants from stimulated PBMCs. IL-6 were significantly upregulated in the saliva of relapsed BD patients compared with HC (p=0.0104) and the capacity for IL-10 secretion from BD was compromised. Phosphorylation of STATs, transcription factors RORγt, T-bet and 48 protein kinases were investigated using a novel PhosphFlow method and by microarray analysis. STATs were upregulated in BD and seven novel kinase proteins showed differential phosphorylation in BD. Conclusion: SOCS 1-3 expression has changed in BD patients with differences in PBMC and Neutrophil expression between the SOCS proteins. Phosphorylation of STATs and several kinases show up-regulation in BD and seven kinases with altered phosphorylation states in BD were identified as novel targets for future investigation.