MYO1E mutations are associated with familial pediatric nephrotic syndrome, a disease in which pharmaceutical treatment is limited to immunosuppressive drugs such as steroids and cyclosporine but still often fails to prevent progression to end-stage renal disease. While Myo1e deficiency leads to glomerular filtration abnormality, including podocyte foot process effacement and glomerular basement membrane thickening, developing a precise treatment for this disease is hindered, as the underlying mechanisms linking MYO1E mutations and nephrotic syndrome are unclear. With the power of whole genome and exome sequencing, multiple novel Myo1e variants have been rapidly identified from SRNS cohorts. To determine whether a MYO1E mutation identified in patients is likely to be pathogenic or benign, we have characterized the functional effects of novel sequence variants in cultured podocytes. Differential protein degradation, localization, endocytic and motor activities of Myo1eT119I and Myo1eD388H have been discovered (Chapter 2). Specifically, even when expressed as a full-length protein, Myo1eT119I is deficient in localization to podocyte junctions and clathrin-coated vesicles (CCVs). Consistent with the hypothesis that Myo1eT119I is a loss-of-function mutant, cells expressing Myo1eT119I exhibit decreased CCV density and prolonged CCV lifetimes. The junctional and CCV localization of Myo1eD388H is not affected but it exhibits increased association with structures in the membrane-actin interface. Most importantly, Myo1eD388H is deficient in ATP hydrolysis and actin filament translocation. We have also characterized other MYO1E variants to provide cell-based evidence to assist in the curation of variants of uncertain significance (Chapter 3). Unexpectedly, while Myo1eD185G may be considered as a likely benign variant based on the population and computational predictions, it exhibits prolonged association with the podocyte junctions, while no junctional localization and dissociation abnormality was found in a likely deleterious variant, Myo1eR523W. We also discovered that localization of Myo1edel3094-7 to the podocyte junctions can be partially restored with a proteasomal inhibitor treatment, which may be considered as a potential treatment for patients with this variant. Finally, to follow up on the questions derived from Chapter 2, we examined our hypothesis that Myo1eD388H tail is constitutively active, we demonstrated the similar junctional exchange but differential endocytic activity of Myo1eD388H and Myo1eTAIL, revealing the critical regulation of Myo1e motor to tail domain when it comes to clathrin-mediated endocytosis (Appendix 1). Overall, the studies documented here have uncovered the differential molecular defects of steroid resistant nephrotic syndrome (SRNS)-associated Myo1e variants and further elucidated the underlying mechanisms of podocyte disease.

Vector-borne diseases affect an estimated 3.9 billion people in over 128 countries annually (WHO, 2017b). Mosquito-borne viruses, such as Chikungunya virus (CHIKV), are transmitted to human hosts through the saliva of female blood-feeding mosquitoes. During blood-feeding, salivary molecules enhance blood-feeding efficiency, dampen specific inflammatory signals, and enhance virus infection and dissemination. Skin is the interface where mosquito salivary molecules and viruses interact with the human immune system. Our long-term goal is to understand the impact of mosquito saliva on the skin and the role of salivary molecules in potentiating virus infection. This thesis aims to model the human skin bite site, identify mosquito salivary molecules present during virus infection, and investigate their impact on virus replication. We developed a human skin arbovirus infection model that was viable for four days ex vivo (Esterly et al., 2022a). Arbovirus infection in human skin ex vivo allowed flavivirus and alphavirus replication and dissemination. Using this model, we will investigate mosquito salivary factors and their impact on virus transmission. Next, we identified mosquito salivary factors highly expressed in Ae. aegypti salivary glands during CHIKV infection. We hypothesize that salivary proteins that contain secretion signals and are found to be highly expressed during CHIKV infection are delivered to the bite site during mosquito feeding. Through a combination of RNA-seq and miRNA-seq analysis, we identified several salivary factors, protein and small non-coding RNAs, which warrant further investigation. Lastly, we assessed the impact of identified and recombinantly expressed proteins or specific small RNA inhibitors on CHIKV replication in vitro relative to Ae. aegypti salivary gland extract (SGE). We found that both protein and small RNAs can influence CHIKV replication in a significant manner; however, the effect observed by mosquito saliva is a combination of multiple factors and will require more investigation to characterize fully. In summary, mosquito salivary factors work in concert to enhance virus replication and dissemination. Although individual factors may not recapitulate the total effect of SGE, the discovery and isolation of these factors further our understanding of arbovirus transmission. These studies highlight the complexity of mosquito salivary secretion and further our knowledge of mosquito-borne virus transmission. The following thesis will describe our most recent findings and contribution to understanding mosquito saliva's role during arbovirus transmission.

Human cytomegalovirus (HCMV) is a ubiquitous member of the betaherpesvirus family, with seroprevalence rates ranging from 40-100% worldwide. Although primary infection is asymptomatic in most immunocompetent patients, HCMV is a significant cause of morbidity and mortality in the immunosuppressed and immunonaїve, including transplant recipients, patients with AIDS, and developing fetuses in utero. The diverse clinical presentations of HCMV are attributable to the pervasive systemic dissemination and extensive cellular tropism of the virus. Peripheral blood monocytes are believed to be the key cells responsible for HCMV dissemination from the initial site of infection to distant organ systems. Monocytes are normally short-lived, surviving for only 48 h in circulation before undergoing apoptosis. Previous work from our lab has shown that HCMV circumvents the short lifespan of monocytes by inducing a noncanonical activation of Akt to upregulate the expression of antiapoptotic proteins, thereby prolonging survival of infected monocytes. HCMV promotes survival in the absence of viral replication and lytic gene expression, rendering current direct-acting antivirals ineffective against quiescently infected monocytes. There are currently no antivirals that target quiescent or latent HCMV infection. We hypothesize that targeting host proteins that are essential for HCMV's induction of monocyte survival mechanisms will reduce viability of infected monocytes, ultimately reducing systemic viral dissemination. The studies in this thesis investigate novel host-directed antivirals targeted at two different cellular factors and their efficacy against quiescent HCMV infection in monocytes. The first host protein under scrutiny as an antiviral target is Sirtuin 2 (Sirt2), an NAD+-dependent deacetylase. Treatment with novel Sirt2 inhibitors promoted death of HCMV-infected monocytes as a cellular antiviral defense response through two concurrent regulated death pathways: apoptosis and necroptosis. HCMV has developed mechanisms to impede both death pathways, but inhibition of Sirt2 relieves the viral obstructions on both pathways by disrupting HCMV's unique phosphorylation on Akt. The second host protein targeted as a potential antiviral strategy is Mcl-1, an antiapoptotic member of the Bcl-2 family of proteins. HCMV-infected monocytes are dependent on Akt-dependent upregulation of Mcl-1 for survival during early infection. Treatment with Mcl-1 inhibitors blocked interaction between Mcl-1 and proapoptotic protein Bak, reducing viability of infected monocytes. Subsequent testing of Mcl-1 inhibitors in an ex vivo skin organ culture system resulted in a decrease in HCMV-infected cells that crawled out of the skin tissue, suggesting that Mcl-1 inhibition may reduce viral dissemination. Our studies lay down the groundwork for the investigation of novel host-directed antivirals, an approach that successfully targets quiescent HCMV infection in peripheral blood monocytes for the first time. Expanding the study of host-directed antivirals may bring the field one step closer to the possibility of a comprehensive antiviral regimen that is effective against all stages of viral infection.

The two SH2-containing inositol 5'-phosphatases , SHIP1 (INPP5D) and SHIP2 (INPPL1), play an essential role in modulation of cellular signaling by transforming the PI3K product PI(3,4,5)P3 into PI(3,4)P2. PI3K signaling triggers activation of downstream signaling cascades that drive survival, effector functions, differentiation, and proliferation. SHIP1 can also mask the cytoplasmic tails of key receptors or their adaptor proteins such as DAP12, thus preventing PI3K recruitment to Trem2, a critical receptor for microglial function. Several GWAS studies correlated single nucleotide polymorphisms (SNPs) in INPP5D with Alzheimer's Disease (AD). However, it remains unclear whether these SNPs are deleterious or protective in AD and how they alter SHIP1 protein expression. SHIP2 overexpression has also been correlated with AD, suggesting that both SHIP1 and SHIP2 might be therapeutic targets. To study how SHIP1 and SHIP2 modulate microglial functions we used small molecule inhibitors and agonists of these enzymes. In our initial study we found that both SHIP paralogs are expressed in murine microglia and that Pan- SHIP1/2 inhibition increases lysosomal size and enhances microglial phagocytosis of Ab1-42 fibrils and dead neurons using both flow cytometry and confocal microscopy. Our lead Pan-SHIP1/2 inhibitor, K161, showed Blood Brain Barrier penetration as detected in the cerebral cortex of treated mice with mass spectrometry. K161 treatment of WT mice showed no difference in microglial frequency or lysosomal content in vivo; however, we observed a significant 2 increase in Ab1-42 and dead neurons phagocytosis ex vivo in microglia in K161- treated mice versus controls. Subsequently, we discovered a novel and highly potent SHIP1 selective agonist (K306) via artificial intelligence guided computational screening. We found that K306 can reduce the release of inflammatory cytokines in macrophages and microglial cells stimulated with LPS or Ab1-42. Interestingly, K306 didn't alter microglial phagocytic uptake of cargo, but did promote degradation of phagocytosed lipid-laden cargo - defining a novel role of SHIP1 in degradation of lipid cargo in microglia. These results highlight the importance of SHIP1 and SHIP2 in microglial biology and their modulation as therapeutics in different stages of neurodegenerative disease where microglia play a major role, such as AD.