The Human Immunodeficiency Virus (HIV) is able to infect CD4+ T cells as well as macrophages. Macrophage-tropism has been linked to determinants in the envelope of HIV. These determinants allow envelopes to exploit low levels of CD4 for infection. Macrophages are an important reservoir of virus, especially during chronic infection, and are likely responsible for the bulk of virus produced after CD4+T cells have declined. Viral factors that may impact the ability to infect macrophages are worth studying because this cell type is so important in infection. It was previously reported that the macrophage-tropic primary isolate AD8 was vpu-independent. The molecular clone YU-2, derived from brain tissue without culture, was also reported to be macrophage-tropic despite having a mutation in the vpu start codon. It was therefore possible that vpu-independent envelopes could evolve in vivo. To examine this possibility, I constructed chimeras containing wild type or defective vpu start codons, and gp160 sequences from AD8, YU-2 or SF162 (a vpu-dependent control). I also used full length AD8 and YU-2 with wild type or defective vpu start codons. I infected macrophages with equal amounts of virus, and measured viral output over two weeks. Viruses with defective vpu start codons were released to lower levels compared to their wild type vpucounterparts. In contrast to previous reports, the AD8 envelope is not vpu-independent for replication in macrophages. The YU-2 envelope is also not vpu-independent. Macrophage-tropic envelopes from late stages of infection can be sensitive to antibodies that bind the CD4 binding site on gp120, implying that macrophage-tropic envelopes have more exposed CD4 binding sites. Neutralizing antibodies may act as modulators of macrophage-tropism over the course of infection. Using chimeras containing gp120 sequences derived from the PBMC of four HIV+patients, I examined the capacity for envelopes to infect macrophages. Three patients (MM1, 4, and 8) had macrophage-tropic envelopes before and after developing autologous neutralizing antibodies. Three patients (MM1, 4, and 23) developed heterologous antibodies against IIIB, an easily neutralized T-cell line adapted strain of HIV-1. This data indicates that macrophage-tropism in these patients is not modulated by the presence of neutralizing antibodies. The macrophage-tropism of envelopes tends to segregate depending on the tissue origin of the virus. Envelopes from two separate tissues from the same patient exhibit very different infectivity characteristics. The B33 envelope, from brain tissue, is very infectious and is macrophage-tropic, while the LN40 envelope, from lymph node tissue, is weakly infectious and is not macrophage-tropic. Replacing the entire gp41 of LN40 with that of B33 restores some infectivity to LN40. The cytoplasmic domain of gp41 contains many motifs important for assembly and infectivity. To examine which motifs are responsible for the weak infectivity of LN40, I made chimeras of gp41, as well as point mutations in gp41. The LN40 chimera containing the entire gp41 of B33 restored the most infectivity. Point mutations in the palmitoylation site, Pr55gagbinding region, and dileucine motif at the C-terminus also restored infectivity when combined. Determinants in the gp41 cytoplasmic domain are responsible for the weak infectivity of LN40; however, it is possible that there are contributing determinants in gp120, such as the ability to use low levels of CD4. Here, I examined how changes in the vpu and env genes of HIV-1 can impact infectivity, especially infectivity of macrophages. Changes that adversely impact the virus’ ability to infect macrophages may also impact the overall course of disease. However, the data here show that retaining the ability to infect, and replicate in, macrophages give HIV an advantage. I speculate that retaining the ability to infect macrophages gives the virus a reservoir for later in disease, when CD4+ T cells have been depleted, as well as way of avoiding neutralizing antibodies. This work further defines the importance of macrophages in HIV-1 infectivity and disease.

T cells play a central role in cellular-mediated immunity and must become activated to participate as effector cells in the immune response. The activation process is highly intricate and involves stimulation of a number of downstream signaling pathways enabling T cells to proliferate and produce cytokines that are vital for proper effector function. This increase in protein production and protein folding activity adds to the normal physiological strain on cellular machinery. One cellular compartment that has generated a mechanism to mitigate the stress induced by increased protein production is the endoplasmic reticulum (ER). In general, an increase in cellular production of proteins that overwhelms a cell’s protein folding capability can alter ER homeostasis and lead to ER stress. To counteract this stress, an adaptive cellular mechanism known as the ER stress response (ERSR) is initiated. The ERSR allows a cell to cope with normal physiological stress within the ER caused by increased protein translation. In this dissertation, we show that in vitro and in vivoT cell activation involving T cell receptor (TCR) ligation in the presence of costimulation initiates the physiological ERSR. Interestingly, the ERSR was also activated in T cells exposed only to TCR ligation, a treatment known to induce the ‘non-responsive’ states of anergy and tolerance. We further identified a key component of the downstream TCR signaling pathway, protein kinase C (PKC), as an initiator of physiological ERSR signaling, thus revealing a previously unknown role for this serine/threonine protein kinase in T cells. Therefore, induction of the physiological ERSR through PKC signaling may be an important ‘preparatory’ mechanism initiated during the early activation phase of T cells. If ER stress is persistent and ER homeostasis is not reestablished, physiological ER stress becomes pathological and initiates cellular death pathways through ER stress-induced apoptotic signaling. We further present data demonstrating that absence of functional Gimap5, a putative GTPase implicated to play a role in TCR signaling and maintenance of overall T cell homeostasis, leads to pathological ER stress and apoptosis. Using the BioBreeding diabetes-prone (BBDP) rat, a model for type 1 diabetes (T1D), we link pathological ER stress and ER stress-induced apoptotic signaling to the observed T cell lymphopenic phenotype of the animal. By depleting the ER stress apoptotic factor CHOP with siRNA, we were able to protect Gimap5-/-BBDP rat T cells from ER stress-induced death. These findings indicate a direct relationship between Gimap5 and maintenance of ER homeostasis for T cell survival. Overall, our findings suggest that the ERSR is activated by physiological and pathological conditions that disrupt T cell homeostasis. TCR signaling that leads to PKC activation initiates a physiological ERSR, perhaps in preparation for a T cell response to antigen. In addition, we also describe an example of pathological ERSR induction in T cells. Namely, we report that the absence of functional Gimap5 protein in T cells causes CHOP-dependent ER stress-induced apoptosis, perhaps initiated by deregulation of TCR signaling. This indicates a dual role for TCR signaling and regulation in the initiation of both the physiological and pathological ERSR. Future research that provides insights into the molecular mechanisms that govern ERSR induction in TCR signaling and regulation may lead to development of therapeutic modalities for treatment of immune-mediated diseases such as T1D.

Topographical and mechanical properties of adhesive substrates provide important biological cues that affect cell spreading, migration, growth, and differentiation. The phenomenon has led to the increased use of topographically patterned and flexible substrates in studying cultured cells. However, these studies may be complicated by various limitations. For example, the effects of ligand distribution and porosity are affected by topographical features of 3D biological constructs. Similarly, many studies of mechanical cues are compounded with cellular deformation from external forces, or limited by comparative studies of separate cells on different substrates. Furthermore, understanding cell responses to mechanical input is dependent upon reliable measurements of mechanical properties. This work addresses each of these issues. To determine how substrate topography and focal adhesion kinase (FAK) affect cell shape and movement, I studied FAK-null (FAK -/-) and wild type mouse 3T3 fibroblasts on chemically identical polystyrene substrates with either flat surfaces or micron-sized pillars, I found that, compared to cells on flat surfaces, those on pillar substrates showed a more branched shape, an increased linear speed, and a decreased directional stability, which were dependent on both myosin-II and FAK. To study the dynamic responses to changes in substrate stiffness without other confounding effects, I developed a UV-modulatable substrate that softens upon UV irradiation. As atomic force microscopy (AFM) proved inadequate to detect microscale changes in stiffness, I first developed and validated a microsphere indentation method that is compatible with fluorescence microscopy. The results obtained with this method were comparable to those obtained with AFM. The UV-modulatable substrates softened by ~20-30% with an intensity of irradiation that has no detectable effect on 3T3 cells on control surfaces. Cells responded to global softening of the substrate with an initial retraction followed by a gradual reduction in spread area. Precise spatial control of softening is also possible - while there was little response to posterior softening, anterior softening elicited a pronounced retraction and either a reversal of cell polarity or a significant decrease in spread area if the cells move into the softened region. In conclusion, these techniques provide advances in gaining mechanistic insight into cellular responses to topographical and mechanical cues. Additionally, there are various other potential applications of the novel UV-softening substrate, particularly in regenerative medicine and tissue engineering.

Cells need to complete DNA replication in a timely and error-free manner. To ensure that replication is completed efficiently and in a finite amount of time, cells regulate origin firing. To prevent any errors from being transmitted to the next generation, cells have the checkpoint mechanism. The S-phase DNA damage slows replication to allow the cell to repair the damage. The mechanism of replication slowing by the checkpoint was not clear in fission yeast, Schizosaccharomyces pombe, at the start of my thesis. The downstream targets of the DNA damage checkpoint in fission yeast were also unclear. I worked on identifying the downstream targets for the checkpoint by studying if Cdc25, a phosphatase, is a target of the checkpoint. Work from our lab has shown that origin firing is stochastic in fission yeast. Origins are also known to be inefficient. Inefficient origins firing stochastically would lead to large stretches of chromosome where no origins may fire randomly leading to long replication times, an issue called the random gap problem. However, cells do not take a long time to complete replication and the process of replication itself is efficient. I focused on understanding the mechanism by which cells complete replication and avoid the random gap problem by attempting to measure the firing efficiency of late origins. Genome-wide origin studies in fission yeast have identified several hundred origins. However, the resolution of these studies can be improved upon. I began a genome-wide origin mapping study using deep sequencing to identify origins at a greater resolution compared to the previous studies. We have extended our origin search to two other Schizosaccharomyces species- S. octosporus and S. japonicus.There have been no origin mapping studies on these fission yeasts and identifying origins in these species will advance the field of replication. My thesis research shows that Cdc25 is not a target of the S-phase DNA damage checkpoint. I showed that DNA damage checkpoint does not target Cdc2-Y15 to slow replication. Based on my preliminary observation, origin firing might be inhibited by the DNA damage checkpoint as a way to slow replication. My efforts to measure the firing efficiency of a late replicating sequence were hindered by potentially unidentified inefficient origins firing at a low rate and replicating the region being studied. Studying the origin efficiency was maybe further complicated by neighboring origins being able to passively replicate the region. To identify origins in recently sequenced Schizosaccharomyces species, we initiated the genome-wide origin mapping. The mapping was also done on S. pombe to identify inefficient origins not mapped by other mapping studies. My work shows that deep sequencing can be used to map origins in other species and provides a powerful tool for origin studies.

High visual sensitivity is a common but important characteristic of animal eyes. It is especially critical for night vision. In animal eyes, photoreceptors are the first to receive the incoming rays of light and they convert the light signals to electrical signals before passing the information to interneurons in the eye and finally to the brain. To function in dim light conditions, photoreceptors have developed high sensitivities to light. It is reported that both mammalian rod photoreceptors and Drosophilaphotoreceptors can detect single photons. The high sensitivities of photoreceptors largely depend on a high content of rhodopsin, a light-stimulated G protein-coupled receptor (GPCR), in light sensory organelles, outer segments in mammals and rhabdomeres in Drosophila. Two shared characteristics, the tightly packed photoreceptive membrane and the high concentration of rhodopsin in the membrane, work together to enable the photoreceptors to achieve the high content of rhodopsin in photosensory organelles in both mammals and Drosophila. In this thesis, I have used the Drosophilaeye as a model system to study the molecular mechanisms required for the maintenance of these two characteristics. In the second chapter, I present a new molecular mechanism of preventing Gq-mediated rhabdomeral degeneration. A new gene named tadr (for torn and diminished rhabdomeres), when mutated, leads to visual sensitivity reduction and photoreceptor degeneration. Degeneration in the tadr mutant is characterized by shrunken and disrupted rhabdomeres. The TADR protein interacts in vitro with the major light receptor Rh1 rhodopsin, and genetic reduction of the Rh1 level suppresses the tadr-induced degeneration, suggesting the degeneration is Rh1-dependent. Nonetheless, removal of phospholipase C (PLC), a key enzyme in phototransduction, and that of Arr2 fail to inhibit rhabdomeral degeneration in the tadr mutant background. Biochemical analyses reveal that, in the tadr mutant, the Gq protein of Rh1 is defective in dissociation from the membrane during light stimulation. Importantly, reduction of Gq level by introducing a hypomorphic allele of Gαq gene greatly inhibits the tadr degeneration phenotype. These results may suggest that loss of a potential TADR-Rh1 interaction leads to an abnormality in the Gqsignaling, which in turn triggers rhabdomeral degeneration independent of the PLC phototransduction cascade. We propose that TADR-like proteins may also protect photoreceptors from degeneration in mammals including humans. In the third chapter, I present a Drosophila CUB- and LDLa-domain transmembrane protein CULD that counteracts the visual arrestin Arr1-mediated endocytosis to retain rhodopsin in rhabdomeral membrane. CULD is mostly localized in rhabdomeres, but is also detected in scarce rhodopsin endocytic vesicles that contain Arr1. An intracellular region of CULD interacts with Arr1 in vitro. In both culdmutant and knockdown flies, a large amount of rhodopsin is mislocalized in the cell body of photoreceptors through lightdependent, Arr1-mediated endocytosis, leading to reduction of photoreceptor sensitivity. Expressing a wild-type CULD protein in photoreceptors, but not a mutant variant lacking the Arr1-interacting site, rescues both the rhodopsin mislocalization and the low sensitivity phenotypes. Once rhodopsin has been internalized in adult mutant flies, it is reversed only by expression of CULD but not by blocking endocytosis, suggesting that CULD promotes recycling of endocytosed rhodopsin to the rhabdomere. Our results demonstrate an important role of CULD in the maintenance of membrane rhodopsin density and photoreceptor sensitivity. We propose that a common cellular function of CUB- and LDLa-domain proteins, in both mammals and invertebrates, is to concentrate receptors including GPCRs in particular regions of cell membrane. In summary, the work addressed in this thesis has identified new molecular mechavii nisms underlying the maintenance of visual sensitivity in Drosophila, either through preventing Gq-mediated rhabdomeral degeneration or through antagonizing arrestin-mediated rhodopsin endocytosis. This work has advanced our understanding of visual biology and the general regulatory mechanisms of GPCR signaling, and may provide valuable clues to pathologic studies of human retinal degeneration disorders.

Innate immunity is the first line of defense against invading pathogens. It functions to eliminate pathogens and also to control infections. The innate immune response is also important for the development of pathogen-specific adaptive immune responses. As a result, the study of innate immune signaling pathways is crucial for understanding the interactions between host and pathogen. Unlike mammals, insects lack a classical adaptive immune response and rely mostly on innate immune responses. Innate immune mechanisms have been widely studied in the fruit fly, Drosophila melanogaster. The genetic and molecular tools available in the Drosophila system make it an excellent model system for studying immunity. Furthermore, the innate immune signaling pathways used by Drosophila show strong homology to those of vertebrates making them ideal for studying these pathways. Drosophila immunity relies on cellular and humoral innate immune responses to fight pathogens. The hallmark of the Drosophilahumoral immune response is the rapid induction of antimicrobial peptide genes in the fat body. The production of these antimicrobial peptides is regulated by two immune signaling pathways-Toll and Immune Deficency (IMD) pathways. The Toll pathway responds to many Gram-positive bacterial and fungal infections , while the IMD pathway is potently activated by DAP-type peptidoglycan (PGN) from Gram-negative bacteria and certain Gram-positive bacteria. Two receptors, PGRP-LC and PGRP-LE, are able to recognize DAP-type PGN at the cell surface or in the cytosol, respectively, and trigger the IMD pathway. Upon binding DAP-type PGN, both PGRP-LC and PGRP-LE dimerize/ multimerize and signal to the downstream components of IMD pathway. It is unclear how the receptor activates its downstream components. My work has focused on understanding the molecular events that take place at the receptors following there activation. In these studies I have identified a common motif in the N-terminal domains of both the receptors, known as the RHIM-like domain. The RHIM-like domain is critical for signaling by either receptor, but the mechanism(s) involved remain unclear. IMD, a downstream component of the pathway, associates with both PGRP-LC and -LE but the interaction of PGRP-LC with IMD is not mediated through its RHIM-like domain. Also, mutations affecting the PGRP-LC RHIM-like motif are defective in all known downstream signaling events. However, the RHIM-like mutant receptors are capable of serving as a platform for the assembly of all known components of a receptor proximal signaling complex. These results suggest that another, unidentified component of the IMD signaling pathway may function to mediate interaction with the RHIM-like motif. I performed a yeast two-hybrid screen to identify proteins that might interact with the receptor PGRP-LC through its RHIM- like domain. With this approach, two new components of the IMD pathway were identified. The first component I characterized is called Rudra and it is a critical feedback inhibitor of peptidoglycan receptor signaling. The other factor is known as RYBP, it includes a highly conserved ubiquitin binding motif (NZF), and RNAi studies suggest it is a critical component of the IMD pathway. The identification and characterization of these two new components of the IMD pathway has provided a new insight into the molecular events that take place proximal to the receptor.

N. meningitidis is a common commensal of the human upper respiratory tract and a leading cause of bacterial meningitis and septicemia worldwide. The classical pathway of complement (C) is essential for both naturally acquired and vaccine induced immunity against N. meningitidis. Qualitative and/or quantitative differences in anti-meningococcal antibodies (Abs) in serum is one reason for variations in C-dependent bactericidal Ab activity among individuals. I showed that IgG isolated from select individuals could block killing of group B meningococci by Abs that were otherwise bactericidal. Ligand overlay immunoblots revealed that these blocking IgG Abs were directed against a meningococcal antigen called H.8, Killing of meningococci in reactions containing bactericidal mAbs and human blocking Abs was restored when blocking Ab binding to meningococci was inhibited (or competed for) using either synthetic peptides corresponding to H.8 or a non-blocking mAb against H.8. Further, genetic deletion of H.8 from target organisms abrogated blocking. The Fc region of the blocking IgG was required for blocking because F(ab)2 fragments alone generated by pepsin treatment were ineffective. Blocking required IgG glycosylation; deglycosylation of blocking IgG with peptide:N-glycanase (PNGase) eliminated blocking. C4 deposition mediated by a bactericidal mAb directed against a meningococcal vaccine candidate, called factor H-binding protein (fHbp), was reduced by blocking Ab. Anti-fHbp-mediated C4 deposition was unaffected, however, by deglycosylated blocking IgG. Although preliminary, our data suggests blocking of serum bactericidal activity by human anti-H.8 blocking antibody may require mannan-binding lectin (MBL), which itself is a complement activator. Also, whether MBL recruits a complement inhibitor(s) that facilitates blocking remains to be determined. In conclusion, we have identified H.8 as a meningococcal target for novel blocking antibodies that are commonly found in human serum. Blocking Ab may reduce the efficacy of meningococcal vaccines. We propose that outer membrane vesicle-containing meningococcal vaccines may be more efficacious if purged of subversive immunogens such as H.8.

Among heat shock proteins, Hsp90 is unusual because it is not required for the proper folding of most cellular proteins but rather is disproportionally linked to the activation of signal transduction proteins including over forty kinases and many steroid hormone receptors. Mutated forms of many Hsp90 clients are causative agents in cancer, making Hsp90 a promising pharmacological target. Many small molecular inhibitors have been identified that competitively bind to the ATP binding site of Hsp90, some of which are in clinical trials as anticancer agents. Although the activation of kinase and hormone receptor clients by Hsp90 and its co-chaperones has been extensively studied, the molecular mechanism of client protein activation is poorly understood. Hsp90 is a dimeric chaperone containing three domains: the N-terminal (N) and middle (M) domains contribute directly to ATP binding and hydrolysis and the C-terminal (C) domain mediates dimerization. At physiological concentration, Hsp90 predominantly forms dimers, but the possibility that full-length monomers might also function in cells has not been tested. In Chapter 3, we used a single-chain strategy to design a full-length Hsp90 monomer (NMCC). The resulting construct was predominantly monomeric at physiological concentration and did not function to support yeast viability as the sole Hsp90. NMCC Hsp90 was also defective at ATP hydrolysis and the activation of kinase and steroid hormone receptor clients in yeast cells. The ability to support yeast growth was rescued by the addition of a coiled-coil dimerization domain, indicating that the parental single-chain construct is functionally defective because it is monomeric. After finding that a full-length Hsp90 monomer containing only one ATPase site was unable to support yeast viability or activate Hsp90 clients, we set out to further explore the role of ATPase activity in client protein activation. Approximately 10 % of the yeast proteome binds to Hsp90 making it important to study Hsp90 function in the cellular environment where all binding partners are present. In Chapter 4, we observed that co-expression of different Hsp90 subunits in Saccharomyces cerevisiae caused unpredictable synthetic growth defects due to cross-dimerization. We engineered super-stabilized Hsp90 dimers that resisted cross-dimerization with endogenous Hsp90 and alleviated the synthetic growth defect. We utilized these super-stabilized dimers to analyze the ability of ATPase mutant homodimers to activate known Hsp90 client proteins in yeast cells. We found that ATP binding and hydrolysis by Hsp90 are both required for the efficient maturation of the glucocorticoid hormone receptor (GR) and v-src confirming the critical role of ATP hydrolysis in the maturation of steroid hormone receptors and kinases in vivo. In addition to its role in the activation of signal transduction client proteins, Hsp90 has been shown to suppress the in vitro aggregation of numerous hard-to-fold proteins. In Chapter 5, we examine the role of charge in Hsp90 anti-aggregation activity. The charge on Hsp90 is largely concentrated in two highly acidic regions. We found that deletion of both charge-rich regions dramatically impaired Hsp90 anti-aggregation activity. Addition of an acid-rich region with a distinct amino acid sequence to our double-deleted Hsp90 construct rescued the anti-aggregation activity of Hsp90 indicating that the net charge contributes to its anti-aggregation activity. The in vitro anti-aggregation activity of Hsp90 studied in Chapter 5 occurs in the absence of ATP. However, all of the biologically important functions of Hsp90 in cells identified to date, including the maturation of kinases and nuclear steroid hormone receptors, clearly require ATP hydrolysis. Why does Hsp90 robustly hinder the aggregation of hard-to-fold proteins without ATP in vitro, but in vivo uses ATP hydrolysis for all of its essential functions? By utilizing separation of function Hsp90 variants (that specifically lack in vitro anti-aggregation activity) we have begun to address this question. We find that anti-aggregation deficient Hsp90 is unable to support yeast growth under stressful conditions, potentially due to reduced cellular expression. Interestingly, the ATP-independent anti-aggregation activity of Hsp90 has no measureable impact on cellular function. Thus, hindering the aggregation of most hard-to- fold proteins by Hsp90 (independent of ATP hydrolysis) does not appear to be important for cell function. These results suggest a cellular model where the Hsp40/60/70 machinery is responsible for hindering the aggregation of most hard-to-fold proteins while Hsp90 assists in the maturation of a select set of clients in an ATP-dependent fashion, potentially aided by its inherent anti-aggregation properties.

The innate immune system is the first line of defense against invading pathogens. Recognition of microbial ligands by the innate immune system relies on germ-line encoded, evolutionarily conserved receptors called pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are one such family of PRRs and are involved in innate defenses to a variety of microbes. At the core of TLR signaling pathways are Toll interleukin-1 receptor (TIR) domain containing adapter proteins. Much of the specificity of TLR pathways arise from the differential use of these adapter proteins. The TLR signaling cascade that ensues upon ligand recognition is marked by finely orchestrated molecular interactions between the receptor and the TIR domain containing adapter proteins, as well as various downstream kinases and effector molecules. Conserving the structural integrity of the TLR components is thus essential for maintaining a robust host defense system. Sometimes, changes in a protein can be brought about by single nucleotide polymorphisms (SNPs). Studies carried out in this thesis focus on polymorphisms in MyD88 adapter-like (Mal) and myeloid differentiation protein 88 (MyD88), two TIR domain-containing adapter proteins, which incidentally are also highly polymorphic. Mal is a 235 amino acid protein that is involved in TLR2 and TLR4 signaling. The known polymorphisms in the coding region of Mal were screened with an aim to identify SNPs with altered signaling potential. A TIR domain polymorphism, D96N, was found to be completely defective in TLR2 and TLR4 signaling. Immortalized macrophage-like cell lines expressing D96N have impaired cytokine production as well as NF-κB activation. The reason for this loss-of-function phenotype is the inability of Mal D96N to bind the downstream adapter MyD88, an event necessary for signaling to occur. Genotyping studies reveal a very low frequency of this polymorphism in the population. Similar SNP analysis was carried out in myeloid differentiation protein 88 (MyD88). MyD88 is a key signaling adapter in TLR signaling; critical for all TLR pathways except TLR3. In reporter assays, a death domain variant, S34Y, was found to be inactive. Importantly, in reconstituted macrophage-like cell lines derived from knockout mice, MyD88 S34Y was severely compromised in its ability to respond to all MyD88-dependent TLR ligands. S34Y mutant has a dramatically different localization pattern as compared to wild type MyD88. Unlike wild type MyD88, S34Y is unable to form distinct foci in the cells but is present diffused in the cytoplasm. IRAK4, a downstream kinase, colocalizes with MyD88 in these aggregates or “Myddosomes”. S34Y MyD88, however, is unable to assemble into Myddosomes, thus demonstrating that proper cellular localization of MyD88 is a feature required for MyD88 function. This thesis thus describes two loss‐of‐function polymorphisms in TLR adapter proteins Mal and MyD88. It sheds light not only on the structural aspects of signaling by these two proteins, but also has implications for the development of novel pharmaceutical agents.

HCV afflicts many millions of people globally, and antiviral therapies are often ineffective and intolerable. The Food and Drug Administration approved the HCV protease inhibitors telaprevir and boceprevir in May 2011, marking an important milestone in anti-HCV research over the past two decades. Nevertheless, severe drug side effects of combination therapy – flu-like symptoms, depression and anemia – limit patient adherence to treatment regimens. The acquisition of resistance challenges the long-term efficacy of antiviral therapies, including protease inhibitors, as suboptimal dosing allows for the selection of drug resistant viral variants. A better understanding of the molecular basis of drug resistance is therefore central to developing future generation protease inhibitors that retain potency against a broader spectrum of HCV strains. To this end, my research characterizes the molecular basis of drug resistance against HCV protease inhibitors. Chapter II defines the mode of substrate recognition by the common volume shared by NS3/4A substrate products – the substrate envelope. Chapter III then correlates patterns of drug resistance to regions where drugs protrude from the substrate envelope. Lastly, Chapter IV elucidates the molecular underpinnings of resistance against four leading protease inhibitors – telaprevir, danoprevir, vaniprevir and MK-5172 – and provides practical approaches to designing novel drugs that are less susceptible to resistance. I ultimately hope my work appeals to the broader biomedical community of virologists, medicinal chemists and clinicians, who struggle to understand HCV and other human pathogens in the face of rapid disease evolution.