The immune system is a complex network of interacting cells and tissues that is designed to protect the body from pathogens and other foreign substances. T cells are a major component of the immune system and consist of two distinct lineages distinguished by the expression of αβ or γδ T cell receptors (TCR). The Tec family kinase, Itk is an important mediator of signaling downstream of the TCR. Past studies on Itk has focused on how Itk regulates development, activation and differentiation of conventional αβ T cells and more recently how Itk regulates the development of innate-like αβ T cells. However, very little is known about the influence of Itk on γδ T cells. My studies show a previously unknown role for Itk in the development and function of γδ T cells. We report in the absence of Itk, γδ T cells were responsible for the spontaneously elevated levels of serum IgE and Itk-/- mice γδ T cells produced high levels of TH2 cytokines. Furthermore, there was an increase in γδ T cells specifically in the Vγ1.1+Vδ6.3+ (V6) subset that represents the dominant population of γδ NKT cells in Itk-/- mice. In addition, the V6 subset had increased expression of PLZF, a transcription factor normally required for αβ iNKT cell development. We further show that V6 cells develop and mature similar to αβ iNKT cells. Similar to defects previously seen in the terminal differentiation of Itk-/- αβ iNKT cell, V6 cells also had impaired maturation in the thymus in the absence of Itk. This data demonstrates a previously unknown role of Itk for the terminal maturation of V6 cells that has been shown to be the cell population that led to spontaneous dermatitis in mice. Given that drug companies have targeted Itk as a potential allergy drug due to Itk’s role in TH2 development and function, our data suggests that further studies on Itk are warranted.

Dengue viruses (DV) are re-emerging mosquito-borne pathogens for which four distinct lineages, grouped based on serology and referred to as serotypes 1-4 (DIV-D4V), have been described. Epidemiological data imply that re-infection with a "heterologous" serotype, i.e, one other than that to which the individual was originally exposed, enhances the risk for development of severe disease, dengue hemorrhagic fever (DHF). The hallmark of DHF is a transient capillary leakage syndrome of rapid onset, temporally associated with the resolution of fever and viremia. In its most grave form, the vascular permeability phenomenon in DHF may progress to dengue shock syndrome (DSS), which is often fatal in the absence of appropriate medical care. Despite the fulminant nature of vascular leakage during DHF/DSS, this phenomenon does not appear to be due to direct cytopathic effects of DV. Rather, inappropriate reactivation and/or regulation of dengue-specific memory are the prevailing theorized (immunopathological) etiologies. Traditional vaccine development techniques have proven insufficient for DV, since any vaccine must offer complete protection against all four serotypes to avoid enhanced pathology on natural viral challenge. Understanding the underlying mechanisms that contribute to dengue disease, particularly the development of dengue-specific memory, is therefore of critical importance. Dengue immunopathology and the specific aspects of immunological memory that determine disease severity are heatedly debated. Previous research in our lab has suggested that T cell responses contribute to the severity of dengue illness. Clinical data indicate enhanced immune activation in more grave cases of DV infection, and serotype cross-reactive T cells from multiple individuals are present after both primary and secondary dengue infections. However, little is known about the conditions under which T cells are primed and dengue-specific memory is generated. Dendritic cells (DCs) are bone marrow-derived cells that play a central role in directing activity within the immune system. DCs shape quantitative and qualitative aspects of adaptive immunity, and therefore the intrinsic characteristics of host memory to a pathogen. DCs are essential in generating primary immune responses, due to their particular effectiveness in stimulating naïve T cells. DCs also play important roles in the reactivation of memory to an infectious agent, and as reservoirs for the dissemination of invading microorganisms. Exposure to pathogens or their products initiates a series of phenotypic and functional changes in DCs, termed maturation. DC maturation involves a coordinated response of immunomodulatory surface molecule elaboration and cytokine production, culminating in antigen presentation to, and co-stimulation of, T cells specific for the invading agent. The DC response is ostensibly tailored to facilitate effective elimination by regulating effective downstream interactions of the DC with T cells. A number of viruses have evolved to infect DCs and alter their functional behavior, facilitating their own survival within the host, and the herd. DV readily infects DCs both in primary cell cultures and in vivo. However, reports on the effects of DV infection on DC maturation vary both with regard to some of the cytokines produced, and the phenotypes of infected versus bystander cells. Although DCs appear to be activated following DV exposure, responses on the single-cell level appear to depend on the infection state of the cell, hypothetically driven by intracellular virus-mediated effects. Therefore, downstream responses to these divergent populations - i.e., actively infected cells versus uninfected bystander cells - are likely to be the consequence of at least two modes of DC behavior. Because DCs play a pivotal role in adaptive immune development, and because the resulting memory response appears to be critical in affecting disease pathology after heterologous DV re-infection, I sought to explore the phenomena of DC maturation in response to dengue exposure, and to begin to answer the question of how active infection alters the functional capabilities of DCs. Notably, primary dengue infection is generally well-controlled with minimal pathology. Therefore, this thesis addresses the hypothesis that DV infection of DCs results in cellular activation and stimulation of antiviral immunity, despite virus-mediated alteration of DC maturation. In order to address this hypothesis, I examined both DV infection-dependent and independent effects on DC functional responses including surface molecule regulation secretory activity, and CD4 T cell allostimulatory priming. DCs derived from human peripheral blood monocytes were readily infected with multiple strains of DV. DV infection of DCs derived from separate donors was dose-dependent, with substantial variability in DC susceptibility to infection. Exposure to live DV activated surface molecule expression in DCs, similar to the effects of defined maturation stimuli including a combination of TNF-α and IFN-α, or LPS. In addition, UV-inactivated DV induced expression of cell surface molecules, albeit to a lesser extent than did live virus demonstrating inherent stimulatory properties of DV particles. Using intracellular staining for DV envelope (E) protein, I detected increased surface molecule expression on both infected DCs and uninfected bystander DCs from the same culture, as compared to mock-infected DCs. These data indicate that activation was not prevented in cells undergoing active viral replication. However, the degree of surface molecule induction depended on the infection state of the cell. Infected DCs had enhanced PD-L2 and MHC II expression relative to uninfected bystander cells, while PD-L1, CD80, CD86, and MHC I expression were suppressed with active infection. Therefore, intracellular DV replication altered the process of cell surface molecule regulation within these cells. DV infection of DCs also resulted in the secretion of a broad array of cytokines and chernokines. These included the antiviral cytokine IFN-α, inflammatory cytokines TNF-α, IL-6, and IL-1α, and inflammatory chemokines IP10, MCP-1, MIP-1α, and RANTES. DV infection did not induce DC production of the IL-12 p70 heterodimer, and secretion of the immunosuppressive cytokine IL-10 was low in most experiments. Similar to the results seen with surface molecule induction, UV inactivation of DV reduced, but did not eliminate, cytokine and chemokine responses. At the single-cell level, TNF-α and IP10 production profiles of infected DCs and uninfected bystander DCs were distinct. DV infection in DCs reduced production of IP10, but stimulated TNF-α as compared to uninfected bystander cells in the same culture. Blocking experiments demonstrated that IFN-α/β produced by DCs in response to infection actively inhibited viral protein expression and drove IP10, but not TNF-α, production. DV infection of DCs did not consistently suppress DC stimulation of allogeneic CD4 T cell proliferation. In cases where infection enhanced DC stimulatory function, T cell proliferation was less pronounced than that induced by DCs activated with exogenous TNF-α plus IFN-α. Increasing multiplicity of infection (MOI) of DCs with DV resulted in increasing DC infection rates, but a statistically significant trend at the highest MOIs for decreased T cell alloproliferation, suggesting that direct infection of DCs reduces their CD4 T cell priming function. MOI-dependent reduction in DC stimulatory function depended on replication-competent virus. Increased MOIs during DV infection of DCs did not cause an elevation in detectable IL-10 in supernatants derived from T-DC co-cultures. In addition, increased DV MOI of DCs was not associated with increased levels of either IL-13 or IFN-γ in supernatants from T-DC co-culture, suggesting that actively infected DC do not skew CD4 T cells towards a specific Th phenotype. These data demonstrate that DV infection induces functional maturation of DCs that is modified by the presence of virus through both IFN-dependent and independent mechanisms. However, the allostimulatory phenotype of DCs was not universally enhanced, nor was it skewed towards antiviral (Th1)-type responses. These data suggest a model whereby dengue infection during primary illness results in controlled immune stimulation through activation of bystander DCs, and the generation of mixed Th-type responses. Direct DV infection of DCs appears to attenuate activation of, and potentially clearance by, antiviral mechanisms. During secondary infection, reduced IP10 production and enhanced TNF-α secretion by infected cells coupled with MHC I downregulation and enhanced PD-L2 expression, would subvert both Th1 CD4 T cell recruitment and result in CD8 T cell suppression and death. Furthermore, DV-specific effects on DCs would allow for continued viral replication in the absence of effective clearance. These DV-mediated effects would modify T cell memory responses to infected DC, and potentially facilitate the expansion of pathologic T cell subsets. Contributing to this pathological cascade, antibody-dependent enhancement of infection in monocytic cells and macrophages would shift antigen presentation and cytokine production paradigms, increasing the risk of DHF.

Carrier mediated nutrient import is vital for cell and tissue homeostasis. Structural insights of carrier mediated transport, particularly the human glucose transporter GLUT1, are essential for understanding the mechanisms of human metabolic disease, and provide model systems for cellular processes as a whole. GLUT1 function and expression is characterized by a complexity unexplained by the current hypotheses for carrier-mediated sugar transport (9). It is possible that the operational properties of GLUT1 are determined by host cell environment. A glucose transport-null strain of Saccharomyces cerevisiae(RE700A) was transfected with the p426 GPD yeast expression vector containing DNA encoding the wild-type human glucose transport protein (GLUT1) to characterize its functional properties. Identical protein sequences generated different kinetic parameters when expressed in RE700A yeast, erythrocytes, and HEK293 cells. These findings support the hypothesis that red cell sugar transport complexity is host cell-specific. Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 sugar export site. Paradoxically, very low concentrations of these inhibitors produce a modest stimulation of sugar transport (16). This result is consistent with the hypothesis that the glucose transporter contains multiple, interacting, intracellular binding sites for e1 ligands CB and FSK. The present study tests this hypothesis directly and, by screening a library of cytochalasin and forskolin analogs, asks what structural features of exit site ligands determine binding site affinity and cooperativity. Our findings are explained by a carrier that presents at least two interacting endofacial binding sites for CB or FSK. We discuss this result within the context of GLUT1 quaternary structure and evaluate the major determinants of ligand binding affinity and cooperativity. Cytochalasin B (CB) inhibits GLUT1 substrate transport at or near the endofacial sugar binding site. N-bromosuccinamide analysis combined with 3H-CB photolabeling implicates the region between Trp388 and Trp412 in ligand binding. Although its structure has been modeled(5), the specific residues comprising the sugar binding site are unknown. A series of alanine point mutants were made, and mutant protein 2-deoxy glucose transport was tested in the presence of increasing [CB]. Arg126Ala and Cys421Ala GLUT1 mutations altered CB affinity but were determined not to be in the e1 site. The Arg400Ala mutation decreased binding affinity for CB, and may comprise part of the e1 binding site. Because point mutations were individually insufficient to abrogate CB binding, Trp388 to Trp412 chimeras were made. GLUT1/GLUT4388-412/GLUT1 and GLUT1/GLUT5388-412/GLUT1 chimeras showed moderately less sensitivity to CB inhibition of transport; these amino acids likely comprise regions determinant of CB binding affinity. Furthermore GLUT1/GLUT5388-412/GLUT1 shows enhancement of 2-DG uptake at 50nM CB, but an overall dose response indistinguishable from WT GLUT1. A multisite fit of the data suggested GLUT1/GLUT5388-412/GLUT1 chimera possesses strong first site affinity for CB but slight negative second-site cooperativity. We conclude that point mutants were insufficient to abrogate CB binding and that the Trp388 to Trp412 sequence is necessary for CB binding affinity but is not the sole determinant of inhibition of 2 deoxyglucose uptake by CB. We discuss these results with their implications for structure-function sequence localization of the CB binding site, and by extension, the e1 sugar binding site.

From the yeast genome completed in 1996 to the 12 Drosophilagenomes published earlier this year; little more than a decade has provided an incredible amount of genomic data. Yet even with this mountain of genetic information the regulatory networks that control gene expression remain relatively undefined. In part, this is due to the enormous amount of non-coding DNA, over 98% of the human genome, which needs to be made sense of. It is also due to the large number of transcription factors, potentially 2,000 such factors in the human genome, which may contribute to any given network directly or indirectly. Certainly, one of the central limitations has been the paucity of transcription factor (TF) specificity data that would aid in the prediction of regulatory targets throughout a genome. The general lack of specificity data has hindered the prediction of regulatory targets for individual TFs as well as groups of factors that function within a common regulatory pathway. A large collection of factor specificities would allow for the combinatorial prediction of regulatory targets that considers all factors actively expressed in a given cell, under a given condition. Herein we describe substantial improvements to a previous bacterial one-hybrid system with increased sensitivity and dynamic range that make it amenable for the high-throughput analysis of sequence-specific TFs. Currently we have characterized 108 (14.3%) of the predicted TFs in Drosophilathat fall into a broad range of DNA-binding domain families, demonstrating the feasibility of characterizing a large number of TFs using this technology. To fully exploit our large database of binding specificities, we have created a GBrowse-based search tool that allows an end-user to examine the overrepresentation of binding sites for any number of individual factors as well as combinations of these factors in up to six Drosophila genomes (veda.cs.uiuc.edu/cgi-bin/gbrowse/gbrowse/Dmel4). We have used this tool to demonstrate that a collection of factor specificities within a common pathway will successfully predict previously validated cis-regulatory modules within a genome. Furthermore, within our database we provide a complete catalog of DNA-binding specificities for all 84 homeodomains in Drosophila. This catalog enabled us to propose and test a detailed set of recognition rules for homeodomains and use this information to predict the specificities of the majority of homeodomains in the human genome.

Studies have demonstrated that under conditions of chronic metabolic stress, GLUT1-mediated sugar transport is upregulated at the blood-brain barrier by a number of mechanisms. Although acute metabolic stress has also been shown to increase GLUT1-mediated transport, the mechanisms underlying this regulation remain unclear. This work attempts to explain how GLUT1-mediated sugar uptake is increased during acute metabolic stress, as well as explore the factors involved in this modulation of sugar transport in blood-brain barrier endothelial cells. Glucose depletion, KCN and FCCP were applied to brain microvascular endothelial cell line bEnd.3 in order to induce acute metabolic stress by ATP depletion. Kinetic sugar uptake measurements in combination with qPCR, whole cell lysate western blots, and cell-surface biotinylation were employed to probe for changes in GLUT1-mediated sugar uptake, GLUT1 expression levels, and GLUT1 localization during metabolic stress. Finally, the role of AMP-activated kinase (AMPK) in the bEnd.3 cell response to acute stress was examined using the specific AMPK activator AICAR and inhibitor Compound C. The data presented in this thesis supports the following two conclusions: 1. GLUT1-mediated sugar transport in bEnd.3 cells during acute metabolic stress is increased 3-7 fold due to translocation of intracellular GLUT1 to the plasma membrane, with no change in expression of total GLUT1 protein, and 2. AMPK plays a direct role in modulating increases in GLUT1-mediated sugar transport in bEnd.3 cells during acute metabolic stress by regulating trafficking of GLUT1 to the plasma membrane.

In the Drosophila germ line, PIWI-interacting RNAs (piRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. We examined the genetic requirements for the biogenesis and function of piRNAs in both female and male germ line. We found that piRNAs function through the PIWI, rather than the AGO, family Argonaute proteins, and the production of piRNAs requires neither microRNA (miRNA) nor small interfering RNA (siRNA) pathway machinery. These findings allowed the discovery of the third conserved small RNA silencing pathway, which is distinct from both the miRNA and RNAi pathways in its mechanisms of biogenesis and function. We also found piRNAs in flies are modified. We determined that the chemical structure of the 3´-terminal modification is a 2´-O-methyl group, and also demonstrated that the same modification occurs on the 3´ termini of siRNAs in flies. Furthermore, we identified the RNA methyltransferase Drosophila Hen1, which catalyzes 2´-O-methylation on both siRNAs and piRNAs. Our data suggest that 2´-O-methylation by Hen1 is the final step of biogenesis of both the siRNA pathway and piRNA pathway. Studies from the Hannon Lab and the Siomi Lab suggest a ping-pong amplification loop for piRNA biogenesis and function in the Drosophila germline. In this model, an antisense piRNA, bound to Aubergine or Piwi, triggers production of a sense piRNA bound to the PIWI protein Argonaute3 (Ago3). In turn, the new piRNA is envisioned to produce a second antisense piRNA. We isolated the loss-of-function mutations in ago3, allowing a direct genetic test of this model. We found that Ago3 acts to amplify piRNA pools and to enforce on them an antisense bias, increasing the number of piRNAs that can act to silence transposons. Moreover, we also discovered a second Ago3-independent piRNA pathway in somatic ovarian follicle cells, suggesting a role for piRNAs beyond the germ line.

The function of transforming growth factor β (TGF-β) in cancer is notoriously complex. Initially TGF-β limits tumorigenesis, but at later stages in tumor progression TGF-β promotes the malignant spread of tumor cells. Past studies to understand the pro-metastasis utility of TGF-β centered upon its ability to regulate protein-coding genes. Recently, a small class of non-coding RNAs known as microRNAs (miRNAs) emerged as novel posttranscriptional regulators of gene expression. The significance of miRNA function in cellular processes from embryonic development to the maintenance of homeostasis in adult tissues is becoming increasingly clear. Also apparent is the strong association between aberrant miRNA expression and human diseases, such as cancer. The contribution of miRNAs to TGF-β-mediated cellular responses remains an open question. Thus, I became interested if miRNAs offered an additional layer of regulation in TGF-β signaling through which this cytokine exerts its pro-metastasis function. To address this inquiry, in the first part of this dissertation I investigated whether miRNAs influenced the ability of TGF-β to induce cellular responses directly involved with carcinoma metastasis, such as epithelial-mesenchymal transition (EMT). Here, I identified two miRNAs, miR-21 and miR-31, that are upregulated during EMT in LIM 1863 organoids, a colon carcinoma model of EMT driven by TGF-β. We performed in vitro studies to characterize the function of miR-21 and miR-31 and found that these two miRNAs positively impact the induction of EMT, migration and invasion by TGF-β. Furthermore, we uncovered TIAM1 (T lymphoma and metastasis gene 1) as a novel target of both miR-21 and miR-31 and show that downregulation of TIAM1 is critical for the pro-migration and pro-invasion activities of miR-21 and miR-31. Together these findings reveal miR-21 and miR-31 as downstream effectors of TGF-β signaling by facilitating EMT, migration and invasion of colon carcinoma cells. How TGF-β regulates miR-21 and miR-31 became important questions and thus the focus of the second part of this thesis. Interestingly, I found that TGF-β and TNF-α synergize to increase miR-21 and miR-31 levels in LIM 1863 organoids and that the synthesis of new factors induced by TGF-β/TNF-α are required for this upregulation. Moreover, I report that regulation of miR-21 by TGF-β/TNF-α occurs at multiple levels of biogenesis. More specifically data provided here show that Smad4 binds to the promoter of miR-21 to upregulate its expression thereby specifying miR-21 as a typical TGF-β target gene. This mechanism is different from one recently observed in smooth muscle cells in which TGF-β did not stimulate miR-21 transcription, but interestingly, Smad4 enhanced the Drosha-mediated processing of the miR-21 precursor. These two mechanisms suggest that TGF-β regulation of miR-21 is contextual and highlight the complexity of TGF-β signaling. As a whole, my findings establish important roles for miR-21 and miR-31 in TGF-β-mediated cellular responses that facilitate the pro-metastasis utility of TGF-β in colon cancer. Also, I describe a novel mechanism by which TGF-β/TNF-α signaling elevates the level of miR-21 and miR-31. Future studies that identify additional targets of miR-21 and miR-31 may offer further insight into the molecular mechanisms underlying cellular regulation by TGF-β. This information will be vital for the design of therapeutic interventions for colon cancer patients.

Glioma therapy relies on induction of cytotoxicity; however, the current combination of surgery, irradiation (IR) and temozolomide (TMZ) treatment does not result in a long-term cure. Our lab previously demonstrated that a small population of glioma cells enters a transient cell cycle arrest in response to chemotherapy. Treatment with TMZ significantly decreases initial neurosphere formation; however, after a short recovery period, a small number of cells resume neurosphere formation and repopulate the culture. This recovery of neurosphere growth recapitulates the inevitable glioma recurrence in the clinic. The focus of our laboratory is to study direct-target therapies that can be combined with TMZ to inhibit neurosphere recovery. The Notch pathway is a promising target because it is involved in cell growth and survival. Here, we demonstrate that blocking the Notch pathway using gamma-secretase inhibitors (GSIs) enhances TMZ treatment. The combination of TMZ and GSI treatments targets the cells capable of recovery. TMZ + GSI treated cells do not recover and are no longer capable of self-renewal. Interestingly, recovery is inhibited when the GSI is administered 24 hrs after TMZ treatment, demonstrating a sequence-dependent mechanism. TMZ + GSI treatment also decreases tumorigenicity. When glioma cell lines were treated in vitro and implanted in NU/NU nude mice, TMZ + GSI treatment extended latency and greatly increased survival. In addition, in vivo TMZ + GSI treatment completely blocked tumor progression and resulted in the loss of a palpable tumor in 50% of mice, while none of the TMZ-only treated mice survived. TMZ + GSI treated cultures and xenografts display a senescent phenotype. Cultures treated with TMZ + GSI have decreased proliferation, but no increase in cell death. We observed an increase in the number of cells expressing senescence-associated β-galactosidase in vitro and in vivo. This demonstrates that inhibition of the Notch pathway shifts TMZ-treated cells from a transient cell cycle arrest into a permanent senescent state. Senescent cells can stimulate the innate immune system. Here we demonstrate that TMZ + GSI treatment increases phagocytosis in vitro. New therapy combinations, such as TMZ + GSI, are arising in the field of therapy-induced senescence (TIS). Overall, this data demonstrates the importance of the Notch pathway in chemoprotection and maintenance of TMZ-treated gliomas. The addition of GSIs to current treatments is a promising target-directed therapy to decrease the rate of brain tumor recurrence by inducing senescence and tumor clearance.

Molecular recognition, defined as the specific interactions between two or more molecules, is at the center of many biological processes including catalysis, signal transduction, gene regulation and allostery. Allosteric regulation is the modification of function caused by an intermolecular interaction. Allosteric proteins modify their activity in response to a biological signal that is often transmitted through the interaction with a small effector molecule. Therefore, determination of the origins of intermolecular interactions involved in molecular recognition and allostery are essential for understanding biological processes. Classically, molecular recognition and allosteric regulation have been associated to structural changes of the system. NMR spectroscopic methods have indicated that changes in protein dynamics may also contribute to molecular recognition and allostery. This thesis is an investigation of the contributions of both structure and dynamics in molecular binding phenomena. In chapter I, I describe molecular recognition, allostery and examples of allostery and cooperativity. Then I discuss the contribution of protein dynamics to function with a special focus on allosteric regulation. Lastly I introduce the hemoglobin homodimer, HbI of Scapharca inaequivalvis and the mRNA binding protein TIS11d. Chapter II is the primary focus of this thesis on the contribution of protein dynamics to allostery in the dimeric hemoglobin of scapharca inaequivalvis, HbI. Thereafter I concentrate on the mechanism of adenine recognition of the Tristetraprolin-like (TTP) protein TIS11d; this study is detailed in Chapter III. In Chapter IV I discuss broader impacts and future directions of my research. This thesis presents an example of the use of protein NMR spectroscopy to probe ligand binding. The studies presented in this thesis emphasize the importance of dynamics in understanding protein function. Measurements of protein motions will be an element of future studies to understand protein function in health and disease.

Two decades after the discovery of the Hepatitis C Virus (HCV), Hepatitis C infection still persists to be a global health problem. With the recent approval of the first set of directly acting antivirals (DAAs), the rate of sustained viral response for HCV-infected patients increased significantly. However, a complete cure has not been found yet. Drug development efforts primarily target NS3/4A protease, bifunctional serine protease-RNA helicase of HCV. HCV NS3/4A is critical in viral function; protease domain processes the viral polyprotein and helicase domain aids replication of HCV genome by unwinding double stranded RNA transcripts produced by NS5B, RNA-dependent RNA polymerase of HCV. Protease and helicase domains can be isolated, expressed and purified separately while retaining function. Isolated domains of HCV NS3/4A have been extensively used in biochemical and biophysical studies for scientific and therapeutic purposes to evaluate functional capability and mechanism. However, these domains are highly interdependent and modulate the activities of each other bidirectionally. Interdomain dependence was demonstrated in comparative studies where activities of isolated domains versus the full length protein were evaluated. Nevertheless, specific factors affecting interdependence have not been thoroughly studied. Chapter II investigates the domain-domain interface formed between protease and helicase domains as a determinant in interdependence. Molecular dynamics simulations performed on single chain NS3/4A constructs demonstrated the importance of interface in the coupled dynamics of the two domains. The role of the interface in interdomain communication was experimentally probed by disrupting the domain-domain interface through Ala-scanning mutations in selected residues in the interface with significant buried surface areas. These interface mutants were assayed for both helicase and protease related activities. Instead of downregulating the activities of either domain, interface mutants caused enhancement of protease and helicase activities. In addition, the interface had minimal effect in RNA unwinding activity of the helicase domain, the mere presence of the protease domain was the main protagonist in elevated RNA unwinding activity. In conclusion, I suspect that the interface formed between the domains is transient in nature and plays a regulatory role more than a functional role. In addition, I found results supporting the suggestion that an alternate domain-domain arrangement other than what is observed in crystal structures is the active, biologically relevant conformation for both the helicase and the protease. Chapter III investigates structural features of HCV NS3/4A protease inhibitors in relation to effects on inhibitor potency, susceptibility to drug resistance and modulation of potency by the helicase domain. Nearly all NS3/4A protease inhibitors share common features, with major differences only in bulky P2 extension groups and macrocyclization statuses. Enzymatic inhibition profiles of different drugs were analyzed for wildtype isolated protease domain and single chain NS3/4A helicase-protease construct, their multi drug resistant variants, and additional helicase mutants. Inhibitor potency was mainly influenced by macrocyclization, where macrocyclic drugs were significantly more potent compared to acyclic variants. Potency loss with respect to resistance mutations primarily depended on the P2 extension, while macrocyclization had minimal effect except for P2-P4 macrocyclic compounds which were up to an order of magnitude more susceptible to mutations A156T and, in lesser extent, D168A. Modulation by helicase domain was also dependent on P2 extension, although opposite trends were observed for danoprevir analogs versus others. In conclusion, this study provides a basis for future inhibitor development in both avoiding drug resistance and exploitation of the helicase domain for additional efficacy. In this thesis, I have provided evidence further supporting and revealing the details of domain-domain dependency in HCV NS3/4A. Lessons learned here will aid future research for dissecting the interdependency to gain a better understanding of HCV NS3/4A function, which can possibly be extended to all Flaviviridae NS3 protease-helicase complexes. In addition, interdomain dependence can be exploited in future drug development efforts to create better drugs that will pave the way to an effective cure.