Discovery of new antifungal chemo-therapeutics for humans is limited by the large degree of conservation among eukaryotic organisms. In recent years, the histone acetyl-transferase Rtt109 was identified as the sole enzyme responsible for an abundant and important histone modification, histone H3 lysine 56 (H3K56) acetylation. In the absence of Rtt109, the lack of acetylated H3K56 renders yeast cells extremely sensitive to genotoxic agents. Consequently, the ability to sustain genotoxic stress from the host immune system is crucial for pathogens to perpetuate an infection. Because Rtt109 is conserved only within the fungal kingdom, I reasoned that Rtt109 could be a novel drug target. My dissertation first establishes that genome stability provided by Rtt109 and H3K56 acetylation is required for Candida albicans pathogenesis. I demonstrate that mice infected with rtt109 -/- cells experience a significant reduction in organ pathology and mortality rate. I hypothesized that the avirulent phenotype of rtt109 -/- cells is due to their intrinsic hypersensitivity to the genotoxic effects of reactive oxygen species (ROS), which are utilized by phagocytic cells of the immune system to kill pathogens. Indeed, C. albicans rtt109 -/- cells are more efficiently killed by macrophages in vitro than are wild-type cells. However, inhibition of ROS generation in macrophages renders rtt109 -/- and wild-type yeast cells equally resilient to killing. These findings support the concept that ability to resist genotoxic stress conferred by Rtt109 and H3K56 acetylation is a virulence factor for fungal pathogens and establish Rtt109 as an opportune drug- target for novel antifungal therapeutics. Second, I report the discovery of a specific chemical inhibitor of Rtt109 catalysis as the initial step in the development of a novel antifungal agent. We established a collaboration with the Broad Institute (Cambridge, MA) to perform a high-throughput screen of 300,000 compounds. From these, I identified a single chemical, termed KB7, which specifically inhibits Rtt109 catalysis, with no effect on other HAT enzymes tested. KB7 has an IC50 value of approximately 60 nM and displays noncompetitive inhibition regarding both acetyl-coenzyme A and histone substrates. With the genotoxic agent camptothecin, KB7 causes a synergistic decrease in C. albicans growth rate. However, this effect is only observed in an efflux-pump mutant, suggesting that this compound would be more effective if it were better retained intracellularly. Further studies through structure-activity relationship (SAR) modifications will be conducted on KB7 to improve its effective cellular concentration.

In this dissertation, the roles of endocytosis and phagocytosis pathways in a variety of clinically relevant scenarios were examined. These scenarios include antibody-mediated internalization of cell surface proteins, titanium wear-particle uptake in failed joint replacements, and polymeric microparticle uptake and immune responses for drug delivery or adjuvant use. The use of antibodies specific for cell surface proteins has become a popular method to deliver therapeutics to target cells. As such, it is imperative to fully understand the ability of antibodies to mediate internalization and endosomal trafficking of the surface protein that it recognizes, so that drug delivery can be optimized. By comparing the internalization and endosomal localization of two different antibody-bound proteins, the transferrin receptor (TfR) and rabies G, we have found that there is a specific antibody-mediated internalization pathway that occurs when an antibody binds to a cell surface protein. Interestingly, the internalization pathway induced by antibody binding is different than that seen with recycling receptor internalization after ligand binding. This may have broad implications for the future development of antibody-based therapeutics. Joint replacement failure is a major clinical problem. Studies have indicated that a large amount of metal and polyethylene wear debris is found in the synovial membrane and tissue surrounding failed replacements. Through examination of the immune response following uptake of titanium particles, our results suggest that titanium wear-particle induced inflammation and subsequent joint replacement failure may be due to activation of the NLRP3 inflammasome, leading to increased IL-1ß secretion and IL-1 associated signaling. These findings introduce IL-1 as a target for potential therapeutics for patients exhibiting significant inflammation. Polymeric microparticles have been widely used in a variety of therapeutic applications, including drug delivery and vaccine adjuvants. It is essential to understand the ability of such particles to either activate or inhibit an immune response following uptake. Through comparison of particles with varying surface morphology, we have determined that particles with regions of high surface curvature (budding) are more immunogenic than particles with low surface curvature (spherical). Budding particles were more rapidly phagocytosed and induced higher levels of the inflammasome-associated cytokine, IL-1ß, when exposed to mouse macrophages. Additionally, budding particles induced a more rapid neutrophil response in vivo, when compared to spherical particles. These findings have broad implications for the development of future targeting vehicles for delivery of vaccines, drugs, proteins, and siRNA therapeutics.

Human Immunodeficiency Virus Type 1 (HIV-1) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS), currently the leading cause of death from infectious diseases. Since HIV-1 co-opts the host cellular machinery, the study of cellular factors involved is a rational approach in discovering novel therapeutic targets for AIDS drug development. In this thesis, we present studies on two such proteins. APOBEC3G is from the family of cytidine deaminases known to keep endogenous retroviruses and retrotransposons at bay to maintain stability of the human genome. APOBEC3G targets Vif-deficient HIV-1 particles and renders them noninfectious, partially through deaminase-dependent hypermutation of the provirus during reverse transcription. APOBEC3G largely localizes in mRNA processing (P) bodies, cytoplasmic structures involved in RNA metabolism. Here we explore the significance of APOBEC3G localization in P bodies. We found that disrupting P bodies does not affect virion incorporation of endogenous APOBEC3G, implying that the APOBEC3G fraction in P bodies is not directly involved in the production of nascent, non-infectious particles. We also study UPF1, another host protein encapsidated by HIV-1. It is an essential protein mainly studied for its role in nonsense-mediated decay (NMD) pathway and belongs to the same helicase superfamily as MOV10, a recently identified antiviral factor. We found that UPF1 is incorporated in HIV-1 virions in a nucleocapsid-dependent manner and is required for single-cycle infectivity at an early, post-entry step of the viral life cycle. This novel function of UPF1 most likely does not involve NMD since depletion of UPF2 does not affect viral infectivity.

Nursing homes are an essential yet understudied provider of cancer-related care for those with complex health needs. Nine percent of nursing home residents have a cancer diagnosis at admission, and it is estimated that one-third of them experience pain on a daily basis. Although pain management is an essential component of disease treatment, few studies have evaluated analgesic medication use among adults with cancer in this setting. Use of opioids, which are the mainstay of pain management in older adults because of their effectiveness in controlling moderate to severe pain, may be significantly related to coverage by the Medicare Part D prescription drug benefit. However, little is known about Medicare Part D’s effects on opioid use in this patient population. A limited body of evidence also suggests that despite known risks of overdose and respiratory depression in opioid-naïve patients treated with long-acting opioids, use of these agents may be common in nursing homes. This dissertation examined access to appropriate and effective pain-related health care services among US nursing home residents, with a special focus on those with cancer. Objectives of this dissertation were to: 1) estimate the prevalence, and identify resident-level correlates, of pain and receipt of analgesic medications; 2) use a quasi-experimental research design to examine the relationship between implementation of Medicare Part D and changes in the use of fentanyl patches and other opioids; and 3) to estimate the prevalence, and identify resident-level correlates, of naïve initiation of long-acting opioids. Data on residents’ health status from the Resident Assessment Instrument/Minimum Data Set (versions 2.0 and 3.0) were linked with prescription drug transaction data from a nationwide long-term care pharmacy (January 2005–June 2007) and the Centers for Medicare and Medicaid Services (January–December 2011). From 2006 to 2007, more than 65% of residents of nursing homes throughout the US with cancer experienced pain (28.3% on a daily basis), among whom 13.5% reported severe pain. More than 17% of these residents who experienced daily pain received no analgesics (95% confidence interval [CI]: 16.0–19.1%), and treatment was negatively associated among those with advanced age, cognitive impairment, feeding tubes, and restraints. These findings coincided with changing patterns in opioid use among residents with cancer, including relatively abrupt 10% and 21% decreases in use of fentanyl patches and other strong opioids, respectively, after the 2006 implementation of Medicare Part D. In the years since Medicare Part D was introduced, some treatment practices in nursing homes have not been concordant with clinical guidelines for pain management among older adults. Among a contemporary population of long-stay nursing home residents with and without cancer, 10.0% (95% CI: 9.4–10.6%) of those who began receiving a long-acting opioid after nursing home admission had not previously received opioid therapy. Odds of naïve initiation of these potent opioids were increased among residents with terminal prognosis, functional impairment, feeding tubes, and cancer. This dissertation provides new evidence on pharmaceutical management of pain and on Medicare Part D’s impact on opioid use in nursing home residents. Results from this dissertation shed light on nursing home residents’ access to pain-related health care services and provide initial directions for targeted efforts to improve the quality of pain treatment in nursing homes.

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.

Background: Up to 55% of patients administered ketamine, experience an emergence phenomena (EP) that closely mimics schizophrenia and increases their risk of injury. While genetics accounts for about 50% of severe adverse drug reactions, no studies have investigated genetic association of ketamine-induced EP in healthy patients. Ketamine is metabolized by CYP 2B6 enzymes and CYP 2B^8^ allele significantly alter ketamine metabolism. In addition, ketamine exerts most of its effects by inhibiting the N-methyl-D-aspartate receptor (NMADR), and NMDAR genes (GRIN2B) are associated with learning and memory impairment and schizophrenia. Purpose: To investigate the relationship between CYP2B6*6 and GRIN2B single nucleotide polymorphisms (SNPs) and ketamine-induced emergence phenomena (EP). Methods: This cross-sectional pharmacogenetic study recruited 75 patients having minor orthopedic, hand, foot, anorectal surgeries from two outpatient surgical centers. EP was measured with the Clinician Administered Dissociative State Scale (CADSS). DNA was genotyped using standard Taqman assays and protocols. Genetic association of CYP2B6*6 and GRIN2B (rs1019385 & rs1806191) SNPs and ketamine induced EP occurrence and severity were tested using multivariate logistic and linear regression, adjusting for age, ketamine dose, duration of anesthesia, and time since ketamine administration. Results: Forty-seven patients (63%) received ketamine and were genotyped. Nineteen EP cases were identified (CADSS > 4), leaving 28 non-EP controls. For our population, CADSS has an internal consistency reliability Cronbach’s alpha of 0.82, and could reliably distinguish ketamine from non-ketamine cases. Occurrence and severity of EP were not associated with CYP2B6*6 or GRIN2B (p > 0.1). Models removing genotype and containing age, ketamine dose, duration of v anesthesia, and time since ketamine administration significantly predicted EP occurrence (p = 0.001) and severity (p = 0.007). Presence and severity of EP did not affect patient satisfaction with care. Discussion: Younger age, higher dose and longer duration of anesthesia significantly predicted EP occurrence and severity among our sample. This study provides effect size estimates useful for the design of adequately powered future genetic association studies. The feasibility of recruitment from patients undergoing elective, outpatient surgeries and ease of post-operative EP assessment with CADSS supports our approach. However, the small sample size may have limited about ability to determine significant differences. Conclusion: Fully powered studies are needed to investigate this important phenomena. Determining factors for anesthesia-related EP symptoms may reduce risks and costs associated with this adverse medication effect.

In an age of biomedicine, technologies, drugs, and treatments are expanding in new and diverse ways. Especially relevant to biomedicalization and this research is how such information is conveyed to the public through the media. Medical information is omnipresent in the media through direct-to-consumer (DTC) advertising and regular coverage of health topics in the news. The accessibility and proliferation of medical information provides an important opportunity to examine the ways in which these topics are framed in the media. This research specifically examines the framing of the HPV vaccine, Gardasil in the mass media. In this study, I explore how Gardasil was framed, how gender and sexuality were utilized within such frames, and what groups influenced these frames. I employ frame analysis to examine the presentation of Gardasil in the mass media. Due to the vaccine's intricate connection to gender and sexuality, I examine how these dimensions are utilized and reproduced in such frames. Gardasil was originally approved only for women, making gender a salient aspect of the vaccine. The current body of research examining Gardasil in the media presents data from the time the vaccine was only available to women. Now that the HPV vaccine is approved for men, this research seeks to contribute to a more comprehensive understanding of how Gardasil was framed in the media now that it is available to men and women. And given that Gardasil prevents a sexually transmitted infection (STI) in men and women, it is uniquely tied to issues of sexuality. To analyze Gardasil in the media, I examine newspaper articles, magazine articles, and media representations from stakeholder groups, including DTC advertising, official statements, and group websites. Analysis of key sources indicates that Merck dominated the framing of the vaccine in DTC advertisements and the news media, illustrating the power of the pharmaceutical industry. Findings indicate that the initial marketing of Gardasil constructed the vaccine as uniquely tied to femininity and later, women's empowerment. However, once the drug was approved for men, messages were reframed to appeal to a wider audience. Overall, the media continued to frame the vaccine specifically for women, further constructing HPV as a "woman's disease." The dominant focus on women concomitantly silenced the sexual health of men and sexual minorities. In conclusion, the marketing, discourse, and structural elements of Gardasil make it less accessible to those most in need, therefore contributing to the ongoing problem of cervical cancer and HPV.

Tumor microenvironmental conditions play a vital role in promoting metastasis and tumor recurrence. Due to inefficient vasculature, cancer cells experience hypoxia, glucose deprivation and low pH even during the early stages of tumor growth. Tumor cells are proposed to adapt to these microenvironmental conditions by acquiring increased migratory and invasion potential and tumor initiating ability. Our research addresses the effect of these biochemical factors of the tumor microenvironment (TME) on motility, epithelial to mesenchymal transition (EMT) and stemness of non-small cell lung cancer (NSCLC). NCI-H292 and NCI-H1650 NSCLC cell lines were used to measure the effect of the above mentioned TME conditions. Apart from acidic pH, low glucose and hypoxia, the effect of high glucose conditions was also measured on H292 and H1650 cell lines. Acidic pH, high and low glucose conditions were observed to have no effect on the motility, EMT and stemness of H1650 cell line. Hence, use of this cell line was discontinued and no further treatment conditions were tested on this cell line. In H292 cell line, acidic pH, low glucose and tumor like conditions combined together (acidic pH + low glucose + hypoxia) [AP+LG+HYP] significantly decreased motility whereas hypoxia significantly increased the motility of H292 cells. High glucose did not affect the motility of H292 cells. Although N-cadherin, a mesenchymal marker, expression was significantly upregulated by acidic pH, high and low glucose conditions, no direct correlation was observed between N-cadherin expression and motility. E-cadherin expression was not affected by acidic pH, high and low glucose conditions. An increase in N-cadherin expression and no change in E-cadherin expression under these conditions might be an indication of partial EMT. Hypoxia and AP+LG+HYP did not alter the expression of E-cadherin and N-cadherin. Although expression of vimentin, another mesenchymal marker, and Sox2, a cancer stem cell marker (CSC), was observed at the mRNA level, no expression of vimentin and Sox2 proteins was observed in H292 cells under any of these treatment conditions. The expression of OCT4, another CSC marker, was also not observed at the protein level in H292 cells. HIF-1α expression was observed in H292 cells under normoxic conditions and was unaffected by hypoxia and AP+LG+HYP. Therefore our research indicates that the effect of these TME conditions might be different on different cancer cell lines or cancer types. Not all cancers may depend on EMT for metastasis. An increase in metastasis under hypoxia may be independent of HIF-1α.

The data presented in this thesis is a comprehensive study on the nature of peptide structure and how subtle and systematic changes in sequence and sidechain affect the basicity, ion stability, and conformation of a peptide. The peptides characterized were acetylated polyalanine di-, tri-, and tetra- peptides containing a proton-accepting probe: lysine and or the non-proteinogenic lysine-homologs: ornithine, 2,4-diaminobutyric acid, and 2,3-diaminopropionic acid. Peptides were studied in isomeric pairs for which the basic amino acid was placed closest to the N-terminus or the C-terminus of each peptide family (A n Probe vs. ProbeA n ). Using a variety of mass spectrometry based techniques and infrared multiphoton dissociation ion spectroscopy, the isomeric families of polyalanine peptides were characterized. Quantum chemical techniques were employed in parallel to provide theoretical predictions of three-dimensional structure, physical properties (dipole moment, polarizability, and accessible surface area), thermochemical values, and vibrational IR spectra, to gain further understanding of the peptides studied and to push the limits of current theoretical models. Overall it was found that the AnProbe peptide was more basic than their ProbeAn isomer. For the dipeptide systems, the greater basicity of AProbe peptides was due to efficiently charge-solvated ions which formed more compact structures compared to their ProbeA counterpart. For the tri- and tetra- peptide systems, greater basicity of the A 2,3 Probe peptides was likely due to formation of α or 3 10 helix-like structures in the protonated forms., introducing the macrodipolar effect, which cooperatively encouraged helical formation while stabilizing the charged site. On the other hand, ProbeA 2,3 peptides formed charge-solvated coils which do not exhibit any kind of dipole effect, resulting in lower basicity than their A2,3Probe counterpart.