The conventional method for quantifying sleep is through the use of Polysomnography (PSG) and a trained human sleep scorer by observing and evaluating the output in 30-second epochs. A PSG device can be rather invasive to one’s regular sleep pattern and therefore can potentially result in irregular sleep patterns. Furthermore, human sleep scoring classification by a trained expert can be rather time consuming and subject to inter/intra rater variability. Nevertheless, human sleep scoring with PSG still remains the gold-standard for sleep measuring and classification for the diagnosis disorders related to sleep. The present pilot study explores the possibility of using a wearable device known as a ByteFlies Sensor Dot to measure signal activity from an individual during a night’s sleep. This validation study focuses on the signal capture of alpha frequency band through a phenomenon known as “the Berger effect.” Participants will be asked to open and close their eyes while being connected to the gold standard PSG device and exploratory ByteFlies Sensor Dot device. The resulting alpha signals will be identified with a machine learning algorithm for cross comparison and analysis. In conclusion, the validation study will discuss methods to improve on the measuring of EEG and sleep stage scoring with the ByteFlies Sensor Dot for sleep monitoring and sleep disorder diagnosis.

African swine fever (ASF) is a disease that affects domestic pigs and wild boars, resulting in up to 100% case fatality rate, and there is currently no effective treatment or vaccine. To date, there are 67 ASFV complete genome sequences available, but most of the sequences represent only genotypes I-V and VII-X of the 24 genotypes identified based on p72 sequencing, limiting inter and intra-genotype comparative studies. ASFVs encode several multigene families (MGFs) involved in virulence and host range which are found at the genomic termini and the majority of genomic differences between isolates are due to the composition of these MGFs. The comparison of the MGFs across ASFV isolates is of utmost importance in understanding genome variability and their contribution to virulence. The p72 gene has historically been used in phylogenetic analysis of ASFV. However, it lacks the capacity for higher resolution between isolates belonging to the same genotype. This study aimed to analyse and characterise six novel ASFV isolates of African origin from a domestic pig, warthog, wild boar and ticks in terms of genomic makeup, MGF composition and phylogenetic relationships, including identification of additional phylogenetic markers, specifically for use in discrimination between closely related isolates. Genomes of six novel isolates were sequenced and annotated by identifying open reading frames (ORFs) with a methionine START codon and performing BLASTx searches of each ORF against the NCBI data base. Differences between the genomes were analysed by generating dotplots and using Base-By-Base which showed them to be mostly collinear, but regions of difference were observed at the termini and the CCR. MGF analysis using sorting and clustering in Morpheus software, based on genotype, serogroup, country, host, virulence, and year, showed that genotype and serogroup play a role in the MGF arrangement patterns. Loci corresponding to regions of difference in the CCR were used for phylogenetic comparison to the previously identified marker p72. The tree topology of all of the alternative phylogenies differed from the current p72 classification. B117L and B169L provided slightly better resolution of genotypes I and II, respectively, and viruses from East Africa that are classified as belonging to genotype IX based on p72 were separated when using EP364R. This data adds to the pool of diverse ASFV isolates available for comparative genomics studies, and to the knowledge of ASFV in Africa. The sequencing of more diverse ASFV isolates of each genotype will help characterise the MGFs arrangement patterns among isolates. The novel alternative phylogenetic markers should further be investigated using more ASFV isolates representing the 24 genotypes described to date.

Background: According to the World Health Organization Global TB report 2018, rifampicin monoresistant tuberculosis (RMR-TB) comprises 22% and 38% of all rifampicin-resistant TB (RR-TB) globally, and within South Africa, respectively. National surveys from South Africa show an increasing proportion of RMR-TB among TB cases compared to multi-drug resistant tuberculosis (MDR-TB) from 2001-02 (0.4% vs 2.9%) to 2012-14 (1.7% vs 2.8%). Data from the 2012-14 survey showed considerable variation in RMR-TB prevalence throughout the nine provinces of South Africa. Despite the above, factors associated with the rise in RMR-TB are unknown; and research is limited. This thesis aims to describe RMR-TB in more detail, by investigating the emergence and transmission of RR-TB strains in Khayelitsha, Western Cape Province, South Africa. This included: conducting a systematic review on temporal trends, transmission and risk factors associated with RMR-TB; describing the overall prevalence of RMR-TB among RR-TB; assessing the relative risk of RMR-TB versus MDR-TB among RRTB patients by HIV status during prior TB treatment; describing the distribution of rpoB mutations among RR-TB strains and assessing minimum inhibitory concentration (MIC) values in RR-TB strains with particular rifampicin-resistance (RIF R) conferring mutations; and investigating potential transmission through whole genome sequence (WGS) derived clusters among RR-TB strains. Methods: Routinely diagnosed RR-TB isolates are stored in a biobank at Stellenbosch University (SU). Clinical data (Médecins sans Frontières and additional data requested from the Western Cape Provincial Health Data Centre), together with stored RR-TB isolates from the biobank across 2013-15 inclusive were used to address research questions in Khayelitsha. To describe the overall prevalence of RMR-TB among all RR-TB over time, epidemiological data from 2008-17 were used. Laboratory techniques (sub-culturing of stored frozen cultures into mycobacterial growth indicator tubes [MGITs] for DNA extraction and quantitative phenotypic DST [q pDST]) involving the handling of live Mycobacterium tuberculosis cultures, were done in a Biosafety Level 3 laboratory at SU. Extracted DNA was sent to the University of Basel in Switzerland for library preparation and whole genome sequencing (WGS) on the Illumina HiSeq. The raw fastq WGS data files of the sequenced DNA were securely transferred to UCT. TB profiler was used to identify RIF R conferring mutations in rpoB and strain lineages. rpoB mutations were classified as high/moderate and minimal confidence in conferring rifampicin-resistance. q pDST (MGIT) was performed for isolates with minimal, moderate and rpoB confidence level mutations that were not classified. q pDST was also performed on isolates found to be rifampicin susceptible TB (RS-TB using WGS) [no RIF R conferring rpoB mutations detected] but were isolated from patients routinely diagnosed with RR-TB. A combination of software packages was used, as well as in-house developed scripts to compile a pipeline for WGS transmission cluster analysis. Computations were performed using facilities provided by UCTs ICTS High Performance Computing (HPC) team. Clusters were identified with Clusterpicker and by generating a single nucleotide polymorphism (SNP) distance matrix (SNP differences found between genomes) using R software; a SNP threshold of 12 was used to suggest recent transmission. Results: i) The overall prevalence of RMR-TB among all RR-TB remained relatively stable (17-31%) with no major temporal trend observed during 2008-17 in Khayelitsha. ii) The proportion of RMR-TB among all RR-TB was significantly higher among patients who were HIV positive during previous TB treatment compared to those who were HIV negative. iii) A high proportion (11%) of discordance was found among RIF R routinely diagnosed RR-TB patients (43% RMR-TB; 57% MDR-TB); resulting from possible mixed infections (43%), false-positive RIF R (18%) or both (39%). iv) The WGS-based DR-TB profile for rpoB mutations were distinctly different between RMR- and MDR-TB strains. The proportion of high/moderate vs minimal confidence levels for rpoB mutations was significantly higher among MDRTB (high confidence rpoB S531L mutation - Lineage 2) than for RMR-TB. v) Among RMR-TB strains, rpoB L511P (described as a disputed mutation, conferring minimal confidence for RIF R or low-level RIF R) was predominantly found among RMR-TB strains compared to MDR-TB strains. All rpoB L511P mutations (including RMR- and MDR-TB) tested phenotypically susceptible to rifampicin with MGIT; causing discrepancies between WGS and q pDST. All RMR-TB strains, including those with rpoB L511P mutations, had no other mutations conferring resistance to any of the other TB drugs. vi) Clustering was higher among MDR-TB strains compared to RMR-TB and more MDR-TB strains were clustered among strains with the rpoB S531L mutation compared to RMR-TB with the same mutation. In contrast, among strains with the rpoB L511P mutation, more RMR-TB strains were clustered compared to no clustering found among MDR-TB strains with the same mutation. Clinical data showed that RMRTB rpoB L511P clusters were due to closely related community acquired or nosocomial transmission; and SNP differences were < 5, suggesting direct transmission between RMR-TB rpoB L511P patients. Conclusion: In Khayelitsha, lower clustering of RMR-TB strains suggests reduced transmission compared to MDR-TB and along with a different rpoB mutation profile, suggests a different evolutionary mechanism of RIF R. Additionally, RMR-TB appears to be associated with HIV positivity during previous TB treatment, suggesting a role for HIV in the generation of RMR-TB. Given the association of rpoB L511P with low-level RIF R among RMR-TB strains, it is possible that different treatment approaches could be effective for these patients, as there are also no clinical trials to optimise treatment of these patients. WGS is beneficial, not only for understanding transmission of RR-TB strains, but also to be used in combination with MIC testing for individualised patient treatment regimens, in order to accurately diagnose RR-TB in future. Recommendations for preventing M.tb transmission; irrespective of HIV status; include early diagnosis and treatment initiation, and implementation of infection control programmes in various settings.

The advent of gene expression profiling studies has allowed for the identification of genes with potential as disease markers and therapeutic targets. Our laboratory identified the eukaryotic nuclear importer protein Karyopherin B1 (KpnB1), to be up-regulated in different cancer cell lines, including cervical and oesophageal as well as transformed cells. Inhibition of KpnB1 in these cells using small interfering RNA (siRNA) resulted in significant cancer cell death via apoptosis, suggesting KpnB1 is essential for cancer cell survival. Within our laboratory, we established that candidate small molecules targeted against KpnB1 identified using a rational drug design approach. The outcome of this research is for examine inhibitors of KpnB1 for potential as future anti-cancer agents using. Based on the long-term goal of this research, this particular project was aimed at investigating a small molecule inhibitor identified in our laboratory, known as Inhibitor of Nuclear Import-43 (INI-43) for its potential to bind to the nuclear importer, KpnB1. Using conventional assays as well as cutting edged techniques including circular dichrosim (CD) and isothermal titration calorimeter (ITC), an examination of INI-43 and its interactions with KpnB1 was made. In vitro analysis showed that INI-43 exhibits cytotoxic effects on cervical cancer cells with an IC₅₀ of ≈10μM and induces apoptotic cell death. The NFAT dual luciferase assay measured nuclear import of KpnB1 associated proteins, showing that INI-43 inhibits nuclear import/activity of NFAT in a dose dependent manner. Confocal microscopy of exogenous FRFP-KpnB1 as well as endogenous KpnB1 in the presence of INI-43 showed a change in the localisation of KpnB1 upon drug treatment. Both FRFP-KpnB1 and endogenous KpnB1 appear to be prevented from entering the nucleus, and is retained in the peri-nuclear space and the cytoplasm suggesting that INI-43 inhibits KpnB1 movement into the nucleus. To investigate KpnB1-INI-43 interactions, purified KpnB1 was prepared and used in biophysical techniques. Purified KpnB1 protein was prepared using GST-tagged purification methods and the tagged protein confirmed by mass spectrometry. Purified GST-KpnB1 was used in drug binding studies including circular dichrosim (CD) isothermal titration calorimetry (ITC). CD showed a drug concentration dependant shift in the spectra at around 233nm, indicative of drug protein interaction possibly occurring in a region of KpnB1 containing aromatic amino acids. The purified GST-KpnB1 was used in ITC, which confirmed an interaction between KpnB1 and INI-43, a relatively weak interaction. In conclusion, our data shows that the small molecule, INI-43 kills cancer cells, likely by interfering with KpnB1 associated nuclear import pathways. We show that INI-43 interferes with the nuclear localisation of KpnB1 itself and biophysical assays provide evidence for possible KpnB1-INI-34 interactions. Small molecules such as INI-43 present as promising tools to studying the potential of KpnB1 as an anticancer target.

Includes bibliographical references. / Vaccinia virus is the most extensively studied, prototype vertebrate poxvirus, which was used as a vaccine in the eradication of smallpox. The genome of this virus has characteristic variable termini encoding open reading frames that are not essential for virus replication in cell culture. One such open reading frame, N1L situated at the left terminal region of the neurovirulent Western Reserve (WR) vaccinia virus strain, encodes a protein 13.8 kDa in size. In vivo studies in mouse brains revealed that a recombinant virus, vGK5, tacking the expression of the 13.8 kDa protein was rendered replication deficient in the brain. An essential requirement of poxviruses for their replication is the energy molecule adenosine triphosphate (ATP). The supply of this molecule in the brain to support replication of a virus is limited due to the high-energy requirements and small energy reserves of this organ. The specific function of the vaccinia virus 13.8 kDa protein in relation to viral replication in the brain was investigated. The South African (SA) Lister vaccinia virus strain was confirmed to encode an identical N1L gene to that of the WR vaccinia virus by amplification, cloning and sequencing of the Lister N1L open reading frame. The Lister vaccinia virus and a 13.8 kDa deletion strain (vGK5) were cultivated and used to intracranially infect mice. Using a luciferin/luciferase bioluminescence assay system the ATP levels in Lister and vGK5 vaccinia virus-infected mouse brains were measured and found to differ significantly after a 5-day infection period. The SA vaccine Lister vaccinia virus strain was found to be a slow growing virus in the brain. Subsequently, a possible role for the vaccinia virus 13.8 kDa protein in influencing ATP levels in the brain was postulated, yet a neurovirulent wild type strain is needed for further studies to consolidate this result. The 13.8 kDa protein was successfully expressed in the P. pastoris yeast expression system and positively identified by immunodetection studies.

Includes bibliographical references (leaves 176-221).

Includes bibliographical references (leaves 172-200).

Fibrosis is characterized by excessive synthesis of type I collagen which impedes the normal function of an affected organ. Type I collagen is the most abundant protein in the human body, produced by the folding of two a1(I) polypeptides and one a2(I) polypeptides into the triple helix. Expression of collagen 1a(I) gene is predominantly regulated at the level of mRNA stability and translation. A conserved stem'Cloop structure is found in the 5' untranslated region of collagen mRNAs. The 5'stem-loop structure which is critical for the coordinated translation encompasses the start codon in collagen mRNAs and regulates collagen synthesis by the binding of RNA binding proteins. Assembly of collagen heterotrimer occurs on the membrane of endoplasmic reticulum (ER) while a1(I) and a2(I) mRNAs are associated with polysomes. I have found that the binding of 5'stem-loop binding proteins LARP6 and nonmuscle myosin II to the 5'stem-loop regulates coordinated translation of collagen mRNAs and is required for collagen triple helix formation. My work describes that the coordinated translation of collagen mRNAs increases local concentration of the chains necessary for productive folding. Chapter 1 presents the identification of 5'stem- loop binding protein and its function in regulating collagen synthesis. To identify 5'stem-loop binding proteins we performed expressional cloning and cloned La ribonucleoprotein domain family member 6 (LARP6) as the protein which binds to both 5'stem- loop of collagen a1(1) and a2(1) mRNAs a sequence specific manner. LARP6 has a distinctive bipartite RNA binding domain (amino acids 32-45 and 218-300) which is not found in other members of the La super family. The RNA binding domain of.LARP6 interacts with the two single-stranded regions of the 5'stem-loop. The Kd for binding of LARP6 to the 5'stem-loop is 1.4 nM. The binding is to the single stranded regions of the bulge of 5'stem-loop RNA, in both the nucleus and the cytoplasm. Recombinant LARP6 has a similar binding affinity and specificity to the 5'stem-loop as the endogenous LARP6. The C-terminal region of LARP6 has a nuclear localization signal, which allows LARP6 to accumulate in the nucleus. Combination of gain of function by adenoviral delivery and loss of function by using either dominant negative forms of LARP6 or siRNA directly against the RNA sequence of LARP6 were employed to determine the function of LARP6. Over expression of LARP6 decreased synthesis of collagen protein, however it did not change collagen mRNA steady state level. Also, collagen mRNAs were redistributed from polysomal fractions to the fractions representing the free polysomes in LARP6 overexpressed cells. This suggests that overexpression of LARP6 blocked ribosomal loading on collagen mRNAs and inhibited collagen mRNA translation. Endogenous LARP6 and overexpressed LARP6 were not associated with polysomes. These results suggested that LARP6 prevents premature translation of collagen mRNAs. Knocking down LARP6 by small interfering RNA also decreased steady state level of collagen polypeptide in the cell as well as the section rate of the protein. However, collagen mRNA stability was not affected, nor was the degradation of collagen protein by proteasome. Therefore, it is likely that collagen mRNA translation had been inhibited by the depletion of LARP6. We describe that collagen protein is synthesized at discrete regions of the endoplasmic reticulum. Using a collagen-GFP (green fluorescent protein) reporter protein, we could reproduce this focal pattern of synthesis, but it was observed only when the reporter was encoded by mRNA with the 5'stem-loop and in the presence of LARP6. When the reporter was encoded by mRNA without the 5' stem-loop, or in the absence of LARP6, it accumulated diffusely throughout the endoplasmic reticulum. This indicates that LARP6 activity is needed for focal synthesis of collagen polypeptides. We postulated that the LARP6-dependent mechanism increases local concentration of collagen polypeptides for more efficient folding of the collagen heterotrimer. In Chapter 2, we describe that nonmuscle myosin IIB plays an important role in regulating collagen synthesis. First, we identified nonmuscle myosin II B as LARP6 binding protein by tobramycin affinity purification and confirmed the binding specificity of myosin II B to LARP6 and to collagen mRNAs. We showed that the C terminus of LARP6 was required for the binding to nonmuscle myosin IIB, and that this binding was not RNA dependent. Secondly, we identify the role of nonmuscle myosin IIB in regulating type I collagen synthesis. Nonmuscle myosin II filaments are required for the secretion of collagen a2(I) peptide and the colocalization of a1(I) and a2(I) peptides in the cell. The motor activity of myosin II as well as the integrity of the filaments is involved in this process. We also discovered that the effect of myosin II on type I collagen synthesis was mediated by 5'stem-loop of LARP6 and through the protein-protein interaction with LARP6. At the end, we determined that nonmuscle myosin IIB filaments are involved in directing collagen mRNAs to polysomes for translation. Therefore, we concluded that nonmuscle myosin II interacts with LARP6 and collagen 5'stem-loop to regulates coordinate translation of collagen mRNAs. In Chapter 3, we explain the role of LARP6 and nonmuscle myosin II in collagen synthesis stimulated by cytokines and growth factors TGF-beta and ouabain. Cytokines and growth factors including TGF-beta and ouabain are stimulators of fibrosis and they increase collagen synthesis in scleroderma skin fibroblasts, hepatic stellate cells, kidney fibroblasts and cardiac fibroblasts. When we down regulated LARP6 or disrupted nonmuscle myosin II filaments, the stimulation of type I collagen secretion by TGF-beta 1 and ouabain was diminished. Therefore, we concluded that LARP6 and nonmuscle myosin II regulate inducible collagen synthesis in fibrosis. This dissertation describes the specific regulation of type I collagen synthesis in fibrosis. The first finding was that collagen protein synthesis took place of discrete regions on the ER membrane through by the binding of LARP6 to 5'stem-loop. The second finding was that coordinated synthesis of type I collagen polypeptide requires nonmuscle myosin II. The third finding discovered that profibrotic cytokines like TGF-beta and ouabain induced collagen synthesis through LARP6 and nonmuscle myosin II mechanism in human scleroderma skin fibroblasts and rat cardiac fibroblasts. In conclusion, we have discovered that LARP6 and nonmuscle myosin II regulated collagen synthesis. This pathway may contribute to excess collagen deposition in fibrosis. This information will help to find future anti-fibrotic therapy. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester, 2010. / March 15, 2010. / Includes bibliographical references. / Branko Stefanovic, Professor Directing Dissertation; Hengli Tang, University Representative; Myra M. Hurt, Committee Member; Yanchang Wang, Committee Member.

The mechanisms underlying the gestational and circadian timing of parturition in humans are not fully understood. Studies of the timing of initiation of spontaneous labor show a peak between 2400 and 0500. This peak in labor onset coincides with peak serum melatonin levels in humans. Melatonin, N-acetyl-5-methoxytryptamine, is the molecular messenger of circadian night. A monoamine hormone produced by the pineal gland, melatonin, is released into the blood directly in a circadian manner controlled by input from suprachiasmatic nuclei (SCN). Peak levels occur several hours after darkness and its release is inhibited by light via photic input transmitted from the eye via the retino-hypothalamic tract to the SCN. Once in the circulation melatonin can act on numerous tissues via its receptors or via antioxidant mechanisms inferred by its indole ring. Our laboratory recently characterized the expression of the melatonin receptors in the human myometrium and showed that the expression of these receptors is suppressed until late pregnancy. In an effort to understand better the significance of melatonin in the human myometrium, we explored the mechanisms through which this hormone influences the expression of the oxytocin receptor in vitro. The stable melatonin analog iodo-melatonin (I-MEL) was presented to cultured telomerase-immortalized smooth muscle myometrial cells of the human telomerase reverse transcriptase line under physiological doses and durations. Pharmacological inhibitors of melatonin binding (4P-PDOT), gene transcription (actinomycin), phospholipase C (U73122), and protein kinase C (C1) signaling were used to define the mechanism of melatonin action. Our results reveal that melatonin significantly reduces oxytocin receptor mRNA levels primarily via the melatonin type 2 receptor, MT2R. We assayed OTR mRNA levels over 24 hours after treatment with the transcriptional inhibitor, Actinomycin, with and without cotreatment melatonin. Our data suggest the melatonin-dependent decrease in oxytocin receptor transcripts involves reduction of the OTR mRNA accumulation rate rather than enhanced rates of transcript degradation. Melatonin effects were abolished by pre-treating the cells with the phospholipase C inhibitor U73122 or the protein kinase C inhibitor C1. Melatonin, like oxytocin, can negatively regulate oxytocin receptor transcription in human myometrial cells via modulation of protein kinase C signaling. Due to the similarities between the melatonin and oxytocin signaling pathways including reduction of OTR mRNA levels, we next sought to determine the effects of melatonin on contractility and the contractile machinery in telomerase-immortalized human myometrial cells. To ascertain the effect of melatonin on myometrial contractility in cell cultures, we performed gel retraction assays with cells exposed to I-MEL, oxytocin and the pharmacological inhibitors 4P-PDOT, U73122, C1 and combinations of ligands and inhibitors. I-MEL was found to synergistically enhance oxytocin-induced contractility via the MT2R, which is coupled to a protein kinase C-dependent increase in phosphorylation of the myosin light chain protein. The effects of I-MEL on gap junctions were also investigated as gap junction proteins have been shown to be upregulated by melatonin in other tissues and have also been shown to be important for coordination of contractions in the laboring uterus. I-MEL increased expression of the gap junction protein, connexin 43. In vitro dye spread assays showed that I-MEL-treated cells displayed substantially increased intercellular coupling. Increases in connexin 43 mRNA and cell to cell coupling were also found to be mediated via the MT2R in a protein kinase C-dependent manner. Additionally, expression levels of the type 2 melatonin receptor (MT2R) were assessed in myometrial biopsies from term pregnant women with or without labor. MT2R expression was markedly elevated in samples from pregnant women who had entered labor, as compared to matched non-laboring pregnant women. To ascertain the signaling pathway of melatonin and leading to its effects on myometrial contractility in vitro, we performed gel retraction assays with cells exposed to I-MEL with or without oxytocin and the Rho kinase inhibitor Y27632. I-MEL effects on IP3/DAG/ Protein Kinase C (PKC) signaling were also investigated as these signaling molecules were implicated by our previous pharmacology experiments. I-MEL was found to activate PKCα via the phospholipase C/IP3/DAG signaling pathway which was confirmed by PKC enzyme assay. I-MEL did not affect myosin light chain phosphatase activity and its effects on contractility were insensitive to Rho kinase inhibition. In order to examine another possible method of contractile sensitization, we assayed for caldesmon phosphorylation and upstream Erk1/2 activation. I-MEL did increase phosphorylation of Erk1/2 and caldesmon, which was inhibited by the MEK inhibitor, PD98059 or the PKC inhibitor, C1. These findings lead us to surmise that melatonin sensitizes myometrial cells to subsequent pro-contractile signals in vitro through activation of the phospholipase C/IP3/DAG signaling pathway resulting in specific activation of PKCα and Erk1/2, thereby phosphorylating caldesmon, which increases actin availability for myosin binding and crossbridging. This research revealed a new role for melatonin in reproductive physiology, sensitizing myometrial cells to a subsequent pro-contractile oxytocin signal. This function would help explain the increased nocturnal uterine contractility and increased incidence of parturition observed in late term human pregnancy. Synergistic actions of melatonin on oxytocin-induced contractility may be of clinical relevance in that it could provide a means to lower the oxytocin dose used in the induction of labor and thus reducing the contraindications associated with oxytocin induction of labor. / A Dissertation Submitted to the Department of Biomedical Sciences in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. / Fall Semester, 2009. / November 4, 2009. / Myometrium, Oxytocin, Parturition, Contractility, Melatonin / Includes bibliographical references. / James M. Olcese, Professor Directing Dissertation; Hank W. Bass, University Representative; Akash Gunjan, Committee Member; Choogon Lee, Committee Member; Branko Stefanovic, Committee Member.

The hepatitis C virus (HCV) is a serious health threat globally. Current therapies are not tolerated well, have a low response rate, and there is no available vaccine. New viral targets for drugs are urgently needed. The aim of this study was to characterize resistance to chemical and non-chemical treatments of the HCV replicon and determine a viral target for new treatment options. Replicon cells were treated with both Cyclosporine A (CsA) and temperature shift treatment (39°C). Resistant replicon cells were attained by double treatment with selection antibiotics and anti-viral treatments, in concert with live cell sorting techniques. Resistant cell lines were analyzed and RNA was extracted. This RNA was electroporated into naïve or cured cells, creating new cell lines. These new cell lines were then tested for resistance. Resistant replicon RNA was also sequenced and compared to non-resistant strains. After cell lines had been attained with high levels of resistance, and RNA was electroporated into naïve or cured replicon cells, these new cell lines also showed resistance. This indicated that the viral RNA was the source of the treatment resistance. There were unique mutations at the amino acid level in both CsA and temperature shift resistant cell lines. These unique mutations in both the CsA and temperature shift resistant replicon genomes indicate changes in the NS5B and NS3 proteins, respectively. Further work on the protein structures with the amino acid substitutions and their interactions could lead to new targets for therapies in patients. / A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science. / Fall Semester, 2009. / July 17, 2009. / Temperature Shift, Hepatitis C Virus. Drug Resistance / Includes bibliographical references. / Hengli Tang, Professor Directing Dissertation; Thomas Keller, Committee Member; Kenneth Roux, Committee Member; Fanxiu Zhu, Committee Member.