published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

Vaccines directed against the influenza virus become ineffective due to continuous mutation. An alternative approach might be to control replication at the genomic level by enzymatic methylation of the polymerase genes. Hence in this study, a method to locate and successfully isolate the H1N1 influenza A polymerase genes was investigated. The virus was cultured in chick embryos via the allantoic route using aseptic techniques. Following incubation, the allantoic fluid was isolated and washed to remove any contaminating blood cells. The allantoic fluid was checked for fungal and bacterial contamination using the blood agar test and the presence of the virus was established by the haemagglutination titration test. Viral particles were pelleted by ultracentrifugation. Electron microscopy verified the morphology and size of these viruses while immunofluorescence studies, using a monoclonal antibody, confirmed the influenza A strain. The ribose test verified the presence of RNA in the samples. Purified viral pellets were pooled and homogenised in buffer containing guanidine thiocyanate, mercaptoethanol and sarkosyl. The samples were incubated on ice before mechanical disruption of the virus. Viral RNA was isolated from the upper aqueous layer after a standard phenol/chloroform extraction procedure. RNA was quantified spectrophotometrically and purity assessed initially by the absorbance ratio readings at 260/280 nm. Electrophoresis of the RNA samples was performed together with RNA molecular weight markers on a 1.5% formamide agarose gel. Five bands were identified and the band containing the polymerase genes was size selected, located and excised. Purification of the polymerase genes from the agarose was achieved by using the BIO 101 RNAid kit. The three isolated polymerase RNAs were reverse transcribed using the Boehringer Mannheim cDNA synthesis kit. The results indicate that the H1N1 influenza virus was successfully grown and isolated from chick embryos. Absence of contamination and verification of viral presence at different stages of the study were indications that asepsis was successfully achieved. The RNA obtained was sufficient and suitable for cDNA synthesis. This cDNA may now be used for further molecular analysis and subsequent DNA methylation studies. Further, transfection studies may then be performed to determine, if any, the the expression of methylated and unmethylated cDNA. / Thesis (M.Med.)-University of Natal, 1992.

Multi-drug resistance among bacterial pathogens can be mediated by a number of mechanisms, including multidrug efflux pumps. One such pump in Bacillus spp. is ykkCD, a heterodimer of the SMR family consisting of C and D subunits. Previous studies suggest that the expression of ykkCD is controlled by a putative riboswitch and that the antibiotic tetracycline binds to the riboswitch in vitro. Additional studies have shown that two derivatives of tetracycline also bind to the putative riboswitch. These findings now need to be validated by an in vivo study. In this study, the effects that tetracycline and its commercially available derivatives—doxycycline, minocycline, anhydrotetracycline, and oxytetracycline—have on the expression levels of the ykkCD gene in Bacillus subtilis were explored. The level of ykkCD expression was quantified using two different methods: (1) ykkCD protein levels was determined using a ykkCD RNA--galactosidase reporter gene construct and (2) ykkCD mRNA levels was quantified by quantitative RT-PCR. Although the findings from method (1) were inconclusive, upregulation was observed for tetracycline and minocycline, in agreement with the results of the previous binding studies. / Department of Chemistry

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Chemistry

The aim of this investigation was to compare the acute anabolic and catabolic responses of male and female vastus lateralis (VL) and soleus (SOL) muscles in response to resistance exercise (RE). Muscle biopsies from the VL of 7 males (26±3 y, 75±8 kg) and 7 females (25±3 y, 59±5 kg) were obtained before, and 2 and 6 h after 4 x 7 supine-squat, and 4 x 14 calf-press exercises at maximal effort using inertial ergometry. The mRNA levels of select myogenic (MyoD, myogenin, MRF4), proteolytic (atrogin-1 , MuRF-1), myostatin, and inflammatory (IL-6, -8, -15) genes were quantified using real-time RT-PCR. Male VL vs SOL: The SOL had higher basal mRNA levels of myogenic, proteolytic, and inflammatory genes. After exercise, the myogenic response was similar between the VL and SOL. Both muscles increased MuRF-1 similarly at 2 h, whereas 6 h post-RE proteolytic gene expression (GE) was suppressed in the VL but not in the SOL. The SOL had a reduction in myostatin GE, and a more robust inflammatory response compared to the VL. These findings indicate a more favorable growth response in the VL. Gender comparisons: VL – Basally, the male VL had higher levels of myogenic, proteolytic, myostatin, and inflammatory mRNA compared to the female VL. After exercise, both genders increased myogenic GE similarly. Both genders increased MuRF-1 initially, with females also increasing atrogin-1 and myostatin post-RE. At 6 h, males decreased proteolytic GE to below basal levels. Females also had a greater inflammatory response than males. These findings indicate a greater growth response to RE in the male VL as compared to the female VL. SOL – After exercise, both genders increased myogenic GE in the SOL, but only males increased MyoD expression. Males increased MuRF-1 mRNA but decreased myostatin GE, while females decreased atrogin-1. The inflammatory response was similar between males and females. Despite the modest differences, the net response of the female and male SOL was similar, and indicated a molecular response slightly favorable for growth. / School of Physical Education, Sport, and Exercise Science

Interleukin-8 is a chemokine which is chemotactic for neutrophils and T-lymphocytes and plays a crucial role in the pathogenesis of inflammatory bowel disease. Intestinal mucosal epithelial cells produce IL-8 in response to pathogens which mediates bidirectional communication between pathogen and host. The objective of this study was to investigate the molecular mechanisms involved in IL-8 gene regulation in T84 human colonic epithelial cells. To determine if IL-8 plays a role in the pathogenesis of intestinal amebiasis, the effect of Entamoeba histolytica on IL-8 gene expression was investigated. E. histolytica secreted components enhanced IL-8 mRNA expression and protein production in the absence of amebae-enterocyte contact. The proinflammatory cytokines IL-1beta and TNF-alpha were not involved in IL-8 protein production. As PGE2 is central in mucosal inflammation, the effect of PGE2 on IL-8 gene expression was determined. Using purified PGE2 and PGE2 receptor agonists, it was shown that PGE2 coupled to the EP4 receptor and triggered cAMP-dependent PKA signaling which upregulated IL-8 mRNA expression at the posttranscriptional level. Elevation of [Ca 2+]i from intracellular Ca2+ stores by A23187 or thapsigargin stimulated IL-8 mRNA transcription and IL-8 protein production through the activation of calcineurin. Moreover, IL-8 3'-UTR had a strong suppressive effect on CAT reporter gene expression in COS7 cells by reducing its mRNA level. A unique fragment (nt 2387-2743) containing AU rich elements was shown to attenuate CAT mRNA expression by destabilizing the transcripts. Secondary structure but not AU rich elements played a major role in CAT mRNA turnover.

Transposable elements (TEs) are mobile DNA sequences that can move from one location to another in the genome. These elements encode regulatory features including transcriptional promotion and termination signals facilitating the production of new transcripts (or elements). The elements thus produced are inserted back into the genome. Due to their insertional capacity and encoded regulatory features, TEs have, in recent years, been recognized as significant contributors to regulatory variation both within and between species. In comparing the human and chimpanzee genomes it has been hypothesized that the genetic basis of the phenotypic differences that distinguish them may be the result of regulatory differences existing between the two species. Since TEs inserted in proximity to genes can significantly alter gene expression patterns, this research aims at exploring the influence of TE sequences and retrotransposons in particular in the evolution of gene regulation between humans and chimpanzees. A first systematic search of one particular class of retrotransposons - endogenous retroviruses (ERVs) was carried out in the chimpanzee genome. Forty two families of ERVs were identified in the chimpanzee genome including the discovery of 9 previously unknown families in humans. The vast majority of these families were found to have orthologs in the human genome except for two (CERV 1/PTERV1 and CERV 2) families. The two CERV families without orthologs in the human genome display a patchy distribution among primates. Nine families of chimpanzee ERVs have been transpositionally active since the human-chimpanzee divergence, while only two families have been active in the human lineage. The genomic differences [INDEL variation (80-12,000 bp in length)] between humans and chimpanzees are laid out. The INDEL variation located in or near genes is categorized in detail and is correlated with differences in gene expression patterns in a variety of organs and tissues. Results indicate that the majority of the INDEL variation between the two species is associated with retrotransposon sequences and that this variation is significantly correlated with differences in gene expression most notably in brain and testes. These findings are consistent with the hypothesis that retrotransposon mediated regulatory variation may have been a significant factor in human/chimpanzee evolution.