Mitochondria provide the main energy required for cardiac excitation-contraction coupling via aerobic oxidative phosphorylation (OXPHOS) process. Accumulation of reactive oxygen species (ROS), by-products of mitochondrial respiration, within dysfunctional mitochondria results in the activation of cardiac cell death pathways and has been associated with heart failure development. Therefore, maintaining mitochondrial homeostasis as a balance between mitochondrial biogenesis and degradation is of great importance toward cardiac proper functioning. In addition to the importance of mitochondrial energy supply, gap junctions, intercellular channels which connect plasma membrane of adjacent cardiomyocytes, by propagating action potential throughout the myocardium maintain cardiac synchronous beating and rhythm. Gap junctions have a rapid turnover and impair of gap junction quality control impacts cell-to-cell communication; resulting in electrical conduction abnormalities and arrhythmogenesis. Therefore, understanding the underlying mechanism the quality control of mitochondria and gap junctions profoundly contributes toward understating the genesis of cardiomyopathy. Furthermore, cardiovascular problems in HIV (Human immunodeficiency virus) positive patients whose viral load is controlled via antiretroviral therapy remains a problem while the underlying mechanism remains elusive. The current study has used an in vitro model of primary neonatal rat ventricular cardiomyocytes (NRVCs) to discover the molecular mechanisms of mitochondrial as well as gap junction quality control under normal and stress conditions. Furthermore, electrical activities of the primary cardiomyocytes were recorded using microelectrode array (MEA) system and important electrophysiological components such as impulse propagation pattern and conduction velocity were extracted from the complex signal recordings. Overall, we have pursued four main aims; Aim 1. Dysregulation of mitochondrial quality control machinery leads to cardiac death; Aim 2. HIV-1 Tat (transcriptional transactivator) dysregulates cardiac homeostasis via mitochondrial pathway; Aim 3. Impairment of protein quality control impacts the quality of gap junction; Aim 4. Inhibition of gap junction quality dysregulates electrical signal propagation within the culture. / Bioengineering

Background: Due to its continuous exposure to food antigens and microbes, the gastrointestinal tract (GIT) is in a constant state of low level immune activation and contains an abundance of activated CCR5+CD4+ T lymphocytes, the primary target HIV-1. As a result, the GIT is a site of intense viral replication and severe CD4+ T cell depletion, a process that begins during primary HIV-1 infection and continues at a reduced rate during chronic infection in association with increased production of pro-inflammatory cytokines, a breakdown in the epithelial barrier, microbial translocation, systemic immune activation and the continued recruitment and infection of new target cells. AntiRetroviral Therapy (ART) is only partially effective in reversing these pathogenic changes. Despite the importance of the GIT in HIV-1 pathogenesis, and as a reservoir of persistent virus during ART, little is known about the diversity of HIV-1 in the GIT, or how different tissues in the GIT respond to ART. Objectives: Primary objectives of this thesis were to: 1) characterize the diversity of HIV-1 RNA variants in different parts of the GIT; 2) determine whether there is compartmentalized evolution of HIV-1 RNA variants in the GIT and whether these variants are likely to have different biological properties; 3) investigate the impact of ART on immune restoration in the GIT. Methods: A prospective study of the duodenum, jejunum, ileum and colon of African AIDS patients with chronic diarrhea and/or weight loss, sampled before and during 6 months of ART. RNA extracted from gut biopsies was reverse transcribed and PCR amplified. Env and gag PCR fragments were cloned, sequenced and subjected to extensive phylogenetic analysis; pol PCR fragments were analyzed for drug resistance. CD4+, CD8+ and CD38+CD8+ T cells levels in biopsies collected at baseline (duodenum, jejunum, ileum and colon) and after 3 (duodenum) and 6 (duodenum and colon) months of ART were quantified by flow cytometry and immunohistochemistry, plasma and tissue VL by the Nuclisens assay. Results: Viral diversity varied in different regions of the GIT with env HIV-1 RNA variants being significantly more diverse than gag variants. Gag HIV-1 RNA variants were widely dispersed among all tissue compartments. Some env variants formed tight monophyletic clusters of closely related viral quasispecies, especially in the colon, a finding that is suggestive of compartmentalized viral replication and adaptive evolution. CD4+ T cell and VL levels were significantly lower, while CD8+ including activated CD38+CD8+ T cell levels were higher in the duodenum and jejunum versus the colon. After 6 months of ART, a significant but incomplete recovery of CD4+ T cells was observed in the colon but not in the duodenum. Failed restoration of CD4+ T cells in the duodenum was associated with non-specific enteritis and CD8+ T cell activation. Conclusions: These results advance our understanding of the GIT as a host-pathogen interface by providing new insights into the diversity, evolution and dissemination of HIV-1 variants in the GIT. Strategies aimed at decreasing immune activation, especially in the small intestine, may be highly beneficial in enhancing the therapeutic efficacy of ART. / Thesis (PhD)--University of Pretoria, 2014. / lk2014 / Immunology / PhD / Unrestricted

Das HIV-1 Tat-Protein hemmt die Peptidase-Aktivität des 20S Proteasoms durch Konkurrenz mit dem 11S Regulator/PA28 um die Bindungsstelle am Proteasom. Aus den kinetischen Daten und durch Strukturvergleiche geht hervor, dass die Aminosäuren Lys51, Arg52 und Asp67 des Tat-Proteins für den Effekt auf das 20S Proteasom verantwortlich sind und die REG/Tat-Proteasom-Bindungsstelle bilden. Eine in der 11S Regulator alpha-Untereinheit (REG alpha) identifizierte vergleichbare Struktur wird von den Aminosäuren Glu235, Lys236 und Lys239 gebildet. Durch eine Mutation der REG alpha Aminosäuren Glu235 und Lys236 zu Ala geht die Fähigkeit des REG alpha die Peptidase-Aktivität des 20S Proteasoms zu stimulieren verloren, während die Bindungsfähigkeit an den 20S Komplex erhalten bleibt. Die Bindungsstelle in REG alpha ist für die verstärkte Präsentation eines Epitops des Cytomegalovirus pp89 durch MHC Klasse I essentiell. Das Tat-Protein und das Tat-Peptid 37-72 unterdrücken die 11S-Regulator vermittelte Antigenpräsentation des pp89 Epitops. Im Gegensatz dazu weist das Tat-Peptid mit Mutation der Aminosäuren Lys51, Arg52 und Asp67 zu Ala keine Reduktion der Antigenpräsentation auf. / The HIV-1 Tat protein inhibits the peptidase activity of the 20S proteasome and competes with the 11S regulator/PA28. Kinetic assays and structural comparison found amino acids Lys51, Arg52 and Asp67 of Tat to be responsible for the effects on proteasomes, forming the REG/Tat-proteasome-binding site. A similar site identified in the 11S regulator alpha subunit (REG alpha) consists of the residues Glu235, Lys236 and Lys239. Mutation of the REG alpha amino acids Glu235 and Lys236 to Ala resulted in a REG alpha mutant that lost the ability to activate the 20S proteasome even though it still binds to the 20S complex. The site in REG alpha is needed to enhance the presentation of a cytomegalovirus pp89 protein-derived epitope by MHC class I molecules. Full-length Tat and the Tat peptide 37-72 suppressed 11S regulator-mediated presentation of the pp89 epitope. In contrast, a Tat peptide with mutation of amino acids Lys51, Arg52 and Asp67 to Ala was not able to reduce antigen presentation.