Acquired immunodeficiency syndrome (AIDS) is a major problem in our current society. There are over 35 million of the population that are currently living with human immunodeficiency virus (HIV), and the number of HIV-infected patients are still rising every year in spite of our efforts to control it. Furthermore, within the AIDS affected population, opportunistic infection is a major cause of complications and is the number one cause of death. The HIV trans-activator (Tat) protein plays a major role in the AIDS pathogenesis. HIV-1 Tat is known to cause dysregulation of cytokines such as TNF-α, IL-6, and IL-10 in AIDS patients. In this study we recognized a proto-oncogene, c-Myc, could regulate the cytokine dysregulation caused by HIV-1 Tat in primary blood monocyte derived macrophages (PBMac). By knocking down the expression of c-Myc with gene specific small-interfering RNA (siRNA), we demonstrated that c-Myc may be critical for the expression of the pro-inflammatory cytokines TNF-α and IL-6. HIV-1 Tat was subsequently found to regulate the expression of c-Myc via the activation of dsRNA-activated protein kinase (PKR), ERK1/2 and p38 mitogen-activated protein kinase (MAPK). Furthermore, c-Myc regulation of the pro-inflammatory cytokines was demonstrated to have a role in AIDS related opportunistic infections. HIV-1 Tat was shown to increase the intracellular growth of Mycobacteria avium complex (MAC) within PBMac. This increase in MAC growth was in turn found to be regulated by TNF-α expression controlled by c-Myc. HIV-1 Tat was also demonstrated to induce the expression of RIG-I, a common pattern recognition receptor of double stranded RNA viruses, in PBMac. RIG-I is known to activate the viral immune responses such as the type-I interferon (IFN) and pro-inflammatory cytokine pathways. This induction of RIG-I by HIV-1 Tat was found to be regulated by c-Myc, as well as through other signalling kinases such as p38 MAPK and PKR. Tat induction of RIG-I ultimately led to the induction of IFN-α2 and IFN-β through the expression and nuclear translocation of the interferon regulatory factor-7 (IRF-7). This alteration in type-I IFN expression regulated by HIV-1 Tat and RIG-I was also found to play a role against AIDS related opportunistic infections. HIV-1 Tat is known to increase the infectivity of Kaposi’s sarcoma-related herpesvirus (KSHV), a common opportunistic viral infection. We were able to demonstrate that this increase in KSHV infectivity was regulated by RIG-I and type-I IFN induced by HIV-1 Tat. Lastly, this study also demonstrated how HIV-1 Tat was able to manipulate the expression of IL-8 induced by KSHV in PBMac. HIV-1 Tat was able to mediate the production of IL-8 induced by KSHV by altering the phosphorylation of the p38 MAPK and the signal transducer and activator of transcription-1 (STAT-1). Taken together, the results of this study showed how c-Myc and RIG-I may be able to play critical roles in HIV-1 Tat induced cytokine dysregulation. Furthermore, the importance of these pathways is further demonstrated in their roles in regulating the immune responses against opportunistic infections in AIDS patients. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

abstract / Pathology / Doctoral / Doctor of Philosophy

Uncoupling proteins (UCPs) constitute a highly conserved subset of mitochondrial solute carriers. Discovered in small rodents in the early 1970’s, UCPs and their homologs have since been found in nematodes, plants, birds, and, most recently, in significant depots within humans (Krauss et al. 2005, Van marken Lichtenbelt 2009). Following activation by long chain fatty acids (LCFA, e.g. oleic acid) and reactive oxygen species (ROS, e.g. 4-hydroxynonenal (4HNE)), UCPs form a proton channel within the inner mitochondrial membrane and permit the influx of hydrogen ions from the inter membrane space into the mitochondrial matrix. UCPs effectively uncouple oxidative phosphorylation (OX-PHOS) from ATP generation, resulting in increasing oxygen consumption and dissipating the chemical energy in the form of heat. Found primarily in brown adipose tissue (BAT) of small hibernating mammals, the canonical role of uncoupling protein 1 (UCP1) in mammalian adaptive thermogenesis has been thoroughly studied. However, UCP1 is not the only member of the uncoupling family found within BAT. Also playing a key role in this tissue is uncoupling protein 3 (UCP3), which is a close homolog to UCP1. However, in spite of the fact that UCP3 shares more than 50% amino acid homology and tissue localization with UCP1, the true function UCP3 is very poorly elucidated. Part of the difficulty in determining this function lies in the expression levels of the UCP3 protein, which are hundreds of folds less than UCP1 in this tissue. In addition, their homologous structure makes teasing apart UCP3-specific phenomena from UCP1-mediated mechanisms very difficult using conventional techniques in cell and molecular biology. While UCP1 is almost exclusively found in BAT, UCP3 is expressed primarily in skeletal muscle (SKM), which lacks UCP1 completely (Krauss et al. 2005). Because UCP3 is so enriched in SKM, many studies have focused on its role in that tissue and have then tried to transpose these functions into BAT. As a result, UCP3 has been implicated in facilitating numerous biological processes, including non-adaptive facultative thermogenesis, affecting SKM oxidative capacity by modulating LCFA export, and ameliorating elevated levels of ROS-mediated stress within the tissue via glutathionine (GSH) interacting moieties. Ultimately, however, little consensus exists on the function of UCP3 within SKM, and subsequently, even less is known about its purpose in BAT. Previous data has shown that murine UCP1 has the capacity to bind to itself and form homo-tetramers when expressed in vitro in recombinant E. coli (Hoang T. et al. 2013). Here we show that UCP1 interacts with UCP3 in BAT in vivo, supporting Hoang’s research above by showing that UCP1 has the capacity to not only homodimerize but potentially oligomerize with other UCP homologs. While many groups using UCP3-null mice have reported no gross changes in physiologic responses, data previously published in the lab showed that mice lacking UCP3 were protected from potentially fatal hyperthermic effects when administered sympathomimetic agents such as 3,4-Methylenedioxymethamphetamine (MDMA), methamphetamine (METH), lipopolysaccharide (LPS), or norepinephrine (NE) (Mills et al. 2003, Kenaston et al. 2010). This implies that UCP3 plays an intimate role in sympathetic nervous system (SNS) mediated thermogenesis. Based upon the foregoing, the primary goal of the research discussed in this thesis was to elucidate the functions of UCP3 within BAT. In this study, we recapitulated results seen by other students in this lab: that global UCP3-null mice do indeed exhibit a blunted thermogenic response when treated with sympathomimetic agonists. In addition, despite the near-ubiquitous expression of UCP2 throughout the mammalian organism, this UCP is not involved in SNS-mediated thermogenesis (Arsenijevic et al. 2000). Our data shows that UCP3 is vital to the catecholamine-mediated thermogenic responses following sympathomimetic drug administration. When challenged by METH, UCP3-null mice were able to respond, albeit with a blunted increase in body temperature. Furthermore, when challenged by NE, a key neurotransmitter involved in mediating the responses initiated by the SNS following METH exposure, UCP3-null mice were able to mount half the hyperthermic response seen in WT littermates. However, UCP1/UCP3 double-null animals exhibited an almost four-fold hypothermic effect compared to WT littermates when challenged with NE. In addition, UCP1/UCP3 double-null mice were unable to restore body temperatures back to baseline values, an effect seen in all the other genotypes. This implies that UCP3 plays an important role in restoring body temperatures to physiological norms. Therefore, while the mechanism underlying the decreased responsiveness to NE remains unclear, it is clear that whether localized to SKM or BAT, UCP3 is a major player in the mammalian response to SNS-mediated thermogenesis and global thermoregulation. / text

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