Pathology & Laboratory Medicine Department Theses and Dissertations
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Item The impact of mTOR, TFEB and Bid on non-alcoholic fatty liver disease and metabolic syndrome(2015-05-18) Zhang, Hao; Yin, Xiao-Ming; Chalasani, Naga P.; Konger, Raymond Lloyd; Murrell, Jill R.Non-alcoholic fatty liver disease and metabolic syndrome induced by high nutrient status have increasingly become a global health concern as it cause multiple complications. The mTOR complex is central in regulating anabolic reactions within cells under growth factors or under high nutrients stimulation. Constitutive and persistent activation of mTOR can impair cellular functions. In the first part of this study, we demonstrate a damping oscillation of mTOR activity during a long-term treatment of high fat diet. TFEB translocation and lysosomal enzyme activity also oscillate, but in an opposite direction. TFEB controls the lysosomal activity, autophagic degradation and lipid metabolism. Overexpression of wild type and mutant TFEB could inhibit NAFLD development in mice. In addition, TFEB location in nucleus inversely correlates with NAFLD severity in patients. mTOR activation under hypernutrition status suppresses TFEB translocation, inhibits lysosomal functions and autophagic degradation of lipid droplets. Inhibition of mTOR activity by rapamycin reverse the above phenotypes. Because mTOR activation also requires normal lysosomal function, the inhibition of TFEB by mTOR leads to decreased lysosomal function and mTOR downregulation. This negative feedback may explain the oscillation pattern of mTOR activation in long term high fat diet regimen and is a novel mechanism for inhibition of mTOR. In the second part of study, we report that Bid protein, previously known for its pro-apoptosis function in promoting mitochondrial permeability, plays an unexpected role in regulating fatty acid beta oxidation. Deletion of Bid in mice reprograms the body's response to hyper-nutrition caused by high fat diet, leading to the resistance to the development of obesity, liver steatosis and metabolic syndrome. These mice present a higher oxygen consumption, a lower respiratory quotient, and an increased beta-oxidation rate. Mechanistically, the high fat diet regimen triggers translocation of the full length Bid molecule to mitochondrial membrane. Genetic deletion of Bid also affects the stability of its binding protein, MTCH2 in the mitochondrial membrane. In summary, we describe in this study a mTOR-TFEB-lysosome feedback loop, which can regulate NAFLD development, and a novel Bid-mediated regulatory mechanism in beta-oxidation, which limits energy expenditure and promotes obesity development.Item Investigation of peptide nucleic acid fluorescence in situ hybridization for diagnosis of ventilator-associated pneumonia in bronchoalveolar lavage specimens(2014-01-03) Phillips, Aaron M.; Davis, Thomas E.; Leland, Diane S.; Relich, Ryan F.Item Polyamines and Alveolar Macrophage Apoptosis during Pneumocystis Pneumonia(2009-10-01T18:06:46Z) Liao, Chung-Ping; Lee, Chao-Hung; Lasbury, Mark E.; Davis, Thomas E.; Gregory, Richard L.Pneumocystis pneumonia (PCP) is the leading opportunistic disease in immunocompromised individuals, particularly in AIDS patients. The alveolar macrophage (AM) is the major type of cell responsible for the clearance of Pneumocystis organisms; however, they undergo a high rate of apoptosis during PCP due to increased intracellular polyamine levels. This study examined the mechanism of this polyamine mediated apoptosis and investigated an alternative therapy for PCP by targeting this mechanism. The elevated polyamine levels were determined to be caused by increased polyamine synthesis and uptake. Increased polyamine uptake was found to be AM-specific, and recruited inflammatory cells including monocytes, B cells, and CD8+ T cells were found to be a potential source of polyamines. The expression of the antizyme inhibitor (AZI), which regulates both polyamine synthesis and uptake, was found to be greatly up-regulated in AMs during PCP. AZI overexpression was confirmed to be the cause of increased polyamine synthesis and uptake and apoptosis of AMs during PCP by gene knockdown assays. Pneumocystis organisms and zymosan were found to induce AZI overexpression in AMs, suggesting that the β-glucan of the Pneumocystis cell wall is responsible for this AZI up-regulation. In addition, levels of mRNA, protein, and activity of polyamine oxidase (PAO) were also found to be increased in AMs during PCP, and its substrates N1-acetylspermidine and N1-acetylspermine were found to induce its up-regulation. These results indicate that the H2O2 generated during PAO-mediated polyamine catabolism caused AMs to undergo apoptosis. Since increased polyamine uptake was demonstrated to be a pathogenic mechanism of PCP in this study, the potential therapeutic activity of five putative polyamine transport inhibitors against PCP was tested. Results showed that compound 44-Ant-44 significantly decreased pulmonary inflammation, organism burden, and macrophage apoptosis, and prolonged the survival of rats with PCP. In summary, this study demonstrated that Pneumocystis organisms induce AZI overexpression, leading to increased polyamine synthesis, uptake, and apoptosis rate in AMs and that targeting polyamine transport is a viable therapeutic approach against PCP.Item The peroxisome proliferator-activated receptor γ antagonist, GW9962, alters UVB-induced inflammatory responses, apoptosis, and delayed hyperproliferation(2009-01-16T17:25:58Z) Martel, Kellie Clay; Konger, Raymond L.; Travers, Jeffrey B.; Spandau, Dan F, 1957-It has recently been shown that the gamma subtype of the peroxisome proliferator-activated receptor (PPARγ) is a target of ultraviolet B (290-320 nm; UVB) irradiation, and that PPARγ activation is necessary for full UVB-induced cyclooxygenase-2 (COX-2) induction. However, the biological significance of PPARγ activation in cutaneous photobiology is unknown. Acute UVB irradiation results in a characteristic series of events in the epidermis which includes: an initial edema response and subsequent inflammation, COX-2 induction, apoptosis, and a delayed hyperproliferative response. Therefore, the regulatory role of PPARγ activation was examined in this acute photoresponse using a topical application of the potent, irreversible PPARγ antagonist, GW9962. GW9662 was applied to the epidermis of SKH1 hairless albino mice at increasing doses (0.01-1.0mM) prior to UVB irradiation. The photobiological responses were examined through RT-PCR, skin thickness measurements, and immunohistochemistry, at 24 and 72 hours after UVB-irradiation. At the highest dose, GW9622 significantly inhibited UVB-induced inflammation, as measured by COX-2 induction at both 24 and 72 hrs. Inflammation assessed by skin thickness measurements indicated that lower doses mildly increased inflammation at 72 hrs, but suppressed inflammation at the highest dose. In contrast, GW9662 treatment dose dependently augmented UVB-induced apoptosis at 24 hours, while affecting the delayed hyperproliferative response at 72 hours in an inverse dose-response manner. The results from this study suggest that PPARγ is a key regulator of these photobiological responses. Because these responses are well known to be involved in tumor development and progression, this study also suggests a potential role for PPARγ in UVB-induced skin cancers.Item A STUDY OF THE BETA-2 TOXIN GENE AND THE BETA-2 TOXIN IN CLOSTRIDIUM PERFRINGENS STRAINS ISOLATED FROM HUMAN SOURCES(2008-10-09T17:36:13Z) Roskens Dalzell, Heidi M.; Allen, Stephen D.Clostridium perfringens is an important human pathogen known to cause a range of diseases including diarrhea, necrotizing bowel disease and gas gangrene. Though potentially pathogenic, this microorganism is commonly identified in the fecal microbiota of healthy individuals. The major clinical findings associated with C. perfringens diseases are linked to production of potent toxins. In 1997, Gibert et al. identified a new toxin, the beta2 toxin, from a C. perfringens strain from a piglet with necrotic enteritis. Subsequently, this new beta2 toxin gene (cpb2) has been identified in C. perfringens from dogs, horses, and other animals. The principal objective of this investigation was to study cpb2 and the beta2 toxin in C. perfringens isolates from human sources. The C. perfringens isolates were grouped into three different populations: 1) fecal samples from patients suspected of having C. perfringens gastrointestinal illnesses (e.g. antibiotic-associated diarrhea or colitis), 2) extraintestinal specimen sources (e.g. wounds, abscesses, blood cultures), 3) a control group of isolates from healthy volunteers. Results of studies using different PCR methods and nucleotide sequencing revealed that cpb2 was present in the genome of isolates from all populations, and that the genetic variation between cpb2 from the different C. perfringens isolates was greater then expected. Using western blotting techniques, it was found that the beta2 protein was not expressed by all cpb2 positive C. perfringens isolates. Finally, different variants of cpb2 were cloned into E. coli, and the recombinant beta2 protein used in cell cytotoxicity assays. Results from these assays demonstrated that recombinant beta2 proteins caused a range of cellular damage at different levels of protein concentration and different lengths of time. Our results from these experiments provided new information regarding cpb2 in C. perfringens isolates from human sources; as well as on the range of variation of cpb2 genes, differences in beta2 toxin expression, and differences in the effects of recombinant beta2 toxin on enterocytes. This information could help to explain differences in virulence between C. perfringens isolates, differences in diseases and disease severity.