Exposure cigarette smoke (CS) during prenatal life is the leading cause of preventable premature death. In this study, we explored the hypothesis that in vitro exposure of fetal lung cells to cigarette smoke extract (CSE) may result in the alteration of apoptosis through activation of caspase-3. Alongside we compared the responses of fetal lung cells with A549 cells and rat periodontal ligament (PDL) fibroblasts exposed to CSE in a dose dependent manner. Caspase-3 activity and inhibition was measured using a fluorometric assay. Cell viability in smoke exposed cells was measured using MTT formazan assay. Caspase-3 expression and cellular localization was detected by western blot analysis and immunofluorescence. Our results indicate that caspase-3 activity was significantly (p < 0.05) elevated and cell viability was significantly inhibited in fetal rat lung cells exposed to 10% or 15 % (v/v) CSE. No significant differences were observed in the caspase-3 activity or cellular viability in A549 cells and rat PDL fibroblasts exposed to 5%, 10% or 15% (v/v) CSE. Activation of caspase-3 in fetal lung connective tissue and alveolar epithelial cells may be one of the reasons for the developmental pulmonary toxicity induced by CSE.

Exposure cigarette smoke (CS) during prenatal life is the leading cause of preventable premature death. In this study, we explored the hypothesis that in vitro exposure of fetal lung cells to cigarette smoke extract (CSE) may result in the alteration of apoptosis through activation of caspase-3. Alongside we compared the responses of fetal lung cells with A549 cells and rat periodontal ligament (PDL) fibroblasts exposed to CSE in a dose dependent manner. Caspase-3 activity and inhibition was measured using a fluorometric assay. Cell viability in smoke exposed cells was measured using MTT formazan assay. Caspase-3 expression and cellular localization was detected by western blot analysis and immunofluorescence. Our results indicate that caspase-3 activity was significantly (p < 0.05) elevated and cell viability was significantly inhibited in fetal rat lung cells exposed to 10% or 15 % (v/v) CSE. No significant differences were observed in the caspase-3 activity or cellular viability in A549 cells and rat PDL fibroblasts exposed to 5%, 10% or 15% (v/v) CSE. Activation of caspase-3 in fetal lung connective tissue and alveolar epithelial cells may be one of the reasons for the developmental pulmonary toxicity induced by CSE.

Dissertation (Ph.D.)--University of South Florida, 2008. / Title from PDF of title page. Document formatted into pages; contains 111 pages. Includes vita. Includes bibliographical references.

Abstract Apoptosis plays a crucial part in human ovarian function from fetal development to the end of reproductive potential. Failures in the regulation of ovarian apoptosis are associated with many pathological conditions such as premature ovarian insufficiency, infertility and cancer. The purpose of the present study was to analyze the factors regulating cell survival in human fetal and adult ovaries. The fetus is exposed to maternal- and placental-derived estrogens and insufficient estrogen action has destructive effects on rodent ovarian development. We detected estrogen receptors and estrogen-converting enzymes in human fetal ovaries after primordial follicle formation, indicating that estrogens participate in human fetal ovarian development, especially after folliculogenesis. The WNT4 gene is crucial for female sexual differentiation, follicle formation and oocyte survival. We detected WNT4 in follicular cells of fetal and adult human ovaries. In addition, Wnt4- knockout mice demonstrated a dramatic loss of oocytes before birth. However, no changes were detected in protein expression patterns of common apoptosis-related proteins. The results support the possible role of WNT4 in human ovarian function and strengthen previous knowledge on the antiapoptotic role of Wnt4. Apoptosis signaling is mediated by extracellular- and mitochondria-associated- pathways, ending in caspase cascade activation and fragmentation of cellular structures. In the present study we analyzed the expression of several apoptosis-related factors and detected TRAIL, TNF, Bcl-XL, Bok and caspase-3 in human ovaries. In addition, TRAIL was found to be a potent and rapid inducer of human granulosa tumor cell (KGN) apoptosis. Lentiviral downregulation of Bok or Bcl-XL protein expression in KGN cells also resulted in significant changes in cell vulnerability to apoptosis. The results show for the first time the spatiotemporal expression patterns of TRAIL, TNF, Bcl-XL, Bok and caspase-3 in human ovaries and suggest an important functional role of TRAIL, Bok and Bcl-XL in regulation of human ovarian apoptosis. The present study offers novel information on the expression and function of cell survival factors in human ovaries. These new findings open possibilities for future clinical research in attempts to understand and treat ovarian diseases caused by imbalanced regulatory pathways of apoptosis.

NAMPT (Nicotinamide phosphoribosyltransferase) catalyses the rate-limiting step in the NAD biosynthesis from nicotinamide and thereby regulates the activity of NAD-dependent enzymes. Cancer cells are highly dependent on NAD for energy and DNA repair processes and are assumed to be more susceptible to an inhibition of NAD synthesis than non-transformed cells. We aimed to investigate whether or not inhibition of NAMPT with its specific inhibitor FK866 can sensitize leukemia cells for chemotherapeutic agents. NAMPT protein abundance, enzymatic activity and NAD concentrations were significantly higher in Jurkat and Molt-4 leukemia cell lines compared to normal peripheral blood mononuclear cells. Combination of etoposide and FK866 caused increased cell death in leukemia cell lines compared to etoposide alone. Etoposide decreased protein abundance of NAD-dependent deacetylases SIRTUIN1. After combining etoposide and FK866 treatment SIRTUIN2 was further decreased and accumulation and acetylation of the downstream target p53 was further enhanced in MOLT4 cells. Concomitantly, protein abundance of p21 and cleaved BAX was increased. Targeting NAMPT could be a novel therapeutic strategy to enhance the efficacy of chemotherapeutic agents such as etoposide against leukemia.

Cancer is the second leading cause of death among men and women after heart disease. Though our knowledge associated with the complexities of the cancer network has significantly improved over the past several decades, we have only recently started to get a more complete molecular understanding of the disease. To better comprehend signaling pathways that prevent disease development, we focused our efforts on investigating endogenous tumor suppression networks in controlling effectors of cancer cell survival and proliferation. Survivin is one such effector molecule that controls both cell proliferation and survival. In order to identify how this protein is overexpressed in cancer cells as opposed to normal cells, we looked at signaling molecules that negatively regulate this inhibitor of apoptosis protein. PTEN and caspase 2 are two of the identified proteins that utilize their enzymatic activity to suppress tumor growth by inhibiting downstream cell survival effectors, namely survivin. PTEN uses its phosphatase activity to suppress the PI3K/AKT pathway and maintain cellular homeostasis. In the absence of AKT activity, FOXO transcription factors are able to target downstream gene expression and regulate cell proliferation and survival. Here we have identified survivin as a novel gene target of FOXO, which binds to a specific promoter region of survivin and suppresses its transcription. Alternatively, caspase 2 uses its catalytic activity to suppress survivin gene expression by targeting the NFκB pathway. Caspase 2 acts by cleaving a novel substrate known as RIP1 that prevents NFκB from entering the nucleus, thus inhibiting target gene transcription. Interestingly, survivin is known to be a direct gene target of NFκB that controls cancer cell survival. In our investigation, we found that survivin is downregulated upon caspase 2 activation via the NFκB pathway, resulting in decreased cell cycle kinetics, increased apoptotic threshold and suppressed tumor growth in mice. These studies conclude that survivin is a common effector molecule that is regulated by tumor suppressors to maintain cellular homeostasis. However, upon deactivation of the tumor suppressor pathway, survivin is deregulated and contributes significantly to disease progression. These observations may lead to potential therapeutic implications and novel targeting strategies that will help eradicate harmful cancer cells and spare surrounding healthy cells; often the most persistent problem of most conventional chemotherapy.

The innate immune system is an evolutionarily conserved primary defense system against microbial infections. One of the central components of innate immunity are the pattern recognition receptors which sense infection by detecting various conserved molecular patterns of pathogens and trigger a variety of signaling pathways. In this dissertation, the signaling pathways of several classes of these receptors were dissected. In chapters II and III, the role of two NOD-like receptors, NLRP3 and NLRC4 were investigated in the context of infection with the fungal pathogen, C. albicans. C. albicans is an opportunistic pathogen that causes diseases mainly in immunocompromised humans and innate immunity is critical to control the infection. In chapters II and III, we demonstrate that a multiprotein-inflammasome complex formed by the NLR protein, NLRP3 and its associated partners, ASC and caspase-1 are critical for triggering the production of mature cytokine IL-1β in response to C. albicans. NLRC4, another inflammasome forming NLR that is activated by intracellular bacterial pathogens, was not required for this process in macrophages. Thus, our data indicates that NLRP3 inflammasome responds to fungal infections in addition to its known stimuli such as bacterial and viral infections, toxic, crystalline and metabolic signals. Interestingly, this NLRP3 dependent inflammasome response was maintained even when the pathogen is not viable, and is either formalin fixed or heat-killed (HK). Hence, in chapter III, we examined β-glucans, a structural cell wall component, as the potential immunostimulatory component of C. albicans and dissected the inflammasome responses to β -glucans. We observed that NLRP3-ASC-caspase-1 inflammasome was critical for commercially obtained particulate β-glucans similar to the case of C. albicans. β-glucan sensing C-lectin receptor dectin-1 and the complement receptor CR3 mediated inflammasome activation, IL-1β production in response to the glucan particles. Interestingly, CR3 which recognizes glucans as well as complement opsonized pathogens was strongly required for HK C. albicans induced IL-1β, and partially required for that of live C. albicans, while dectin-1 was not required. Consistent with the receptor studies, blocking of β -glucan receptors by pre-incubating cells with nonstimulatory, soluble glucans led to decreased IL-1β production in response to HK C. albicanswith no effect on IL-1β in response to the live fungus. Dectin-1, CR3 and β-glucan sensing also triggered a moderate dendritic cell death response to β-glucans and HK C. albicans. Live C. albicans induced cell death requires phagocytosis but not the inflammasome, β-glucan sensing, dectin-1 or CR3. The Drosophila caspase-8 like molecule DREDD plays an essential, nonapoptotic role in the Drosophila NF-κB pathway called the ‘IMD’ pathway. Owing to the remarkable evolutionary conservation between Drosophila and mammalian innate immune NF-κB pathways, we explored the potential role of caspase-8 in inflammasomes and in TLR signaling. Using casp8-/- Rip3-/- macrophages and dendritic cells, we observed that caspase-8, specifically augments β-glucan and HK C. albicans induced IL-1β as well as cell death in a caspase-1 independent manner, but not that of live C. albicans, in chapter III. We also found that caspase-8 differentially regulates TLR4 and TLR3 induced cytokine production (chapter IV). Caspase-8 specifically promotes TLR4 induced production of cytokines such as TNF, IL-1β in response to LPS and E. coli. On the other hand, caspase-8 negatively regulates TRIF induced IFNβ production in TLR4 and TLR3 signaling in response to LPS and dsRNA. Caspase-8 executed a similar mode of regulation of the cytokine RANTES in MEFs, in part, by collaborating with RIP3. Strikingly, caspase-8 deficiency alone triggers higher macrophage death and IL-1β production in response to TLR ligands, due to the presence of RIP3. Thus, in addition to its conventional roles in apoptosis, caspase-8 modulates TLR4 and TLR3 induced cytokine production and prevents RIP3 mediated hyper inflammation in response to TLR signals. Together, our findings provide valuable information on fungal pattern recognition and inflammasome pathways and define the contribution of β-glucan sensing to C. albicans induced inflammasome responses. In addition, we demonstrate how caspase-8 adds a layer of specificity to inflammasome as well as TLR signaling. Overall, these results also shed light on the cross talk between death signaling components and innate immune pathways to mount a specific and potentially effective innate immune response against microbial pathogens.

Previously it has been shown that cultures of Plasmodium falciparum died following exposure to a febrile temperature of 40°C, as demonstrated by a decrease in parasitemia of the following generation. In the current study, the effect of 40°C treatment on culture media, erythrocytes, and parasite glucose consumption, were ruled out as possible influences on parasite death, demonstrating that 40°C impacted the parasites directly. Metabolic profiling of DNA synthesis, protein synthesis, and glucose utilization during exposure to 40°C clearly indicated that febrile temperatures had direct effect on major metabolic pathways and parasite development, beginning 20-24 hr after erythrocyte invasion. The ring stages were relatively refractory to heat and recovered completely if returned to 37°C. The mechanism of parasite death was investigated for evidence of an apoptosis-like pathway in cells treated with 40°C, chloroquine, and staurosporine. Lack of typical physiological hallmarks, namely, caspase activation, characteristic mitochondrial membrane potential changes, and DNA degradation as indicated by DNA laddering, eliminated ‘classical’, apoptosis as a mechanism of parasite death. Parasites dying under the influence of 40°C, staurosporine, and chloroquine initially appeared pyknotic in light and electron microscopy, as in apoptosis, but eventual swelling and lysis of the food vacuole membrane led to secondary necrosis. Initially, chloroquine did induce DNA laddering, but it was later attributed to occult white blood cell contamination. While not apoptosis, the results do not rule out other forms of temperature-induced programmed cell death.

No description available.

No description available.