Severe bloody diarrhea and subsequent serious post-diarrheal illnesses, including the hemolytic uremic syndrome and central nervous system complications, may develop following infections with Shiga toxin (Stx)-producing bacteria. The cytotoxic actions of Stxs destroy the microvasculature of organs, preventing function. A role for the cytokines tumor necrosis factor-alpha (TNF-[alpha]) and interleukin-1 beta (IL-1[beta]) in exacerbating disease may lie in their ability to up-regulate the Stx receptor, Gb3, on endothelial cell surfaces. A main source of proinflammatory cytokines is the macrophage, thus leading us to utilize the monocytic/macrophage-like cell line, THP-1, as a model for cytokine production in Stx pathogenesis. In addition to treating THP-1 cells with purified Stx1, cells were also treated with lipopolysaccharides (LPS), since bacterial LPS are known to be potent inducers of cytokines, and may be present during infection. Undifferentiated THP-1 cells are sensitive to Stx1 and do not produce TNF-[alpha] or IL-1[beta], while differentiated THP-1 cells, a better model for resident tissue macrophages, are less sensitive to Stx1 and produce TNF-[alpha] and IL-1[beta]. Prolonged expression of TNF-[alpha] mRNA over a 12 h time course experiment led us to inquire whether the extended elevation of transcripts involved Stx1induced mRNA stability. Our data suggest that the presence of Stx1 increases the stabilities of TNF-[alpha] and IL-1[beta] transcripts. In contrast to TNF-[alpha], the level of secreted IL-1[beta] protein does not correlate with the level IL-1[beta] mRNA, suggesting an alteration of post-translational processing and/or secretion of IL-1[beta]. Differentiated THP-1 cells produce chemokines in response to Stx1 and/or LPS treatments. Chemokines may enhance the destruction of tissue cells during an infection by mediating an inflammatory cell influx. Comparison of cytokine and chemokine mRNA and protein kinetics suggests that the regulation of expression may differ between individual cytokines and chemokines. Extension of experimental time courses demonstrated THP-1 cell sensitivity to killing by Stx1, especially in the presence of LPS. Further experiments revealed that undifferentiated and differentiated THP-1 cells were induced to undergo apoptosis following treatment with Stx1, LPS, and Stx1+LPS, and that caspase activation was involved. Collectively, these results allowed us to propose a model of the role of macrophages in Stx1 pathogenesis.

Apoptosis in testicular germ cells has been demonstrated in many species. Features of apoptosis reported in other species were used to confirm use of the TUNEL assay in stallion testes. Eight stallions with normal testicular size and semen quality were evaluated to determine the germ cell types and stages where apoptosis most commonly occurs. Mean numbers of TUNEL-positive germ cells per 100 Sertoli cell nuclei were highest in stages IV and V of the seminiferous epithelial cycle corresponding to meiotic divisions of primary spermatocytes and mitotic proliferation of B1 and B2 spermatogonia. Round and elongated spermatids were labeled less frequently by the TUNEL assay. To examine the relationships between germ cell apoptotic rate and spermatogenic efficiency, seminal traits were assessed to classify stallions into normal or reduced quality semen groups. Apoptotic rates were higher for stages IV-VI and stage VIII seminiferous tubules in stallions with reduced semen quality. Daily sperm production (DSP) per gram and per testis were lower for stallions with reduced semen quality. Regression analyses revealed negative linear relationships for germ cell apoptotic rate with DSP/g, DSP/testis, daily sperm output, progressively motile sperm and morphologically normal sperm in ejaculates. Mean circulating concentrations of inhibin were lower for stallions ejaculating reduced quality semen. Apoptotic rate was negatively correlated with concentrations of inhibin and estradiol-17b and positively correlated with concentrations of LH and FSH. To study germ cell apoptosis and formation of the Sertoli cell barrier during the initiation of spermatogenesis, tubule development was classified based on lumen score. Formation of a seminiferous tubule lumen was consistent with events leading to development of a Sertoli cell barrier. A primary wave of apoptosis removed early differentiating germ cell types prior to the formation of a tubule lumen facilitating both the formation of a tubule lumen and a Sertoli cell barrier. A second wave of apoptosis occurred after the formation of a lumen but before seminiferous tubule cross-sections contained a full complement of germ cells. In conclusion, apoptosis is an essential mechanism during normal spermatogenesis. Apoptosis also accounts for low numbers of normal sperm seen in the ejaculates of some stallions.

Thesis (M.S.)--Marshall University, 2009. / Title from document title page. Includes abstract. Document formatted into pages: contains : 89 p. Includes bibliographical references : p. 44-48.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 152-196).

Thesis (M.S.)--California Polytechnic State University, 2009. / Title from PDF title page; viewed on January 6, 2010. Major professor: Scott Hazelwood, Ph.D. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Engineering With a Specialization in Biomedical Engineering." "December 2009." Includes bibliographical references.

Regensburg, Universität, Thesis (doctoral), 2001.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 127-155). Available also in a digital version from Dissertation Abstracts.

Apoptosis is a biologically essential phenomenon executed in large part by caspases. Members of the caspase family are activated at different points during apoptosis to proteolyze specific substrates. Given that both excessive and insufficient apoptosis is related to the pathogenesis of various diseases, proper regulation of caspases and apoptosis is necessary for the health of living organisms. Inhibitor of apoptosis (IAP) proteins are endogenous inhibitors of caspases, and since XIAP, the prototypical IAP, binds to and inhibits caspases, all IAPs have been speculated to engage in similar inhibition mechanisms. However, in this dissertation, I demonstrate that cIAP1 binds to the effector caspases-3 and -7, through distinct mechanisms. cIAP1 readily binds to and ubiquitinates, but dos not directly inhibit the activity of fully mature caspase-7. By contrast, cIAP1 does not bind to caspase-3. cIAP1 binding to caspase-7 is mediated primarily by the N-terminus of the large subunit of caspase-7. An AKPD motif located on the N-terminus of caspase-7 is involved in the proteasome-mediated degradation of caspase-7 in cells, thereby decreasing the sensitivity of these cells to apoptosis. Thus, I demonstrate for the first time that cIAP1 is capable of inhibiting caspase-dependent apoptosis through indirect regulation of caspase activity. / text

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Autophagy is a major catabolic pathway for maintaining cell homeostasis through degradation and recycle of macromolecules and organelles. Autophagy can be activated under environmental stress conditions, including reactive oxygen species (ROS). TRPM2, a non-selective trans-membrane calcium channel, can be activated by ROS that, in turn, leads to intracellular 〖Ca〗^(2+) increase through 〖Ca〗^(2+) influx. It is well known that ROS regulates autophagy, and vice versa. Yet, the molecular mechanisms underlying the interplay between ROS and autophagy remain elusive. Here we studied the role of TRPM2-mediated 〖Ca〗^(2+) influx in interplay between ROS and autophagy. From our study, we found that ROS activated TRPM2 for 〖Ca〗^(2+) influx via ADPR to inhibit early autophagy induction, which ultimately led to apoptosis in TRPM2 expressing cancer cell lines. On the other hand, ROS induced autophagy, not apoptosis, for cell survival in cancer cell lines which do not express TRPM2, and autophagy inhibition, either by ATG5 knockdown or by treating cells with bafilomycin A1 (an autophagy inhibitor), converted cells to apoptosis upon ROS treatment. In addition, ROS dramatically changed mitochondrial morphology, increased mitochondrial 〖Ca〗^(2+) content, and abolished mitochondrial membrane potential in TRPM2 expressing cells. Moreover, we found that ROS-induced Ca2+ influx via TRPM2 actually activated calmodulin-dependent protein kinase II (CaMKII) to phosphorylate Ser295 on Beclin1. Phosphorylated Beclin1, in turn, decreased the association between Beclin1 and VPS34, but induced the binding between Beclin1 and BCL-2. In summary, our data demonstrated that the TRPM2/〖Ca〗^(2+)/CaMKII/ Beclin1 cascade is the molecular switch between autophagy and apoptosis in response to ROS. Since dysregulation of ROS and autophagy has been associated with a variety of human diseases, e.g. cancer, neurological disorders, heart diseases, and liver diseases, manipulating the TRPM2/〖Ca〗^(2+)/CaMKII/ Beclin1 cascade should provide novel treatment option for these diseases. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy