Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Jul 7.

Thesis (Ph.D.)--Ohio University, November, 2006. / Title from PDF t.p. Includes bibliographical references.

Thesis (M.S.)--University of Massachusetts Amherst, 2008. / Includes bibliographical references (p. 33-40).

The obligate human pathogen Neisseria gonorrhoeae is responsible for the sexually transmitted disease, gonorrhea. This pathogen colonizes mucosal surfaces, and is most commonly found in the urogenital tract. The genital mucosa is comprised of various cells from epithelial to immune cells including the macrophage. Macrophages are abundant immune cells within the genital submucosa. Though the cytokine response of macrophages following N. gonorrhoeae infection is well characterized, survival of these cells following infection has not been well described. In this study, we examined the ability of N. gonorrhoeae strain FA1090B to modulate cell death in differentiated THP-1 cells (dTHP-1) and human monocyte-derived macrophages (MDMs) harvested from peripheral blood. N. gonorrhoeae was demonstrated to induce cell death in both macrophage types in a dose-dependent manner as measured at 6 hours post-stimulation. Cell death did not proceed via classical apoptosis but was associated with activation of immune caspases-1 and -4, required for the canonical and non-canonical pyroptotic pathways, respectively. MDM cell death was found to be dependent on immune caspase activity and associated with intracellular bacteria. Furthermore, caspase-4-associated MDM cell death was also observed with cytosolic N. gonorrhoeae-purified lipooligosaccharide (LOS). We did not however observe differences in the induction of pyroptosis by a penta-acylated non-immune stimulating LOS mutant strain, 1291ΔmsbB, as compared to the isogenic wild type strain 1291, or strain FA1090B. Activation of pyroptosis correlated with increased production of the pro-inflammatory mediators IL-1β, IL-6 and TNF-α. Pre-treatment of dTHP-1 cells with conditioned media from bacterial stimulated samples had little effect on N. gonorrhoeae induced cell death. Collectively, our results demonstrate that N. gonorrhoeae induces pyroptosis in human macrophages due, in part, to LOS. We postulate that N. gonorrhoeae induced pyroptosis of macrophages may partially contribute to lack of immunological memory and continual neutrophil recruitment, a hallmark of N. gonorrhoeae infection.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Apoptosis, or programmed cell death, is a genetically controlled form of cell suicide used to rid an organism of superfluous or damaged cells and serves as one of the major mechanisms for patterning during the development of complex animal structures. The antenna and ovary of the fruitfly, Drosophila melanogaster, were chosen as model systems in which to examine the molecular mechanisms of developmental apoptosis. The caspases are a family of cysteine proteases required for the execution of cell death, while the inhibitor of apoptosis protein Diap-1 is responsible for repressing the function of caspases. Diap-1, also known as Thread, was discovered in 1922 with the isolation of thread^1, a homozygous viable mutant whose molecular nature is unknown. thread was given its name due to its branchless or thread-like arista, the featherlike structure at the tip of the antenna. thread^1 antennal imaginal discs show excessive cell death during a brief period of larval development, which corresponds to a significant decrease in levels of Thread protein. Caspase activity is found more broadly than apoptotic DNA fragmentation in thread1 imaginal discs, suggesting that the mutant fails to inhibit caspases in many cells, but only a fraction succumb to apoptosis. These findings point to a narrow window of development in which regulation of programmed cell death is essential to the formation of the arista. Additionally, a candidate mutation has been discovered in a transcriptional regulatory region of the thread gene. Proof that this mutation is responsible for the branchless aristal phenotype may have important implications for understanding tissue-specific gene regulation during organogenesis. In the ovary, apoptotic roles for the DP subunit of the E2F transcription factor and the actin binding protein Profilin were investigated. While mutants for DP affect the regulation of Cytochome c and caspase activity in nurse cells, Profilin mutants show milder effects. To further characterize the role of apoptosis in the ovary, an ethylmethane sulphonate (EMS) mutagenesis screen was conducted and has led to the identification of several new genes. Future study of the apoptotic pathway will assist in developing treatments for diseases associated with its misregulation. / 2031-01-02

Thesis (MTech (Biomedical Technology))--Cape Peninsula University of Technology, 2017. / Apoptosis is the dominant mechanism of cell death induced by radiation and is the key mechanism used to remove cells with significant DNA damage. Previous research investigated the feasibility of using the Leukocyte Apoptosis Assay (LAA) to determine individual sensitivity to radiation and it was found that an apoptotic response could be loosely linked to age, race and gender. Apoptosis is controlled by the Bcl-2 proteins and therefore the balance between Bax and Bcl-2 protein expression is important. With this background it would be relevant to know why certain individuals are more sensitive to radiation than others. The objectives of this study was to evaluate the effect of ionising radiation on apoptotic proteins, Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) expression and to explore if there is a relationship between radiation induced apoptosis (RIA) and Bcl-2 or Bax expression.

Abstract All-trans retinoic acid (ATRA) is a derivative of vitamin A. It is able to stimulate neutrophilic differentiation of normal progenitors and acute promyelocytic leukemia (APL) cells. Although ATRA-induced differentiation is not observed in any other acute myeloblastic leukemia (AML) subtypes, ATRA is known to be able to inhibit AML blast cell proliferation. The present in vitro study using AML cell lines representing subtypes other than APL focuses on the following questions: (1) Is the inhibitory effect of ATRA on AML cell growth related to apoptosis of cells? (2) Are the effects of ATRA dependent on two important regulators of apoptosis, p53 and Bcl-2? (3) Do mitochondria have any role in mediating the effects of ATRA? ATRA-induced apoptosis in AML cells was observed by morphology, DNA fragmentation, phosphatidylserine externalization, and poly(ADPribose)polymerase (PARP) cleavage. It was a slow event, manifested as DNA cleavage after 48 hours exposure and as morphological apoptosis after 72 hours exposure. The AML cells expressed constitutively p53 as determined by immunohistochemistry, Western blotting and flow cytometry. However, no mutation of TP53 was observed in exons 5 to 8 as analysed with a single strand conformation polymorphism technique. As the flow cytometer analysis showed, most of p53 was in a aberrant conformation, which was not changed into a wild type conformation by ATRA. Two of the cell lines were analysed more specifically in relation to Bcl-2 and mitochondral function: ATRA-induced apoptosis of the cell lines was associated with down-regulation of Bcl-2. Western blotting showed ATRA-induced apoptosis also to be related to the release of cytochrome c from mitochondria into cytosol, resulting in the activation of caspase-3, an apoptotic effector, which was manifested as a cleavage of its substrate PARP. The process was also accompanied by disruption of the mitochondrial membrane potential as determined fluoricytometrically. These results show that ATRA is able to induce apoptosis in AML cells other than APL, and ATRA-induced apoptosis in the AML cells studied is related to the down-regulation of Bcl-2 and the disruption of mitochondrial function, but is independent of the p53 pathway.

M.Tech. (Biomedical Technology) / The uncontrolled growth of cells in the body is often associated with cancer. It constitutes a major health problem and is one of the leading causes of death in the world. Cancers of the lung, breast, colon/rectum and prostate are no longer only associated with developed countries but are the most common occurring cancers worldwide. Breast cancer is the leading cancer faced by women in South Africa as well as in the world. Conventional cancer therapies often result in uncertain outcomes with numerous side effects and may be associated with limited therapeutic advantage. This has led to the development of safer and better treatment regimes with improved therapeutic outcomes. Photodynamic therapy (PDT) is a treatment used for a wide range of conditions, including cancer. This treatment utilises a photosensitiser (PS), a light activated chemotherapeutic agent, and light of a specific wavelength and power density. It is based on the selective tumour localisation of the PS and the ability to generate high levels of reactive oxygen species (ROS) in the presence of light. The generation of ROS causes permanent damage to the tumour cells resulting in cancer cell death. The distinctive criteria when comparing different PDT modalities is the choice of PS as the treatment outcomes are greatly influenced by the light dependent properties of the chemotherapeutic agent. Phthalocyanines are second generation PSs used in PDT. Effects of members of this PS family have been studied and they exhibited good photosensitising properties including lack of cytotoxicity in the absence of light, extended retention times in the tumour and high triplet lifetime of singlet oxygen species.

Artemisia afra is one of the oldest, most well known and widely used traditional medicinal plants in South Africa. It is used to treat many different medical conditions, particularly respiratory and inflammatory ailments. There is no reported evidence of its use for the treatment of cancer but due to its reported cytotoxicity, an investigation of the mode of cell death induced by an ethanol A. afra extract using two cancer cell lines was done. IC50 values of 18.21 and 31.88 μg/mL of ethanol extracts were determined against U937 and HeLa cancer cells, respectively. An IC50 value of the aqueous extract was greater than 250 μg/mL. The ethanol extract was not cytotoxic against confluent control cell lines, Chang Liver and Vero cells. The effect of the cytotoxic ethanol A. afra extract on U937 and HeLa cells and their progression through the cell cycle, apoptosis and mitochondrial membrane potential was investigated. After 12 hours of treatment with A. afra a delay in G2/M phase of the cell cycle was evident. Apoptosis was confirmed using the TUNEL assay for DNA fragmentation, as well as fluorescent staining with annexin V-FITC. Apoptosis was evident with the positive control and A. afra treatment at 24 and 48 hours. JC-1 staining showed a decrease in mitochondrial membrane potential at 24 hours. It was deduced that A. afra ethanol extract induces caspase-dependent apoptosis in a mitochondrial dependent manner. Plants harbour many compounds that are not only useful to the plants but also to mankind. Many metabolites have been isolated from A. afra and their biological activity characterised. Due to observed apoptosis induction, isolation of cytotoxic compounds was done and a new sesquiterpene lactone from A. afra was isolated. Structural elucidation of the compound was done by IR, 1D and 2D NMR, CD and mass spectrometry and it was identified as isoalantolactone. HeLa cancer cells were treated with isoalantolactone and cytotoxicity was exhibited in a dose-dependent manner. A low IC50 value of 8.15 ± 1.16 μM was achieved. This study showed that isoalantolactone is partly responsible for the observed A. afra cytotoxicity. Due to the evidence of G2/M arrest, the anti-mitotic potential and the possible onset of mitotic catastrophe by A. afra and isoalantolactone was investigated. It was evident from various flow cytometric analysis of cyclin B1 and phospho-H3 and confocal microscopy that A. afra does possess anti-mitotic activity by causing hyperpolymerisation of tubulin and cells progress into the mitotic phase where M arrest is experienced. The anti-inflammatory activity of sesquiterpene lactones is well documented; however, the anti-inflammatory activity of A. afra is not. Here, it is reported that the production of NO and COX-2 protein levels in RAW 264.7 cells decrease in the presence of A. afra and isoalantolactone after stimulation with LPS. The activated NF-κB subunit, p65 was also investigated. The results suggest that A. afra and isoalantolactone inhibit p65 activation as a decrease in the activated subunit was evident. Thus, the results indicate that exposure to A. afra and isoalantolactone induces an anti-inflammatory response. In conclusion, this study shows, for the first time, the mechanism of induced apoptosis, the anti-mitotic and anti-inflammatory activity of A. afra and its isolated compound, isoalantolactone. It also proves that although extensive research may have been done on a particular plant, as with A. afra, more can be discovered leading to the identification of new compounds and integration of signalling pathways that can be exploited for the treatment of various diseases and ailments.

Maternal tolerance during pregnancy increases the risk of infection with certain intracellular pathogens such as Salmonella enterica serovar Typhimurium (S.Tm). Systemic S.Tm infection during pregnancy in normally resistant 129X1/SvJ mice, with a functional natural resistance-associated macrophage protein-1 (Nramp1), leads to severe placental infection followed by fetal and maternal death. We hypothesized infection-induced inflammatory trophoblast cell death contributes to adverse pregnancy outcomes. We therefore investigated the kinetics of systemic and oral S.Tm infection in wild-type and gene deficient mice with defects in specific inflammatory pathways. Systemic infection with S.Tm resulted in preferential placental replication compared to other tissues in Nramp1+/+ mice. At 24 hours, <25% of individual placentas per mouse were infected, progressively increasing to >75% by 72 hours which correlated with a steady increase in resorption rates. Moreover, placental infection was associated with increased neutrophils, macrophages and natural killer cells whereas neutrophil numbers in the spleen remained unchanged, suggesting dichotomous modulation of inflammation in the systemic compartment compared to the feto-maternal interface. Oral infection resulted in systemic dissemination of the bacteria, substantial placental colonization and fetal loss five days post-infection in C57BL/6J mice. Systemic infection in pregnant cell death deficient Rip3-/-Nramp1+/+ mice (with defective necroptosis) resulted in decreased fetal demise relative to Nramp1+/+ and Caspase-1,11-/-Nramp1+/+ mice (with defective pyroptosis) suggesting a role for necroptotic inflammation. This study provides insight into the kinetics and mechanism of inflammation and cell death during placental S.Tm infection. Such studies may assist in the rational management of foodborne pathogens contracted during pregnancy.