Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Sphingolipids are a complex family of molecules that participate in many aspects of cell structure and function, including an essential cellular process known as autophagy. Autophagy is a degradation and recycling pathway whereby intracellular components are sequestered into double-membrane vesicles, known as autophagosomes, for subsequent fusion with lysosomes and degradation. Autophagy takes part in cell survival, host immune defense against pathogens, and other biological processes, but is also sometimes lethal. Ceramide, sphingosine 1-phosphate, and more recently dihydroceramide have been shown to induce autophagy, which opens an interesting new field of cell regulation by sphingolipids. This dissertation describes two new cases in which sphingolipids participate in the induction of autophagy: a) RAW264.7 cells treated with Kdo2-Lipid A, a lipopolysaccharide sub-structure with endotoxin activity equal to LPS; and b) MCF7 cells treated with fenretinde, a chemotherapeutic agent which has shown success in clinical trials. It also analyzes the structural properties of fenretinide that contribute to its ability to modulate sphingolipid metabolism through inhibition of dihydroceramide desaturase, thereby elevating dihydroceramide and induction of autophagy. Autophagy was monitored by following the redistribution of GFP-LC3 into discrete punctate vesicles in response to the agents and by Western blotting; in parallel, the sphingolipid composition of the cells was monitored by liquid chromatography, electrospray ionization tandem mass spectrometry. These analyses revealed that Kdo2-Lipid A and fenretinide induce profound changes in sphingolipid metabolism in RAW264.7 and MCF7 cells, respectively, and that one of the purposes for increased de novo biosynthesis is to enable the production of autophagosomes, as the autophagic response was inhibited by myriocin. These studies have uncovered a direct link between sphingolipid metabolism and autophagy, which could pave the way for new therapeutic interventions for the treatment of pathogenic infection and be clinically useful in enhancing the efficacy of current cancer treatment strategies.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Medical Sciences - Medical Genetics. Title from pdf file main screen (viewed on March 19, 2010). Includes bibliographical references.

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The goal of the current research was to examine the expression, signaling and function of the membrane progestin receptors (mPRs) in the ovarian follicular cells of the Atlantic croaker (Micropogonias undulatus) and in human breast cancer cells. Multiple studies have examined the role of mPRs in the germ cells of several vertebrate classes, yet few studies have examined the role of the mPRs in the somatic cells of reproductive tissues. Therefore this research examines the mechanism of mPR action and its function in somatic cells of female reproductive tissues. Results from studies on the expression, localization and signaling of the mPR[alpha] in co-cultures of granulosa and theca cells from the croaker suggest that the mPR[alpha] is localized to the plasma membrane of both cell types and that the mPR[alpha] is associated with and signals via pertussis toxin-sensitive inhibitory G proteins to decrease intracellular cAMP and activate ERK. In addition, exposure of follicular co-cultures to progestins that activate the mPR[alpha] results in a decrease in serum starvation-induced cell death which is not replicated by progestins which activate the nuclear progestin receptor (nPR), indicating mPR mediation. Similar studies in two immortalized human breast cancer cell lines, MDA-MB-468 and SKBR3, suggest that the mPR[alpha] is also present in the membranes of these cells and signals in human breast cancer cell lines via activation of a pertussis toxin-sensitive G protein to significantly decrease in intracellular cAMP and activate ERK. Progesterone exposure also decreased serum starvation-induced cell death in SKBR3 cells which are nPR positive and in MDA-MB-468 cells which are nPR negative. Synthetic progestins which activate the nPR but not the mPR were ineffective in inhibiting death in either cell type suggesting that the mPR is the mediator of this progestin action. mPR[alpha], mPR[beta] and mPR[gamma] expression analysis of paired normal and malignant breast tissue biopsies from thirteen women revealed that at least one mPR isoform was upregulated in the malignant tissue of 70% of the women. In addition the expression of mPR[gamma] was positively correlated with the expression of the nPR and CK19, a breast epithelial cell marker. / text

This thesis reports that checkpoint adaptation occurs in human brain cancer cells. M059K cells, after treatment with camptothecin (CPT), recruited γ-histone H2AX, phosphorylated Chk1 and arrested in the G2 phase. Strikingly, cells escaped the checkpoint, became rounded and entered mitosis as measured by phospho-histone H3 signals. Lamin A/C immunofluorescence microscopy revealed that 48% of the cells that survived checkpoint adaptation contained micronuclei. These data suggest that brain cancer cells undergo checkpoint adaptation and may have an altered genome. This thesis also explored if phosphatases participate in checkpoint adaptation. Human colon cancer cells were treated with CPT and the PP2A inhibitor cantharidin. Following treatment the cells became rounded and 65% were positive for phospho-histone H3 signals indicating that cantharidin caused cells to be in mitosis following CPT treatment. These data suggest that PP2A might have a role in checkpoint adaptation, or participate in a pathway that bypasses checkpoint adaptation. / xi, 114 leaves : ill. (some col.) ; 29 cm

Bibliography: p. 69-81. / 81, [52] p., [16] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis demonstrates that string (a homologue of the mitotic initiator cdc 25 from Schizosaccharomyces pombe) is a downstream target of the patterning genes, making a direct connection between patterning information and morphogenesis, which suggests that mitotic timing forms an independent and important part of morphogenesis. / Thesis (Ph.D.)--University of Adelaide, Depts. of Biochemistry and Genetics, 1997

[Truncated abstract] Neuregulins (NRG’s) are pleiotropic growth factors that participate in a wide range of biological processes. The family of membrane-bound growth factors bind to and activate ErbB receptors on adjacent target cells, mediating multiple biological processes. NRG-1, NRG-2 and NRG-3 are all highly expressed in the nervous system, where it has been shown that NRG-1 is important for neuronal development, migration, synapse formation and glial cell proliferation. Little is known, however, on the specific roles of NRG-2 and NRG-3, although it is apparent that despite similar expression patterns and overlapping receptor specificity, NRG-2 and NRG-3 do not compensate for the loss of NRG-1 and mediate their own distinct activities. … Subcellular localisation experiments showed that this domain is important for trafficking of the fulllength protein to various intracellular compartments in an activity dependent manner. In addition, the ICD is required to elicit a cell death response in cultured cells and provoke an elevated α-amino-3-hydroxyl-5-methylisoxazole-4-propionate (AMPA) response in organotypic neuronal cultures following transient expression of NRG-3. A yeast two-hybrid screen identified 14-3-3ζ and PICK1 as two proteins that interacte with the human NRG-3 ICD. These interactions were confirmed both in vitro and in vivo, and were further characterised at a molecular level. This study demonstrates the ability of NRG-3 to mediate signal transduction through a biologically active ICD; a conclusion supported by identifying cytoplasmic proteins that interact with the ICD. These observations point to an additional layer of complexity where bi-directional signalling contributes to the full repertoire of NRG-3 functions.

[Truncated abstract] A range of biotic and abiotic stresses increase levels of reactive oxygen species (ROS) in plants due to perturbations of chloroplast and mitochondrial metabolism and the generation of ROS in defence responses. The polyunsaturated fatty acids of membrane lipids are susceptible to ROS induced peroxidation yielding various aldehydes, alkenals and hydroxyalkenals including the cytotoxic compound 4-hydroxy- 2-nonenal (HNE). HNE has the potential to cause substantial oxidative damage in cells via its reactivity with sulfhydryl groups of cysteine (Cys) and lipoic acid, the imidazole group of histidine (His) and the ?-amino group of lysine (Lys) protein residues. Analysis of the components of the plant respiratory electron transport chain to HNE revealed a particular susceptibility to inhibition of activity of the alternative oxidase (Aox). Incubation with HNE prevented dimerisation of Aox protein, suggesting that one site of modification was the conserved cysteine residue involved in dimerisation and activation of this enzyme (Cys1). However, a naturally occurring isoform of Aox lacking Cys1 and unable to dimerise, LeAox1b from tomato, was equally sensitive to HNE inhibition, showing that other amino acid residues in Aox also interact with HNE and are likely responsible for inactivation of the enzyme. ... The broader impact of HNE on the whole Arabidopsis mitochondrial proteome was examined by use of various 2-dimensional gel separation techniques coupled with use of HNE-adduct antibodies. 32 proteins involved in a number of mitochondrial functions were found to be susceptible to modification by HNE, including components of the electron transport chain, the TCA cycle, as well as proteins involved amino acid metabolism and stress-responses. Implications of modification of these proteins by HNE are discussed. As HNE is produced in vivo during oxidative stress, the profile of mitochondrial targets of HNE was examined from Arabidopsis cell cultures exposed to various oxidative stress inducers. Menadione and hydrogen peroxide induced oxidative stress throughout the cell, while antimycin A initiated a mitochondrial targeted stress. A differential profile of mitochondrial proteins was observed to be modified by HNE in the various treatments. These results also showed that induction of stress within a whole cell can impact lipid peroxidation within the mitochondria. Overall, this work showed the presence and production of HNE in plant cells, and that HNE, both exogenous and endogenous, has the ability to modify a specific subset of mitochondrial proteins. In several cases this HNE modification was shown to have functional or structural consequences.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.