The precise structure of the adult Drosophila eye results from a coordinated process of cell sorting, differentiation and selective cell death in the retinal epithelium. Mutations in the gene echinus cause supernumerary pigment cells due to insufficient cell death. This study reports the identification of echinus and the characterization of its role in Drosophila retinal development. Using a combination of deletion mapping, gene expression analysis and genomic sequencing, echinus was cloned and several alleles were sequenced. echinus encodes a ~180kDa protein containing an ubiquitin hydrolase domain at its N-terminus and a polyglutamine tract at its C-terminus. echinus is expressed in the retina during pupal development and mutants of echinus have decreased levels of apoptosis during several stages of retinal development. Defects in the cell sorting process that precedes cell death are also observed in echinus loss-of-function mutants and echinus overexpression can cause defects in ommatidial rotation and the morphology of cone cells. echinus is a positive regulator of DE-cadherin and Enabled accumulation in adherens junctions of retinal epithelial cells. Genetic interactions were observed between echinus and the genes wingless, enabled and expanded. An immunofluorescence assay in Drosophila S2 cell cultured demonstrated that Echinus localizes to intracellular vesicles that do not appear to be endocytic in nature, and the C-terminal region of Echinus was shown to be necessary for this association. A protein interaction screen using an immunoprecipitation and mass spectrometry approach identified interactions between Echinus and the vesicle coat protein Clathrin, the scaffolding protein RACK1 and the casein kinase I epsilon (Dco). Co-immunoprecipitation additionally identified an interaction between Echinus and Enabled. This work has revealed echinus to be an important regulator of cell sorting and adherens junction formation in the developing retina and has identified multiple interactions between echinus and enabled, a regulator of the actin cytoskeleton.

Doctor of Philosophy / Rice (Oryza sativa L.) is very susceptible to water deficit at any time during its life cycle as a semi-aquatic cereal crop. However, the consequential damage is particularly severe if water deficit occurs during reproductive phases. The conspicuous injury often observed in rice plants exposed to water stress during meiosis of the pollen mother cell is the reduction of grain set, which is attributed to the decline of male fertility. In spite of much research on drought-induced male sterility in rice, the underlying mechanisms of the problem are poorly understood. This project was therefore conducted to investigate the molecular mechanisms of water deficit-induced pollen sterility in rice. In this study three consecutive days of water deficit treatment at -0.5 MPa osmotic potential during anther development effectively reduced the leaf water potential (leaf) and the number of viable pollen which later led to a decrease in grain set. Moreover, this thesis demonstrates that the immediate deleterious effects of water deficit to plant fertility could be estimated using a young microspore viability index, which showed a strong correlation with mature viable pollen and grain set. The present work has also illustrated that oxidative stress appears to be a plausible cause for the decline of male fertility and grain set. Water deficit has induced the excessive production of reactive oxygen species (ROS) above the redox balance, which in turn caused detrimental effects to cellular DNA and might result in programmed cell death (PCD) in the anthers. Moreover, ROS accumulation effectively influenced ATP synthesis leading to a decrease in the level of ATP in the anthers. Excessive ROS accumulation after drought could be the consequence of insufficient activity of the antioxidant system, which has been illustrated by qRT-PCR expression analysis of major antioxidant genes. Down-regulation of those genes would increase the incidence of oxidative damage. In contrast, stable or up-regulated expression of these genes resulted in less oxidative damage. Detailed investigations of sugar metabolism in anthers has provided supplemental data to develop a model of sugar unloading and transport within anther using in situ hybridisation to mRNA techniques. Analysis of sugar transportation within the cellular compartments of anther has unveiled the role of sugar metabolism on pollen sterility in rice. qRT-PCR assays of genes associated with the sugar metabolic pathway has demonstrated that the supply of both sucrose and hexoses from the anther walls to the locules was not restricted after water deficit stress. The results indicate that water deficit might not cause sugar starvation for developing microspores as previously thought, nor inhibit the initial steps of sugar utilisation such as glycolysis. This thesis has suggested new ideas regarding the role of rising sugar levels to cope with oxidative stress in anthers. Sugar accumulation might have provided protection against oxidant damage by strengthening the antioxidant system. However, the interplay between sugar and oxidative stress is not straightforward and needs to be further characterised. In-depth investigations on the interaction between sugar signalling and oxidative stress responses may help indentify the role of sugars in protecting anthers under water deficit. Although many studies on drought and chilling stresses in rice anthers have been performed, the causal mechanism of male sterility still remains to be elucidated. Findings presented in this thesis may contribute to understanding molecular mechanisms of male sterility in rice as a response to drought stress. A more detailed investigation of mitochondrial respiration in rice anthers is required to further examine this problem. Finally, this thesis suggests that signalling molecules such as 14-3-3 proteins and abscisic acid (ABA) might act upstream of ROS production and antioxidant defence in plants. Further work on these molecules might therefore further illustrate how they influence plant fertility under water shortage conditions.

x, 83 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Programmed cell death (PCD) is a critical process during development and maturity of vertebrates and invertebrates. Aberrations in PCD are responsible for numerous developmental abnormalities and diseases in humans. Cell death pathways are surprisingly similar across species, so the study of PCD in simpler organisms such as insects provides important insight into the roles of cell death in higher animals including humans. Metamorphosis of the fruit fly, Drosophila melanogaster , provides an excellent model system in which to study PCD. During metamorphosis, many obsolete larval structures undergo PCD, largely in response to changes in circulating levels of steroid hormones known as ecdysteroids. These effects of ecdysteroids are particularly striking in the nervous system, where many larval neurons undergo PCD or functional remodeling during metamorphosis. One wave of neuronal PCD takes place during the first 24 hours of metamorphosis while a second follows adult emergence. Studies in another insect, Manduca sexta , suggested that the rise in ecdysteroids that initiates metamorphosis, the prepupal pulse, may trigger the first wave of neuronal PCD in Drosophila . This dissertation investigated steroid-regulated neuronal PCD in Drosophila by studying an individually-identified larval motoneuron, RP2. Using molecular genetics, ïmmunocytochemistry and primary cell culture, I showed that abdominal RP2s undergo PCD within the first 24 hours of Drosophila metamorphosis; identified a role for previously-identified PCD genes and ecdysteroid receptors in RP2's demise; and demonstrated that the prepupal pulse of ecdysteroids acts directly and cell-autonomously on RP2s to activate PCD. These experiments advance our understanding of hormonally-induced cell death and its regulation within the developing nervous system. This dissertation includes unpublished co-authored material. / Adviser: Janis C. Weeks

Le travaux présenté dans cette thèse concerne le rôle des canaux ioniques de la membrane plasmique en réponse à des stress salins et non salins ainsi qu’aux interactions possibles avec d’autres événements de signalisation conduisant à la mort cellulaire programmée (PCD). Nous avons montré que les réponses cellulaires précoces: tels que l`augmentation du calcium cytosolique et la production de ROS, classiquement impliqués lors de la PCD, ne semblaient pas être impliqué dans la mort cellulaire induite par les stress hyperosmotiques chez les cellules en culture de tabacco BY2 ou d’A. thaliana. Nous avons montré que, dans les cas de stress salin chez les cellules de BY2 un influx précoce de sodium à travers des canaux cationiques non spécifiques participe au développement de la PCD en entraînant un disfonctionement mitochondrial et la production de O2• - par des NADPH oxydases. Dans le cas de stress hyperosmotique non-ionique, nous avons observé une diminaution précoce de l’intensité des courants anioniques. Afin de poursuivre l’étude du rôle des canaux anioniques lors du stress hyperosmotique non salin, nous avons utilisé des cellules A.thaliana nous permettant de travailler avec le mutant de canal anionique SLAC1. Nous avons constaté que l’activation retardée des canaux SLAC1 participait au développement de la PCD induite par un stress hyperosmotique non salin. La réduction précoce de l'activité des canaux anioniques pourrait participer à la signalisation ou l'ajustement osmotique permettant l'adaptation et la survie cellulaire alors que des évènements retardés, à savoir la production d'anion superoxyde (O2• -) par les NADPH-oxydases et l'activation des canaux anioniques pourraient participer au développement de la PCD d'une partie de la population cellulaire. Nous avons aussi étudié le rôle potentiel des petits peptides appartenant à la famille des peptides FMRFamide décrite chez les métazoaires à l'osmorégulation chez des cellules d’A. thaliana. Des génes susceptibles de coder de tels peptides sont en effet présent dans le génome d’A. thaliana. En utilisant des peptides synthétiques, nous avons montré que ces FLPS putatifs pourraient participer aux réponses induites losr de stress hyperosmotique chez les plantes. Ce travail illustre la complexité et l'importance de la régulation des canaux ioniques dans les voies de signalisation et les processus conduisant à la PCD / The work presented in the present thesis relates to the role of ion channels in response to (ionic and non-ionic) hyperosmotic stresses and their interactions with signaling events leading to PCD in plant. Early cell responses such as cytosolic calcium increase and ROS production classically involved in PCD process, seems not to be involved in hyperosmotic-induced cell death in BY2 tobacco and A. thaliana cultured cells. When BY2 tobacco cells were subjected to hyperosmotic stress, an early influx of sodium through non-selective cation channels participates in the development of PCD through mitochondrial dysfunction and NADPH-oxidase-dependent O2•– generation. On the contrary, non-ionic hyperosmotic stress resulted in an early decrease in anion currents. To further investigate the role of anion channels in non-ionic hyperosmotic stress further experiments were conducted by using A.thaliana cells of the anion channel mutant SLAC1. Results showed that the delayed activation of SLAC1 channels was involved in the non-ionic hyperosmotic stress induced pathway leading to cell death. Interestingly, the early anion channel activity decrease could participate to signalisation or osmotic adjustment allowing cell adaptation and survival, when a second set of events, namely superoxide anion (O2•-) generation by NADPH-oxidase and anion channel activation could participate in PCD development of a part of the cell population. In addition, the potential role of small peptides belonging to the FMRFamide-like peptide (FLP) family described in metazoan in osmoregulation in A. thaliana was investigated. By using synthetic peptides, based on FLPs homolog genes existing in A. thaliana, it was possible to demonstrate that these putative FLPs are involved in hyperosmotic stress response. Overall, the present work shed light on the importance and the complexity of ion channels regulation in the signaling pathways and the processes leading to PCD

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-01-26T18:42:50Z No. of bitstreams: 0 / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-01-29T11:01:37Z (GMT) No. of bitstreams: 0 / Made available in DSpace on 2018-01-29T11:01:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-06-09 / - / Bacteria are important components of the food web structure in aquatic ecosystems in which they influence the flow of carbon and energy. Populations of bacteria in these ecosystems comprise a diverse spectrum of individual cells able to respond to many factors such as nutrient supply, temperature and virus infection, which regulate bacterial life and death. Bacterial death is a key cellular event involved in the control and production of bacteria in aquatic ecosystems with functional meaning in the carbon and nutrient cycles. Therefore, the study of bacterial structural features and cellular mechanisms underlying bacterial death is crucial to understand processes affecting the entire population. However, both bacterial structure and cellular events of death in aquatic ecosystems are still poorly understood. In the present work, we used single cell approaches to study the structural organization of bacteria as well as to characterize cellular processes of death in these organisms. First, by using fluorescence and transmission electron microscopy (TEM), we provided a general panorama of how microscopy techniques, especially TEM, are powerful tools to understand bacterial structure and their responses to environmental stresses. We showed that bacteria from aquatic ecosystems have remarkable ultrastrutural diversity with components such as bacterial envelope of individual cells differing in structure within the same population. Second, we sought to identify and characterize mechanisms of bacterial cell death. Because our TEM analyses revealed morphological signs of apoptosis, a type of program cell death (PCD), in aquatic bacteria directly collected from natural ecosystems, we applied different techniques to detect apoptosis in bacteria cultured from natural samples. We used TEM as well as different probes to detect this type of PCD in cultured bacteria exposed to increased temperature and viral infection, which are recognized inducers of bacterial death. TEM showed, in both situations, ultrastructural changes indicative of apoptosis, such as cell retraction and condensation, similar to those reported for eukaryotic cells. Assays for membrane permeability, DNA fragmentation, phosphatidilserine exposition and caspase activation were significantly increased in treated bacteria compared to the control group. Altogether, our data demonstrate, for the first time, that PCD occur in aquatic bacteria, and that this event may be a basic mechanism for regulation of bacterial communities in these ecosystems.

A transição materno-embrionária é um fenômeno complexo caracterizado pela iniciação da transcrição no embrião e a substituição do mRNA materno pelo mRNA embrionário. O mRNA e as proteínas estocadas no oócito são utilizados nas primeiras clivagens e, posteriormente, o embrião deve iniciar a transcrição dos genes necessários ao seu desenvolvimento, que ocorre no estádio de oito células em embriões bovinos. Nesta etapa, podem ser observados embriões competentes a continuar o desenvolvimento, enquanto embriões incompetentes sofrem bloqueio. Pelo fato do bloqueio ocorrer na fase de ativação do genoma embrionário, formulou-se a hipótese de que o bloqueio estaria associado a genes transcritos neste momento, contrariamente à hipótese mais aceita de bloqueio passivo. O objetivo deste trabalho foi de identificar, categorizar e avaliar transcritos diferencialmente expressos entre embriões bovinos de desenvolvimento rápido e lento, além de elucidar possíveis vias de sinalização de morte ou sobrevivência celular. Para isso, foi feita uma hibridação em membrana de macro arranjo contendo genes humanos relacionados a ciclo celular, hibridada com aRNA marcado radioativamente oriundo de embriões bovinos produzidos in vitro de acordo com sua velocidade de desenvolvimento, seguido por RT-PCR e análise de vias de sinalização para validação da hibridação. A média de similaridade entre estes genes humanos e bovinos de 89,3%. Pelas membranas de macro arranjos foram identificados 120 genes com modulação diferencial entre embriões lentos e rápidos, sendo 100 genes com regulação superior nos embriões lentos. Entre os genes com modulação positiva nos embriões rápidos, 40% foram primariamente identificados como ligantes a proteínas e 25% têm atividade catalítica, com resultados similares no grupo de genes com modulação positiva nos embriões lentos. Por um lado, as diferenças de transcrição entre embriões de desenvolvimento rápido e lento não foram confirmadas pelo RTPCR. Mas os genes diferencialmente modulados estão associados e constitutivamente presentes em algumas vias de sinalização para morte celular. Os resultados sugerem que a ativação do genoma embrionário é necessária para a sinalização de vias de sobrevivência ou morte celular programada. Assim, o bloqueio do desenvolvimento não é um processo passivo, mas sim um processo ativo de transcrição de genes, ativando tanto a cascata de sobrevivência quanto a cascata de morte em embriões com baixo potencial de desenvolvimento. / Maternal-zygotic transition is a complex phenomenon characterized by the initiation of transcription in the embryo and the transition of maternal mRNA with embryonic mRNA. It is believed that the mRNAs and proteins synthethized by the oocyte during its growth and final maturation allow the zygote to develop during the early stages of embryo development up to the 8 cell-stage, the moment when the bovine embryo acquires transcriptional competence. Competent embryos are able to develop until blastocyst, while incompetent embryos block. Since the blockage occurs during embryo genome activation, we developed the hypothesis that gene transcription in incompetent embryos is associated with the blockage, instead of passive blockage. The aim of this work was to identify, categorize and analyze gene expression differences between fast cleavage and slow cleavage embryos, and discover possible signaling pathways to cell death or cell survival. We used a macroarray membrane spotted with human genes related to cell cycle and hybridizated with a radioactive labeled aRNA from fast or slow in vitro produced embryos. Real-time PCR and signaling pathways analysis were designed for further validation of the array. The mean similarity between human and bovine genes was 89,3%. According to the array membranes, it was possible to identify 120 genes differentially expressed between slow and fast cleavage embryos. Hence, the majority of the genes were more expressed in slow embryos (100 genes versus 20 genes in fast group). Among genes more expressed at fast embryos, 40% were identified as protein binding and 25% have catalytic activity, with similar results in slow embryos. In one hand, differences between fast and slow embryos transcripts were not confirmed by real-time PCR analysis. But on the other hand, the differentially expressed genes are somehow related to and constitutively present in some recognized death pathways. Together, these results presented herein suggest that embryonic genome activation is necessary for survival or cell death signaling. Moreover, developmental block is not a passive pathway, but rather a very active transcriptional pathway, leading to activation of cell survival genes prior to genes related to death in slow-developing embryos.

Diversos tipos celulares nas mais variadas condições têm sido usados como doadores de núcleo para a TN. Ainda não está claro se o estado fisiológico destas células afeta o posterior desenvolvimento dos embriões. Neste trabalho, testou-se a hipóese que fibroblastos bovinos em processo de MCP podem ser reprogramados na transferência nuclear. Fibroblastos foram cultivados até atingirem 60% de confluência, sincronizados por restrição de soro durante 24h e em seguida a MCP foi analisada por citometria de fluxo com a ténica da Anexina V/Iodeto de propídeo. Células Anexina positivas (MCP) e Anexinanegativas (Vivas) foram separadas por citometria de fluxo e utilizadas para a TNS. Céulas não coradas e não separadas no citômetro serviram como controle (Controle). Os embriões reconstruídos foram avaliados quanto à fusão, clivagem (2º dia de cultivo), blastocisto (7º dia) e prenhez (D30, D60 e nascimento). O índice de MCP dos blastocistos obtidos foi determinado. Os resultados foram analisados pela ANOVA ou teste de X2 com nível de significância de 5%. Não houve efeito nas taxas de fusão (p>0,05). Embriões reconstruídos com células MCP tiveram menor taxa de clivagem e formação de blastocistos (72,7% e 18,8%, respectivamente) em comparação ao grupo reconstruído com células Vivas (83,4% e 34,7%, respectivamente; p<0,05), não diferindo dos embriões Controle (77,3% e 27,3%, respectivamente; p>0,05). O índice de MCP do grupo de embriões MCP foi similar aos índices dos embriões clonados a partir de células Vivas e Controle (p>0,05). Após a transferência para receptoras, os grupos MCP, Vivas e Controle não diferiram com relação à taxa de prenhez aos 30d (18,1%, 13,3% e 27,5%, respectivamente; p>0,05). Entretanto aos 60d, a perda gestacional no grupo MCP (25%) foi inferior a do grupo Vivas (100%) e Controle (62,5%). Somente um nascimento, do grupo MCP (4,5% dos embriões transferidos), foi obtido no experimento. Conclui-se que células em processo de MCP, podem ser reprogramadas quando utilizadas como doadoras de núcleo na técnica de transferência nuclear, podendo estabelecer gestações e nascimentos, entretanto houve um efeito prejudicial nas taxas de desenvolvimento embrionário até o estádio de blastocisto assim como houve aumento do índice de MCP nos embriões reconstruídos. / It is not clear if the physiological status of the cells can affect further embryonic development in NT. We hypothesized that adult bovine fibroblasts in PCD can be reprogrammed when used as nuclear donors for cloning. Fibroblasts were cultivated until 60% confluency, synchronized by serum starvation for 24 h and stained with Annexin V and Propidium iodide (PI) by flow citometry. Annexin positive cells (PCD cells) and Annexin negative cells (Live cells) were sorted and used for NT. Unsorted, unstained cells were used as control (Control cells). After reconstruction, fusion, cleavage (day 2 of culture), blastocyst (day 7) and pregnancy rates (day 30, 60 and birth) were recorded. Apoptotic index of the embryos was determined by TUNEL. Data were analyzed with ANOVA and Chi-square test with 5% of significance level. There was no effect on fusion rates (p>0.05). Embryos reconstructed with PCD cells had lower cleavage and blastocyst rates (72.7 and 18.8%, respectively) compared with embryos reconstructed with Live cells (83.4 and 34.7%, respectively; p<0.05). Apoptotic index in embryos produced from cells in PCD was similar compared to embryos produced from Live and Control cells (p>0.05). Pregnancy rates were similar between cloned groups on day 30 after embryo transfer (p>0.05). However it was observed a reduced pregnancy loss in PCD group on day 60 (25%) compared with Control (62.5%) and Live (100%) groups. Only one calf, from PCD cells (4.5% of the transferred embryos), has been obtained in this experiment. In conclusion, it was showed that cells in PCD process can be reprogrammed when used as nuclear donors after NT producing even live animals. However, a negative effect on embryonic development and an increase in the apoptotic index of these embryos was observed.

The precise structure of the adult Drosophila eye results from a coordinated process of cell sorting, differentiation and selective cell death in the retinal epithelium. Mutations in the gene echinus cause supernumerary pigment cells due to insufficient cell death. This study reports the identification of echinus and the characterization of its role in Drosophila retinal development. Using a combination of deletion mapping, gene expression analysis and genomic sequencing, echinus was cloned and several alleles were sequenced. echinus encodes a ~180kDa protein containing an ubiquitin hydrolase domain at its N-terminus and a polyglutamine tract at its C-terminus. echinus is expressed in the retina during pupal development and mutants of echinus have decreased levels of apoptosis during several stages of retinal development. Defects in the cell sorting process that precedes cell death are also observed in echinus loss-of-function mutants and echinus overexpression can cause defects in ommatidial rotation and the morphology of cone cells. echinus is a positive regulator of DE-cadherin and Enabled accumulation in adherens junctions of retinal epithelial cells. Genetic interactions were observed between echinus and the genes wingless, enabled and expanded. An immunofluorescence assay in Drosophila S2 cell cultured demonstrated that Echinus localizes to intracellular vesicles that do not appear to be endocytic in nature, and the C-terminal region of Echinus was shown to be necessary for this association. A protein interaction screen using an immunoprecipitation and mass spectrometry approach identified interactions between Echinus and the vesicle coat protein Clathrin, the scaffolding protein RACK1 and the casein kinase I epsilon (Dco). Co-immunoprecipitation additionally identified an interaction between Echinus and Enabled. This work has revealed echinus to be an important regulator of cell sorting and adherens junction formation in the developing retina and has identified multiple interactions between echinus and enabled, a regulator of the actin cytoskeleton. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

A biochemical characterization of ubiquitin (Ub)-proteasome pathway activity was conducted in <I>Zinnia</I> mesophyll cell cultures to examine potential differences between differentiating cells of tracheary element (TE) cultures and non-differentiating cells of control cultures. The pathway is highly active throughout development of differentiating TEs, a programmed cell death (PCD) process during which the majority of cellular proteins and biochemical processes are expected to be down-regulated in activity and/or expression. Addition of the proteasome inhibitors <I>clasto</I>-lactacystin Beta-lactone (LAC) and carbobenzoxy-leucinyl-leucinyl-leucinal (LLL) at culture initiation prevented TE differentiation in this system. Proteasome inhibition at 48h did not alter the final percentage of TEs compared to controls. However, proteasome inhibition at 48 h delayed the differentiation program by approximately 24 h, as indicated by examination of morphological markers and the expression of putative autolytic cysteine proteases.These results suggest that proteasome activity is required both for induction of TE differentiation and for progression of the TE program in committed cells. Treatment at 48 h with LLL resulted in partial uncoupling of autolysis from differentiation. Results of protease activity gel analysis suggest that incomplete autolysis was due to the ability of LLL to inhibit TE cysteine proteases. A characterization of phytohormone-stimulated growth of non-differentiating cultured <I>Zinnia</I> cells is also presented. Differential effects on radial cell expansion versus cell elongation were observed for the four plant growth regulators examined. Auxin (naphthaleneacetic acid, NAA) and a brassinosteroid (2,4-epibrassinolide, BI) stimulate only cell elongation. Cytokinin (N-6-benzyladenine, BA) has a greater effect on growth in cell girth rather than length. Gibberellic acid (GA₃) has equivalent effects on expansion in both dimensions. These results demonstrate that radial cell expansion and cell elongation can be uncoupled, and therefore, may be controlled by different mechanisms. Additionally, this study establishes the utility of <I>Zinnia</I> suspension cultures as a valuable model for studies of cell expansion. Finally, we modified <I>Arabidopsis</I> plant growth conditions to promote proliferation of secondary tissues, permitting the separation of secondary xylem from bark (phloem plus nonvascular) tissues using hypocotyl-root segments. Dissected tissues were used for semi-quantitative and quantitative RT-PCR and for the construction of bark and xylem cDNA libraries for PCR-based screening of several Ub pathway components, including Ub-conjugating enzymes (<I>UBCs</I>), deubiquitinating enzymes (DUBs), and an Alpha (<I>PAF1</I>) and Beta (<I>PAF1</I>) subunit of the proteasome. All targeted <I>UBC</I> families, candidate <I>UBCs</I> and DUBs, and proteasome subunits are expressed in secondary xylem and bark in this system. / Ph. D.

Programmed cell death (PCD) is a major regulatory mechanism employed during development and homeostasis. The term PCD was coined to describe the death of the intersegmental muscles (ISMs) of moths at the end of metamorphosis. The timing of ISM death in the Tobacco Hawkmoth, Manduca sexta, is regulated by a fall in the titer of the steroid molting hormone 20-hydroxyecdysone (20E) late on day 17of pupal-adult development. This triggers the release of the peptide hormone, Eclosion Hormone (EH), which mediates its effects via the secondary messenger cGMP. It has been previously demonstrated that ISM death requires de novo gene expression. One induced gene in the ISMs encodes the novel protein Acheron. However, Acheron’s role in PCD is unknown. Acheron is a novel member of the Lupus-Antigen family of RNA binding proteins. In humans, Acheron is expressed in many tissues including the myoepithelial cells in mammary ducts. Analysis of the mammary gland revealed that Acheron mRNA levels were elevated in some basal-like breast cancers in women. Ectopic expression of Acheron in human MDA-MB-231 breast cancer cells results in dramatic elevations in proliferation, angiogenesis and metastasis. Moreover, Acheron expressing MDA-MB-231 cells in mouse xenographs resulted in tumors that were five times larger than control cell tumors. These data suggests that Acheron enhances the growth of some human breast cancers. This thesis describes two primary studies. The first tested the hypothesis that Acheron functions as a survival protein for cells in vitro. MDA-MB-231 cells engineered to express Acheron were challenged with various death-inducing treatments, which act via different signaling pathways, to determine if Acheron expression confers survival. Acheron protects cells from apoptosis induced by nutrient withdrawal, proteosome inhibition, heat stress, mitochondrial toxins, inhibiting cellular respiration, DNA damage, and oxidative stress. The second study tested the hypothesis that Acheron is phosphorylated by a cGMP-dependent kinase in the ISMs when the cells initiate death following adult eclosion. Using a non-radioactive in-vitro kinase assay I observed that Acheron is phosphorylated via a cGMP-dependent kinase, presumed via kinase binding motif predictions to be Protein Kinase G. Furthermore I show that phosphorylation is coupled to Acheron degradation.