Thesis (Ph. D.)--Florida State University, 2005. / Advisor: Dr. Richard Hyson, Florida State University, Program in Neuroscience. Title and description from dissertation home page (viewed Sept. 19, 2005). Document formatted into pages; contains ix, 64 pages. Includes bibliographical references.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 98-112). Also available in electronic version.

Thesis (Ph.D.)--Montana State University--Bozeman, 2006. / Typescript. Chairperson, Graduate Committee: Frances Lefcort. Includes bibliographical references (leaves 60-70).

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed June 30, 2007). Includes bibliographical references (p. 70-72).

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Apoptosis represents a finely orchestrated and highly conserved natural form of cell death. It exhibits unique morphological and biochemical characteristics which culminate in the controlled dismantling of a cell from within followed by its discreet removal by phagocytic cells. Apoptosis is vital for the preservation of cell and tissue homeostasis but also performs several defensive and protective functions. Owing to its importance, apoptosis is highly regulated and a large number of proteins have been shown to mediate and safeguard the process. Furthermore, deregulated or altered levels of apoptosis can have severe pathological consequences; indeed, apoptosis has been shown to play a central role in several diseases, including neurological and autoimmune diseases as well as a variety of cancers. Consequently, the search for apoptotic-based therapies has received much attention and of vital importance to this quest is the characterisation of the specific mediators of apoptosis and their regulation as well as the identification of novel genes or proteins that can have a regulatory effect on apoptosis. It is thus the aim of this study to assist in this characterisation and also to identify novel candidate genes potentially involved in apoptosis. In a previously performed pilot study, three novel candidate genes potentially involved in apoptosis were identified by performing promoter-trap mutagenesis experiments. These genes were lipoic acid synthetase (LIAS), cyclophilin A (CYPA) and ribosomal protein L9 (RPL9). Since the methodology for this pilot study involved the use of functionally haploid cells, it was aimed in this study to verify these results in a diploid mouse cell line. Candidate gene knockdown was achieved by means of RNA interference and apoptosis assays were performed. A potential role for LIAS and CYPA in apoptosis was successfully verified in this study; however this could not be achieved for RPL9 and the gene was thus excluded from further study. In addition, nucleotide sequences isolated during the promoter-trap mutagenesis experiments in the pilot study were also investigated in order to identify additional novel candidate genes involved in apoptosis. By performing nucleotide BLAST searches, two potential candidate genes were identified, namely AHNAK nucleoprotein (AHNAK) and serum amyloid A-like 1 (SAAL1). Further bioinformatic analyses were performed with the four candidate genes in order to ascertain possible associations with apoptosis or cancer. Lastly, to further characterise the four candidate genes, the relative gene expression was investigated by means of quantitative PCR in two cancer and control cell lines. The results revealed significant differential expression for the majority of genes in the cancer cell lines when compared to the control cell lines. In conclusion, this study identified and characterised four novel genes potentially involved in apoptosis. Results obtained during this study can aid in the complete characterisation and functional annotation of these genes. Potential ties to apoptosis and associations with cancer are discussed for all four candidate genes and the possibilities of therapeutic strategies for anticancer treatments involving these candidate genes are noted. / AFRIKAANSE OPSOMMING: Apoptose verteenwoordig ‘n fyn georganiseerde en hoogs gekonserveerde natuurlike vorm van seldood. Dit vertoon unieke morfologiese and biochemiese eienskappe wat uitloop in die beheerde afbreek van ‘n sel vanuit die binnekant waarna dit onopsigtelik deur fagositiese selle verywder word. Apoptose is uiters belangrik vir die bewaring van sel en weefsel homeostase, maar dit vervul ook menigde afwerende and beskermde funksies. Vanweë sy noodsaaklikheid is apoptose hoogs gereguleer and ‘n groot aantal proteïene is al aangewys as bemiddelaars en beskermers van die proses. Verder, wangereguleerde en veranderde vlakke van apoptose kan ernstige patalogiese nagevolge hê; inderdaad, ‘n sentrale rol vir apoptose in verskeie siektes is al bevestig, insluitend neurologiese en outo-immuun siektes asook ‘n verskeidenheid van kankers. As gevolg hiervan ontvang die soektogte vir apoptose-gebaseerde terapieë vele aandag en uiters noodsaaklik vir hierdie soektogte is die karakterisering van die spesifieke bemiddelaars van apoptose en hul regulering asook die identifisering van nuwe gene of proteïene wat ‘n regulerende effek kan hê op apoptose. Dit is dus die doel van hierdie studie om by te dra tot hierdie karakterisering en ook om nuwe kandidaat gene wat moontlik betrokke kan wees in apoptose te identifiseer. In ‘n vorige loodsprojek is drie gene moontlik betrokke in apoptose geïdentifiseer deur middel van promoter-strik mutagenese eksperimente. Hierdie gene is lipoic acid synthetase (LIAS), cyclophilin A (CYPA) en ribosomal protein L9 (RPL9). Aangesien die metodiek in the loodsprojek gebruik gemaak het van funksionele haploïede selle, was dit die doel van hierdie studie om die resultate te bevestig in ‘n diploïede muis sellyn. Ribonukleïensuur (RNS) steuring is uitgevoer vir die uitklopping van die kandidaat gene en apoptose toetse is ook gedoen. Die bevestiging van ‘n moontlike rol vir LIAS en CYPA in apoptose was suksesvul in hierdie studie; alhoewel dit was nie bereikbaar vir RPL9 nie en hierdie geen is dus uitgesluit in verdere studies. Bykomend is nukleotied volgordes wat geïsoleer is tydens die promoter-strik mutagenese eksperimente in die loodsprojek ook nagesien om moontlike addisionele nuwe kandidaat gene te identifiseer wat moontlik betrokke kan wees by apoptose. Twee potensiële kandidaat gene, naamlik AHNAK nucleoprotein (AHNAK) en serum amyloid A-like 1 (SAAL1), was geïdentifiseer deur middel van nukleotied BLAST soektogte. Addisionele bioinformatiese analises is uitgevoer op die vier kandidaat gene om moontlike redes vir ‘n assosiasie met apoptose of kanker vas te stel. Laastens, om die kandidaat gene verder te karakteriseer, is daar ondersoek ingestel op die relatiewe geen uitdrukking van die kandidaat gene in twee kanker en twee normale sellyne. Die resultate het betekenisvolle differensiële regulering getoon vir meeste van die gene in die kanker sellyne in vergelyking met die normale sellyne. Ten slotte, vier kandidaat gene moontlik betrokke in apoptose is in die huidige studie geïdentifiseer en gekarakteriseer. Die resultate verwerf in hierdie studie kan moontlik bydra tot die volkome karakterisering en funksionele annotering van die kandidaat gene. Moontlike skakels met apoptose en assosiasies met kanker is bepreek vir die vier kandidaat gene en die moontlikheid van terapeutiese strategieë gebaseer rondom die kandidaat gene word ook genoem. / National Research Foundation (NRF)

Programmed cell death (PCD) and removal of cell corpses are important processes in animal development and homeostasis. Typically, engulfment of cell corpses is performed by professional phagocytes, such as macrophages. In tissues with limited accessibility to circulating cells, engulfment is carried out by neighboring non-professional phagocytes, such as epithelial cells. Compared to professional phagocytosis, the mechanisms that govern non-professional phagocytosis are not well characterized. The Drosophila ovary provides a powerful in vivo model for the study of PCD and engulfment by non-professional phagocytes. This dissertation identifies genetic pathways that govern non-professional phagocytosis during starvation-induced PCD and elucidates the major mechanism promoting non-apoptotic developmental PCD. During mid-oogenesis, germline nurse cells can be induced to die by starvation and their remnants are engulfed by surrounding epithelial follicle cells. We show that the engulfment receptor Draper is enriched and required in engulfing follicle cells. Additionally, we demonstrate that the JNK pathway is activated by Draper and required in engulfing follicle cells. Overexpression of Draper or the JNK pathway is sufficient to induce death of the germline, suggesting that there is coordination between the germline and follicular epithelium to promote cell death. Furthermore, activation of JNK bypasses the need for Draper in engulfment. These data demonstrate that JNK and Draper are crucial regulators of engulfment by non-professional phagocytes. During late oogenesis, nurse cells transfer their contents into the oocyte and undergo developmental PCD. Disruption of apoptosis or autophagy only partially inhibits PCD, indicating that other mechanisms contribute to the process. We demonstrate that the large-scale non-apoptotic developmental PCD in the Drosophila ovary occurs by a novel cell death program where follicle cells non-autonomously promote death of the germline. The phagocytic machinery of follicle cells, including Draper, JNK, and Ced-12, is essential for death and removal of nurse cells. Cell death events including acidification, nuclear envelope permeabilization, and DNA fragmentation are impaired when phagocytosis genes are inhibited. Moreover, elimination of a subset of follicle cells prevents nurse cell death and cytoplasmic dumping. Developmental PCD in the Drosophila ovary is an intriguing example of non-apoptotic, non-autonomous PCD, providing insight on the diversity of cell death mechanisms.

Cell death is an integral part of oogenesis in the fruit fly, Drosophila melanogaster. When the fly is starved of protein, some pre-vitellogenic egg chambers die apoptotically. As egg chambers mature, excess germline cells die via a non-apoptotic, developmentally programmed death. Overexpression of the transcription factor escargot was found to block both death events in the ovary, which is very unusual. escargot overexpression blocked starvation-dependent death upstream of caspases, but still needed a death signal to produce undead egg chambers. In maturing egg chambers, escargot overexpression blocked death more effectively than disrupting both apoptosis and autophagy, indicating that it must affect non-apoptotic, non-autophagic death mechanisms. RNA-Seq and a genetic modifier screen were used to identify potential escargot targets that inhibit cell death. Studies were also undertaken to characterize the loss-of-function phenotype of escargot in the ovary. escargot is a member of the Snail family of transcription factors that play integral roles in development and gene regulation throughout Bilaterian organisms. In Drosophila melanogaster, the genes snail, escargot, and worniu are critical for stem cells in neuroblasts, gut, and testis, but a role in the ovary had not been shown. To analyze Snail family function in the ovary, I made a triple deficiency that removed the three Snail family members, called ΔSF. Surprisingly, ΔSF homozygous follicle stem cells are rapidly lost. Follicle stem cell loss was rescued by the expression of escargot or worniu but not snail, indicating that there is shared capability between genes. Moreover, follicle stem cells did not linger in the germarium, and their loss was not prevented by blocking apoptosis, indicating that the ΔSF defect is a failure of stem cell maintenance. Together, the results described in this dissertation show that Snail genes are needed for the normal function of the Drosophila ovary, and that escargot can regulate multiple kinds of cell death. Understanding Snail family genes is particularly important for the study of cancer, as they are implicated in mechanisms underlying the cancer stem cell state. Analysis of the highly conserved Snail family genes in Drosophila illuminates their function and dysfunction in human health and disease.

abstract: Cell death is a powerful tool through which organisms can inhibit the spread of viruses by preventing their replication. In this work, I used viral and chemical stressors to elucidate the mechanisms by which one anti-viral system might be activated over another, focusing on the programmable death pathway necroptosis and Protein Kinase R (PKR). PKR can detect viral dsRNA and trigger antiviral effects such as cessation of translation and induction of programmed death. Necroptosis is a rapid cellular death that can be induced via sensors such as DNA-dependent activator of IFN-regulatory factors (DAI), also known as Z-DNA-binding protein 1 (ZBP1). DAI contains a Z-form nucleic acid (ZNA) binding domain. E3, the primary vaccinia virus (VACV) interferon resistance protein, contains a similar domain in its amino terminus. We have previously reported this domain to be necessary for the inhibition of both PKR activation and DAI/ZBP1-mediated necroptosis. Monkeypox virus is a reemerging human pathogen. Despite a partial amino-terminal deletion in its E3 homolog, it does not activate PKR. In chapter 2, I show that MPXV produces less dsRNA than VACV, which could explain how the virus avoids activating PKR. The amino-terminus of vaccinia is associated with ZNA binding, inhibition of PKR, and inhibition of necroptosis. To determine the roles of PKR inhibition and ZNA binding in necroptosis inhibition, I characterized the VACV mutants Za(ADAR1)-E3, which binds ZNA but does not inhibit PKR, and E3:Y48A, which cannot bind ZNA. I found that while Za(ADAR1)-E3 fails to induce necroptosis, E3:Y48A does not activate PKR but does induce necroptosis. This suggests that Z-form nucleic acid binding is not necessary for vaccinia E3-mediated inhibition of PKR, nor is the inhibition of PKR sufficient for the inhibition of necroptosis. Finally, all known ZNA-binding proteins have immune functions and home to stress granules. I asked if stress granule formation alone could lead to necroptosis. I found that in L929 cells sodium arsenite, a known inducer of stress granules, could trigger DAI-dependent necroptosis. This suggests that DAI/ZBP1 is not necessarily a sensor of viral ligands but perhaps is a sensor of stress signals brought about by infection. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2018

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / As quinonas sÃo metabÃlitos de ampla distribuiÃÃo na natureza que possuem diversas atividades farmacolÃgicas de importÃncia clÃnica. A naftoquinona α-lapachona demonstrou potencial como protÃtipo para o desenvolvimento de substÃncias com propriedades anticÃncer, como relatado pelo nosso grupo de pesquisa, em que o seu derivado tetrahidropirano (THP) apresentou citotoxicidade e seletividade significante contra a linhagem de melanoma MDA-MB-435. Assim, o objetivo deste trabalho foi avaliar o mecanismo de aÃÃo envolvido na citotoxicidade da α-lapachona e derivado THP em cÃlulas de melanoma MDA-MB-435. Inicialmente, avaliou-se a citotoxicidade da alfa-lapachona e derivado THP em 8 linhagens tumorais de mama e melanoma, atravÃs do ensaio do MTT, mostrando CI50 de 1,37 e 8,18 ÂM, respectivamente, apÃs 72 horas de incubaÃÃo. A seletividade do derivado THP no ensaio de Alamar Blue, demonstrou que este se apresentou 2,6 vezes menos citotÃxico para cÃlulas normais quando comparado Ãs cÃlulas tumorais. Estudos do mecanismo de morte celular na linhagem tumoral MDA-MB-435 indicaram que o derivado THP causou reduÃÃo de cÃlulas viÃveis associado com o aumento de cÃlulas nÃo-viÃveis por induÃÃo da perda de integridade da membrana plasmÃtica nas concentraÃÃes de 5 e 10 ÂM apÃs 24 horas de incubaÃÃo. A atividade citotÃxica do derivado THP nÃo està relacionada a uma fase especÃfica do ciclo celular, ativaÃÃo de caspases efetoras e formaÃÃo de espÃcies reativas de oxigÃnio, sugerindo a ocorrÃncia de um processo necrÃtico a partir de 6 horas de tratamento, demonstrado pela avaliaÃÃo da integridade de membrana. Assim, os resultados exibidos sugerem que a introduÃÃo do radical tetrahidropirano na molÃcula da α-lapachona aumenta a citotoxicidade em cÃlulas de melanoma MDA-MB-435, via necrose, o que reforÃa a importÃncia de naftoquinonas, como protÃtipo para o desenvolvimento de novos compostos sintÃticos com atividade antitumoral / Quinone metabolites are widely distributed in nature showing various pharmacological activities of clinical importance. The naphthoquinone α-lapachone has been shown to be suitable as a prototype for the development of substances with anticancer properties, as reported by our group to tetrahydropyran derivative (THP), which demonstrated significant cytotoxicity and selectivity against MDA-MB-435 melanoma line. The aim of the present work was to evaluate the mechanism of action involved in the cytotoxicity of α-lapachone and its THP derivative against MDA-MB-435 melanoma cells. Initially, we evaluated the cytotoxicity of α-lapachone and its THP derivative against 8 cell lines, by the MTT assay, showing IC50 of 1.37 and 8.18 ÂM to breast and melanoma lines, respectively, after 72 hours of incubation. The selectivity of the THP derivative in Alamar Blue assay, demonstrated that THP is 2.6 times less cytotoxic to normal cells as compared to tumor cells. Studies on the mechanism of cell death in MDA-MB-435 tumor line showed that the THP derivative caused a reduction on viable cells associated with an increase of non-viable cells by inducing loss of membrane integrity in concentrations of 5 and 10 ÂM. The cytotoxic activity of THP was independent of cell cycle, activation of effector caspases and formation of reactive oxygen species, suggesting the occurrence of a necrotic process after 6 hours of treatment, demonstrated by evaluation of membrane integrity. Thus, the data suggest that a tetrahydropyran group introduction in α-lapachone molecule enhances the cytotoxicity of MDA-MB-435 in melanoma cells, via necrosis, which reinforces the importance of naphthoquinones as prototypes for the development of new synthetic compounds with antitumor activity

Orientadores: Aníbal Eugênio Vercesi, Róger Frigerio Castilho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-17T07:09:51Z (GMT). No. of bitstreams: 1 Ronchi_JulianaAparecida_M.pdf: 2592293 bytes, checksum: f14b83c15cfc0b7b79f5b89218adf498 (MD5) Previous issue date: 2010 / Resumo: A transição de permeabilidade mitocondrial (TPM) é caracterizada pela abertura de um poro protéico não seletivo na membrana mitocondrial interna, o qual é induzido por Ca2+ e sensível à ciclosporina A. Trabalhos anteriores do nosso laboratório demonstram que o poro de transição de permeabilidade ocorre devido a ligações entre tióis (S-S) de proteínas, ocasionando a agregação de proteínas de membrana interna. Contrariamente, dados da literatura propõem a participação de proteínas da membrana mitocondrial externa na composição do poro, como o canal aniônico voltagem dependente (VDAC), hexoquinase e receptor benzodiazepínico. Para verificar a participação de proteínas da membrana externa no poro TPM, realizamos experimentos utilizando mitocôndrias desprovidas de membrana externa (mitoplastos). Mitoplastos de fígado e rim de rato foram obtidos através do tratamento de mitocôndrias com o detergente digitonina (seletivo para colesterol) para eliminar a membrana externa. Esses mitoplastos de fígado apresentaram menos que 5% e 10% dos marcadores de membrana externa monoamino oxidase e VDAC, respectivamente. Os mitoplastos de rim apresentaram depleção parcial de hexoquinase (73%). Os mitoplastos tiveram redução de 70% na velocidade de fosforilação do ADP, o que foi parcialmente recuperado pela adição de citocromo c exógeno. Estes mitoplastos foram capazes de gerar e sustentar potencial elétrico de membrana suficiente para fosforilar ADP. Como previsto, os mitoplastos sofreram TPM induzido por Ca2+ de modo semelhante às mitocôndrias. Os mitoplastos foram sensíveis a conhecidos indutores de TPM como Ca2+ e tert-butil hidroperóxido ou inibidores como ciclosporina A, quelantes de Ca2+, ADP, redutor ditiol e ainda catalase. Conjuntamente, nossos resultados indicam que a membrana mitocondrial externa não é necessária para a formação/abertura do poro de TPM. / Abstract: Mitochondrial permeability transition (MPT) is a Ca2+-induced opening of a nonselective proteinaceous membrane pore sensitive to cyclosporin A in the inner membrane. Data from our laboratory provided evidence that MPT is assembled by the aggregation of inner membrane proteins due to the formation of thiol cross-linking. Alternatively, literature data propose the participation of a number of other outer membrane proteins in the composition of the MPT pore, such as, voltage-dependent anion channel (VDAC), hexokinase and the benzodiazepine receptor. In order to ascertain the independence of outer membrane proteins to generate the MPT we performed experiments with mitochondria devoid of outer membrane (mitoplasts). Liver and kidney mitoplasts were obtained by use of cholesterol-specific detergent digitonin to eliminate the outer membrane. Compared with the mitochondria, liver mitoplasts retained less than 5% and 10% of the markers of the outer membrane monoamine oxidase and VDAC, respectively. Kidney mitoplasts also showed a partial depletion of hexokinase (~73%). Mitoplasts presented with a 70% lower oxygen consumption rate during ADP phosphorylation ,which was partially reverted by the addition of exogenous cytochrome c. Mitoplasts were able to generate and sustain a membrane potential high enough to phosphorylate ADP. As hypothesized, these mitoplasts undergo Ca2+ induced MTP with similar properties as compared to intact mitochondria. Mitoplast MPT was sensitive to known permeability transition inducers such as Ca2+ and tert-butyl hydroperoxide and to inhibitors such as cyclosporin A, Ca2+ chelators, ADP, dithiol reductant and catalase. Altogether, the data indicate that the outer membrane is not necessary for MPT assembling or opening. / Mestrado / Mestre em Fisiopatologia Médica