Дисертації з теми "Yeast apoptosis"
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Ilina, Yulia. "Functions of the yeast protein Stm1 and its involvement in apoptotic cell death." [S.l. : s.n.], 2005.
Знайти повний текст джерелаNargund, Amrita Mohan. "Mechanism (S) of Metal-Induced Apoptosis in Saccharomyces Cerevisiae." Digital Archive @ GSU, 2010. http://digitalarchive.gsu.edu/biology_diss/80.
Повний текст джерелаCosta, Ana Margarida Pinto e. ""The Role Of Ceramide Pathway In Yeast Apoptosis Induced By Acetic Acid"." Master's thesis, Instituto de Ciências Biomédicas Abel Salazar, 2009. http://hdl.handle.net/10216/26276.
Повний текст джерелаCosta, Ana Margarida Pinto e. ""The Role Of Ceramide Pathway In Yeast Apoptosis Induced By Acetic Acid"." Dissertação, Instituto de Ciências Biomédicas Abel Salazar, 2009. http://hdl.handle.net/10216/26276.
Повний текст джерелаKritsiligkou, Paraskevi. "Peroxiredoxins : yeast redox switches that regulate multiple cellular pathways." Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/peroxiredoxins-yeast-redox-switches-that-regulate-multiple-cellular-pathways(fbb44664-5021-4dbc-88c7-64aef8a6c045).html.
Повний текст джерелаYang, Hui. "Chromosome dynamics and chromosomal proteins in relation to apoptotic cell death in yeast." Laramie, Wyo. : University of Wyoming, 2008. http://proquest.umi.com/pqdweb?did=1594496261&sid=1&Fmt=2&clientId=18949&RQT=309&VName=PQD.
Повний текст джерелаKoduru, Rupa. "Study of Cellular Activities in Response to Metal-Induced Apoptosis in Saccharomyces Cerevisiae using FTIR." Digital Archive @ GSU, 2011. http://digitalarchive.gsu.edu/biology_theses/30.
Повний текст джерелаBrezniceanu-Mehedinti, Marie-Luise Ligia. "Identification of mammalian proteins inhibiting apoptosis downstream of cytochrome c release in a yeast survival screen." [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=969077343.
Повний текст джерелаBenzing, Jörg. "Identifikation intrazellulärer Interaktionspartner der Rezeptortyrosinkinasen UFO und MET im Two-Hybrid-System." Ulm : Universität Ulm, Medizinische Fakultät, 2001. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB9394024.
Повний текст джерелаLigr, Martin. "Apoptosis in the yeast Saccharomyces cerevisiae a novel cell death process regulated by the Ubiquitin-Proteasome system /." [S.l. : s.n.], 2001. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB9203728.
Повний текст джерелаWright, Michael Eugene. "Development, characterization, and use of a novel yeast expression system to identify inhibitors of the caspase-3 cell death protease /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/5018.
Повний текст джерелаRing, Giselle Natasha. "Identification and characterization of TMEM 85, a novel suppressor of bax-mediated cell death in yeast." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=112352.
Повний текст джерелаRoset, i. Huguet Ramon. "Study of the regulation and signalling of cdk2-Cyclin o complexes during apoptosis." Doctoral thesis, Universitat Pompeu Fabra, 2008. http://hdl.handle.net/10803/7199.
Повний текст джерелаL'objectiu d'aquesta tesi és la caracterització d'una proteïna involucrada en l'apoptosi. El nostre grup ha identificat un pas primerenc comú en diversos estímuls apoptòtics de la ruta intrínseca. Aquest pas requereix la síntesi de novo d'una nova Ciclina, Ciclina O, que quan s'indueix apoptosi en cèl·lules limfoides forma complexes actius majoritàriament amb Cdk2. L'expressió de la Ciclina O és prèvia a l'apoptosi induïda per glucocorticoids i radiació gamma i la seva sobreexpressió indueix apoptosi en cultius cel·lulars. La baixada dels nivells d'expressió de la Ciclina O endògena amb shRNA provoca una inhibició de l'apoptosi induïda per glucocorticoids o agents que danyen el DNA, mentre que l'apoptosi mediada pel receptor CD95 es manté intacta. Aquests resultats demostren que la inducció d'apoptosi en cèl·lules limfoides és una de les funcions fisiològiques de la Ciclina O i que no es deu a una pertorbació de processos cel·lulars normals com ara el cicle cel·lular. A més a més, hem identificat c-Myb com a substrat dels complexes Cdk2-Ciclina O i demostrem que els nivells de c-Myb baixen durant l'apoptosis de cèl·lules limfoides.
Stratico, Valerie Anne. "CHARACTERIZATION OF A NOVEL INTERACTOR/SUBSTRATE FOR THE PRO-APOPTOTIC SERINE PROTEASE OMI/HTRA2." Master's thesis, University of Central Florida, 2004. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4400.
Повний текст джерелаM.S.
Department of Molecular Biology and Microbiology
Health and Public Affairs
Molecular Biology and Microbiology
Wilkinson, Derek. "Proteases and programmed cell death in fungi." Thesis, University of Exeter, 2011. http://hdl.handle.net/10036/3629.
Повний текст джерелаFerté-Chaudoy, Marion. "Virus host interactome du polyomavirus à cellules de Merkel." Thesis, Tours, 2017. http://www.theses.fr/2017TOUR3805/document.
Повний текст джерелаThe Merkel cell polyomavirus is now recognized as the etiologic agent of Merkel cell carcinoma (MCC). The viral cycle and viro-induced oncogenesis mechanisms are not fully understood and the knowledge is mainly based on the studies carried out particularly on the SV40 polyomavirus. The aim of our work is to identify interactions between viral proteins and cellular proteins during productive infection or in MCC context. To identify these interactions, we performed yeast two hybrid screens on MCPyV and BKPyV oncogenes, as control. To validate the interactions obtained in yeasts, we used an orthogonal method of validation by complementation in mammalian cells based on the restoration of Gaussia princeps luciferase. The combination of these two orthogonal techniques allowed us to validate interactions with cellular partners involved in cell cycle regulation or Akt-mTOR pathway. Previous lab work on VP2/VP3 minor capsid proteins allowed the identification of interactions with NF-kB pathway involved proteins. We examined the interactions between oncogenes, VP2, with the cellular proteins involved in this pathway. This work led us to evaluate pathway activation, genes expression under the control of NF-kB and apoptosis regulation. These results evidenced an action of the VP2 protein on the activation of NF-kB pathway and an induction of apoptosis
Khoury, Chamel Michael. "Identification of novel anti-apoptotic sequences by screening for suppressors of the effects of Bax in yeast." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=18762.
Повний текст джерелаLa détermination de nouveau cheminements de normalization anti-apoptotique est essentiel à la compréhension de plusieurs pathologies au niveau moléculaire, incluant le cancer et les maladies du coeur. La famille de protéines Bcl-2 inclu des agents de normalization centrales du processus apoptotique présents dans des cellules mammifères, dont l'anti-apoptotique Bcl-2 et le pro-apoptotique Bax. La description des phénotypes apoptotique contenus dans la levure induit par un stimulus aggressif, incluant l'expression hétérologue des protéines Bcl-2, indique la présence d'un programme apoptotique conservé durant l'évolution de ce dernier. Précédemment, nous avons reporté sur l'identification de plusieurs cADNs plutôt efficaces envers la prévention des éffets néfastes concernant l'expression hétérologue d'un BAX cADN pro-apoptotique dans la levure (Yang, Khoury et al., FEMS Yeast Research 2006; 6:751-762). Içi, je reporte le fait qu'un abrogeur Bax est en mesure d'encoder une nouvelle variante d'acide aminé 156 de la protéine humaine Vps24, nommé Vps24ß, qui n'est pas muni du domaine caractéristique d'aggripage de lipides à N-bornes qui se retrouve généralement dans le résidue 222 du Vps24 (Vps24a). Je démontre que le Vps24ß cADN représente une transcription qui est probablement produit par le processus alternatif d'épissement du gène humain Vps24. C'était le Vps24a, et non le Vps24ß, qui a empêché la croissance des défauts sensible au sel et à la température observés dans une affectation de levure qui ne possédait pas un gène Vps24 fonctionnel. Par contre, le Vps24ß, et non le Vps24a, a supprimé les éffets négatives du Bax envers la croissance de la levure. De plus, la protéine Vps24ß a aussi supprimé les éffets du Bax dans des échantillons le levure mutant manquant d'autres gènes VPS, qui suggèrent qu'une voie ESCRT fonctionnelle, ou le Vps24p est essentiel, n'est pas requis pour le Vps24ß. Pris ensembles, no
Yang, Zhao 1970. "Identification of a novel anti-apoptotic protein and characterization of mammalian regulators of G protein signaling (RGSs) in yeast." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111875.
Повний текст джерелаBased on the observation that human RGS1 causes yeast cell growth arrest, I therefore used RGS1 expressing yeast cells to screen a mouse T cell cDNA library in order to find potential interacting proteins. From the screen, I identified a mouse sphingomyelin synthase 1 (SMS1) cDNA. By using a series of different apoptotic stimuli, such as hydrogen peroxide, osmotic stress, exogenous ceramide and its precursors, high temperature etc., SMS1 expression was found to suppress cell growth arrest and prevent viability decline, indicating that SMS1 represents an anti-apoptotic protein that functions by decreasing the intracellular level of pro-apoptotic ceramide.
Gene analysis further indicated that the SMS1 gene consists of 16 exons spread over a 256kb portion of mouse chromosome 19. It is alternatively spliced to produce 4 different transcripts (SMS1alpha1, SMS1alpha2, SMS1beta and SMS1gamma) and encode 3 different proteins (SMS1alpha, SMS1beta and SMS1gamma). Notably, I found that SMS1beta protein does not interfere with SMS1alpha anti-apoptotic function, although both of these two proteins contain the protein-protein interaction domain, sterile alpha motif (SAM), at their N-terminus.
I also carried out a study to examine GPCR-RGS interactions using the yeast expression system. Our lab had noticed that there was an extra RGS5 related protein that was detected by western blot analysis in the protein extracts prepared from yeast and HEK293 cells expressing RGS5. The size of the band was approximately 2 times the molecular weight of RGS5, indicating the possibility that RGS5 forms a dimer. To further examine this hypothesis, I, therefore, performed a series of experiments, included yeast 2 hybrid assays, to demonstrate that RGS5 does interact with itself. This is the first report that RGS can form a dimer. The implications for this finding are discussed in detail.
Pereira, Clara Isabel Ferreira. "Involvement of mitochondrial proteins in yeast apoptosis." Doctoral thesis, 2008. http://hdl.handle.net/1822/8243.
Повний текст джерелаIn the yeast Saccharomyces cerevisiae, acetic acid triggers a mitochondria-mediated death pathway with apoptotic characteristics. In mammalian cells, the mitochondrial outer membrane permeabilization (MOMP), necessary for the release of pro-apoptotic proteins, is a pivotal event for the activation of the apoptotic cascade in numerous cell death pathways. MOMP is thought to be mediated by a complex of proteins that constitute the permeability transition pore (PTP). Since S. cerevisiae possesses orthologues of the components believed to be involved in mammalian PTP composition/regulation, it was used herein to study such proteins both concerning their role in mitochondrial permeabilization and involvement in the course of cell death. The proteins studied were Por1p (yeast voltage-dependent anion channel, VDAC), Cpr3p (mitochondrial cyclophilin) and Aac1/2/3p (ADP/ATP carrier, AAC). We found that during apoptosis triggered by acetic acid deletion of CPR3 has no effect. Absence of Por1p enhances and absence of AAC proteins decreases acetic acidinduced apoptosis indicating an anti- and pro-apoptotic role, respectively, for these proteins. Moreover, the pro-death role of AAC does not require the ADP/ATP translocase activity. Absence of AAC proteins impairs MOMP and release of cytochrome c, which is degraded along with other mitochondrial inner membrane proteins. We observe that, during acetic acid-induced apoptosis, caspase activation is independent of AAC proteins but strongly dependent on the growth phase of the culture. In addition, a strain deleted for the yeast metacaspase YCA1 shows decreased overall caspase activation but still died exhibiting apoptotic features, supporting the existence of an Yca1p-independent apoptotic pathway. Fragmentation and degradation of mitochondria are common events in mammalian apoptosis. Both fragmentation and degradation are able to strongly affect the course of cell death and a relation has been proposed between these events and MOMP/cytochrome c release. Interestingly, por1Δ cells exhibit fragmented mitochondrial reticulum in the absence of any death stimulus. This phenotype however, does not contribute to the apoptosis stimulation observed in por1Δ mutant. We observe that during acetic acidinduced apoptosis the absence of AAC proteins leads to aggregation of fragmented mitochondria and a slower degradation of these organelles. Degradation of mitochondria in response to acetic acid is not due to classical autophagy or mediated by Uth1p-dependent mitophagy. We show that mitochondrial degradation during acetic acid-induced apoptosis is dependent on the protease Pep4p that is released from the vacuole to the cytosol. pep4Δ cells, which are strongly impaired in mitochondria degradation, are sensitized to acetic acid and mainly die by necrosis. This suggests that mitochondria degradation in response to acetic acid helps to sustain the apoptotic process. Taken together, the results show that vacuolar and mitochondrial proteins interfere with mitochondria morphological remodelling and subsequent degradation, suggesting that there is a complex interplay between these organelles in the regulation of yeast cell death. In conclusion, we were able exploring the distinctive ability of yeast to survive without respiration-competent mitochondria to study the involvement of mitochondria and mitochondria-interacting proteins in cell death. Additional studies using this model will undoubtedly further our understanding of the complex cell death processes.
Na levedura Saccharomyces cerevisiae, o ácido acético desencadeia uma via de morte dependente da mitocôndria com características apoptóticas. Nos mamíferos, a permeabilização da membrana mitocondrial externa (PMME), necessária para a libertação de proteínas mitocondriais pró-apoptóticas, constitui um passo crítico na activação do processo de morte apoptótico. Pensa-se que a PMME seja mediada por um complexo proteico que constitui o poro de transição de permeabilidade (PTP). Uma vez que S. cerevisiae possui ortólogos de algumas das proteínas que se pressupõe compor/regular o PTP de mamíferos, o principal objectivo desta tese foi estudar essas proteínas, tanto ao nível do seu papel na permeabilização mitocondrial bem como do seu envolvimento na execução do processo de morte apoptótico. As proteínas estudadas incluem o canal Por1p (canal de aniões dependente da voltagem, VDAC), a ciclofilina mitocondrial Cpr3p e as três isoformas do transportador Aac1/2/3p (antiportador mitocondrial de ATP/ADP, AAC). A interrupção do gene CPR3 não afecta a morte induzida pelo ácido acético. A interrupção do gene POR1 estimula e a ausência dos genes AAC1/2/3 protege as células da morte apoptótica induzida pelo ácido acético indicando um papel anti- e pró-apoptótico, respectivamente, para estas proteínas. A função das proteínas AAC na morte celular apoptótica não depende da actividade de antiporte. Em células tratadas com ácido acético a ausência das proteínas AAC afecta negativamente a PMME e a libertação de citocromo c, o qual juntamente com outra proteína da membrana mitocondrial interna sofre degradação. Observamos que durante o processo apoptótico induzido pelo ácido acético ocorre a activação de caspases e que esta activação é independente das proteínas AAC e fortemente dependente da fase de crescimento da cultura. Adicionalmente, uma estirpe interrompida na metacaspase de levedura, YCA1, que exibe um decréscimo na activação total de caspases, desencadeia uma morte celular apoptótica. Esta observação suporta a existência de uma via de morte apoptótica independente de Yca1p. A fragmentação e a degradação mitocondrial são eventos comuns no processo apoptótico em mamíferos. Estes eventos podem ter um forte impacto no decurso da morte celular tendo sido proposta uma relação entre estes e a PMME/libertação de citocromo c. Curiosamente, a ausência da Por1p origina uma elevada percentagem de células com o retículo mitocondrial fragmentado mesmo na ausência de qualquer estímulo externo. Este fenótipo, contudo, não parece contribuir para a estimulação da apoptose exibida pela estirpe por1Δ. Durante o processo de morte apoptótico induzido pelo ácido acético, a ausência das proteínas AAC leva à formação de agregados mitocondriais associada a uma menor degradação destes organelos. A degradação mitocondrial durante a apoptose induzida pelo ácido acético não é devida à activação da autofagia clássica, nem mediada pela mitofagia dependente de Uth1p. A degradação mitocondrial induzida pelo ácido acético é dependente da protease Pep4p, que é libertada do vacúolo para o citosol. A estirpe pep4Δ que exibe uma degradação mitocondrial manifestamente diminuída é sensível ao ácido acético, e direcciona o processo de morte para uma forma necrótica. Estes dados sugerem que a degradação mitocondrial favorece a manutenção do processo apoptótico. Globalmente, os resultados evidenciam que proteínas vacuolares e mitocondriais interferem com a morfologia mitocondrial e subsequente degradação e sugerem uma interacção e regulação complexa entre estes organelos durante o processo apoptótico em leveduras. Concluindo, a capacidade de sobrevivência da levedura em condições em que a respiração mitocondrial está afectada ou ausente permitiu a sua utilização como um excelente modelo para a pesquisa do papel de proteínas mitocondriais, ou de proteínas que interactuam com a mitocôndria no processo de morte celular. Estudos adicionais utilizando este modelo irão seguramente contribuir para uma melhor compreensão dos processos de morte celular.
Fundação para Ciência e a Tecnologia (FCT) - grant SFRH/BD/13497/2003
Davidich, Maria I. [Verfasser]. "Boolean network models of the fission yeast cell cycle and apoptosis / Maria I. Davidich." 2009. http://d-nb.info/993836208/34.
Повний текст джерелаRodrigues, Andreia Dóris Pedras. "Functional characterization of purified vacuoles and evaluation of their role in yeast apoptosis induced by acetic acid." Master's thesis, 2010. http://hdl.handle.net/1822/35187.
Повний текст джерелаThe vacuole is the largest organelle of yeast cells and is functionally equivalent to animal lysosome and plant vacuole. Vacuoles are the most acidic organelles of the cell, and play major roles in protein degradation, ion and metabolite storage, as well as in ion homeostasis, response to nutrient deprivation, osmotic and ionic stress, autophagy and even in apoptosis. Lysosome membrane permeabilization and the consequent release of lysosomal proteases, namely cathepsins, are now widely accepted to trigger apoptosis. Of all lysosomal cathepsins, the aspartic cathepsin D was the first identified protein with apoptogenic properties. Acetic acid was shown to induce apoptosis in yeast, associated with changes in mitochondria and release of pro-apoptotic proteins. Pep4p, a vacuolar yeast protease orthologue of the lysosomal human cathepsin D was also shown to be involved in acetic acid-induced apoptosis. The ultimate objective of the present study was to contribute to the understanding of the crosstalk between the vacuole and mitochondria in yeast programmed cell death induced by acetic acid. For these purposes the effect of acetic acid in isolated vacuoles and whole cells with different genetic backgrounds, namely on vacuole membrane permeabilization as well on the release of Pep4p and their relation with alterations in vacuole function, was investigated. The functional characterization of isolated vacuoles as well the effect of acetic acid on vacuolar function was monitored with fluorescent probes combined with flow cytometry and fluorescence microscopy. Functional characterization of isolated vacuoles was also monitored through the activity of V-H+-ATPase by spectrofluorimetry. Epifluorescence microscopy imaging showed that the vacuolar membrane stains strongly with the styryl dye FM1-43 and that most of the vacuoles accumulate Ca2+, as assessed with Fluo-4 AM. Flow cytometry analysis of vacuole samples incubated with these fluorescent probes confirmed a well-defined population of intact and functional vacuoles. Consistently spectrofluorimetric assays with the pH-sensitive probe ACMA suggested that the isolated vacuoles were intact and functional, the vacuolar membrane being able to generate and maintain a pH gradient through a concanamycin A-sensitive V-H+-ATPase. The addition of acetic acid induced the release from the vacuole lumen of an EGFP-Pep4p fusion protein. Changes in fluorescence of vacuoles stained with acridine orange and Fluo-4 AM suggest that the acid induces a transient perturbation of vacuolar pH and Ca2+ release. It has been described that release of Ca2+ is an event involved in cell death, as well as the release of H+ and consequent cytosol acidification. The main novelty of the present study with isolated vacuoles is the finding that acetic acid is able to directly induce a partial permeabilization of the vacuolar membrane, similar to LMP in mammalian cells, without the involvement of other organelles or triggering upstream pathways.
O vacúolo é o maior organelo da levedura e é funcionalmente equivalente ao lisossoma das células animais e ao vacúolo das células vegetais. Este organelo, cujo lúmen tem o pH mais ácido, participa em processos celulares importantes tais como degradação proteica e armazenamento de iões e metabolitos, bem como na homeostase iónica, resposta à privação de nutrientes, stress osmótico e iónico, autofagia e mesmo na apoptose. Sabe-se que a permeabilização da membrana lisossomal (LMP) e a consequente libertação de proteases lisossomais, nomeadamente de catepsinas, induz apoptose. De todas as proteases lisossomais, a catepsina D foi a primeira proteína a ser identificada com propriedades apoptóticas. Foi demonstrado que o ácido acético induz apoptose em leveduras, associada a alterações mitocondriais e à libertação de proteínas pró-apoptóticas. Foi igualmente demonstrado que a protease vacuolar Pep4p, ortóloga da catepsina D humana, está envolvida na apoptose induzida por ácido acético. O presente estudo teve como principal objectivo contribuir para a compreensão da interligação entre o vacúolo e a mitocôndria na morte celular programada induzida por ácido acético na levedura Saccharomyces cerevisiae. Para tal foi investigado o efeito do ácido acético em vacúolos isolados e em células inteiras de leveduras com diferentes backgrounds genéticos, nomeadamente no que respeita à permeabilização da membrana vacuolar bem como à libertação de Pep4p e sua relação com alterações nas funções do vacúolos. A caracterização funcional de vacúolos isolados bem como o efeito da adição de ácido acético na função vacuolar foram realizadas por citometria de fluxo e microscopia de fluorescência associada à utilização de diferentes sondas fluorescentes. A caracterização funcional dos vacúolos isolados foi complementada pela determinação da actividade da V-H+-ATPase por espectrofluorimetria. Os estudos de microscopia de fluorescência mostraram uma marcação forte da membrana vacuolar com a sonda lipofílica FM1-43 e que a maioria dos vacúolos acumulavam Ca2+ com base na marcação pela sonda Fluo-4 AM. A análise por citometria de fluxo de amostras de vacúolos incubadas com estas sondas fluorescentes evidenciou uma população bem definida de vacúolos intactos e funcionais. Consistentemente os ensaios de espectrofluorimetria com a sonda ACMA, sensível ao pH, sugeriram que os vacúolos isolados se encontravam intactos e funcionais, dado que a membrana vacuolar era capaz de gerar e manter um gradiente de pH através da actividade da bomba V-H+-ATPase, sensível à concanamicina A. A adição de ácido acético promoveu a libertação da proteína de fusão EGFP-Pep4p em vacúolos isolados e induziu variações de fluorescência de vacúolos marcados com laranja de acridina e com Fluo-4 AM indicativas de uma perturbação transitória do pH vacuolar e de uma libertação de Ca2+. Está descrito que a libertação de Ca2+ e de H+, com a consequente acidificação do citosol, está envolvida na morte celular. Estes novos resultados em vacúolos isolados permitem concluir que o ácido acético é capaz de induzir directamente uma permeabilização parcial da membrana vacuolar, semelhante à LMP em mamíferos, sem o envolvimento de outros organelos ou sem a activação de vias a montante.
This work was supported by the following projects: (PTDC/BIA-BCM/69448/2006) and by PTDC/AGR-ALI/100636/2008
Pereira, Helena Paula Fernandes. "The role of Pep4p, the vacuolar yeast protease ortholog of human cathepsin D, in mitochondria-dependent apoptosis." Doctoral thesis, 2015. http://hdl.handle.net/1822/38434.
Повний текст джерелаLysosomal cathepsins play a crucial role in cell homeostasis by participating in the degradation of heterophagic and autophagic material. Additionally, following their release into the cytosol, these proteases are involved in pro-apoptotic and anti-apoptotic processes, particularly the aspartic cathepsin D (CatD). Indeed, CatD released into the cytosol triggers a mitochondrial apoptotic cascade. However, CatD can have anti-apoptotic effects in some cellular types and specific contexts. Therefore, targeting this apoptosis regulator in therapies for apoptosis deficiency-associated diseases, such as cancer, requires detailed elucidation of its mechanisms of action. Understanding the molecular mechanisms connecting lysosomal to mitochondrial membrane permeabilization is thus particularly relevant. More recently, vacuolar membrane permeabilization and consequent release of vacuolar proteins into the cytosol was also observed in yeast. It was demonstrated, that Pep4p (yeast CatD), a pepsin-like aspartic protease found in the yeast vacuole and ortholog to human CatD, is released from the vacuole during hydrogen peroxide- or actin stabilization-induced apoptosis. It also translocates into the cytosol during acetic acidinduced apoptosis, and is required for autophagy-independent degradation of mitochondria and for increased cell survival in response to this acid. Furthermore, acetate in colorectal carcinoma (CRC) cells seems to behave as acetic acid in yeast, triggering lysosomal membrane permeabilization (LMP), CatD release and mitochondria-dependent apoptosis. Recently, we found that CatD is involved in autophagy-independent degradation of damaged mitochondria, which renders CRC cells more resistant to apoptosis induced by acetate. These observations, combined with the hints provided by the yeast cell model, support the idea that LMP associated with the release of CatD protects CRC cells from mitochondrial dysfunction during acetate-induced apoptosis through its involvement in degradation of damaged mitochondria. Thus, it has become apparent that the approaches with yeast have already provided and can further offer new perspectives for an enhanced understanding of the role of CatD in mammalian apoptosis, as well of the molecular basis of the crosstalk between the lysosome and mitochondria. Thereafter, we set out to exploit acetic acid-induced apoptosis in Saccharomyces cerevisiae to study the yeast vacuolar protease Pep4p, both concerning its role in mitochondrial degradation and its involvement in the course of apoptosis. In this thesis, it is shown that the protective role of Pep4p in acetic acid-induced apoptosis is independent of the yeast voltage dependent channel Por1p (which has no role on mitochondrial degradation) but dependent on AAC proteins, the yeast adenine nucleotide translocator. Moreover, it has shown that both the Pep4p anti-apoptotic function and its role in mitochondrial degradation depend on Pep4p proteolytic activity. In this study, we also demonstrated that the pro-survival role of Pep4p in acetic acid-induced apoptosis is dependent on mitochondrial respiratory function, and that deficiency in mitochondrial respiration suppresses its role in mitochondrial degradation. Altogether, these results contributed to unveil a novel pro-survival function of CatD in autophagy-independent mitochondrial degradation, which can lead to enhanced cell survival in CRC cells undergoing acetate-induced apoptosis. Moreover, these studies reinforce the use of yeast as a valuable model to elucidate the role of CatD in mammalian apoptosis, as well as the molecular mechanisms involved in the crosstalk between the lysosome and mitochondria.
As catepsinas lisossomais têm um papel crucial na homeostasia celular, participando na degradação de material hetero- e autofágico. Adicionalmente, estas proteases estão envolvidas em processos pró- e anti-apoptóticos após a sua libertação para o citosol, particularmente a catepsina aspártica D (CatD). Com efeito, uma vez no citosol, a CatD desencadeia a cascata apoptótica mitocondrial. Contudo, esta protease pode ter um papel anti-apóptótico. A utilização deste regulador apoptótico como alvo molecular na terapia de doenças associadas a deficiências no processo apoptótico requer portanto uma elucidação detalhada dos seus mecanismos de acção. Por este motivo, a compreensão dos mecanismos moleculares que conectam a permeabilização da membrana lisossomal (PML) à permeabilização da membrana mitocondrial é particularmente relevante. Mais recentemente, observou-se que a permeabilização da membrana vacuolar e consequente libertação de proteases vacuolares para o citosol também ocorre na levedura. Foi demonstrado que a Pep4p, a protease aspártica encontrada no vacúolo da levedura e ortóloga da CatD humana, é libertada do vacúolo para o citosol durante a apoptose induzida pelo peróxido de hidrogénio ou estabilização da actina. Esta protease também é translocada para o citosol durante a apoptose induzida pelo ácido acético desempenhando um papel crucial na degradação mitocondrial independente da autofagia e na sobrevivência celular em resposta a este ácido. Adicionalmente, o acetato em linhas celulares derivadas do carcinoma colorectal (CRC) comporta-se de modo análogo ao ácido acético na levedura, induzindo PML, libertação da CatD e apoptose dependente da mitocôndria. Nós demonstrámos recentemente que a CatD está envolvida na degradação mitocondrial independente da autofagia, o que torna as células do CRC mais resistentes à apoptose induzida pelo acetato. Estas observações, mais as indicações obtidas através do modelo de levedura, reforçam a ideia de que a PML associada à libertação da CatD protege as células do CRC de uma disfunção mitocondrial durante a apoptose induzida pelo acetato, através do seu envolvimento na degradação de mitocôndrias danificadas. Tornou-se então aparente que as abordagens na levedura forneceram informação importante e podem vir a oferecer perspectivas adicionais, contribuindo assim para uma melhor compreensão do papel da CatD na apoptose em mamíferos, bem como das bases moleculares do “crosstalk” entre o lisossoma e a mitocôndria. Por conseguinte, decidimos explorar o modelo da apoptose induzida pelo ácido acético na Saccharomyces cerevisiae para estudar a protease vacuolar da levedura Pep4p, relativamente ao seu papel na degradação mitocondrial e consequentemente seu envolvimento na apoptose. Nesta tese, mostra-se que o papel protector da Pep4p na apoptose induzida pelo ácido acetico é independente do canal de aniões dependente da voltagem de levedura Por1p (que por sua vez não desempenha um papel na degradação mitocondrial), mas é dependente das proteínas AAC, o antiportador mitocondrial de ATP/ADP da levedura. Também foi demonstrado que a função anti-apoptótica da Pep4p, bem como o seu papel na degradação mitocondrial dependem da sua actividade proteolítica. Neste estudo, foi também demonstrado que o papel protector da Pep4p na apoptose induzida pelo ácido acético é dependente da função respiratória mitocondrial, e também que a deficiência na respiração mitocondrial suprime o papel da Pep4p na degradação mitocondrial. Em conjunto, os resultados aqui descritos contribuíram para revelar uma nova função da CatD na degradação mitocondrial independente da autofagia, que pode conduzir a um aumento da sobrevivência nas células do CRC durante a apoptose induzida pelo acetato. Além disso, estes estudos reforçam o uso da levedura como modelo para elucidar o papel da CatD na apoptose de mamíferos, bem como os mecanismos moleculares envolvidos no “crosstalk” entre o lisossoma e a mitocôndria.
A autora deste trabalho usufruiu de uma bolsa da Fundacao para a Ciencia e a Tecnologia (FCT), com a referencia SFRH/BD/73139/2010 co-financiada pelo Programa Operacional Potencial Humano (POPH) do Quadro de Referencia Estrategico Nacional (QREN), comparticipado pelo fundo Social Europeu e por fundos nacionais
Ligr, Martin [Verfasser]. "Apoptosis in the yeast Saccharomyces cerevisiae : a novel cell death process regulated by the Ubiquitin-Proteasome system / vorgelegt von Martin Ligr." 2001. http://d-nb.info/961924918/34.
Повний текст джерелаSarras, Haya. "A Role for Bclaf1 in mRNA Processing and Skeletal Muscle Differentiation." Thesis, 2012. http://hdl.handle.net/1807/35158.
Повний текст джерелаBrezniceanu-Mehedinti, Marie-Luise Ligia [Verfasser]. "Identification of mammalian proteins inhibiting apoptosis downstream of cytochrome c release in a yeast survival screen / by Marie-Luise Ligia Brezniceanu-Mehedinti." 2003. http://d-nb.info/969077343/34.
Повний текст джерелаChibi, Moredreck. "A yeast 2-hybrid screen to identify and characterize interaction partners of the cancer associated protein retinoblastoma binding protein 6." Thesis, 2009. http://hdl.handle.net/11394/3190.
Повний текст джерелаRetinoblastoma binding protein 6 (RBBP6) is a 250 kDa protein that is implicated in mRNA processing and ubiquitination functions and has been shown to be highly up-regulated in a number of cancers. In humans and mice,RBBP6 interacts with both tumour suppressors p53 and pRb, suggesting that it is involved in regulation of transcription, induction of apoptosis and cell cycle control. Knock-out of an RBBP6 homologue PACT resulted in p53 dependent cell cycle arrest and apoptosis. Although the biological functions of RBBP6 remain largely unclear, it is possible that its functions are mediated through interaction with other cellular proteins. Since it is possible to unveil novel functions of a target protein through identifying its interacting protein partners,this study aims to further characterize the functions of RBBP6 through identifying novel protein interacting partners using a yeast 2-hybrid screen.In order to identify interaction partners of RBBP6, two well characterized domains of RBBP6, the N-terminal ubiquitin-like DWNN domain and RING finger domain, were used as baits in a yeast 2-hybrid screen of a human testis cDNA library. Putative interactors were verified using in vitro and in vivo immunoprecipitation assays. The RING finger domain was shown to interact with transcriptional factors Y-Box binding protein 1 (YB-1) and zinc finger and BTB containing protein 38 (zBTB38), resulting in their ubiquitination. In the case of YB-1 ubiquitination was correlated with a decrease in the intra-cellular levels of YB-1, suggesting that ubiquitination leads to degradation in the proteosome. The DWNN domain was shown to interact with a splicing associated small nuclear ribonucleoprotein polypeptide G (snRPG) and heat shock protein 70 (Hsp70).The results of this work suggest that, at least in the case of YB-1 and zBTB38,RBBP6 plays a role in the regulation of gene expression by ubiquitination of transcription factors, causing them to be degraded in the proteosome. The study provides further evidence of RBBP6’s involvement in mRNA splicing through its interaction with snRPG. The interaction with Hsp70 suggests a possible role in protein quality control similar to that played by other E3 ligases such as Parkin and CHIP.
Guérin, Renée. "La calnexine: un élément clé dans l'apoptose chez la levure Schizosaccharomyces pombe." Thèse, 2008. http://hdl.handle.net/1866/2896.
Повний текст джерелаProgrammed Cell Death (PCD) is an essential process to the cells. PCD was first characterized in cell development and can be separated in sub-groups depending of cell death characteristics observed. Apoptosis is one of the PCD sub-groups that was first associated to complex organisms for its roles in cell development and in maintenance of tissues integrity. The apoptotic pathway is characterized by specific morphological and signalization characteristics. In the last ten years, numerous studies demonstrated the existence of apoptosis in unicellular organisms such as yeast. This apoptotic program was extensively studied in the two yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe and share characteristics with the mammalian one. However, yeast apoptosis is distinctive at many points as yeast do not encodes all the mammalian homologues of the apoptotic pathway. Although yeast and mammalian apoptosis seems to differs, the interest about yeast apoptosis is growing given that yeast is an excellent and easily tractable model system. External and internal stimuli can induce apoptosis by different ways. Accumulation of unfolded or incompletely folded proteins in the endoplasmic reticulum (ER) causing ER stress is a well-known inducer of the apoptotic pathway. Signalization of ER-stress induced apoptosis involves the same transducers than the UPR (Unfolded Protein Response) pathway. It was recently shown that calnexin, a transmembrane chaperone of the ER, is implicated in ER-stress apoptosis in mammalian cells. In this particular case, it was demonstrated that calnexin acts as a scaffold in the cleavage of the apoptotic protein Bap31 by caspase 8. We demonstrated that ER stress and inositol deficiency, a precursor of many important signalization molecules, are two situations leading to apoptosis in the yeast S. pombe. These two pathways leading to apoptosis seems to differ as only inositol deficiency is dependant of the yeast metacaspase Pca1p. We also demonstrated that S. pombe calnexin, essential for cell viability of this yeast, takes part in these two apoptotic process. ER stress induced apoptosis needs a calnexin anchors to the ER membrane to be efficient. However, apoptosis induced by inositol starvation needs the calnexin C-terminal tail with the transmembrane domain to be delayed. These two opposite actions from the same protein lead to the hypothesis that calnexin encodes both pro and anti-apoptotic functions. By the discovery that calnexin is cleaved under normal culture conditions, a model was elaborated explaining the distinctive roles of calnexin in these two apoptotic pathways. This model proposed a role of calnexin cleavage to apoptosis.
Sharom, Jeffrey Roslan. "A Global Kinase and Phosphatase Interaction Network in the Budding Yeast Reveals Novel Effectors of the Target of Rapamycin (TOR) Pathway." Thesis, 2011. http://hdl.handle.net/1807/29864.
Повний текст джерелаBaptista, Vitória da Cunha. "Exploring yeast as a tool to study the regulation of the human pro-apoptotic protein Bax." Master's thesis, 2018. http://hdl.handle.net/1822/65182.
Повний текст джерелаCell death has long been considered crucial for proper tissue shaping during embryonic development and for normal turnover of the cells in several tissues. Moreover, mis-regulation of apoptosis has been implicated in a diversity of abnormal functions and in the progression and development of diseases. Indeed, a decrease in apoptosis has been associated with ageing and tumour progression, whereas an increase in apoptosis is mainly associated with ageing-related neurodegenerative diseases including Alzheimer’s, Parkinson’s and Huntington’s diseases. The life and death switch is among others regulated by interactions between pro- and anti-apoptotic Bcl-2 family members, including Bax and Bcl-xL, respectively. Bax plays a central role in apoptosis, as it is involved in the formation of pores within the mitochondria outer membrane through which apoptogenic factors, including cytochrome c, are released and originate a cascade of events that culminate in cell death. Given the importance of this pro-apoptotic protein in cell death, its potential as a therapeutic target was quickly recognized, and its mode of action and regulation have been studied extensively. However, many questions still remain, and thus further understanding of Bax regulation was the general purpose of this thesis. To accomplish this, we took advantage of the genetically tractable yeast Saccharomyces cerevisiae, whose genome is devoid of genes coding apparent homologues of the human Bcl-2 family. Therefore, the heterologous expression of human Bax allows its study without interference of the apoptotic network. To achieve our goal, we first optimized the conditions for expression of human Bax (Bax α) in yeast and characterized the phenotype of expression of Bax α and of an active form, Bax c-myc. Then, conditions that led to Bax α activation were found, and its regulation through different proteins was explored. Herein, we describe for the first time that sub-lethal concentrations of acetic acid trigger Bax α-mediated cell death without disturbing plasma membrane integrity and mitochondrial mass but increasing superoxide anion accumulation and release of cytochrome c, which is partially reverted by the anti-apoptotic protein Bcl-xL. This finding allows us to mimic what happens in human cells, without the need to use non-natural mutants of Bax, such as phosphomimetic or non-phosphorylatable, or mitochondrial tagged versions. Thus, yeast continuously proves to be a valuable tool to express human Bax and perform studies regarding its function, regulation and interaction with other members of the Bcl-2 protein family and other partners.
A morte celular é um evento fundamental para a correta formação dos tecidos durante o desenvolvimento embrionário e para o normal funcionamento das células. Devido ao seu papel fundamental a nível fisiológico, desregulações neste processo estão associadas a uma grande diversidade de funções anormais e, consequentemente, ao desenvolvimento e progressão de várias doenças. De facto, uma diminuição da morte celular relaciona-se com envelhecimento e a progressão de tumores, enquanto que um aumento está associado a doenças neurodegenerativas incluindo a doença de Alzheimer, Parkinson e Huntington. O destino de uma dada célula é regulado, entre outras formas, por interações entre membros pro-apoptóticos e anti-apoptóticos da família de proteínas Bcl-2, nomeadamente a Bax e a Bcl-xL, respetivamente. A proteína Bax está envolvida na formação de poros na membrana mitocondrial externa, através do qual são libertados fatores apoptóticos, incluindo o citocromo c, originando uma cascata de eventos que culmina na morte da célula. Considerando o papel importante desta proteína na morte celular, as suas capacidades como alvo terapêutico emergiram rapidamente. Por este motivo, o seu modo de ação e regulação têm sido extensivamente estudados. Contudo, várias questões permanecem eminentes e, por isso a melhor compreensão da sua regulação é o foco principal desta tese. Assim, escolhemos a levedura Saccharomyces cerevisiae, cujo genoma não possui genes codificantes de homólogos da família de proteínas humanas Bcl-2. Deste modo, a expressão heteróloga da proteína Bax humana na levedura permite o seu estudo sem interferência dos restantes membros da família. Para alcançarmos o nosso objetivo, primeiramente otimizamos as condições para expressão da proteína na levedura, e caracterizamos o seu fenótipo de expressão, bem como o de uma forma ativa, Bax c-myc. De seguida, determinamos condições que nos permitiram a ativação da proteína Bax humana e a sua regulação por outras proteínas. Nesta tese, descrevemos pela primeira vez que concentrações sub-letais de ácido acético desencadeiam um processo de morte celular mediado por Bax, sem provocar alterações na integridade da membrana plasmática e na massa mitocondrial, mas aumentando a acumulação de anião superóxido e causando a libertação de citocromo c, parcialmente revertida pela proteína anti-apoptótica Bcl-xL. Estes resultados permitiram-nos mimetizar o que acontece em células humanas, sem a necessidade de usar mutantes de Bax artificiais, tais como os mutantes fosfomiméticos e não fosforiláveis ou versões endereçadas para a mitocôndria. Deste modo, a levedura continua a revelar o seu valor como modelo celular para expressar a proteína Bax humana e realizar estudos no que toca à sua função, regulação e interação com outros membros da família Bcl-2 e outros parceiros.
Fundação para a Ciência e Tecnologia (FCT) através do projeto UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) e dos financiamentos PD/BD/128032/2016 no âmbito do Programa Doutoral em Biologia Aplicada e Ambiental (DP_AEM) e SFRH/BPD/89980/2012.
Ilina, Yulia [Verfasser]. "Functions of the yeast protein Stm1 and its involvement in apoptotic cell death / vorgelegt von Yulia Ilina." 2005. http://d-nb.info/979741963/34.
Повний текст джерелаCosta, Verónica Sofia Leite Salazar e. "Searching for New Potential Small-Molecule Modulators of Pro-Apoptotic Proteins Using the Yeast-Based Screening Assay." Dissertação, 2015. http://hdl.handle.net/10216/80207.
Повний текст джерелаCosta, Verónica Sofia Leite Salazar e. "Searching for New Potential Small-Molecule Modulators of Pro-Apoptotic Proteins Using the Yeast-Based Screening Assay." Master's thesis, 2015. https://repositorio-aberto.up.pt/handle/10216/84495.
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