Le réticulum endoplasmique (RE) joue un rôle majeur dans la conformation des protéines. L’accumulation de protéines non- ou mal-conformées dans le RE induit un stress qui peut être résolu par la réponse aux protéines mal-conformées (UPR). Un stress chronique du RE entraine une apoptose dépendante de l’UPR et se traduit par un déséquilibre de l’homéostasie tissulaire. Bien que l’apoptose dépendante d’un stress du RE soit observée et à l’origine d’un grand nombre de maladies humaines, les mécanismes pro-apoptotiques ainsi que ceux favorisant l’homéostasie tissulaire en réponse à un stress chronique du RE restent à ce jour méconnus. Cette thèse apporte une meilleure compréhension de ces mécanismes grâce à un nouveau modèle d’induction de stress du RE chez la drosophile basé sur la surexpression de la préséniline. L’apoptose observée dans ce modèle dépend d’une répression au moins transcriptionnelle du gène anti-apoptotique diap1 par la branche PERK/ATF4 de l’UPR, alors que les voies pro-apoptotiques classiquement impliquées dans l’apoptose en réponse à un stress du RE chez les mammifères ne semblent pas être impliquées. Par ailleurs, la branche PERK/ATF4 active la voie JNK par l’intermédiaire de la petite GTPase Rac1 et de la MAP3K Slipper qui sont activées dans les cellules apoptotiques. Cette activation aboutit à l’expression de Dilp8, un peptide ressemblant à l’insuline qui cause un retard de développement et permet ainsi de remplacer partiellement les cellules éliminées par apoptose. Dans notre modèle, les mécanismes classiquement décrits dans le maintien de l’homéostasie tissulaire chez la drosophile tels que la prolifération compensatoire ou la réparation des tissus ne semblent pas avoir de rôle majeur. Ces résultats établissent, une nouvelle voie qui participe à l’homéostasie tissulaire dans un nouveau modèle de stress chronique du RE / The Endoplasmic Reticulum (ER) plays a major role in protein folding. The accumulation of unfolded proteins in the ER induces a stress which can be resolved by the Unfolded Protein Response (UPR). The chronicity of ER-stress leads to UPR-induced apoptosis and in turn to an unbalance of tissue homeostasis. Although ER stress-dependent apoptosis is observed in a great number of devastating human diseases, how cells activate apoptosis and promote tissue homeostasis after chronic ER-stress remains poorly understood. During my thesis we have established of a novel model of chronic ER-stress using the Drosophila wing imaginal disc as a model system. We have validated that Presenilin (Psn) overexpression induces chronic ER-stress in Drosophila associated to a PERK/ATF4-dependent apoptosis requiring the down-regulation of the anti-apoptotic diap1 gene. Interestingly, the classical pro-apoptotic pathways described in mammals do not seem implicated in Psn-overexpression-dependent apoptosis. PERK/ATF4 also activated the JNK pathway through the small GTPase Rac1 and the MAP3K Slipper activation in apoptotic cells, leading to the expression of Dilp8. This insulin-like peptide caused a developmental delay, which partially allowed the replacement of apoptotic cells. The other mechanisms involved in tissue homeostasis in Drosophila, i.e. compensatory homeostasis and wound healing, do not seem to have a major role in our model. These results establish a new pathway that participates in tissue homeostasis thanks to a novel chronic Drosophila ER stress model

No description available.

Dihydronicotinamide riboside (NRH) quinone oxidoreductase 2 (NQO2) is involved in quinone metabolism reducing quinone to hydroquinone. Quinones are products of oestrogen metabolism and are responsible for the oestrogen-initiated breast carcinogenesis. It has been demonstrated that oestrogen quinones are endogenous biological substrates of NQO2 which acting as a detoxification enzyme catalyses the reduction of oestrogen quinones to hydroquinone. Hydroquinone can then be removed by conjugation to glutathione or glucuronic acid. In this study, the oestrogen dependent and oestrogen independent effects of NQO2 in a variety of networks implicated in breast tumorigenesis were investigated aiming to understand the potential role of NQO2 overexpression in mammary carcinomas. The use of NRH as a cofactor for NQO2 is being studied in parallel with the Î2-oestradiol and tamoxifen treatments. The MCF-7, T47D, MDA-MB-231 and MDA-MB-468 breast cancer cells were transfected with increasing amounts of NQO2 and its biological activity in regulating ERα transcriptional activity, reactive oxygen species (ROS) generation, cell cycle control, mitochondrial membrane potential and antioxidant activities including catalase activity, glutathione (GSH) levels and glutathione peroxidase (GPx) activity were studied. NQO2 overexpression in MDA-MB-231 and T47D cells reduced ROS generation. Increasing amounts of transfected NQO2 induced the ERα transcriptional activity in Î2-oestradiol treated MCF-7 and T47D cells and decreased cyclin D1 protein levels in these cells treated with Î2-oestradiol compared to untransfected cells. Reduction of catalase activity was detected in tamoxifen treated T47D cells overexpressing NQO2, an effect that was not evident in Î2-oestradiol treated cells, whereas NQO2 mediated reduction of GSH levels was detected in these cells treated with Î2-oestradiol but not with tamoxifen. Finally, NQO2 affected mitochondrial membrane depolarization in Î2-oestradiol treated MDA-MB-231 cells. Given the fact that NRH is not physiologically synthesized in humans, the results presented in this study are valuable from the fundamental science point of view indicating the existence of a potential link between NQO2 and estrogens affecting a number of biological pathways important for breast carcinogenesis and as such from the clinical angle it could be assumed that NQO2 effects could impact the design of personalised breast cancer treatment of oestrogen receptor positive and negative breast cancers.

Submitted by PPG Letras (letraspg@pucrs.br) on 2018-12-06T10:40:57Z No. of bitstreams: 1 TESE - Vanessa Zucchi.pdf: 1547346 bytes, checksum: cbb6bfd4500d5459dee05c728c45bc48 (MD5) / Approved for entry into archive by Sheila Dias (sheila.dias@pucrs.br) on 2018-12-10T10:44:19Z (GMT) No. of bitstreams: 1 TESE - Vanessa Zucchi.pdf: 1547346 bytes, checksum: cbb6bfd4500d5459dee05c728c45bc48 (MD5) / Made available in DSpace on 2018-12-10T11:04:28Z (GMT). No. of bitstreams: 1 TESE - Vanessa Zucchi.pdf: 1547346 bytes, checksum: cbb6bfd4500d5459dee05c728c45bc48 (MD5) Previous issue date: 2018-03-27 / Conselho Nacional de Pesquisa e Desenvolvimento Cient?fico e Tecnol?gico - CNPq / This thesis aims to elucidate the development of Brasilian erotic literature's polysystem in the decades from 1960 to 1980, besides it discusses the representations of eroticism in this repertoire. The main support of the thesis are the concepts of 1) erotic literature, which is defined as any literary narrative with the eroticism in the center; 2) eroticism, as an inner e subjective experience, according to George Bataille's philosophical approach; 3) the theory of polysystems, by Itamar Even Zohar. The literary corpus was constituted from the intersection of these concepts, in order to create an integrated panorama, internally and externally. In the highlighted period, it is possible to realize the formation of the erotic polysystem, its subsequent consolidation and renovation. In the first moment, the focus is on Cassandra Rios and Adelaide Carraro. Subsequently it is made an analysis of two specific cases that illustrate the movement of the polysystem: the publication of mass Literature by L?Oren Publishing House and the short story contest by Status magazine. Finally, regarding the 1980s, it is studied how the erotic literature reacts to "pornographic hangover", characteristic of the period. / Essa tese analisa o desenvolvimento do polissistema da Literatura er?tica brasileira nas d?cadas de 1960 a 1980, ao mesmo tempo em que discute as representa??es de erotismo que perpassam esse repert?rio. A base te?rica organiza-se em torno de tr?s conceitos: 1) o g?nero liter?rio er?tico, entendido aqui como toda obra liter?ria que coloca o erotismo como elemento central da narrativa; 2) o erotismo, como um uma experi?ncia interior e subjetiva segundo a concep??o filos?fica de George Bataille; e 3) a teoria dos polissistemas, de Itamar Even Zohar. A partir do entrecruzamento desses conceitos operacionais foi elaborado o corpus liter?rio, de modo a compor um panorama integrado organicamente entre si e entre outros sistemas culturais. No per?odo destacado, ? poss?vel perceber a forma??o do sistema de literatura er?tica, sua posterior consolida??o e renova??o. Dessa forma, no primeiro momento, o foco reca? nas escritoras Cassandra Rios e Adelaide Carraro, protagonistas do novo impulso empreendido pelo g?nero. Posteriormente, abordam-se os principais eixos que movimentam o polissistema atrav?s de dois casos espec?ficos: a literatura de massa publicada pela editora L'Oren e o Concurso de contos er?ticos da Revista Status. Por fim, na d?cada de 1980, examina-se de que forma a literatura er?tica lida com a chamada "ressaca pornogr?fica" que caracteriza o per?odo.

Sizhu was written for the standard Pierrot ensemble though with percussion replacing the singer. This particular ensemble is capable of producing a multitude of colors while maintaining the balance inherent to a chamber group. The Chinese name, si’zhu, is a literal and figurative metaphor for these elements of the ensemble. Literally translated “silk” (sī) and “bamboo” (zhú), the word is a generalization for Chinese classical music developed in the Jiangsu province (Jiāngnán sīzhú) that utilizes strings, or “silk” instruments, and flutes, or “bamboo,” instruments in combination. A typical work involves two or more players of either ilk. In reference to the work presented here, Sizhu is a metaphor for the western instruments (flute and clarinet as “bamboo,” and violin and cello as “silk”) that are employed within the piece. It also refers to my use of a Chinese melody in the compositional process. The song, Er Quan Ying Yue (The Moon Reflected In Second Spring), was composed and performed regularly on the streets by the blind erhu player A Bing. The song has been fragmented, stretched, and varied to the point of near inscrutability, though it becomes more comprehensible following the mid-point. It inspires both structural and local events. The work also employs aspects of the spectral style. The first section is a slow distortion and transformation of the A harmonic spectrum; specific partials are emphasized as the spectrum expands and contracts. Additionally, fragments of the Chinese melody appear within the confines of each specific harmonic structure. The second part completely diverges utilizing assimilated pentatonic scale permutations. Finally, the third section synthesizes these two elements of musical material within the piece as the instruments morendo into silence.

Docetaxel (DTX)-based regimen is the mainstay treatment against castration resistant prostate cancer (CRPC). However, significant side-effects of DTX mandate that strategies to chemosensitize CRPC cells be utilized. We investigated whether physiologically achievable concentrations of nelfinavir (NFR) and curcumin (CUR), known to target the endoplasmic reticulum (ER) stress and AKT pathways, can increase DTX cytotoxicity. A significant reduction (~70%) in survival of a CRPC cell line, C4-2B, was evident within 24 hrs post-exposure to a combination of DTX (10 nM), NFR (5 µM) and CUR (5 µM), as compared to DTX alone (~34%). This rapid cytotoxicity was not seen in non-tumorigenic RWPE-1 cells as well as in primary prostate epithelial cells (PrEC) and bone-marrow mesenchymal stem cells (BM-MSC). A significant increase in apoptosis was seen in C4-2B cells but not RWPE-1 cells, as indicated by DNA-fragmentation, caspase-3 assay, and PARP cleavage. A significant reduction in C4-2B-derived colony forming units (CFU) was observed following exposure to DTX-NFR-CUR combination (92%), as compared to DTX alone (34%). In C4-2B cells, immunodetection and real-time PCR studies showed that exposure to 3-drug combination drastically reduced AKT activation, increased unfolded protein response (UPR) markers, such as XBP-1 mRNA and phosphorylated eIF-2α, and increased ER-stress induced pro-apoptotic markers such as CHOP, ATF4 and TRIB3. In RWPE-1 cells, upregulation of CHOP was observed with DTX-NFR-CUR combination, but no increase in ATF-4 and TRIB3 were observed. In vivo studies using C4-2B tumor xenografts showed a significant reduction in tumor volume following 4 week exposure to the 3-drug combination, as compared to DTX alone. Immunohistochemistry (IHC) of tumor sections revealed decreased Ki-67 staining indicating reduced cell proliferation and increased TUNEL staining indicating apoptosis, in DTX-NFR-CUR treated mice as compared to DTX alone. Therefore, our studies show that NFR and CUR can provide a promising approach as an adjuvant therapy to chemosensitize CRPC to DTX therapy. / acase@tulane.edu

<p>Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an incidence of 1.5-2.7/100000 people/year. Today there is no cure for the disease and only symptomatic treatments are available. ALS progresses rapidly and only 50% of the patients are alive three years after the symptom debut. In ALS, the upper and lower motor neurons undergo degeneration in a process resembling apoptosis. This leads to muscle atrophy and paralysis. The causes of neuronal death are however unknown. In this thesis we have studied transgenic mice carrying human mutant superoxide dismutase, as a model for familial ALS. These mice develop ALS-like symptoms after four months of age with degeneration of the motor neurons. Our results show an involvement of endoplasmic reticulum stress, caspase-12, -9, -3 and procaspase-7 in the ALS mice spinal cord. Overexpression of the antiapoptotic protein XIAP in spinal cord neurons inhibited the activation of caspase-12 and reduced caspase-3 and calpain activity. Calpastatin, the regulator of calpain activity, was kept intact in the ALS-XIAP mice. These mice showed a 12% increase in the mean survival suggesting a beneficial effect of XIAP in ALS. The reason for the ultimate cell death of motor neurons in the ALS-XIAP mice may be due to the activation of additional cell death pathways. Thus, we observed that lysosomal proteases particularly, cathepsinB, -D, and -L were activated in the ALS mice spinal cord together with a less marked upregulation of the inhibitors, cystatinB and -C. We also found activation of astrocytes and microglial cells in the spinal cord of ALS mice indicating their involvement in the disease. The results show that both caspase-dependent and -independent pathways are activated during neuronal degeneration in the ALS spinal cord. The results obtained may help to identify novel drug targets for future treatments of ALS.</p>

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an incidence of 1.5-2.7/100000 people/year. Today there is no cure for the disease and only symptomatic treatments are available. ALS progresses rapidly and only 50% of the patients are alive three years after the symptom debut. In ALS, the upper and lower motor neurons undergo degeneration in a process resembling apoptosis. This leads to muscle atrophy and paralysis. The causes of neuronal death are however unknown. In this thesis we have studied transgenic mice carrying human mutant superoxide dismutase, as a model for familial ALS. These mice develop ALS-like symptoms after four months of age with degeneration of the motor neurons. Our results show an involvement of endoplasmic reticulum stress, caspase-12, -9, -3 and procaspase-7 in the ALS mice spinal cord. Overexpression of the antiapoptotic protein XIAP in spinal cord neurons inhibited the activation of caspase-12 and reduced caspase-3 and calpain activity. Calpastatin, the regulator of calpain activity, was kept intact in the ALS-XIAP mice. These mice showed a 12% increase in the mean survival suggesting a beneficial effect of XIAP in ALS. The reason for the ultimate cell death of motor neurons in the ALS-XIAP mice may be due to the activation of additional cell death pathways. Thus, we observed that lysosomal proteases particularly, cathepsinB, -D, and -L were activated in the ALS mice spinal cord together with a less marked upregulation of the inhibitors, cystatinB and -C. We also found activation of astrocytes and microglial cells in the spinal cord of ALS mice indicating their involvement in the disease. The results show that both caspase-dependent and -independent pathways are activated during neuronal degeneration in the ALS spinal cord. The results obtained may help to identify novel drug targets for future treatments of ALS.

The biogenesis and maintenance of the entire endomembrane system is dependent upon membrane fusion proteins. Mounting evidence indicates that the integral membrane GTPase Atlastin is a membrane fusion protein involved in the homotypic fusion of the endoplasmic reticulum (ER) membrane suggesting a role in the biogenesis and maintenance of ER structure. I helped show that recombinant Drosophila atlastin is able to promote the fusion of synthetic membranes in vitro and that this fusion is dependent upon atlastin GTPase activity. The structure-function experiments presented here assist in elucidating domains required in the mechanism of atlastin mediated membrane fusion. ER homotypic fusion is dependent upon the self-association of Atlastin subunits in adjacent membranes to bring the bilayers into close molecular contact. Atlastin dimerization occurs in the presence of GTPγS but not GDP and stable dimerization is dependent upon a juxtamembrane middle domain three-helix bundle (3HB). The atlastin GTPase domain and 3HB form a potent soluble domain inhibitor of atlastin homotypic fusion, while the GTPase domain alone shows little inhibition. Designed GTPase domain mutations show that GTP binding and atlastin dimerization is insufficient to support fusion without GTP hydrolysis. Additionally, domain analysis of atlastin reveals that the C-terminal cytoplasmic domain of atlastin is absolutely required for membrane fusion, possibly through a protein-lipid interaction of an amphipathic alpha-helix. Genetic lesions in the human Atlastin-1 gene, SPG3A, result in a form of autosomal dominant hereditary spastic paraplegia (HSP). A better understanding of Atlastin function should lend significant insight into normal ER biogenesis and maintenance, as well as the pathology of human disease.

Advanced digital modulation schemes used in the wireless applications, result in the modulated RF signals with high peak to average power ratio which requires linear amplification. On the other hand, the demand for a longer talk time with less battery volume and weight, especially in hand-held radio units, necessitate more power efficient methods to be utilized in power amplifier design. But improved linearity and efficiency have always been contradicting requirements demanding innovative power amplifier and linearizer design techniques. Dynamically varying the load impedance and bias point of a transistor according to the varying envelope of the incoming RF signal also known as Dynamic Load Modulation (DLM) and Dynamic Supply Modulation (DSM), respectively, are two separate methods for improving the efficiency in power amplifier design. In this dissertation, a combination of both variable gate bias and tunable load concepts is applied in an amplifier structure consisting of two transistors in parallel. A novel computer aided design methodology is proposed for careful selection of the load and biasing points of the individual transistors. The method which is based on load-pull analysis performs sweeps on the gate bias voltages of the active devices and input drive level of the amplifier in order to obtain ranges of biases that result in the generation of IMD sweet spots. Following that, the amplifier is designed employing the load line theory and bias switching at the same time in order to enhance the efficiency in reduced drive levels while extending the output 1 dB compression point to higher values at higher drives. Tunable matching networks are implemented utilizing varactor stacks in a &Pi / con