Thèses sur le sujet « PDIA6 »

Environ 80% des cancers du poumon sont des carcinomes du poumon non à petites cellules (NSCLC). Les NSCLC posent un enjeu médical majeur en raison de leur forte incidence et du taux de mortalité qu’ils induisent. Ils sont généralement traités avec le cisplatine (CDDP), l'un des agents anticancéreux les plus largement utilisés aujourd'hui. Cependant, après plusieurs mois, des cellules cancéreuses résistantes à l'apoptose induite par le CDDP apparaissent et compromettent le succès de la chimiothérapie. Le CDDP est un composé pro-apoptotique qui se lie l'ADN nucléaire pour former des liaisons inter et intra-chaîne, entraînant une inhibition de la synthèse de l'ADN. Toutefois, des cellules anucléées restent sensibles au CDDP et seulement 1% du CDDP intracellulaire réagit avec l'ADN nucléaire, ce qui suggère que le CDDP peut affecter d'autres cibles. Ainsi, les mécanismes par lesquels le CDDP conduit à la mort cellulaire sont mal définis et de nouvelles cibles intracellulaires sont encore à déterminer. Notre thèse élucide quelques mécanismes de résistance cellulaires et moléculaires à l'apoptose induite par le CDDP dans les cellules cancéreuses du poumon (A549). Un criblage du protéome de réticulum endoplasmique, nous a permis d'identifier un ensemble de protéines, qui sont régulées à la hausse dans les trois lignées cellulaires A549 résistantes au CDDP. Parmi ces protéines, plusieurs isoformes de protéines disulfure isomérases (PDI) ont été confirmées par spectrométrie de masse dans la lignée cellulaire de cancer du poumon A549 et retrouvées par western-blot dans plusieurs lignées cellulaires résistantes au CDDP. L'invalidation pharmacologique et génétique de deux isoformes de PDI restaure la sensibilité au CDDP et rétablit la mort cellulaire dans des lignées cellulaires résistantes. Fait intéressant, nous avons constaté que l'inhibition de PDIA4 induit l'apoptose mitochondriale classique, tandis que celle de PDIA6 déclenche une voie non-canonique de la mort cellulaire. Enfin, nous avons confirmé le rôle de ces PDI dans une lignée cellulaire de cancer ovarien, ce qui indique que les mécanismes de résistance initiés par les PDI peuvent être conservés dans différentes lignées cellulaires cancéreuses. En conclusion, nous avons identifié deux nouveaux acteurs de la résistance au CDDP, dans les cellules cancéreuses du poumon non à petites cellules, qui pourraient devenir de nouveaux biomarqueurs en vue de thérapies et de diagnostics
About 80% of lung cancers are non-small-cell lung carcinoma (NSCLC). These NSCLC are a major clinical problem because of their high incidence and mortality rate. They are usually treated with cisplatin (CDDP), one of the most widely used anticancer drugs today. However, following several months, cancer cells, that are resistant to CDDP-induced apoptosis appear, compromising the chemotherapy success. CDDP is a pro-apoptotic compound that binds nuclear DNA to form inter- and intra-chain bonds, causing inhibition of DNA synthesis. However, only 1% of intracellular CDDP reacts with nuclear DNA, suggesting that CDDP can affect other molecules. Thus, mechanisms by which CDDP leads to cell death are poorly defined and novel intracellular targets are still to be determined. Our thesis elucidated some cellular and molecular resistance mechanisms to CDDP-induced apoptosis in lung cancer cells (A549). Using a systematic screen of the endoplasmic reticulum proteome, we have identified a set of proteins, which are up-regulated in three CDDP-resistant A549 cell lines. Among these proteins, several isoforms of proteins disulfide isomerase (PDI) have been confirmed by mass spectrometry in an A549 lung cancer cell line and by western-blot in several CDDP-resistant cell lines. Pharmacological and genetic invalidation of two PDI isoforms rescued the sensitivity to CDDP and restored cell death in resistant cells lines. Interestingly, we found that PDIA4 silencing induce classical mitochondrial apoptosis, whereas PDIA6 triggered a non-canonical cell death pathway. Finally, we confirmed the role of these PDIs in an ovarian cancer cell line, indicating that the PDI-mediated resistance mechanisms may be conserved in various cancer cell lines. In conclusion, we identified two novel actors of CDDP resistance in non-small cell lung cancer cell lines that might be novel biomarkers in therapeutic and diagnosis perspectives

2010 - 2011
Mapping of the interaction between STAT1 and flavonoids Abstract An experimental approach is described, in the first part of this Ph.D. work, for determining protein-small molecule non-covalent ligand binding sites and protein conformational changes induced by ligand binding. The methodology utilizes a combination of multiple technical approaches: limited proteolysis, MALDI TOF MS, circular dichroism and Surface Plasmon Resonance (SPR) to determine the binding sites in signal transducer and activator of transcription 1, STAT1 (87kDa)-flavonoid (Epigallocatechin-3-gallate, Myricetin and Delphinidin, about 500 Da) non-covalent complex. Comparing relative ion abundances of peptides released from the limited proteolysis of STAT1 and the STAT1-flavonoid complex after 0, 5, 15 and 30 minutes of digestion revealed that the binding of flavonoid induced a significant change in surface topology of STAT1. An increase in ion abundance and a different peptide profile suggest that the flavonoids obstruct the access of the proteases to one or both termini of specific peptides, identifying flavonoids binding region. Taken together, MALDI MS and SPR data led us to assume that the binding sites are close to Tyrosine 701 and that the flavonoids probably act disturbing the phosphorylation of TYR701 and the following dimerization and activation of STAT1. PDIA6-BiP complex: role in the regulation of the unfolded protein response Abstract The unfolded proteins response (UPR) induced in many experimental settings is an extremely strong response that usually leads to cell death rather than to restoration of the ER homeostasis. Because the outcome of UPR signaling determines cell fate, a key unresolved molecular question is how UPR signaling is attenuated. Indeed, it is often under-appreciated that UPR signaling in response to stress is transient and is attenuated. Recently it has been proved that yeast UPR matches its output to the magnitude of the stress by regulating the duration of IRE1 signaling. An ER protein, known as binding immunoglobulin protein (BiP), binding to UPR sensors regulates their deactivation. Our idea, described in the second part of this Ph.D. work, is that there is another luminal ER factor, which interacts with the UPR sensors and is involved in attenuation of their activities. This factor is protein disulphide isomerase 6, PDIA6 (also known as P5), a poorly understood member of the protein disulfide isomerase (PDI) family, whose absence, according to our data, confers hypersensitivity to ER stress because one of its main action is tied to the sensing of UPR, rather than to the consequences of UPR signaling. We thought that PDIA6 uses its protein disulfide isomerase activity to interact specifically with UPR sensors in the ER lumen and attenuate their activities, thus regulating the duration of ER stress signaling. [edited by author]
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AbstractProtein disulfide isomerases (PDIs) family members are essential for proper folding of proteins entering the secretory pathway. Studies of these remarkable enzymes have shown that PDIs catalyze the oxidation, reduction and isomeration of disulfide bonds in the endoplasmic reticulum (ER). PDIs are found and conserved in a wide range of species and are ubiquitously expressed.In all eukaryotes, the ER redox potential must be tightly controlled since a large fraction of proteins within the cell require disulfide-bond formation. How PDIA1 maintains a balance between oxidation and reduction remains an important open question. Previous studies have revealed the tendency of PDIA1 to dimerize, but little is known about the functional relevance of PDIA1 dimerization. Also a recent crystal structure of a dimeric form of human PDIA1 shows that the formation of dimers inhibits substrate binding and therefore may function as a mechanism to regulate PDIA1 activity in the ER. This thesis has found that PDIA1 dimerizes in vivo and proposes that the dimerization of PDIA1 has physiological relevance by auto-regulating its activity. This mechanism would allow the ER to maintain a balance between oxidation and reduction necessary for native disulfide formation. Another enzyme, PDILT, is a testis-specific PDI. PDILT shares a high sequence similarity (32% identity) with PDIA1. Previous studies suggest that they share the same mechanism for binding substrates, which is mediated by a highly conserved hydrophobic pocket. In this thesis, the crystal structure of the PDILT b'-domain is reported and reveals a hydrophobic pocket that contains interesting features for substrate binding. Structural studies of this new PDI-member will help to understand the important role that PDILT plays in the differentiation and maturation on spermatozoids. The study of ER protein folding in the testis may lead to the identification of proteins and pathways associated with male infertility.
Résumé Les membres de la famille des protéines disulfures isomérases (PDI) sont essentiels pour le repliement des protéines entrant dans les voies de sécrétion. Les études sur ces enzymes remarquables ont montré que les PDI catalysent l'oxydation, la réduction et l'isomérisation des liens disulfures dans le réticulum endoplasmique (RE). Les PDI sont présentes et conservées chez une large gamme d'espèces et sont exprimées de manière omniprésente.Chez tous les eukaryotes, le potential rédox du RE doit être contrôlé fermement puisqu'une grande fraction des protéines dans la cellule nécessite la formation de liens disulfures. Le mécanisme avec lequel la PDIA1 maintient un équilibre entre l'oxydation et la réduction reste une question importante ouverte. Des études précédentes ont révélé que la PDIA1 a tendance à se dimériser, mais on sait peu de choses sur la pertinence fonctionnelle de la dimérisation de la PDIA1. De plus, une structure cristalline récente d'une forme dimérique de la PDIA1 humaine montre que la formation des dimères inhibe la liaison au substrat et ainsi peut agir comme un mécanisme de régulation de l'activité de la PDIA1 dans le RE. Cette thèse a trouvé que la PDIA1 se dimérise in vivo et propose que la dimérisation de la PDIA1 a une pertinence physiologique en auto-régulant son activité. Ce mécanisme permettrait au RE de maintenir un équilibre entre l'oxydation et la réduction nécessaires pour la formation de liaisons disulfures natives.Une autre enzyme, la PDILT, est spécifique pour le testicule. La PDILT partage une similitude de séquence importante (32%) avec la PDIA1. Des études précédentes suggèrent qu'elles partagent le même mécanisme de liaison des substrats qui est médiée par une poche hydrophobe hautement conservée. Dans cette thèse, la structure cristalline du domaine b' de la PDILT est reportée et révèle une poche hydrophobe qui contient des caractéristiques intéressantes pour la liaison du substrat. Des études structurelles de ce nouveau membre de PDI aideront à comprendre le rôle important que joue la PDILT dans la différentiation et la maturation des spermatozoïdes. L'étude du repliement des protéines du RE dans le testicule pourrait mener à l'identification de protéines et voies associées à l'infertilité masculine.

1a,25-Dihydroxyvitamin D3 (1a,25(OH)2D3) is a major functional metabolic form of vitamin D. 1a,25(OH)2D3 has drawn increasing attention due to its functions in addition to maintaining calcium phosphate homeostasis. It directly regulates mineralization by osteoblasts, matrix production and remodeling by chondrocytes, and contraction of cardiomyocytes. 1a,25(OH)2D3 and its analogues have shown beneficial effects in treating multiple sclerosis, diabetes and various types of cancer. In order to maximize the pharmaceutical potential of 1a,25(OH)2D3, a better understanding its cell signaling pathway is necessary. 1a,25(OH)2D3 regulates osteoblasts through both classical nuclear vitamin D receptor (nVDR) mediated genomic effects and plasma membrane receptor-mediated rapid responses. The identity of the plasma membrane receptor for 1a,25(OH)2D3 is controversial. Protein disulfide isomerase associated 3 (Pdia3) has been hypothesized as one of the putative plasma membrane receptors for 1a,25(OH)2D3. The overall goal of this thesis was to understand the general role and the molecular mechanism of Pdia3 in 1a,25(OH)2D3-initiated rapid responses, and to determine the role of Pdia3 and its dependent signaling in osteoblast biology. The results show that Pdia3 is required for membrane-mediated responses of 1a,25(OH)2D3. Moreover, both Pdia3 and nVDR are critical components of the plasma membrane receptor complex for 1a,25(OH)2D3. Finally, Pdia3 and signaling via Pdia3 regulate osteoblast differentiation and mineralization. Taken together, this study demonstrates the role of Pdia3 in rapid responses to 1a,25(OH)2D3 and osteoblast biology, reveals the unexpected complexity of the 1a,25(OH)2D3 plasma receptor complex and opens the new target, Pdia3, for pharmaceutical application and tissue engineering.

Dissulfeto isomerase protéica (PDIA1 ou PDI) é uma chaperona e ditiol-dissulfeto oxido-redutase residente do reticulo endoplasmático (RE). PDI é essencial à regulação da proteostase por ter função no enovelamento oxidativo de proteínas e na via de degradação associada ao RE (ERAD). Além disso, PDI interage fisicamente e regula a atividade de NADPH oxidases, e fora da célula é um regulador redox essencial à atividade de proteínas extracelulares. Este pool epi/pericelular da PDI (pecPDI) regula função de proteínas de membrana/secretadas, como integrinas, glicoproteínas gp120 do virus HIV e outras, com múltiplas funções que incluem: trombose, ativação plaquetária, adesão celular, infecção viral e remodelamento vascular. A rota de externalização da PDI permanece obscura, e seu conhecimento pode indicar mecanismos dos efeitos (fisio)patológicos da PDI. A secreção da PDI pela rota RE-Golgi foi sugerida em células endoteliais infectadas pelo vírus da dengue, células pancreáticas e tireoideanas. No entanto, uma varredura sistemática das possíveis rotas de externalização da PDI não foi previamente realizada. Neste estudo, mostramos que células endoteliais (EC) externalizam constitutivamente, por rotas distintas, dois pools de PDI, de superfície celular e solúvel, enquanto na EC não estimulada PDI não foi detectada significativamente em micropartículas. PDI externalizada corresponde a ca.1,4% do pool total de PDI celular. Tanto a PDI de superfície celular como a solúvel foram majoritariamente secretadas pela via de secreção não-convencional do tipo IV independente de GRASP. Contudo, a via de secreção clássica também contribui para externalização basal da PDI de superfície celular, mas não da solúvel basal ou estimulada por PMA, ATP e trombina indicando que todas envolvem escape do Golgi. Além disso, a externalização constitutiva da PDI de superfície em célula muscular lisa vascular também ocorre por via independente de Golgi. Externalização da PDI não foi detectavelmente mediada pela secreção não-convencional do tipo I, II, III, lisossomos secretórios, endossoma de reciclagem e transporte ativo (dependente de ATP) em EC. Considerando que chaperonas são vias essenciais de resposta a estresses, investigamos o efeito de estresse do RE e choque térmico na pecPDI. Estresse do RE não altera a PDI de superfície celular, mas aumenta PDI solúvel. Ambos os pools de PDI não foram alterados por choque térmico, embora a recuperação desse estresse diminua a secreção de PDI. Estes dados sugerem que a liberação de PDI é um processo regulado, dependente da natureza do estresse. Bloqueio da síntese de proteínas com cicloheximida não altera pecPDI, indicando que PDI recém-sintetizada não é preferencialmente externalizada e que o tráfego da PDI independe de outras proteínas recém-sintetizadas. Um aspecto importante do estudo foi indicar uma resiliência da pecPDI à modulação individual de distintas vias secretoras, consistente com uma estrita auto-regulação e possibilidade de vias sinérgicas e complementares. Estes resultados indicam que a externalização da PDI de superfície e PDI secretada possam ser externalizadas por mecanismos independentes. Estes processos compõem um processo regulado estritamente, consistente com papel homeostático da pecPDI
Protein disulfide isomerase (PDIA1 or PDI) is dithiol-disulfide oxireductase chaperone resident in the endoplasmic reticulum (ER). PDI is essential for proteostasis, due to its support of oxidative protein folding and ER-associated protein degradation (ERAD). In addition, PDI associates with NADPH oxidase(s) and regulate its activity, while outside of the cell, PDI redox-dependently modulates extracellular proteins. This epi/pericellular PDI (pecPDI) pool is known to regulate membrane/secreted proteins such as integrins, HIV glycoprotein gp120 and others, with functions that involve thrombosis, platelet function, cell adhesion, viral infection and vascular remodeling. PDI externalization route remains enigmatic and its elucidation can help understand some (patho)physiological PDI effects. An ER-Golgi route for PDI secretion has been as described on dengue virus-infected endothelial cells pancreatic and thyroid) cells. However, none of these papers addressed PDI secretion routes in a systematic fashion. Here, we show that endothelial cells (EC) constitutively externalize, through different routes, two PDI pools, a cell-surface and a secreted one, while in nonstimulated ECs PDI was not significantly detected in microparticles. Externalized PDI corresponds to < 2% of total cellular PDI pool. Both cell-surface and soluble PDI were predominantly externalized through unconventional type IV GRASP-independent pathway(s). However, the classical secretory pathway also contributes to basal cell-surface, but not soluble, PDI externalization, as PMA, ATP or thrombin-stimulated secretion also involve Golgi bypass. Furthermore, constitutive cell-surface PDI externalization in vascular smooth muscle cells also occurs in a Golgi-independent way. PDI externalization was not detectably mediated by non-conventional type I, II and III secretion routes, secretory lysosomes, recycling endosomes and ATP dependent active transport in EC. Since chaperones are essential for cellular stress response, we assessed the effects of ER stress and heat-shock on pecPDI. ER stress did not affect cell-surface PDI but increased the soluble pool. Both PDI pools were unaltered by heat shock, while stress recovery decreased PDI secretion. These data suggest that PDI release is finely tuned and dependent on the type of stress. Blockade of protein synthesis with cycloheximide did not change pecPDI levels, suggesting that newly-synthesized PDI is not preferentially externalized and that PDI traffic does not require newly-synthesized proteins. An important aspect of the study was the evidence for pecPDI resilience to individual modulation of distinct secretion routes, consistent with strict auto-regulation and possible synergic or complementary pathways. Overall, our data suggest that cell-surface and secreted PDI pool externalization are regulated through independent mechanisms, which in both cases involve Type IV non-conventional routes, with some minor contribution of Golgi-dependent secretory pathway. These patterns compose a strictly regulated process, consistent with an important homeostatic role for pecPDI

Intro: Hypovitaminosis D (vitamin D deficiency) has been observed in ageing patients with brain calcification and loss of the vitamin D receptor leads to abnormal calcification of the basal ganglia and thalamus. We have found that vitamin D can reverse calcification of human osteosarcoma SaOs-2 cells in vitro, in apparent contrast to its known effects of increasing bone strength in patients with Rickets and Osteomalacia. Vitamin-D functions through binding to two Vitamin-D responsive proteins; the vitamin D receptor (VDR) and Protein Disulfide isomerase A3 (PDIA3). The aim of this project was to establish VDR and PDIA3 knockout SaOs-2 cells using CRISPR-Cas9 technology. Methods: We designed guide RNA (gRNA) sequences against PDIA3 and VDR using ChopChop, selecting only gRNAs with low predicting non-specific binding probabilities. These gRNA sequences were ordered as oligonucleotides and dimerised before directional cloning into a Cas-9 plasmid. Plasmids were amplified in DH5 E. coli and purified before transfection into SaOs-2 cells together with a plasmid containing the puromycin resistance gene. Cells were treated with puromycin (1 ug/ml) for 4 days to eliminate non-transfected cells. SaOs-2 cells were maintained for 7 days before being passaged and plated for colony selection. Results: Real Time quantitative PCR showed 1 SaOs-2 clone had non-detectable levels of PDIA3 while 4 out of 6 clones had no detectable VDR mRNA relative to wild type cells. Two clones were selected for further analysis. Western blotting of these two clones probing for VDR and PDIA3 confirmed there were no detectable levels of these two proteins. Conclusion: We successfully knocked out expression of the Vitamin-D receptors VDR and PDIA3 in SaOS2 cells. These cells will be used for further study of Vitamin-D related signaling.

O período de puberdade, em equinos, define-se pela aparição de espermatozóides maduros no ejaculado de animais jovens, bem como a maturação das funções endócrinas. Os espermatozóides precisam passar por um processo de maturação no epidídimo. Para se obter marcadores moleculares que permitam estabelecer a fertilidade de um garanhão, é necessário um melhor conhecimento das proteínas presentes em espermatozóides imaturos e maduros, uma das quais é a PDI (proteína dissulfeto-isomerase). A PDI foi descrita também como importante marcador de fertilidade no plasma seminal e espermatozóides de diversas espécies. O objetivo deste trabalho foi identificar a presença da PDI no epidídimo equino durante a puberdade, e quantificá-la no fluido e espermatozóides epididimários. Visou verificar a presença da PDI no plasma seminal e espermatozóides ejaculados, em garanhões férteis e subférteis. Foram realizados dois experimentos: Experimento 1: utilizou-se 22 equinos Crioulos saudáveis, castrados e divididos em três grupos: G1: potros até 24 meses, G2: de 25-36 meses e G3: a partir de 36 meses. Imediatamente após a castração, foi efetuada a dissecação do epidídimo para a coleta de fluido epididimário, o qual foi separado dos espermatozoides por centrifugação a 800 g por 10minutos. O sobrenadante foi removido, e criopreservado a -196º C. Os espermatozóides foram ressuspendidos em PBS e armazenados a -196º C. Para a dosagem de proteína das amostras, utilizou-se o método de Kit BCA espectrofotômetro e a eletroforese em gel de poliacrilamida a 10% SDS- Page.Para imunodetecção das proteínas, efetuou-se a incubação do anticorpo primário específico por no mínimo, 6 horas a 4º C, e incubação com anticorpo secundário conjugado com peroxidase anti-igG de camundongo ou anti-IgG de rato. Para a visualização das bandas foi utilizado o Kit de ECL em filmes de raio-X, e as bandas quantificadas pela utilização do software livre ImageJ. A PDI foi identificada nos três grupos avaliados na análise proteômica do fluido , e espermatozoides epididimários, sendo sua quantidade inferior no G1 em relação aos grupos dois (2) e três (3). Em conclusão, a expressão da PDI nos espermatozóides e fluido epididimários de potros castrados cirurgicamente, aumenta conforme o animal atinge a maturidade sexual. Experimento 2- Utilizou-se 12 garanhões adultos que já haviam sido submetidos à pelo menos duas temporadas de monta na região da Campanha do Rio Grande do Sul, efetuando-se quatro (4) coletas de cada garanhão, fora da estação de monta, respeitando-se um intervalo de 48h entre coletas. Imediatamente após a coleta, analisou-se motilidade, vigor e concentração com auxílio de microscópio óptico e retirou-se uma alíquota de sêmen para avaliação das patologias espermáticas. A partir dos dados obtidos, os garanhões foram divididos em dois grupos: Grupo 1: motilidade não inferior a 70%, tendo-se uma média de 76,04±5,89% e histórico reprodutivo de temporadas anteriores com índice de prenhez mínimo por temporada de 80%. Grupo 2: motilidade igual ou inferior a 30%, com média de 11,83±11,21%, e histórico reprodutivo de temporadas anteriores de índices de prenhez inferiores a 35%. Efetuadas as análises, as mostras foram centrifugadas a 800 g por 10 minutos para separar o plasma seminal, de mesma forma que no Exp. 1. Os pellets passaram por ressuspensão em PBS gelado e posteriormente armazenados a -196º C. As amostras foram submetidas à dosagem das proteínas, para serem submetidas à eletroforese, utilizando-se géis de poliacrilamida a 10% SDS-Page.Para imunodetecção das proteínas, efetuou-se a incubação do anticorpo primário específico por no mínimo, 6 horas a 4º C, e incubação com anticorpo secundário conjugado com peroxidase anti-igG de camundongo ou anti-IgG de rato. Na visualização das bandas foi utilizado o Kit de ECL em filmes de raio-X, e as bandas foram quantificadas pela utilização do software livre Image J. Ao analisar a presença da PDI no plasma seminal dos garanhões, verificou-se sua expressão em ambos os grupos; porém não houve diferença de expressão da PDI entre eles(p˂0,05). Não houve também relação da PDI com motilidade, nem com a concentração espermática. Considerando os dados obtidos no presente experimento, não foi possível relacionar a PDI com qualidade espermática e assim considerá-la como um potencial marcador de fertilidade em equinos. São necessários mais estudos que possam envolver outros fatores moleculares inclusive as demais proteínas da família das PDIs.
Puberty, in the equine species, may be defined by the appearance of mature spermatozoa in young animals´ ejaculates, as well as endocrine function maturation. One of the proteins found in immature and mature spermatozoa is PDI (protein dissulfide-isomerase). PDI was also described as an important fertility marker both in seminal plasma and sperm of many species. is responsible for rearranging dissulfide bonds, necessary for sperm adhesion proteins to link to the oocyte. The aim of this work was to identify PDI in equine epididymis during puberty, and quantify it in epididymal sperm and fluid of fertile and subfertile sperm. Two experiments were performed. Experiment 1-twenty-two healthy Crioulo colts were surgically castrated, and divided in three groups: G1: until 24 months; G2: from 25-36 months and G3: more than 36 months. Immediately after castration, testicles were measured, weighed, and the epididymis was dissecated for epididymal fluid collection, which was centrifuged at 800 g for 10minutes to separate epididymal fluid from sperm. Supernatant was removed, and cryopreserved at -196º C. Sperm were re-suspended in PBS and stored at -196º C. Protein dosing of samples was performed with BCA Kit and electrophoresis at 10% SDS-Page. To detect proteins, primary antibody was incubated for at least 6 hours at 4º C, and then incubation with secondary antibody conjugated with anti-mouse IgG or anti-rat IgG. To see bands, ECL Kit in X-ray films was used, and the bands quantified with softwareImageJ. In the three groups PDI was identified, in epididymal fluid and epididymal sperm, but in smaller amount in G1 when compared with Groups 2 and 3. In conclusion, expression of PDI in epididymal fluid and sperm of surgically castrated colts, increases as the animal attains sexual maturity. Experiment 2- The aim of this work was to verify the presence of PDI in equine seminal plasma and sperm, quantify it and to compare its expression on seminal plasma from fertile and subfertile stallions. Twelve adult stallions with at least two breeding season were used. For the study, four collections of each animal were performed. Immediately after collection, analysis of motility, velocity, concentration and sperm morphology were performed. Stallions were divided in two groups, according to the semen analysis and previous breeding history: Group 1: motility greater than 70% and previous history of pregnancy rates higher than 80%; Group 2: sperm motility less or equal than 30% and breeding history of less than 35% of pregnacy per season. After the analysis, samples were centrifuged at 800 g/10minutes to remove seminal plasma. Samples were prepared as described in Exp. 1. The expression of PDI in seminal plasma was seen in both groups, but with no statistical difference between them. There was no correlation of PDI with sperm motility or concentration. According to these findings, it is not possible to consider PDI as a fertility marker in stallions. More research is needed, involving other mollecular factors, including other PDIs family proteins.

Bien que le mélanome malin cutané (MMC) ne représente que 10% des cancers cutanés, il en est la forme la plus agressive et est responsable de 90% des décès dus aux cancers de la peau. Son incidence ne cesse d'augmenter ces dernières années et atteint 10.8 cas pour 100 000 habitants chez les hommes et 11 chez les femmes. La survie médiane des patients atteint de mélanome à un stade avancé est de 6.2 mois et seulement 1 patient sur 4 est encore en vie à 1an. Le diagnostic du mélanome est uniquement histopathologique et seul l'examen de la biopsie permet de le poser définitivement. De la même façon, il n'existe pas de marqueurs pronostiques associés au mélanome malin cutané. Seuls des facteurs histologiques tels que l'épaisseur de la tumeur, le degré d'invasion ou encore l'envahissement ganglionnaire ont été validés par l'American Joint Committee on Cancer et servent de facteurs pronostiques. Cependant, bien qu'ils permettent d'évaluer le risque métastatique, leur valeur pronostique est encore très insuffisante. L'objectif de ma thèse est d'identifier et de caractériser les protéines associées à la progression tumorale du MMC. Grâce à une approche protéomique quantitative de type iTRAQ par nanoLC MS/MS, j'ai pu identifier et caractériser 2242 protéines dans un modèle cellulaire de progression tumorale du mélanome. La surexpression de huit de ces protéines dans les lignées tumorales par rapport à la lignée normale a été validée par western blot. Deux d'entre elles, TRAP1 et PDIA4 ont ensuite été validées en immunohistochimie comme marqueurs associés à la progression tumorale dans le MMC. Dans un second temps, l'implication de ces deux protéines dans les mécanismes de carcinogénèse du mélanome a été étudiée. L'inhibition de TRAP1 et de PDIA4 induit une diminution de la migration et de la viabilité de la lignée cellulaire métastatique de mélanome 1676. Enfin, la sous-expression dePDIA4 induit une inhibition du complexe Cycline D/CDK4 provoquant un blocage des cellules en phase G0/G1. Ce blocage passerait par la voie PERK liées au stress du réticulum endoplasmique
Although cutaneous malignant melanoma (CMM) represents only 10% of skin cancers, it is the most aggressive form with 90% of deaths from skin cancer. The Incidence rate is increasing in recent years to 10.8 cases and 11 cases per 100000, in men and women respectively. The prognosis of metastatic melanoma is poor, with a median survival of only 6.2 months. Histopathologic examination remains the gold standard for melanoma diagnosis. There are no prognostic markers associated with CMM. Only histological factors such as tumor thickness, level of invasion, or lymph node involvement have been validated by the American Joint Committee on Cancer and are currently used as prognostic factors. However, although these histological factors are used to assess the risk of metastasis development, their prognostic value is still very low. The aim of my PhD work is to identify and characterize biomarkers associated with tumor progression of CMM. Using a quantitative proteomics approach (iTRAQ and nanoLCMS/MS), I was able to identify and characterize 2242 proteins in a cellular model of melanoma tumor progression. The overexpression of eight proteins in tumor cell lines compared to the normal cell line was validated by immunoblotting. Among these proteins, TRAP1 and PDIA4 were then validated by immunohistochemistry as markers associated with tumor progression in the CMM. Secondly, the involvement of these two proteins in the mechanisms of melanoma carcinogenesis has been studied. The inhibition of TRAP1 and PDIA4 induces a decrease in migration and viability of the metastatic melanoma cell line. Finally, PDIA4 under expression induce an inhibition of cyclin D/Cdk4 complex leading to G0/G1 cell-cycle arrest dependant of endoplasmic reticulum stress by the PERK pathway

La 1α,25(OH)2 vitamine D3 (1,25-D3) est synthétisée à partir du cholestérol par l'exposition solaire de la peau. Les effets de cette hormone sont médiées par le récepteur de la vitamine D (VDR) dans le noyau et à la membrane plasmique, et avec le récepteur PDIA3 ils médient des effets génomiques et non-génomique. La vitamine D joue un rôle important dans la reproduction, puisque la réduction de la fertilité a été observée chez les rats déficients en vitamine D. L'estradiol (E2) est synthétisé à partir de la testostérone par l'enzyme aromatase (CYP19). L’E2 a des effets génomiques et non génomiques médiée par les récepteurs ESR1, ESR2 et GPER. L’objectif de ce travail a été d’étudier l’effet de l’E2 sur le métabolisme et les voies de signalisation de la vitamine D dans des testicules des rats à différents âges ainsi qu’une perturbation éventuelle initiée par un perturbateur endocrinien à activité estrogénique, le Bisphénol A (BPA). Dans le première axe de travail, trois protocoles expérimental (PE) ont été réalisés, où l’E2 et le BPA ont été administrés: traitement de J15pp à J30pp et euthanasie immédiate à J30 (PE1), traitement de J15 à J30 et euthanasie différée à J75 (PE2) et traitement à l’âge adulte de J60 à J75 et euthanasie immédiate à J75 (PE3). Dans le PE1, le traitement avec l’E2 a diminué l'expression du CYP27A1. L’E2 et le BPA ont diminué l'expression du VDR. Cet effet n'a pas été vérifié dans l'expression de la protéine VDR. Dans le PE2, l’E2 a augmenté l'expression des gènes VDR, PDIA3 et CYP27A1, et l'expression de la protéine VDR et CYP27A1. Les traitements n’ont eu aucun effet dans le PE3, ce qui indique qu’un traitement en période prépubère entraîne à la fois un effet immédiat et différé alors que le traitement à l’âge adulte semble sans effet. Dans le deuxième axe de travail, des effets non-génomiques du BPA ont été étudiés par la technique d’afflux de 45Ca2+ dans les testicules de rat prépubères. Le BPA a stimulé l’afflux de 45Ca2+ de manière un peu pareille avec les effets de l’E2. Cet effet semble ne pas impliquer les récepteurs classiques des estrogènes, mais semble se produire de manière compatible avec l'activation d'un récepteur couplé à la protéine G, comme le GPER. Cet effet se produit par la modulation de la fonction des canaux ioniques, comme des canaux potassiques, TRPV1 et des canaux chlorés. Aussi le BPA module le calcium du stock intracellulaire par l’inhibition de la SERCA et l’activation du récepteur IP3. Également des protéines kinases PKA, PKC, MEK et p38MAPK participent de l’effet du BPA, qui pourrait déclencher un cross talk avec les voies de signalisation nucléaires résultant la médiation des réponses génomiques. Dans le troisième axe de travail, l'expression de certains gènes impliqués dans le métabolisme et la signalisation de 1,25-D3 et E2 a été analysée dans des cellules de Leydig. La 1,25-D3 a diminué l'expression du CYP27A1, un effet qui a également été observé lorsque les cellules étaient co-incubées avec l'E2. L’E2 a diminué l'expression des gènes ESR1 et CYP19. Les deux hormones ont démontré un mécanisme de retours négatifs sur leur métabolisme dans ces cellules. Des effets non génomiques ont été étudiés dans ces cellules, où l’E2 semble avoir un effet inhibiteur tandis que la 1,25-D3 a stimulé l'afflux de 45CA2+. A partir de ces résultats, nous pouvons affirmer que la 1,25-D3, l’E2 et le BPA ont des effets moléculaires importants dans le système reproducteur masculin, par l'expression génique des récepteurs et des enzymes impliqués dans le métabolisme des hormones 1,25-D3 et E2. De plus, les résultats obtenus renforcent la théorie selon laquelle il existe une relation entre les voies de signalisation de la 1,25-D3 et l’E2. Comme la 1,25-D3 et l'E2, le BPA stimule également les effets non-génomiques impliqués dans la signalisation du calcium
1α,25-dihydroxyvitamin D3 (1,25-D3), the active form of vitamin D, is synthetized from cholesterol by skin exposure to the sun. This hormone’s actions are mediated by vitamin D receptor (VDR) in the nucleus and in the plasma membrane, resulting in genome actions like gene expression regulation. VDR can also be found in the plasmatic membrane, and together with PDIA3 receptor they mediate 1,25-D3 non-genomic actions. Vitamin D has an important role in reproductive function, since fertility reduction was observed in vitamin D deficient rats, as well as VDR and 1α-hydroxylase deficiency. In these animals, calcium and estrogen supplementation was able to reverse the deleterious effects in reproductive function, indicating that there is a relation between 1,25-D3 and estrogens signalling pathways. Estradiol (E2) is synthetized from testosterone by aromatase enzyme (CYP19). E2 is found in high levels in the male reproductive function, and like 1,25-D3 can induce genomic and non- genomic actions, mediated by ESR1, ESR2 and GPER receptors. Bisphenol A (BPA) is a xenoestrogen utilized in plastic industry, capable of modulating the endocrine system through E2 receptors. The aim of this work was to study metabolism and signaling pathways interactions between 1,25-D3 and E2, as well as BPA influence in testicular cells. In the first line of work, three treatment protocols (TP) were realized, where E2 and BPA were administrated in rats between 15th and 30th days, were a portion of the animals were euthanized at the last day of treatment (TP1) and another portion was kept alive after the treatment until euthanized at adult age with 75 days (TP2). A third animal group also received the same treatments when adults (TP3). In TP1, E2 treatment decreased CYP27A1 gene expression. E2 and BPA decreased VDR gene expression. This effect was not verified in VDR protein expression. In TP2, E2 increased VDR, PDIA3 and CYP27A1 gene expression, and VDR and CYP27A1 protein expression, indicating a compensatory effect over gene expression inhibition in prepubertal age. In TP3, treatments did not change gene expression, indicating that prepubertal age is more susceptible to estrogen exposure. In the second line of work, non-genomic effects of BPA were studied through 45Ca2+ influx in prepubertal rat testis. BPA stimulated 45Ca2+ influx in a similar manner to E2. This effect was independent of classical ERs, consistent with a G protein-coupled receptor mechanism, probably GPER. This effect involves the modulation of ionic channels, such as K+, TRPV1 and Cl- channels. Furthermore, BPA is able to modulate calcium from intracellular storages by inhibiting SERCA and activating IP3 receptor/Ca2+ channels at the endoplasmic reticulum and activate kinase proteins, such as PKA and PKC. The rapid responses of BPA on calcium influx could, in turn, trigger a cross talk by MEK and p38MAPK activation and also mediate genomic responses. In the third line of work, the expression of some genes involved in 1,25-D3 and E2 metabolism and signalling were analysed in Leydig cells. 1,25-D3 decreased CYP27A1 gene expression, an effect that was also observed when cells were coincubated with 1,25-D3 and E2. E2 decreased ESR1 and CYP19 gene expression. Both hormones demonstrated an negative feedback mechanism over their on metabolism in these cells. Non-genomic effects were also studied in these cells, where E2 seems to have an inhibitory effect while 1,25-D3 was able to stimulate calcium influx. From these results we can conclude that 1,25-D3, E2 and BPA have important molecular effects in the male reproductive system, through gene expression control over receptors and enzymes involved in the metabolism of the steroid hormones studied. These results also reinforce the theory that there is a relationship between 1,25-D3 and E2 signalling pathways, as well as 1,25-D3, E2 and BPA also have non-genomic actions in calcium signalling

The vitamin D metabolite 1,25-dihydroxyvitamin D3 [1α,25(OH)2D3] plays an important role in the regulation of musculoskeletal growth and differentiation. 1α,25(OH)2D3 mediates its effects on cells, including chondrocytes and osteoblasts, through the classical nuclear 1α,25(OH)2D3 receptor. Additionally, recent evidence indicates that several cellular responses to 1α,25(OH)2D3 are mediated via a rapid, calcium-dependent membrane-mediated pathway. These actions of 1α,25(OH)2D3 can be blocked by antibodies to protein-disulfide isomerase family A, member 3 (Pdia3), indicating that it is part of the receptor complex; however, the pathway which is activated by this receptor is not fully understood. The overall goal of this thesis was to examine the roles of phospholipase A2 activating protein (PLAA) and calcium calmodulin-dependent kinase II (CaMKII) in 1α,25(OH)2D3 rapid membrane-mediated signaling. We further investigated the interaction between two pathways regulating growth plate cartilage and endochondral bone formation, 1α,25(OH)2D3 and Wnt5a, at the receptor complex level. Results indicated that PLAA was required for mediating 1α,25(OH)2D3 signal from Pdia3. Furthermore, CaM and CaMKII were identified as mediators of 1α,25(OH)2D3-stimulated PLAA-dependent activation of cPLA2 and PKCα, and downstream biological effects. Wnt5a and 1α,25(OH)2D3 are important regulators of endochondral bone formation. This study demonstrated that 1α,25(OH)2D3 and Wnt5a mediate their effects via similar receptor components in osteoblasts and chondrocytes suggesting that these pathways may interact.

Local economic development successes and failures at municipal level, and specifically in secondary cities in South Africa, is deeply influenced by the constitutional imperatives for establishing developmental local government. The local planning, economic development and policy frameworks introduced between 1999 and 2006 were largely based on, and moulded according to, the wave of new public management paradigms and public sector reform 'good governance best practises' implemented in South Africa post the 1994 democratic elections. The study makes two claims about municipal designs and practises, one that the governance design for these expressions of developmental local government in South Africa has been driven by solution based and theoretical mechanisms rooted in primarily new public management frameworks and development approaches. The second claim is that this development approach manifested in practise in specific plans and frameworks which municipal governments and entities are required to implement and this implementation is characterised by mimicry and isomorphism through compliance, specifically in intermediate cites The motivation for the study, and the third claim which the study investigates, is that the implementation of these plans in practise is not doing so well in terms of delivering the results as envisaged, and secondary cities and towns are often in economic, social and service delivery crises and exhibit very high levels of spatial exclusion despite the local economic development profiles and governance arrangements in these settings increasingly being a matter of policy discussion and debate. The study then introduces a proposed alternative by focusing on implementation at local level and explores how things might be done differently. It looks at the possible contribution of the current search for more effective public service reform, generally referred to as 'doing development differently' or 'smart(er) development', to this local economic development debate. Through a conceptual analysis and application of the approaches and methodologies introduced by problem driven iterative adaption, the study identifies possible different approaches for local economic development in secondary cities and explains what it looks like. The study concludes that doing local economic differently in intermediate settings in South Africa can provide more realistic expectations for the results of local economic development initiatives through fundamentally rephrasing the problem as one that matters, and make recommendations for approaches through which problem driven iterative adaptation processes and practises can be introduced in the context of the institutional constraints present in these intermediate settings.

Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2010.
Committee Chair: Dr. Barbara Boyan; Committee Co-Chair: Dr. Zvi Schwartz; Committee Member: Dr. Hanjoong Jo. Part of the SMARTech Electronic Thesis and Dissertation Collection.

PDIA3 protein is a prominent member of the PDI family that has attracted a significant attention by the research community. It is also becoming increasingly evident that PDIA3 could be a pharmacological target in different pathological conditions, though the mechanisms in which it is involved are not completely understood. For this reason, my PhD project puts the spotlight on the PDIA3 involvement in disease processes, focusing on Alzheimer disease and platelet aggregation, and then characterizing its new features and roles in the development of the aforementioned diseases. At present, no selective compounds are known to modulate the PDIA3 biological functions. The discovery of specific PDIA3 interactors could be used in repressing or stimulating PDIA3 in biological model, thus providing useful information on PDIA3 functions.

Introduction PDIA3, also known as ERp57 or GRP58, is a member of the protein disulfide isomerase family; its structure is characterized by four thioredoxin-like domains: a, b, b’ and a’; a and a’ domains contain the redox active site while b and b’ domains are redox inactive. PDIA3 is localized predominantly in the ER (Endoplasmic Reticulum), where it is involved in the correct folding of newly synthesized glycoproteins and in the assembly of the MHC class I complex, but it is also present in the cytosol, in the nucleus and on the cell surface. PDIA3 is a multifunctional protein disulfide isomerase with a wide range of functions. It has been shown that this protein is involved in the cellular response to stress as well as in several diseases such as cancer, prion disorders, Alzheimer’s and Parkinson’s diseases. Considering this and given that PDIA3 is able to interact with a number of macromolecules and small ligands, such as green tea catechins, in the first part of my research I focused my attention on finding molecules that can interact with and modulate PDIA3 activity. This study, previously started in our lab analyzing the major catechins present in the extracts of green tea, has been expanded to various classes of flavonoids to verify if their activity was in some way connected to the modulation of PDIA3 functions. Flavonoids are a large class of plant secondary metabolites of low molecular weight present in fruits, vegetables and in products such as tea and red wine. Their basic structure shows two benzene rings (A and B) linked by the heterocyclic pyran ring, and, according to type and position of substituents on the central structure, they can be divided into different classes. These molecules show antioxidant, anti-inflammatory, antithrombotic, antiviral and antitumor activity. However, for many of them the molecular and cellular basis of their activities are not well known. They can act on different targets affecting regulation of cell signalling and cell cycle, free radical scavenging, inhibition of angiogenesis, initiation of DNA repair mechanisms, apoptotic induction and inhibition of metastasis. For this reason, we undertook a screening study for assessing the interaction and impact on PDIA3 protein activity of several types of flavonoids. Alzheimer’s disease (AD) is a common neurodegenerative disorder in humans, characterized by deposition in the brain of β amyloid (Aβ) plaques and neurofibrillary tangles (NFTs). Aβ plaques are constituted by Aβ1-40 and Aβ1-42 peptides, which are the results of the APP (amyloid precursor protein) proteolytic cleavage and are thought to be the main cause of the Alzheimer development. Aβ is present in the plaques of Alzheimer’s patients only as a naked peptide, while it is complexed to PDIA3 and calreticulin in the cerebrospinal fluid (CSF) of healthy individuals. It has also been reported the beneficial effect of diosgenin on the memory deficit in an AD mice model and on retrieval of axonal and presynaptic degeneration in the cerebral cortex and hippocampus, detecting PDIA3 as a target of diosgenin. Interestingly, the diosgenin-induced axonal growth was significantly inhibited in primary cortical neurons after PDIA3 knockdown. Given the evidences of PDIA3 involvement in AD, in the second part of my PHD I decided to better investigate PDIA3 role in amyloid beta deposits and in Alzheimer’s disease. The last part of my research, started in the Children’s Hospital of Philadelphia in Yair Argon’s laboratory, has been focused on ER stress and UPR (Unfolded Protein Response). In particular I studied one of the sensors of UPR, IRE1. IRE1 is a transmembrane receptor kinase located on the surface of the ER. This protein consists of different domains: the luminal domain, the transmembrane domain (TM), the linker domain, the kinase domain and the endoribonuclease domain (KEN). When ER stress occurs, BIP dissociates from the luminal domain of IRE1; interactions of the stress-sensing luminal domains of two IRE1 monomers promotes trans-autophosphorylation of the kinase and RNase domains on the cytoplasmic side of the ER membrane Phosphorylation triggers conformational changes in IRE1, which stabilize the dimer. This rearrangement in the RNase domain places the residues necessary for the catalysis in the correct orientation. Moreover IRE1 can also arrange in higher-order oligomers. The result is the cleavage of an intron from the XBP1 mRNA, leading to the translation of a potent transcription factor, which regulates the expression of several genes encoding for ER chaperones. Activated IRE1 can also cleave a subset of mRNAs encoding proteins targeted to the ER, leading to mRNA degradation; this process has been called regulated IRE1-dependent decay (RIDD). RIDD regulates many physiological processes, including degradation of mRNAs encoding a subset of ER or secretory proteins prone to misfolding, and regulation of lipid metabolism genes. RIDD is also responsible for the activation of apoptosis through degradation of several miRNAs’ precursors, such as miR-17. Moreover IRE1, through its kinase domain, activates the c-Jun N-terminal kinases (JNKs) via the formation of a complex with the E3 ubiquitin ligase TRAF2 (TNF receptor-associated factor 2) and the apoptosis signal regulating kinase 1 (ASK1), leading to apoptosis. It has been demonstrated in several studies that alteration in IRE1 function occurs in different diseases such as cancer, diabetes, inflammatory and neurodegenerative diseases. It still not entirely known how IRE1 regulates cell fate; the common thinking is that XBP1 splicing has mostly a prosurvival effect, whereas RIDD shows a proapoptotic output. Nevertheless, the precise mechanisms of these actions need to be further investigated. For this reason the main goal of this research was to understand how the different IRE1 activities are related to each other. Moreover we wanted to investigate if they are related to protein dimerization and clustering, and how phosphorylation affects those activities. METHODOLOGIES PDIA3-flavonoids interaction was investigated by quenching analysis of protein intrinsic fluorescence. This analysis was extended to the PDIA3 a’ domain. Disulfide reductase activity of PDIA3 was monitored by sensitive fluorescent assay using dieosin glutathione disulfide (DiE-GSSG) as fluorogenic probe. Flavonoid’s effect on the DNA binding properties of PDIA3 was also evaluated by EMSA analysis. To investigate PDIA3 involvement in AD, the neuroblastoma cell line SH-SY5Y, a model of neuronal cell, was treated with the 25-35 fragment of the amyloid β peptide. PDIA3 protein levels were analyzed through western blot analysis; mRNA levels through real time-PCR. Immunofluorescence studies were conducted to follow PDIA3 localization under Aβ treatment. To assess PDIA3 levels in the culture media proteins were precipitated with trichloroacetic acid (TCA) and analyzed through western blot. To study IRE1 activities we used an IRE1GFP construct in a TetOn inducible expression system. We established a stable IRE1-/- HAP1 cell line, in which the expression of WT-IRE1GFP and its mutants was regulated by adjusting the doxycycline concentration. IRE1-complemented IRE1 -/-HAP1s were generated by two consecutive transductions with lentiviral particles carrying pLVX-Tet-On™ or pLVX-Tight-Puro™ plasmids (Clontech). XBP1 splicing assay and cell living image were performed under ER stress. RESULTS AND CONCLUSIONS In the first part of this study, the interaction of different flavonoids with PDIA3 and their effect on protein reductase activity were evaluated. Two molecules, eupatorin and eupatorin-5-methyl ether, showed the highest affinity for PDIA3 with a Kd near to 1.0x10-5 M. They also showed a noticeable inhibitory effect on disulphide reductase activity of PDIA3, but they did not significantly affect its DNA binding activity. The backbone structure of these two flavones is characterized by a more stable conformation where B and A rings are almost parallel. This structure, associated with a definite degree of polarity, due to the presence of several methoxyl- groups, seems to be an important feature to determine a good affinity toward PDIA3. We can hypothesize that flavones interact with a region of the protein involving the tryptophan residues close to the redox site and given that PDIA3 does not contain any evident deep cavity or slot where this kind of ligands can bind, the binding of flavonoids may occur mainly via a flat interaction with the protein surface. Therefore, the planarity of the molecule as well as the number and specific position of its functional groups (hydroxyl-, methoxyl- and carbohydrates) will definitively play a major role to determine the affinity for the protein. In conclusion, eupatorin and eupatorin-5-methyl ether represent leading compounds for the binding to PDIA3 and for the inhibition of its redox activity. Further experiments are required to better characterize the effect of flavonoids on PDIA3 and to understand if some of the biological activities of these compounds are depending on the interaction with PDIA3. Since these flavones and PDIA3 are both involved in proliferative and carcinogenic processes, our in vitro findings on their interaction suggest that some of the biological effects of flavones could be mediated by modulation of PDIA3 activity. Additionally, this study will help to define and identify compounds to be used as selective inhibitors/modulators of PDIA3 biological activities. Regarding the implications of PDIA3 in β-amyloid deposits and Alzheimer’s disease, we observed that β-amyloid peptide fragment 25-35 induced a decrease in PDIA3 protein but not in its mRNA levels. We demonstrated that this decrease was not a consequence of ER stress, since we proved that this specific fragment of the amyloid β peptide did not cause activation of the unfolded protein response. We also proved that this decrease was not due to protein degradation through proteasome. Moreover we observed a delocalization of PDIA3 toward the plasma membrane following Aβ treatment. Considering our data and since in literature evidences about PDIA3 extracellular presence can be found, we hypothesized that PDIA3 can be secreted under Aβ treatment. In this study we indeed showed that PDIA3 is secreted by SH-SY5Y, with a significant increase after 1 hour of Aβ25-35 treatment. We also observed that PDIA3 secretion seemed not to be dependent on the classical secretion pathway Golgi-mediated. An explanation for this observation could be that PDIA3 is released from the cell within exosomes. This is not totally unlikely since PDIA3 is present in the ExoCarta Database as an exosome-associated protein. From data presented in this work, our hypothesis is that β-amyloid peptide induces a PDIA3 delocalization and secretion in the extracellular fluid as a defence mechanism carried out by the cell to counteract the toxic action of Aβ. Considering the work of Erickson et al., it could be that PDIA3 pursues this aim through direct binding to amyloid β peptide in order to prevent its aggregation and keep it in solution. In the study of IRE1 activities we observed that, in order to have activation of the RNase domain of IRE1 and clustering, a functional luminal domain is required. Interestingly we found out that if the RNase domain of IRE1 is somehow inhibited, with a mutation or using a chemical inhibitor, IRE1 persists in clusters in the ER membrane. This means that IRE1 clustering does not require the endoribonuclease activity of IRE1. Moreover this led us to hypothesize that IRE1 clustering can be related to other activities, such as RIDD or activation of the JNK pathway. Another interesting finding in this study was that a stimulus coming from the cytosol induces XBP1 splicing but not IRE1 clustering. Indeed, the flavonoid luteolin, through direct binding to the interphase between the kinase and endoribonuclease domains, triggers the splicing of XBP1 mRNA but it does not cause IRE1 redistribution in clusters in the ER membrane. This could be explained by the finding that luteolin induces a different degree in IRE1 phosphorylation if compared with the one induced by a common ER stress activator, such as thapsigargin. Our hypothesis is that luteolin induces phosphorylation only in the activation loop, which is sufficient to have XBP1 splicing, but in order to have IRE1 clustering, phosphorylation in additional residues is required. More studies are needed to confirm our thinking and, more important, the next step will be to investigate the other IRE1 activities, RIDD and JNK-pathway activation, in order to relate every single IRE1 activity to its dimeric or oligomeric state. Moreover we want to better understand how the phosphorylation state of IRE1 affects its activities. Last, during the course of this study on IRE1, we discovered by chance a new IRE1 mutant, L827P. This mutation, not present in any of the catalytic domains of IRE1, has proven to led to the complete suppression of its endoribonuclease activity. We are now interested in better characterizing the mechanism of action of this mutant with the intent to develop, in the future, small peptides that can inhibit IRE1. Since it has been proved that IRE1 is involved in pathologies, such as multiple myeloma, it can be a valid and promising pharmacological target.

The complex multiphase flow physics of spray-swirl interaction in both reacting and non-reacting environment is of fundamental and applied significance for a wide variety of applications ranging from gas turbine combustors to pharmaceutical drug nebulizers. Understanding the intricate dynamics between this two phase flow field is pivotal for enhancing mixing characteristics, reducing pollutant emissions and increasing the combustion efficiency in next generation combustors. The present work experimentally investigates the near and far-field break-up, dispersion and coalescence characteristics of a hollow cone spray in an unconfined, co¬annular isothermal swirling air jet environment. The experiments were conducted using an axial-flow hollow cone spray nozzle having a 0.5 mm orifice. Nozzle injection pressure (PN = 1 bar) corresponding to a Reynolds number at nozzle exit ReN = 7900 used as the test setting. At this setting, the operating Reynolds number of the co-annular swirling air stream number (Res) was varied in four distinct steps, i.e. Res = 1600, 3200, 4800 and 5600. Swirl was imparted to the co¬axial flow using a guided vane swirler with blade angle of Ф=45° (corresponding geometric swirl number SG = 0.8). Two types of laser diagnostic techniques were utilized: Particle / Droplet imaging analysis (PDIA) and shadowgraph to study the underlying physical mechanisms involved in the primary breakup, dispersion and coalescence dynamics of the spray. Measurements were made in the spray in both axial and radial directions and they indicate that Sauter Mean Diameter (SMD) in radial direction is highly reliant on the intensity of swirl imparted to the spray. The spray is subdivided into two zones as function of swirl in axial and radial direction: (1) near field of the nozzle (ligament regime) where variation in SMD arises predominantly due to primary breakup of liquid films (2) far-field of the nozzle where dispersion and collision induced coalescence of droplets is dominant. Each regime has been analyzed meticulously, by computing probability of primary break-up, probability of coalescence and spatio-temporal distribution of droplets which gives probabilistic estimate of aforementioned governing phenomena. In addition to this, spray global length scale parameters such as spray cone angle, break-up length, wavelength of liquid film has been characterized by varying Res while maintaining constant ReN.

The extensive growth of nanotechnology has necessitated the development of economical and robust methods for large scale production of nanomaterials. It requires detailed quantitative understanding of lab-scale processes to enable effective scale-up and development of new contacting strategies for their controlled synthesis. In this thesis, attempts are made in both the directions using experimental and modelling approaches for synthesis of different nanoparticles. The two-phase Brust--Schiffrin protocol for the synthesis of gold nanoparticles was investigated first. The mechanism of transfer of reactants from aqueous to organic phase using phase transfer catalyst (PTC) was investigated using the measurement of interfacial tension, viscosity, SLS, SAXS, 1H NMR, DOSY-NMR, and Karl-Fischer titration. The study shows that the reactants are transferred to organic phase through the formation of hydrated complexes between reactants and PTC rather than through the solubilization of reactants in water core of inverse micelles of PTC, proposed recently in the literature. The particle synthesis reactions thus occur in the bulk organic phase. The extensive body of seemingly disparate experimental findings on Brust--Schiffrin protocol were put together next. The emerging picture ruled out both thermodynamic considerations and kinetics based arguments as exemplified by the classical LaMer's mechanism with sequential nucleation growth capping for size control in Brust--Schiffrin protocol. A new model for particle synthesis was developed. The model brought out continued nucleation--growth--capping based size control, an hitherto unknown mechanistic route for the synthesis of monodisperse particles, as the main mechanism. The model not only captured the reported features of the synthesis but also helped to improve the uniformity of the synthesized particles, validated experimentally. The two-step mechanism of Finke--Watzky---first order nucleation from precursor and autocatalytic growth of particles---proposed as an alternative to LaMer model to explain an induction period followed by a sigmoidal decrease in precursor concentration for the synthesis of iridium nanoparticles was investigated next. The mechanism is tested using an equivalent population balance model for its ability to explain the experimentally observed near constant breadth of the evolving size distribution as well. The predictions show that while it captures precursor conversion well, it fails to explain particle synthesis on account of its inability to suppress nucleation. A minimal four-step mechanism with additional steps for nucleation from reduced iridium atoms and their scavenging using particle surface is proposed. The new mechanism when combined with the first or second order nucleation, or classical nucleation with no scavenging of reduced atoms also fails to suppress nucleation. A burst like onset of nuclei formation with homogeneous nucleation and the scavenging of reduced atoms by particles are simultaneously required to explain all the reported features of the synthesis of iridium nanoparticles. A new reactor is proposed for continuous production of CaCO3 nanoparticles in gas-liquid reaction route. The key feature of the new reactor is the control of flow pattern to ensure efficient mixing of reactants. A liquidliquid reaction route for production of CaCO3 nanoparticles is also optimized to produce nanoparticles at high loading. Optimum supersaturation combined with efficient breakup of initial gel-like structure by mechanical agitation and charge control played a crucial role in producing nano sized CaCO3 particles.

Vegetable oil methyl esters obtained by transesterification of vegetable oils are considered to be suitable alternative fuels for diesel engines. However, higher viscosity, surface tension and boiling temperatures of biodiesels may adversely affect spray characteristics as compared to those of diesel. Thus, spray characteristics of Jatropha Methyl Ester (JME) are studied by comparing them to those of diesel in a high-pressure chamber with optical access to simulate the actual in-cylinder conditions. Also, the effect of inner-nozzle cavitation on JME and diesel sprays is studied by utilizing two nozzles, one with sharp entry-radius and the other with larger entry-radius. Finally, spray characteristics of surrogate fuels such as n-dodecane and n-hexadecane are also studied. The first part of the work concerning precise measurements of inner-nozzle geometry revealed that one of the nozzles has a hole diameter of 190-µm and entry-radius of around 70-µm, while the other has a hole diameter of 208-µm and entry-radius of around 10-µm. Injection rate-shape and coefficient of discharge for JME and diesel flow through the two nozzles were then measured. It was observed that while the coefficients of discharge (Cd) are almost identical for JME and diesel, the nozzle with entry radius of 10-µm exhibited around 20% lower Cd than that of the entry-radius of 70-µm. This observation coupled with insight from complementary CFD simulations of inner-nozzle flow showed that the lower Cd of the nozzle with entry-radius of 10-µm could be attributed to inner-nozzle cavitation. The second part of the work involved measurement of non-evaporating spray characteristics including spray-tip penetration, spray-cone angle and droplet size measurement under realistic operating conditions using techniques such as Shadowgraphy and Particle/Droplet Imaging Analysis (PDIA). The non-evaporating spray of the fuels are studied by injecting them using a common-rail fuel injection system into the high-pressure chamber maintained at room temperature. Experimental results show that JME is associated with a slightly faster spray-tip penetration and narrow spray-cone angle indicating inferior spray atomization which is confirmed by around 5% larger droplet sizes. Slower spray-tip penetration, wider spray-cone angle and around 5% smaller droplet sizes are observed for the spray from the cavitating nozzle. Thus, the inner nozzle cavitation is observed to improve the atomization of diesel and JME sprays. The differences in spray characteristics of JME and diesel reduce as the injection pressure increases. The spray-tip penetrations of both surrogates are observed to almost match that of diesel. The third part of the work involved measurements of evaporating spray liquid length, vapour penetration and spread angle for JME, diesel and surrogates at conditions of 50 bar chamber pressure and 900 K temperature. It is observed that the JME exhibits around 16% longer liquid length than that of diesel. The liquid length of n-dodecane is significantly lower than that of diesel and liquid length of n-hexadecane is around 20% higher than that of n-dodecane mimicking the trend of JME and diesel. The liquid length of n-hexadecane is very close to that of diesel at all the three test conditions. Interestingly, the vapour penetration and spread angle for all the fuels is observed to be almost identical. As the cold spray and evaporating spray characteristics of n-hexadecane match well with those of diesel, n-hexadecane can be chosen as a pure component surrogate for diesel. Finally, an analytical model for predicting the spray vapour penetration is assessed with the experimentally-observed trends of penetration and spray spread angle. The model indicated that the effect of fuel density variation is compensated by the corresponding variation in injection velocity for a given injection pressure to result in a similar vapour penetration. Overall, the present work, in addition to studying the effect of fuel physical properties and cavitation on sprays, has generated a comprehensive experimental database on non-evaporating and evaporating sprays of biodiesel, diesel, and pure component surrogates, which would aid significantly in validation of CFD simulations.

The phenomenon of spray in crossflow is of relevance in gas turbine combustor development. The current work focuses on spray in crossflow rather than liquid jet in crossflow from the standpoint of enhancing fuel dispersion and mixing. Specifically, the first part of the work involves study of spray structure, droplet sizing, and velocimetry for sprays of water and ethanol in a crossflow under ambient conditions. Laser-based diagnostic techniques such as Particle/Droplet Image Analysis (PDIA) and Particle Tracking Velocimetry (PTV) are utilized. Using spray structure images, trajectory equations are derived by multi-variable regression. It is found that the spray trajectory depends only on the two-phase momentum ratio and is independent of other flow parameters. A generalized correlation for the spray trajectory is proposed incorporating the liquid surface tension, which is found to be effective for our data, with water and ethanol, as well as data on Jet-A from the literature for a wide variety of operating conditions. An interesting phenomenon of spatial bifurcation of the spray is observed at low Gas-to-Liquid ratios (GLRs). The reason for this phenomenon is attributed to the co-existence of large and highly deformed ligaments along with much smaller droplets at low GLR conditions. The smaller droplets lose their vertical momentum rapidly leading to lower penetration, whereas the larger ligaments/droplets penetrate much more due to their larger momentum leading to a spatial separation of the two streams. The second part of the study focuses on evaporating sprays in preheated crossflow. Experiments are conducted using ethanol, decane, Jet-A1 fuel, and a two-component surrogate for Jet-A1 fuel. The crossflow air is heated up to 418 K and the effect of evaporation is studied on spray trajectory and droplet sizes. Measured droplet sizes and velocities at two successive locations are used to estimate droplet evaporation lifetimes. Evaporation constant for the d2 law derived from the droplet lifetimes represents the first-ever data for the above-mentioned liquids under forced convective conditions. This data can be used to validate multi-component droplet evaporation models. The last part of the study focuses on Large Eddy Simulations (LES) of the spray in crossflow. The near-nozzle spray structure is investigated experimentally to obtain droplet size and velocity distributions that are used as inputs to the computational model. For the spray in crossflow under ambient conditions, trajectory and droplet sizes at different locations are compared with experimental results. While the predicted trajectory is found to be in good agreement with data, the predicted droplet sizes are larger than the measured values. This is attributed to the implicit assumption in the secondary breakup model that the droplets are spherical, whereas the experimental data in the near-nozzle region clearly shows presence of mostly ligaments and non-spherical droplets, especially for the low GLR cases. A modified breakup model is found to lead to improved agreement in droplet sizes between predictions and measurements. Overall, the experiments and computations have provided significant insight into spray in crossflow phenomenon, and have yielded useful results in terms of validated spray trajectory correlations, droplet evaporation lifetimes under forced convective conditions, and a methodology for simulation of airblast sprays.