Thèses sur le sujet « ITGAL »

Les déficits immunitaires primitifs sont un groupe de troubles causés par des mutations monogéniques dans des gènes jouant un rôle clé dans le développement et la fonction du système immunitaire. Dans cette thèse, une approche multiomique a été adoptée pour étudier deux gènes associés à ces conditions, afin d'élucider davantage les mécanismes par lesquels les variants pathogènes nuisent au système immunitaire. Le premier sujet était HYOU1, défini comme un gène dont les défauts causent un déficit immunitaire primitif. Nous avons observé une hypogranularité dans les neutrophiles du patient et révélé un arrêt de maturation dans la lignée des cellules B avant le stade des cellules B pro. Le deuxième sujet était ITGAL, un gène candidat potentiel non précédemment décrit en relation avec les déficits immunitaires primitifs. Nous avons démontré que le variant étudié est hérité selon un mode autosomique récessif, et les analyses de voies ont révélé un dysfonctionnement de plusieurs voies liées à l'adhésion et à la motilité. De plus, nous avons montré une élévation de l'expression d'autres intégrines, suggérant une réponse compensatoire visant à contrebalancer les intégrines défectueuses
Primary immunodeficiencies, or inborn errors of immunity, are a group of disorders caused by monogenic mutations in genes playing a key role in the development and function of the immune system. In this thesis, a multiomics approach was taken to study two genes associated with these conditions, further elucidating the mechanisms by which pathogenic variants impair the immune system. The first subject was HYOU1, defined as a gene whose defects cause primary immunodeficiency. We observed hypogranularity in the patient's neutrophils and revealed a maturation arrest in the B cell lineage before the pro-B cell stage. The second subject was ITGAL, a potential candidate gene not previously described in relation to primary immunodeficiencies. We demonstrated that the studied variant is inherited in an autosomal recessive pattern, and pathway analyses revealed impairment of multiple adhesion and motility-related pathways. Moreover, we showed an elevation in the expression of other integrins, suggesting a compensatory response to counterbalance the defective integrins

Epithelial ovarian cancer (EOC) is a relatively rare disease usually diagnosed in advanced stages (III and IV stage). Standard of care includes radical surgery followed by platinum-based chemotherapy. Yet, 15% to 30% of EOC patients have a primary platinum-resistant or refractory disease and more than 70% of originally platinum-sensitive advanced stages patients will develop a resistant disease disseminated in the abdomen and pelvis. For these reasons, the net five-year survival by stage is only of 26% and 13% for stage III and IV, respectively. The resistance to chemotherapy is one of the major challenges cancer research faces nowadays. In our lab, we have established several Platinum (PT) resistant (PT-res) cellular models to better understand the molecular pathways that could guide PT-resistance in ovarian cancer. We observed that one of the common features of these PT-res cells was the higher ability to adhere on the mesothelial cells respect to their sensitive counterpart, a fundamental capacity for driving cell dissemination into the peritoneal cavity. My PhD project focused the attention on the dissection of the molecular basis of the higher adhesion capability of PT-res cells to possibly identify new therapeutic targets to overcome EOC peritoneal dissemination. We found all tested PT-res cells over-expressed the integrin alpha 6 (ITGA6) protein that in turn mediated their higher ability to adhere and migrate on the specific substrate laminin (LM) and on mesothelial cells. Using pharmacological and genetic tools, we showed that ITGA6 expression was linked to a higher capacity of PT-res cells to form ovaryspheres in vitro and to grow and disseminate in vivo. Molecular analyses next demonstrated that under PT treatment positively regulated ITGA6 gene promoter activity by via the SP1 transcription factor, possibly explaining the higher ITGA6 expression in PT-res cells. Moreover, PT-treatment also induced an active secretion of ITGA6. Once secreted, ITGA6 on one side primed mesothelial cells to form a pro-metastatic niche and, on the other, favoured the spreading of neighbour tumour cells. Mechanistically, the engagement of ITGA6 with LM positively regulated Snail expression favouring cell adhesion and spreading. These in vitro data were recapitulated in the human pathology since we found higher ITGA6 levels in the ascitic fluids of EOC patients with a PT-resistant disease and also demonstrated that higher levels of circulating ITGA6 could be found in the plasma of EOC after PT-based chemotherapy. Altogether, our collected data suggested that ITGA6 could be a reasonable druggable target to prevent EOC metastatization and dissemination in the peritoneal cavity, for instance by the use of specific blocking antibodies. Moreover, since ITGA6 is easily quantifiable in the circulation of EOC patients it could be used as predictive biomarkers to identify patients that could not respond to the standard PT-based chemotherapy
Epithelial ovarian cancer (EOC) is a relatively rare disease usually diagnosed in advanced stages (III and IV stage). Standard of care includes radical surgery followed by platinum-based chemotherapy. Yet, 15% to 30% of EOC patients have a primary platinum-resistant or refractory disease and more than 70% of originally platinum-sensitive advanced stages patients will develop a resistant disease disseminated in the abdomen and pelvis. For these reasons, the net five-year survival by stage is only of 26% and 13% for stage III and IV, respectively. The resistance to chemotherapy is one of the major challenges cancer research faces nowadays. In our lab, we have established several Platinum (PT) resistant (PT-res) cellular models to better understand the molecular pathways that could guide PT-resistance in ovarian cancer. We observed that one of the common features of these PT-res cells was the higher ability to adhere on the mesothelial cells respect to their sensitive counterpart, a fundamental capacity for driving cell dissemination into the peritoneal cavity. My PhD project focused the attention on the dissection of the molecular basis of the higher adhesion capability of PT-res cells to possibly identify new therapeutic targets to overcome EOC peritoneal dissemination. We found all tested PT-res cells over-expressed the integrin alpha 6 (ITGA6) protein that in turn mediated their higher ability to adhere and migrate on the specific substrate laminin (LM) and on mesothelial cells. Using pharmacological and genetic tools, we showed that ITGA6 expression was linked to a higher capacity of PT-res cells to form ovaryspheres in vitro and to grow and disseminate in vivo. Molecular analyses next demonstrated that under PT treatment positively regulated ITGA6 gene promoter activity by via the SP1 transcription factor, possibly explaining the higher ITGA6 expression in PT-res cells. Moreover, PT-treatment also induced an active secretion of ITGA6. Once secreted, ITGA6 on one side primed mesothelial cells to form a pro-metastatic niche and, on the other, favoured the spreading of neighbour tumour cells. Mechanistically, the engagement of ITGA6 with LM positively regulated Snail expression favouring cell adhesion and spreading. These in vitro data were recapitulated in the human pathology since we found higher ITGA6 levels in the ascitic fluids of EOC patients with a PT-resistant disease and also demonstrated that higher levels of circulating ITGA6 could be found in the plasma of EOC after PT-based chemotherapy. Altogether, our collected data suggested that ITGA6 could be a reasonable druggable target to prevent EOC metastatization and dissemination in the peritoneal cavity, for instance by the use of specific blocking antibodies. Moreover, since ITGA6 is easily quantifiable in the circulation of EOC patients it could be used as predictive biomarkers to identify patients that could not respond to the standard PT-based chemotherapy

L’élucidation des mécanismes permettant l’optimisation de la réponse immunitaire antitumorale correspond à un enjeu majeur pour le développement de stratégies d’immunothérapie efficace. En effet, les réponses immunitaires antitumorales se traduisent rarement par l’éradication de la tumeur. Dans ce contexte, les travaux antérieurs de mon équipe ont démontré que l’interaction de l’intégrine αE(CD103)β7, souvent exprimée par les lymphocytes infiltrant la tumeur (TIL), avec son ligand E-cadhérine, à la surface des cellules tumorales épithéliales, joue un rôle majeur dans la potentialisation de l’activité lytique des cellules T en induisant la polarisation et l’exocytose des granules cytotoxiques. Nos résultats ont indiqué aussi que le TGF-β1, souvent abondant dans les tumeurs, joue un rôle déterminant dans cette induction suite à l’engagement du récepteur des cellules T. Dans ce contexte, nous avons cherché à comprendre les mécanismes de régulation du gène ITGAE qui codent la sous-unité alphaE de l’intégrine CD103. Nos résultats ont montré que les facteurs transcriptionnels Smad2, Smad3 et NFAT-1 sont impliqués dans la régulation de l’expression de la sous-unité αE(CD103). En effet, une costimulation avec du TGF-β1 recombinant et un anticorps anti-CD3 d’un clone T CD103- induit l’expression de cette intégrine qui est accompagnée d’une translocation dans le noyau de Smad2, Smad3 et NFAT-1 qui sont cytoplasmiques à l’état basal. L’inhibition spécifique de ces facteurs transcriptionnels inhibe l’expression de CD103 et abroge le potentiel lytique du clone T vis à vis de sa cible tumorale autologue. De plus, nous avons identifié deux séquences régulatrices du gène ITGAE humain, un promoteur proximal et un enhancer. Par ailleurs, mon équipe a récemment montré que l’interaction de CD103 à la surface des TIL avec une molécule E-cadhérine recombinante est suffisante pour induire la polarisation des granules cytolytiques par un mécanisme dépendant de la PLC-g1 et ERK et que cette intégrine possède non seulement une fonction d’adhérence, mais aussi une fonction de costimulation du signal TCR des TIL antitumoraux. Nous avons cherché à mieux comprendre la signalisation de l’intégrine CD103, en identifiant les domaines intracytoplasmiques de la sous-unité αE impliqués dans son activation. Nous avons ainsi construit une protéine de fusion CD103-GFP et plusieurs mutants du domaine intracytoplasmique de la sous-unité αE qui ont été ensuite transfectés dans la lignée Jurkat Tag CD103-/beta7+. Nos résultats ont montré que le domaine intracytoplasmique de la chaîne alphaE n’est pas nécessaire à la reconnaissance du ligand, la E-cadhérine. Par contre, nous avons montré que ce domaine est impliqué dans le phénomène de clustering de l’intégrine et dans sa polarisation à la zone de contact avec des billes couvertes avec la E-cadhérine-Fc. Nous avons identifié un domaine de 8 acides aminés (ESIRKAQL), contenant une sérine en position 1163 potentiellement phosphorylable, et qui est indispensable pour la signalisation de l’intégrine. De plus, nos travaux ont montré que ce domaine ESIRKAQL, est nécessaire pour la phosphorylation de la ERK1/2 et PLC-g1. Ainsi, une meilleure compréhension des mécanismes moléculaires régulant les fonctions de CD103 pourrait contribuer au développement et à l’amélioration de la réponse antitumorale exercée par les CTL
The elucidation of mechanisms for optimizing the antitumor immune response is a major challenge for the development of strategies for effective immunotherapy. Indeed, the anti-tumor immune responses rarely result in the eradication of the tumor. In this context, the previous work of my team have shown that the interaction of integrin αE(CD103)β7, often expressed by tumor infiltrating lymphocytes (TIL) with its ligand E-cadherin at the cell surface tumor epithelial cells, plays a major role in the potentiation of the lytic activity of T cells by inducing polarization and exocytosis of cytotoxic granules. Our results also indicated that TGF-β1, often abundant in tumors, plays a key role in the induction due to the commitment of the T cell receptor. In this context, we sought to understand the mechanisms regulating ITGAE gene encoding the subunit αE of integrin. Our results showed that the transcription factors Smad2, Smad3 and NFAT-1 are involved in regulating the expression of subunit αE(CD103)β7. Indeed, costimulation with recombinant TGF-β1 and anti-CD3 antibody induces on T cell clone CD103- the expression of this integrin ant the translocation into the nucleus of Smad2, Smad3 and NFAT-1 that are cytoplasmic at baseline. Specific inhibition of these transcription factors inhibits the expression of CD103 and repeals the lytic potential of cloned T with respect to the autologous tumor target. In addition, we identified two regulatory sequences of human ITGAE gene, proximal promoter and enhancer. In addition, my team has recently shown that the interaction of CD103 on the surface of TIL with a recombinant molecule E-cadherin is sufficient to induce the polarization of cytolytic granules by ERK and PLC-γ1 pathway thus this integrin has not only a function of adherence, but also a function of costimulatory signal TCR of TIL. We sought to better understand the signaling of integrin CD103, by identifying the cytoplasmic domains of the subunit αE involved in its activation. We have constructed a fusion protein CD103-GFP and several mutants of intracytoplasmic domain of the subunit αE which were then transfected into the Jurkat Tag cell line CD103-/ β7+. Our results showed that the intracytoplasmic domain of CD103 is not necessary for ligand recognition, E-cadherin. By cons, we have shown that this area is involved in the phenomenon of clustering of integrin and its polarization to the contact area with balls covered with E-cadherin-Fc. We have identified a range of 8 amino acids (ESIRKAQL) containing a potentially phosphorylatable serine in position 1163, which is essential for integrin signaling. In addition, our work has shown that this area ESIRKAQL is necessary for the phosphorylation of ERK1/2 and PLC-g1. Thus, a better understanding of the molecular mechanisms that regulate the functions of CD103 may contribute to the development and improvement of the antitumor response exerted by CTL

Questa tesi si pone l’obiettivo di studiare la fortuna e la circolazione nel Veneto dei "Fatti di Cesare", con una proposta di edizione critica delle inedite "Zesarie batalie romane contenute nel ms. Oxford, Bodleian Library, Canonicianus Italicus 136. Nel fare questo si è anzitutto offerto, nella parte I di questo lavoro, una disamina attenta dei numerosi volgarizzamenti italiani dei "Faits des Romains", con l’ampia discussione della bibliografia esistente sulla questione e un riesame complessivo delle diverse testimonianze manoscritte, unito anche a un controllo sulla tradizione del testo francese. Ѐ stata quindi proposta, sulla base di nuovi dati ricavati dalle nostre indagini, una nuova classificazione di tutti i volgarizzamenti italiani dei "Faits des Romains" in otto diversi raggruppamenti. La parte II è invece focalizzata sulla tradizione manoscritta dei "Fatti di Cesare", uno dei più diffusi e fortunati volgarizzamenti dei "Faits des Romains", contraddistinto per essere una versione abbreviata del testo francese che godette, tra i secc. XIV e XV, di una consistente circolazione, attestata anche nell’Italia settentrionale e, in particolare, nel Veneto. A partire da una nuova ricognizione dell’intero testimoniale manoscritto dei Fatti di Cesare, che ha permesso inoltre di incrementare la tradizione con alcuni codici finora mai segnalati, è stata dapprima studiata da un punto di vista materiale la diffusione e la circolazione di questo testo in spazi e tempi diversi. Tutti i codici di questa tradizione sono stati visionati direttamente e descritti tramite l’esame codicologico di ogni singolo testimone, supportato inoltre da un controllo sui cataloghi delle principali biblioteche, sui database digitali disponibili e sulla più aggiornata bibliografia filologica. Si è quindi proceduto con una recensio codicum dell’intera tradizione manoscritta, che, condotta tramite la collazione di loci critici opportunamente selezionati, ha permesso una precisa classificazione dei numerosi codici dei Fatti di Cesare, operazione indispensabile, in attesa di un’edizione critica del testo, per poterne studiare, in una prospettiva ecdotica, la circolazione nel Veneto. Ѐ stato così possibile dimostrare da quale snodo della tradizione abbiano avuto origine le "Zesarie batalie romane" – adattamento quattrocentesco in dialetto veneziano dei Fatti di Cesare –, il cui studio è stato dedicato alla terza ed ultima parte di questo lavoro. L’edizione del testo è stata preceduta da alcune annotazioni sulla lingua delle "Zesarie batalie romane, con uno studio della grafia, della fonetica e della morfologia, che permetta di apprezzarne la particolare stratigrafia linguistica, caratterizzata da un diasistema in cui, alla lingua primaria del modello toscano, si sovrappone la patina linguistica del veneziano. Il testo critico è stato infine presentato, come è norma comune nell’edizione di testi veneti antichi, secondo criteri prudentemente interpretativi, che, pur tenendo conto delle peculiarità del manoscritto, testimone, nella sua specificità, di una precisa modalità di riuso dei "Fatti di Cesare" nel Veneto, non rinuncia a intervenire laddove necessario ai fini di una migliore fruibilità e leggibilità del testo. Naturalmente, si è sempre dato conto di ogni singolo intervento, segnalando in un apposito apparato la lezione del manoscritto, in modo da consentire, a chi lo desideri, la costante ricostruzione della facies originaria. L’edizione critica delle "Zesarie batalie romane", che qui si pubblica per la prima volta, costituisce dunque un’importante acquisizione per lo studio dei volgarizzamenti italiani dei "Faits des Romains", permettendoci di apprezzare le complesse modalità di ricezione, circolazione e riuso nei secoli di un testo che ebbe una tale fortuna nel corso di tutto il Medioevo e che significò molto per la storia dei volgarizzamenti in Italia.
This work aims to study the fortune and circulation in the Veneto region of the "Fatti di Cesare" with a critical edition of the unpublished "Zesarie batalie romane" of the ms. Oxford, Bodleian Library, Canonicianus Italicus 136. The first part of this work offers a careful examination of the numerous Italian vernaculars of the Faits des Romains, with an extensive discussion of all the bibliography on the issue and a comprehensive review of the various manuscripts, together with a check on the tradition of the French text. The second part focuses on the manuscript tradition of the "Fatti di Cesare", one of the most widespread and successful vernaculars of the "Faits des Romains", distinguished by being an abbreviated version of the French text that enjoyed, between the 14th and 15th centuries, a consistent circulation, also attested in northern Italy and, in particular, in the Veneto region. A recensio of the entire manuscript tradition was then carried out through the collation of suitably selected loci criciti, allowing a precise classification of the numerous codices of the "Fatti di Cesare", an indispensable operation, pending a critical edition of the text, in order to study, from a textual perspective, its circulation in the Veneto region. It has thus been possible to identify the origin of the" Zesarie batalie romane" – fifteenth-century adaptation in Venetian dialect of the "Fatti di Cesare" –, whose study has been dedicated to the third and last part of this work. The edition of the text was preceded by some notes on the language of the "Zesarie batalie romane", with a study of handwriting, phonetics and morphology, As is the common norm in the edition of ancient Venetian texts, the critical text was presented according to prudently interpretative criteria, considering the peculiarities of the manuscript, which testifies, in its specificity, to a precise willingness to reuse the material of the "Fatti di Cesare" in Veneto region. By the way, in order to the readability of the text, we do not renounce to correct it where necessary. Every single intervention on the text has always been considered, pointing out the lesson of the manuscript in a special apparatus, so as to allow the constant reconstruction of the original facies. The critical edition of the Zesarie batalie romane, which is published here for the first time, is therefore an important acquisition for the study of the Italian vernaculars of the Faits des Romains, allowing us to appreciate the complex ways of receiving, circulating and reusing over the centuries of such a significant text in the history of vernaculars in Italy.
L’objectif principal de ce travail a été d’étudier la fortune italienne des "Faits des Romains" et, notamment, leur réception en Vénétie; mon étude est complétée par l’édition critique des "Zesarie batalie romane" du ms. Oxford, Bodleian Library, Canonicianus Italicus 136, jusqu’alors inédites. Celles-ci constituent une transposition en vernaculaire vénitien librement adaptée d’une version toscane des "Faits des Romains" connue sous le nom de "Fatti di Cesare" et dotée d’une grande fortune même hors de Toscane, devenant la source de beaucoup d’autres textes. Dans la première partie de ce travail, je présente un examen attentif des nombreuses traductions italiennes des "Faits des Romains". Sur la base de mes enquêtes, conduites à partir d’une discussion approfondie de toute la bibliographie existante sur la question et d’une analyse des différents témoignages manuscrits, aussi bien français qu’italiens, je propose une nouvelle classification de toutes les versions italiennes des "Faits des Romains". La deuxième partie est centrée sur la tradition manuscrite des "Fatti di Cesare". À partir d’une nouvelle recognitio codicum des "Fatti di Cesare", qui m’a permis de dénombrer 49 manuscrits survivants de cette tradition, parmi lesquels deux manuscrits jamais mentionnés auparavant, j’ai d’abord étudié d’un point de vue matériel la diffusion et la circulation de ce texte dans différents espaces et époques. J’ai ensuite procédé à une recensio codicum de toute la tradition manuscrite, qui, grâce à une collation pour loci critici choisis, m’a permis de classer avec précision les nombreux manuscrits des "Fatti di Cesare", opération indispensable, en attendant l’édition critique du texte, pour en étudier même dans une perspective textuelle sa circulation en Vénétie. La troisième partie de ce travail a été finalement dédiée à l’étude des "Zesarie batalie romane". L’édition du texte a été précédée de quelques annotations sur la langue du texte avec une étude de la graphie, de la phonétique et de la morphologie. A cause de la stratigraphie linguistique de ce texte, caractérisée par un diastème dans lequel la patine linguistique du vénitien se superpose à la langue primaire du modèle toscan, il n’a pas toujours été possible d’isoler avec certitude les éléments attribuables au système primaire ou secondaire. De plus, en raison de la datation du ms. Canonicianus Italicus 136, à savoir l’année 1454, il était légitime d’y attendre, également au niveau du système linguistique vénitien-padan, un degré plus ou moins élevé de phénomènes typiquement toscans, et pour cette raison superposable au système linguistique de l’antigraphe toscan. Pour tout cela, j’ai décidé de proposer une analyse linguistique de nature purement descriptive, qui sans viser une analyse linguistique complète, peut présenter un examen attentif des phénomènes les plus importants retraçables dans le texte. Le texte critique a été présenté, comme d’ordinaire pour l’édition des anciens textes vénitiens, selon des critères prudemment interprétatifs, qui, tout en tenant compte des particularités du manuscrit Canonicianus, ne renonce pas à intervenir là où cela est nécessaire pour une meilleure lisibilité du texte. Naturellement, j’ai rendu compte de chaque intervention dans un apparat, où j’ai toujours indiqué la leçon du manuscrit, afin de permettre aux lecteurs la reconstruction constante de la facies original. L’édition critique des "Zesarie batalie romane", qui est publiée ici pour la première fois, est donc une acquisition importante pour l’étude des versions italiennes des "Faits des Romains", nous permettant d’apprécier les méthodes complexes de réception, de circulation et de réutilisation au cours des siècles d’un texte qui a connu un tel succès tout au long du Moyen Âge.

Orientador: Luis Henrique Souza Guimarães
Banca: Daniela Alonso Bocchini
Banca: Valéria de Carvalho Santos Ebinuma
Resumo: A prospecção de enzimas fúngicas tem sido amplamente estudada nos ultimos anos, principalmente utilizando a Fermentação em Estado Sólido (FES). A procura por enzimas microbianas, principalmente de fungos filamentosos, capazes de degradar material lignocelulósico, tem despertado grande interesse para processos biotecnológicos como, por exemplo, na produção de bioetanol, a partir do bagaço de cana-de-açúcar. Fungos do gênero Aspergillus são reconhecidos como ótimos produtores de enzimas do complexo celulolítico e hemicelulolítico, e a busca por novas linhagens com potencial de produção destas torna-se um grande desafio. Aspergillus labruscus ITAL 22.223 é um fungo filamentoso recentemente isolado no sul do Brasil e, por este motivo, não há estudos na literatura sobre seu potencial de produzir enzimas celulolíticas e hemicelulolíticas. Diante do exposto, este estudo visou a produção e quantificação de enzimas do complexo celulolítico (celulase total, endoglucanase e β-glicosidase) e hemicelulolítico (xilanase), a partir de fermentação em estado sólido utilizando resíduos/produtos agroindustriais como substratos. Neste contexto, a maior atividade enzimática de xilanase foi obtida na presença de farelo de trigo (74,83 U/g de substrato) e de β-glicosidase em farelo de aveia (6,35 U/g de substrato) como substratos/fontes de carbono. Sendo a produção de xilanase em FES a que mais se destacou, algumas características da enzima contida no extrato bruto foram determinadas. Apresentou te... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Prospecting of fungal enzymes has been largely studied in recent years, especially by means of Solid-State Fermentation (SSF). The search for microorganisms' enzymes able to degrade lignocellulosic material, mainly those of filamentous fungi, has attracted interest for application in biotechnological processes, such as in production of cellulosic ethanol from sugarcane bagasse. Aspergillus species are well known for their capability to produce cellulosic and hemicellulosic enzymes complex and the search for strains with this potential is an important challenge. Aspergillus labruscus ITAL 22.223 is a filamentous fungus recently isolated in south of Brazil with unknown potential to produce cellulolytic or hemicellulolytic enzymes. This study aimed the production and quantitation of enzymes of the complex cellulolytic (total cellulose, endoglucanase and and β-glucosidase) and hemicellulolytic (xylanse) obtained under solid-state fermentation using agroindustrial waste and product as substrates. According to this, the greatest enzymatic productions of xylanase (74.83 U/g of substrate) and β-glucosidase (6.35 U/g of substrate) were obtained using wheat bran and oat bran as substrates during SSF fermentation, respectively. Taking in account the best production of xylanase in SSF, some biochemical characteristics were determined for the enzyme contained in the crude extract. Optimal of temperature for enzyme activity was 55ºC and optimal pH was 5.5. Regarding its thermal stability, ... (Complete abstract click electronic access below)
Mestre

El objetivo principal de la presente investigación es idear un sistema que logre disminuir el costo y el tiempo de elaboración de muros no portantes de albañilería confinada. Para lograrlo se ha diseñado un sistema de bloques de arcilla apilables en seco y autoencajables que cumple con todas las normas imputables. Para determinar el rendimiento del sistema diseñado se fabricaron las piezas y se construyó un muro con medidas típicas. Los resultados para dicho caso de estudio se resumen en un ahorro de 17% en el costo directo (considerando todo el muro en su conjunto) y 76% en el tiempo empleado (solo en el asentado de bloques).
The principal objective of this research is to design a system that manage to reduce the cost and time in the construction of non-structural masonry walls. To achieve this, it has been designed a Mortarless Dry-Stacked Interlocking Masonry Clay Bricks which verifies all the imputable codes. To determine the performance of the system, the bricks were made and a wall with typical measurements was built. The results for this study are 17% of reduction in the direct cost (considering the whole wall) and 76 % less time used (alone in the brick seating).
Tesis

xv, 76 p. : ill. (some col.) Includes 4 video files.
Improved understanding of pharyngeal arch (PA) patterning and morphogenesis can reveal critical insights into the origins of craniofacial diseases, such as Fraser syndrome. PAs contain mesenchymal condensations, which give rise to most of the facial skeleton in vertebrates. Studies of Endothelin1 signaling reveal that the skeleton derived from the first two PAs are patterned into dorsal, intermediate, and ventral domains. Previous work has indicated that endothelin targets, including the Dlx genes, homeotically pattern dorsal versus ventral PA identity. I show that the Dlx gene family plays a vital role in PA intermediate-domain identity establishment. In WT fish, the PA intermediate domain is delineated by combined expression of all Dlx genes. Reduction of Dlx gene function results in loss of intermediate-domain identity. Conversely, ventral expansion of Dlx expression, seen in hand2 mutants, results in ventral expansion of intermediate-domain identity. Hence, PA intermediate-domain identity is defined by co-expression of Dlx genes. Epithelial-mesenchymal interactions play an important part in PA intermediate-domain morphogenesis. Zebrafish fras1 (epithelially expressed) and itga8 (mesenchymally expressed) mutants also show specific defects within intermediate-domain skeleton and epithelia. Facial phenotypes in fras1;itga8 double mutants look extremely similar to either single mutant, suggesting that fras1 and itga8 might participate in the same epithelial-mesenchymal interaction during PA intermediate-domain formation. Our developmental studies reveal that fras1 - and itga8 -dependent epithelial segmentation of the PA intermediate domain stabilizes developing skeletal elements. Lesions in human FRAS1 underlie many cases of Fraser syndrome, and this work provides an excellent developmental model for the craniofacial defects found in Fraser syndrome. Loss of either Dlx or fras1 function produces defects in the PA intermediate domain, yet seemingly during different developmental periods. Nonetheless, combined reduction of both Dlx and fras1 function synergistically increases skeletal defects, implying a molecular connection between early (Dlx -mediated) pattern formation and later (fras1 -mediated) pattern stabilization. Elucidation of the Dlx-fras1 interaction is an interesting topic which may unveil new molecules pertinent to Fraser syndrome. Supplemental movies highlighting skeletal and epithelial morphogenesis accompany this dissertation.
Committee in charge: Judith S. Eisen, Chairperson; Charles B. Kimmel, Advisor; John H. Postlethwait, Member; Chris Q. Doe, Member; Kennith E. Prehoda, Outside Member

El Rabdomiosarcoma (RMS) es el tumor de partes blandas más común en la infancia. Los pacientes con enfermedad diseminada continúan teniendo un mal pronóstico a pesar del uso de terapias intensivas. En este trabajo se evidencia la expresión y activación notable de la vía Notch en RMS y se demuestra la implicación de la vía como elemento clave en la regulación de mecanismos de adhesividad, movilidad e invasividad en células de RMS. Además se propone un mecanismo de regulación de estos procesos, mediado por Notch, a través de la regulación positiva de varias moléculas de adhesión, tales como la N-Caderina e Integrina-α9. Así mismo, se describen los efectos observados de la inhibición de la vía Notch in vivo y se profundiza en la caracterización del estado muscular inmaduro propio del RMS según los niveles de expresión de varios genes claves para el proceso de la miogénesis, tales como la vía Notch, la N-Caderina y la Integrina-α9 en un amplio abanico de tumores de RMS.
Rhabdomyosarcoma (RMS) is the commonest type of soft-tissue sarcoma in children. Patients with metastatic RMS continue to have very poor prognosis. This work provides evidence of Notch pathway expression and activation in RMS and it demonstrates a critical role of the Notch pathway in the activation of the process that leads to cell mobility and invasiveness in RMS cells. Furthermore N- cadherin and α9-integrin are postulated as leading actors in the association between the Notch pathway and promotion of cell adhesion, motility and invasion, pointing to these proteins and the Notch pathway itself as interesting putative targets for new molecular therapies against metastases in RMS. It also describes the observed effects of the inhibition of the Notch pathway in vivo.

Prostate cancer is the most commonly diagnosed cancer in males and is the second leading cause of cancer deaths in men after skin cancer. It is still associated with significant mortality and morbidity and statistics indicate that 80% of prostate cancer deaths have metastatic bony lesions. Previous work from our group has identified integrin alpha 2 (ITGA2) as a putative prostate cancer susceptibility gene through a familial genetic study. There is an increasing body of evidence suggesting it is involved in prostate cancer progression, particularly contributing to the preferential metastasis of prostate cancer cells to the bone. Integrins are surface receptors which play important roles in cell migration, invasion, survival and angiogenesis. Altered expression of integrins has been found to mediate tumour cell invasion and metastasis in a range of cancers including prostate cancer. Recently, it has become evident that epigenetic mechanisms play important roles in the progression of prostate cancer, affecting a large number of genes associated with the disease, which leads to the hypothesis that deregulation of ITGA2 expression by epigenetic alterations may be associated with prostate tumour progression and metastasis. To examine this hypothesis, regulation of the ITGA2 gene was investigated in a panel of prostate cell lines which represents different aspects of prostate cancer biology. The ITGA2 promoter is associated with a large CpG island and reduced methylation at the ITGA2 promoter was observed in the bone metastatic cell line, PC3 when compared to the non-tumorigenic cell line, LNCaP and the benign prostate cell line, PWR-1E. Reduced methylation correlated with increased ITGA2 expression levels in these cell lines. Chromatin accessibility and histone acetylation at the ITGA2 promoter was found to be higher in the more highly expressing PC3 cell line. A lower percentage of nucleosome occupancy at the transcription start site also correlated with the higher expression of ITGA2 in 22Rv1 as compared to LNCaP cells. In addition, inhibition of DNA methylation and histone acetylation in combination increased ITGA2 expression in LNCaP cells. These data are consistent with DNA methylation, nucleosome occupancy and histone acetylation contributing to ITGA2 regulation. Higher ITGA2 gene expression levels were also found to correlate with higher cell migration capacity. This was consistent with LNCaP cells displaying an epithelial-like phenotype while the highly expressing PC3 cells displayed a mesenchymal-like phenotype. Knockdown of ITGA2 in the metastatic prostate cancer cell line, PC3, resulted in reduced cell migration, without affecting epithelial to mesenchymal transition (EMT). However, selection of cells with increased ITGA2 expression by serial passaging of cells on collagen matrix correlated with altered expression of transcription factors known to modulate EMT. Higher ITGA2 expression correlated with increased Twist and decreased Snail expression. Further examination of the regulation of ITGA2 gene by transcription factors suggests that the Sp1 transcription factor activates ITGA2 expression. However, Sp1 is expressed at equivalent levels in both LNCaP and PC3 cells and thus, can not account for the differential ITGA2 expression observed. While Twist showed inconsistent activity, androgen treatment and Snail overexpression repressed ITGA2 promoter activity. Since Snail is more highly expressed in LNCaP as compared to PC3 cells, this could explain the lower ITGA2 expression observed in LNCaP compared to PC3 cells. Overall, ITGA2 has been shown by others to be involved in the selective metastasis of prostate cancer to the bone, which is the major cause of prostate cancer related death. ITGA2 may therefore represent a potential therapeutic target in metastatic prostate cancer. This study has shown that both epigenetic factors involving DNA methylation, histone acetylation and nucleosome occupancy and the transcription factors, Sp1, Snail and androgen receptor cooperate to regulate ITGA2 gene expression. Further, the data presented suggest that alterations to epigenetic factors and/ or the EMT transcription factor Snail may contribute to aberrant ITGA2 expression during prostate cancer metastasis.

Breast cancer (BC) is the most commonly diagnosed and second greatest contributor to cancer deaths in women. Mortality is usually associated with the development of metastases with breast, like prostate cancer, having higher levels of bone metastases relative to other cancers. In fact 70% of terminal BC patients have bone lesions. Integrin α2β1, and specifically the α2 subunit (ITGA2), have altered expression in cancers, including BC, with implications in metastasis, angiogenesis, cell adhesion and survival. The integrin’s main function is ligation to collagens and laminin. Despite some regulatory pathways for ITGA2 being described, the contributions of these in BC progression are unclear, as are downstream pathways activated by ligation. ITGA2 has anti-metastatic effects in BC due to its promotion of adhesion in mammary gland epithelia. ITGA2 loss in BC metastases was hypothesised to promote motility while re-expression in bone may promote lesion formation and activate osteolytic/pro-invasive pathways. Furthermore there is emerging, albeit limited, evidence that ITGA2 is epigenetically regulated in cancer and it was hypothesised that this may be important in BC. To examine this hypothesis the expression of the ITGA2 gene and protein, as well as epigenetic modifications and transcription factor expression was investigated in BC cell lines representing different subtypes. ITGA2 expression correlated poorly with subtype; basal/metastatic lines MDA-MB-231 and MDA-MB-453 displayed high and low expression, whereas luminal/non-metastatic T-47D and MCF-7 also displayed comparably high and low expression respectively. Differential expression indicated that regulatory mechanisms affect ITGA2 expression within similar BC cell lines. Notably elevated ITGA2 mRNA and protein expression in MDA-MB-231 cells implied that highly metastatic BC cell lines that can be bone-seeking, do upregulate ITGA2 relative to other metastatic cell lines. This may support implications of ITGA2 in metastatic regulation and bone lesion formation. Protein expression reflected mRNA expression, however with less pronounced differences. This study was the first to examine ITGA2 promoter methylation in BC. Previous studies in prostate cancer (PC) concluded that methylation of the CpG island within the ITGA2 gene promoter was capable of regulating its expression, with hypermethylation reducing ITGA2 expression in PC tumours and hypomethylation elevating ITGA2 expression in normal prostate and bone-seeking PC cell lines. In BC cell lines the promoter was hypomethylated with no correlation to cell line subtype or ITGA2 expression. This novel finding suggests that while BC and PC have some similarities in regards to ITGA2 expression they have fundamentally different means of regulating ITGA2 from an epigenetic standpoint. Interestingly MCF-7 cells with low ITGA2 expression relative to the literature did have a consistently methylated CpG site within a putative NF-κB recognition site. As NF-κB can upregulate ITGA2, this implies that blocking of NF-κB – ITGA2 promoter interaction by specific epigenetic marks, may downregulate ITGA2 in BC. Epigenetic post transcriptional regulation of ITGA2 by micro RNA has also been reported for BC, notably with miR-373-3p downregulating ITGA2 protein expression to promote metastasis. A large number of potential miRNA recognition sequences in the ITGA2 mRNA 3’UTR were identified and preparations for future quantitative analysis of specific miRNAs were made. A brief examination of transcription factors regulating ITGA2 gene expression was also conducted. The TWIST1 gene, associated with EMT, was elevated in luminal BC cell lines, contrary to the literature and suggesting that it has no regulatory effect on ITGA2 expression. By contrast EGR-1 showed an inverse correlation to ITGA2 expression and thus possibly represses ITGA2 expression in BC. In other studies, androgens and transcription factors Sp1, SNAI1, AP-1/2 and EGR-3 have also been shown to regulate ITGA2. Overall this study supported ITGA2 downregulation as a mediator of BC metastasis and subsequent upregulation as a promoter of bone metastases. More importantly for the first time this study showed that ITGA2 is likely not regulated by promoter methylation in BC and its expression may instead be more dependent on regulation by miRNA or differential expression of transcription factors with expression varying greatly regardless of subtype. The data presented also suggest that select methylation may interrupt NF-κB interactions, with the ITGA2 promoter in luminal MCF-7 cells, indicating a novel cooperation of epigenetic regulation and transcription factors in BC. Additionally EGR-1 may downregulate ITGA2 in different basal and luminal BC cell lines. Overall a variety of distinct epigenetic and transcription factor regulatory pathways may contribute to ITGA2 expression changes in BC progression and metastasis and provide insight into both future research directions and BC therapies.

Disruption to regulatory mechanisms controlling gene expression is a hallmark of leukaemia, with disruption to transcription factors being one of the most prevalent. By identifying the gene expression profile under the control of these transcription factors, and understanding how the target genes are regulated, critical insight can be gained into the role of these transcription factors in haematopoiesis, as well as their role in leukaemia development. Evidence presented here suggests that the RUNX1 transcription factor regulates the expression of the α6β4 integrin receptor in haematopoietic cells by controlling the integrin genes ITGA6 and ITGB4. Engagement of integrin receptors with extracellular matrix components of the bone marrow and haematopoietic tissues plays an essential role in haematopoiesis. Integrin expression is also altered in many leukaemias, however the regulation of integrin gene expression both in normal and disease states has remained largely unexplored. Data presented here identified ITGA6 and ITGB4 as novel target genes of the RUNX1 transcription factor in myeloid cells. RUNX1 was found to bind to the promoter regions of ITGA6 and ITGB4 in myeloid cells in ChIP assays. Furthermore, RUNX1 had a functional effect on both of the promoters in reporter assays. RUNX1 increased the activity of both promoters, while RUNX1-ETO, which is produced by a common chromosomal translocation in leukaemic cells, repressed promoter activity, consistent with its well-characterised role as a transcriptional repressor. While RUNX1 is commonly described as a sequence-specific DNA binding protein that binds to the consensus motif TGT/cGGT, it is becoming evident that the regulation of genes by RUNX1 is more complex than this and RUNX1 can regulate its target genes through a variety of mechanisms. Evidence presented here suggests that RUNX1 regulates the ITGA6 and ITGB4 integrin genes via two distinct mechanisms. RUNX1 was found to regulate the ITGA6 promoter through a consensus RUNX1 binding motif and RUNX1 activation of the promoter was dependent on this motif, in keeping with the traditional model of RUNX1 function described in the literature. In contrast, RUNX1 does not target the ITGB4 promoter through a consensus sequence motif and may be recruited indirectly to the promoter by other haematopoietic transcription factors. Furthermore, the data presented here suggest that efficient regulation of the ITGB4 gene may require interactions between the promoter and an upstream enhancer. RUNX1 was also found to interact with the ITGB4 enhancer, and similarly to the promoter, these interactions do not require a RUNX1 consensus binding motif and may involve recruitment by other transcription factors. Recent evidence suggests that the traditional model of RUNX1 function through a consensus binding motif may represent only a small proportion of RUNX1 target genes. Genome-wide analysis suggests that a significant proportion of RUNX1 recruitment to DNA occurs in the absence of consensus binding motifs, as shown here for ITGB4. To regulate gene expression, transcription factors must operate in the context of the nuclear chromatin environment. RUNX1 influence on gene expression is therefore also dependent on the chromatin environment at its target genes and its interactions with this environment. In the present study, epigenetic mechanisms were also found to contribute to the regulation of ITGA6 and ITGB4 gene expression in myeloid cell lines. ITGB4 expression was inversely correlated with DNA methylation of a large CpG island located at the promoter in KG-1a and Kasumi-1 myeloid cells. Furthermore, low levels of histone H3 and high levels of histone H3 acetylation at both ITGA6 and ITGB4 promoter regions was associated with higher expression of the genes in these cells. Expression of the ITGA6 and ITGB4 genes is likely to be a result of the interplay between transcription and epigenetic factors and in support of this, data presented here show that despite the presence of RUNX1 in KG-1a cells, ITGB4 is expressed at very low levels in these cells possibly due to high levels of DNA methylation at the promoter. This study has advanced our understanding of the mechanisms by which RUNX1 regulates its target genes and has identified distinct molecular mechanisms by which it operates. These findings may also be relevant to the mechanisms by which other transcription factors operate. Additionally, these findings suggest that RUNX1 disruption in leukaemia may have different effects on its target genes depending on how they are regulated normally by RUNX1. Additional studies are therefore required to further dissect the mechanisms by which RUNX1 regulates its target genes, and to further elucidate the repertoire of RUNX1 controlled genes. In addition, this study has provided insight into the regulation of integrin genes in myeloid cells, which is likely to have relevance to the regulation of these genes in other cell types and disease states.

El proyecto de disertación “PLAN DE MARKETING PARA EL POSICIONAMIENTO DE ITALPARQUET EN COLOMBIA”, es un proyecto basado en una empresa constituida desde 2011 en Bogotá, Colombia. Una empresa importadora y comercializadora de pisos en Madera, laminado, linoleum, Pvc, exteriores y guarda escobas. Durante estos dos primeros años la empresa ha venido desarrollando toda su estructura organizacional para poder empezar a introducir su marca como Importadora y Comercializadora de pisos en Madera italianas en Colombia. Sin embargo, al no tener una experiencia en el mercado colombiano esas barreras de entrada se le ha dificultado. Esta es una de las razones del por qué el desarrollo de este proyecto. Desarrollar todo el posicionamiento a esta empresa mediante una investigación de mercados para conocer el perfil del consumidor y posteriormente plantear recomendaciones estratégicas a corto y mediano plazo que se puedan implementar realmente aprovechando que en Colombia es un país emergente en el desarrollo del sector de la construcción. Teniendo en cuenta el comportamiento del mercado, en este proyecto de disertación se van a realizar estrategias de marketing mix donde logre el cumplimiento del objetivo principal, buscando las mejores estrategias de mercadeo tradicional y online para el posicionamiento de la marca en el mercado colombiano.

Die Mehrheit chronischer Nierenerkrankungen wird durch glomeruläre Defekte hervorgerufen. In dieser Arbeit wurde deshalb im Mausmodell die Bedeutung der Kollagenrezeptoren DDR1 (Discoidin Domain Rezeptor 1) und ITGA2 (Integrin Alpha 2) in der Pathogenese von glomerulären Nierenerkrankungen untersucht. Von zentralem Interesse waren neben der Betrachtung des renalen Phänotyps, die Analyse der glomerulären Basalmembran sowie die Prüfung auf Vorhandensein nierenschädigender Faktoren. Zur Orientierung angefertigte H.E.-Färbungen waren lichtmikroskopisch unauffällig, jedoch ließ sich mittels Gelelektrophorese eine Mikro-, Makro- und Albuminurie mit einem Maximum zum Zeitpunkt von 100 Lebenstagen nachweisen, die mit 200 Tagen wieder stark sank. Auf dem Boden der nierenschädigenden Proteinurie, zeigten die Western-Blot-Analysen das Vorhandensein der Zytokine TGF-ß und CTGF auf. Die zur Detektion von Narbengewebe durchgeführte Fibronektinfärbung, erbrachte keinerlei weiterführende Anhaltspunkte. In der Elektronenmikroskopie ließ sich vereinzelt eine Mehrschichtung der GBM nachweisen, was als Ausreifungsstörung interpretiert wurde. Der Wegfall der beiden Kollagenrezeptoren ITGA2 und DDR1 scheint somit die Interaktion der Podozyten mit der GBM zu stören. Dies hat eine Proteinurie zur Folge. In Folge dessen werden profibrotische Zytokine sezerniert. Das Fehlen der beiden Kollagenrezeptoren DDR1 und ITGA2 führte jedoch nicht zur Ausbildung einer renalen Fibrose, wie in der Fibronektin-Färbung gezeigt werden konnte. Gross und Girgert zeigten, dass nierenkranke Mäuse nach dem Verlust von DDR1 oder ITGA2 einen verzögerten Verlauf der Nierenfibrose entwickelten. Vielversprechend scheinen Untersuchungen z.B. am Mausmodell Col4A3/DDR1/ITGA2 -/- oder an einer diabetischen ITGA2/DDR1 -/- Maus. Gesetzt dem Fall, dass eine renale Fibrose im Vergleich zum Einzelknockout noch später eintritt, eignen sich diese beiden Kollagenrezeptoren als therapeutisches Ziel. Aktuell stehen nur wenige nephroprotektive Medikamente, wie ACE-Hemmer, zur Verfügung. Anti-Integrine und Inhibitoren gegen Tyrosinkinase-Rezeptoren, wie DDR1, haben bereits Einzug in den klinischen Alltag gehalten und stellen eventuell einen wirksamen Ansatzpunkt zur Verhinderung einer renalen Fibrose dar.

Il lavoro definitivo risultante dalla ricerca svolta nel corso triennale di Dottorato consiste in un’indagine sulla figura del poeta Simone Serdini da Siena, detto il Saviozzo (1360 ca.-1410 ca.) concretizzatasi in un’ipotesi di lettura generale della sua produzione poetica e di commento delle sue rime, e più in particolare dei testi del ms. Marciano Ital. IX. 347 (=6483).

Defence date: 15 October 1999
Examining Board: Prof. Robert Rowland, ISCTE, Lisbona (supervisor) ; Prof. Antonio Rotondó, Università di Firenze (co-supervisore) ; Prof. Ottavia Niccoli, Università di Trento ; Prof. Gérard Delille, IUE
PDF of thesis uploaded from the Library digitised archive of EUI PhD theses completed between 2013 and 2017