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CD4+ and CD8+ T cells, the essential mediators of cellular immune responses, are produced in the thymus following sequential maturation stages. Hematopoietic progenitors first seed the thymus and make T cell lineage specification and commitment decisions within the CD4−CD8− double negative (DN) compartment. Thymocytes then mature to the CD4+CD8+ double positive (DP) stage, followed by vigorous negative and positive selection processes. The positively selected DP thymocytes first give rise to CD4+CD8lo intermediate (IM) cells which then differentiate into MHC class II-restricted CD4+ and MHC class I-restricted CD8+ T cells, a crucial decision known as CD4+ vs. CD8+ lineage choice. The lineage choice decision is influenced by the timing, intensity, and duration of signals derived from the TCR and cytokines, and recent studies have identified a number of transcriptional factors that intrinsically regulate this critical fate decision. Among these, Th-POK (encoded by Zbtb7b, called Thpok here for simplicity and consistency with the literature) is specifically required for CD4+ differentiation while Runx factors promote CD8+ T cell production and repress Cd4 in CD8+ lineage committed cells. Upregulation of Thpok is most evident in the CD4+8lo IM cells and is required to antagonize Runx3 activity and expression to promote CD4+ lineage commitment. Collectively, the Th-POK-Runx3 axis appears to be a critical convergence point in the CD4+ vs. CD8+ lineage choice. After committing to either CD4+ or CD8+ thymocytes, lineage-inappropriate genes are silenced to ensure the distinct identity and functional divergence between these two cell types. Repression of the Cd4 gene on CD8+ lineage committed cells is mediated by a ~430 bp silencer sequence in its first intron. Likewise, Thpok is repressed in CD8+ T cells by a ~560 bp sequence upstream of the Thpok exon 1a, and both Cd4 and Thpok silencers contain consensus binding motifs for Runx factors, which are necessary for CD8+ lineage commitment. T cell factor 1 (TCF-1) and lymphoid enhancer binding factor 1 (LEF-1) are members of the TCF-LEF family transcription factors and abundantly expressed in T lineage cells, and known to be necessary for the maturation of DN T cells to the DP stage. However, because germline deletion of TCF-1 and LEF-1 causes severe early T cell developmental block and embryonic lethality, respectively, their roles beyond the DP stage are unknown. In my thesis work, I overcame these obstacles by conditionally ablating both TCF-1 and LEF-1 in DP thymocytes using CD4-Cre. We observed impaired differentiation of CD4+ T cells from the bipotent DP precursors in the absence of TCF-1 and LEF-1. Mechanistically, TCF-1 promotes CD4+ T cell development by positively regulating the expression of Thpok. TCF-1 and LEF-1 deficiency also results in derepression of the CD4 co-receptor in CD8+ lineage committed cells. In CD8+ T cells, TCF-1 interacts with Runx3 to repress expression of Cd4. These findings not only broaden the spectra of TCF-LEF-mediated regulatory activities in late stages of T cell development, but also reveal new paradigms in T cell fate decision and identity maintenance.

This thesis addresses the mechanisms involved in cellulosedegradation in general and Totally Chlorine Free (TCF) bleachingof pulp in particular. The thesis shows that the cellulosedegradation during high consistency ozone bleaching is explainedby free radical chain reactions. By simulation, it has been shown that the number, weight andviscosity average of liner polymer chain length can be used tocalculate the number of random scissions in a linear polymer ofany molecular weight distribution, provided that there is acalibrated Mark-Houwink equation. A model describing partialdegradation of molecular weight distributions of linear polymersmeasured with viscometry was developed and verifiedexperimentally. The model predicts viscometric measurement ofchemical cellulose degradation by a rapidly reacting reagent tobe strongly dependent on cellulose accessibility. The role of free radical reactions in cellulose degradationwas studied by varying the amount of ferrous ions and ozone addedto the cotton linters. The result was compared to the resultsobtained from cellulose of lower crystallinity (cellulose beads)by measuring average chain length. When a ferryl ion reacted withcotton linters in the presence of ozone, the very formation ofone glycosidic radical was more significant to degradation thanthe final step of forming one oxidised glycoside. The inefficientdegradation observed of the oxidation step is explainable by theamount of accessible glycosides being too small to influenceviscometry. The efficient degradation observed in associationwith the glycosidic radical formation is explained by initiationof free radical chain reactions that are propagated as long asthere is ozone in the system. As none of these phenomena werefound in the less crystalline cellulose, cellulose structureappears to be important for how free radical mediated cellulosedegradation develops. The theory of free radical chain reactions coupled withdiffusion suggests a concentric expansion of the chain reactionsoutwards from the initial site of radical formation duringozonation of carbohydrates. This was confirmed by demonstratingfree radical chain reactions spreading from a spot of initiationoutwards during ozonation of a filter paper, using a pH-indicatorto monitor acid formation. Furthermore, the interior and exteriorof cellulose fibres doped with initiator were shown to bepermeated by small holes after ozonation. Ethylene glycol was shown to improve the selectivity duringozone bleaching of oxygen bleached kraft pulp at pH 3. Optimalconditions were obtained at pH 3 for 25 wt% ethylene glycol. Theinfluence of ethylene glycol on selectivity is explained by aproportion of the free radical chain reactions being carried bythe ethylene glycol instead of the cellulose during ozonebleaching. The observations were summarised in the form of amodel where the observed degradations for pulp, bleached pulp andcotton fibres during both ozone bleaching and ethylene glycolassisted ozone bleaching were shown to agree with each other. From g-irradiation of ozonised aqueous solutions of alcohol,the rate constant of superoxide formation from the peroxylradical of methanol was estimated to be 10 s-1. Rate constants of the reactions between ozone andalkylperoxyl radicals were determined to be around 104M-1s-1. The possibility of the reaction betweenalkylperoxyl radicals and ozone contributing significantly tofree radical chain reactions during ozonation of carbohydratesand alcohols could therefore be ruled out. Cellulose, degradation, free radical, ozone, selectivity,ethylene glycol, alcohol, bleaching, kraft pulp, cotton linters,delignification, fibre, fibril, crystallinity, ferryl ion, freeradical chain reactions, TCF, viscometry, molecular weightdistributions, random scissions.

La presente tesis parte del interés de blanquear pasta de lino (Linum usitatissimum) mediante procesos más respetuosos con el medio ambiente, a partir de secuencias totalmente libre de cloro (TCF) y de la utilización de fibras no madereras para la fabricación de papeles especiales de alta calidad.Asimismo, se ha estudiado la aplicación de enzimas en el blanqueo de pasta de eucalipto (Eucalyptus saligna y Eucalyptus grandis) en la fábrica de Jacareí de Votorantim Celulose e Papel (Brasil) para conseguir una mejora de las propiedades de las pastas y un ahorro de reactivos en la secuencia de blanqueo industrial.Los estudios realizados permiten obtener dos tipos de secuencias para el blanqueo de pasta de lino TCF, ambas con reactivos químicos utilizados individualmente (secuencias químicas) o combinados con tratamientos biotecnológicos (secuencias bioquímicas). Las secuencias químicas incluyen una etapa de ozono y las secuencias bioquímicas incluyen un tratamiento enzimático. Las secuencias bioquímicas constan de etapas que son fácilmente aplicables en la industria, donde la primera de ellas es un sistema lacasa mediador con lacasa comercial de Trametes versicolor y el mediador HBT.En cuanto a los tratamientos biológicos, en primer lugar, se ha estudiado la influencia de la adición de oxígeno y del tiempo de tratamiento en el sistema enzimático y se ha comprobado como influyen ambas variables en las propiedades de la pasta de lino. También se ha estudiado la formación de compuestos cromóforos en la pasta a partir de la determinación de las propiedades ópticas. Posteriormente, se ha realizado un estudio estadístico de las variables de proceso del sistema lacasa mediador y su influencia en las propiedades de las pastas y los efluentes después de cada etapa de blanqueo. La optimización de las variables de tratamiento en el bioblanqueo de lino permite determinar las condiciones de aplicación más adecuadas para conseguir unas determinadas propiedades de la pasta a un menor coste y que resulten fácilmente adaptables a los procesos existentes. Gracias a este estudio, se consigue reducir las dosis de reactivos y el tiempo de tratamiento. El estudio sistemático de cada una de las etapas de blanqueo permite determinar cuál es el efecto sobre la pasta de cada reactivo en cada una de las etapas de las secuencias bioquímicas. Por otro lado, también se ha comprobado como el sistema lacasa mediador es capaz de oxidar ciertos extractivos lipofílicos presentes en la pasta de lino.La optimización de las dosis y el tiempo de tratamiento en el bioblanqueo también se deben tener en cuenta por su influencia en las propiedades de los efluentes, debido a la potencial toxicidad del mediador, como también, a la posible pérdida de actividad enzimática durante el tratamiento. Como novedad, cabe decir, que los valores máximos de toxicidad medidos se encuentran por debajo de los límites que fija la legislación en Cataluña. En este estudio se determina que la pérdida de actividad enzimática en el tratamiento depende básicamente de la dosis de HBT utilizada, por lo que la aplicación de dosis de reactivos superiores a las óptimas, no sólo encarece el proceso de blanqueo, sino que además, una sobredosificación puede dificultar la posible reutilización de estos efluentes de blanqueo.En una segunda parte de la tesis, se estudia la influencia del tratamiento con diversas xilanasas comerciales en las propiedades de la pasta de eucalipto, en las condiciones de blanqueo industriales, a pH y temperatura elevados, en la fábrica de Jacareí de Votorantim Celulose e Papel (Brasil).La aplicación de xilanasas permite una mayor flexibilidad de la secuencia de blanqueo. También, permite ahorrar reactivos químicos, así como incrementar la productividad de aquellas plantas en las que la capacidad de producción de dióxido de cloro es un factor limitante. La aplicación de las xilanasas en la torre de almacenamiento de pasta de la línea de fibras B de la unidad de Jacareí permitiría la mejora de las propiedades de índice kappa y blancura de la pasta en la secuencia de blanqueo industrial. Para ello, no es necesario modificar el proceso existente, lo que es favorable desde el punto de vista industrial, ya que no se requiere una inversión en nuevos equipos. La efectividad del tratamiento enzimático disminuye considerablemente al aumentar la cantidad de licor negro y cuando el pH se encuentra por encima de 10. Por lo que, en el supuesto de que se aplicara un tratamiento enzimático en la torre de almacenamiento, se haría necesario un control, tanto de la cantidad de licor negro presente en la pasta, como del pH antes del punto de aplicación de enzima. / The aim of this thesis is to bleach flax pulp (Linum usitatissimum) by more friendly environmental processes, using totally chlorine free sequences (TCF) and non-wood fibers for the manufacture of high quality specialty papers. This work is framed by one of the research topics in the Textile and Paper Engineering Department of the Technical University of Catalonia. Part of this research topic is based on bleaching fibers, focused basically on the study of new bleaching agents and biotechnology application. Likewise, enzyme application has been studied in eucalyptus pulp bleaching (Eucalyptus saligna and Eucalyptus grandis) in Jacareí Mill of Votorantim Celulose e Papel (Brazil) in order to obtain an improvement of the pulp properties and reagent savings in the industrial bleaching sequence. Performed studies allow to obtain two kinds of TCF sequences for flax pulp bleaching, both with chemical reagents used individually (chemical sequences) or combined with biotechnological treatments (biochemical sequences). Before beginning the bleaching sequences studies, the ozonation pilot plant has been modified. This plant was designed by the research group CIPAGRAF. A new design has been carried out improving the distribution of the equipments, pipelines and valves, basically to minimize delay times in ozone measurements. Chemical sequences include an ozone stage and biochemical sequences include an enzymatic treatment. The biochemical sequences include stages that are easily applicable in the industry. The first one is a laccase-mediator system with a commercial laccase from Trametes versicolor and mediator HBT.Regarding biological treatments, firstly, the influence of oxygen addition and treatment time has been studied in the enzymatic system. Both variables have been proven to have influence on flax pulp properties. The formation of chromophores compounds in pulp has been also studied from the determination of optical properties. Later, it has been carried out a statistical study of the process variables in laccase-mediator system and their influence in pulp and effluent properties after each bleahing stage. References have not been almost found on pulp properties modeling as a function of the variables in an enzymatic process in this enclosed operation conditions regarding a future industrial application. Process variables optimization in flax pulp biobleaching allows to establish the application conditions most appropriated to obtain certain pulp properties to a lower cost and that turn out to be easily adaptable to existing processes. The results show that a reduction of reagent doses and treatment time can be obtained, and the study allows establishing those conditions which the system is not effective. The systematic study of each one of the bleaching stages allows determining the effect on the pulp of each reagent in each of the stages of the biochemical sequences. On the other hand, it has been proved that laccase-mediator system is capable of oxidize certain lipophilic compounds on flax pulp.Besides, it must be also considered the influence of the process variables in effluent properties, due to the potential toxicity of mediator and possible enzymatic activity loss during the treatment. It is necessary to emphasize that studies on effluent characterization depending on process variables in laccase mediator system have not been found. As a novelty, maximum values of toxicity measured are below the legal limit that determines legislation in Catalonia. It has been also observed, that effluent toxicity in L stage is not only due to the presence of mediator HBT, but by-products of degradation show a major toxicity than the HBT in its initial form. In this study, enzymatic activity loss depends basically on the HBT dose. Therefore, a reagent dose application higher to the optimal doses, not only increases bleaching cost, but in addition, an overdosage can avoid the possible reutilization of bleaching effluents.In the second part of the thesis, the study is based on the influence of an enzymatic treatment with commercial xylanases on eucalyptus pulp properties at the industrial bleaching conditions, high pH and temperature, in Jacareí Mill of Votorantim Celulosa e Papel (Brazil). Xylanase application allows a major flexibility of the bleaching sequence. Moreover, it allows chemical reagents saving, as well an increase of productivity in those mills in which the production capacity of chlorine dioxide is a limiting factor, as it is the case of Jacareí's Mill. Xylanase application in storage tower would allow pulp properties improvement, like kappa number and brightness, in the industrial bleaching sequence. For this, it is not necessary to modify the existing process, which is an advantage from an industrial point of view, since an investment in new equipments is not needed. Enzymatic treatment efficiency decreases considerably when black liquor amount is increased and when the pH is over 10. For this reason, if an enzymatic treatment was applied in the storage tower, a control would become necessary for black liquor amount in pulp and for pH before the enzyme application point.

The aim of this thesis is to modify flax pulp fibres (Linum usitatissimum) by more friendly environmental processes. Pulp and paper research is focussing through enzyme systems investigation for developing green chemistry technologies due to existing environmental concerns and to legal restrictions. Moreover, it exists also an increasing strategic interest in using flax fibres to obtain high-quality specialty papers. That is why we study the application of biotechnology as an efficient alternative to traditional industrial processes based on the use of chemical agents. This work is framed by two of the main research topics of the Paper and Graphic Specialty Laboratory in the Textile and Paper Engineering Department of the Universitat Politècnica de Catalunya. One research line is based on pulp bleaching and is focused basically on the study of enzymatic systems as biobleaching agents; the other research topic that has been recently introduced in our investigation group is the use of enzymes as functionalisation agents by promoting the grafting of several compounds. Laccase is the main enzyme used in this thesis; it is an oxidoreductase that can assist reactions in an eco-friendly way since laccase uses air and produces water as the only by-product. Moreover, laccase can work under mill conditions and has wide application potential. The first part of this thesis involved the use of enzymes to bleach flax pulp. The aim was to explore the potential of various natural mediators (lignin-derived compounds) for delignifying flax fibres in order to identify the most efficient and ecofriendly choice among them. Afterwards, we assessed the use of various enzyme delignification stages in an industrial bleaching sequence. The ensuing totally chlorine free (TCF) sequence comprised various laccase-mediator system treatments (L stage) followed by a by a chelating stage (Q stage) and a subsequent bleaching step with hydrogen peroxide (Po stage). A xylanase pretreatment was additionally carried out. Laccases used came from the fungi Pycnoporus cinnabarinus and Myceliophthora thermophila; the performance of several natural mediators was compared with the obtained with the application of various synthetic mediators. In addition, the lack of studies on the properties of effluents from the treatment of non-wood pulp with laccase and natural mediators led A-1 A-2 us to examine effluent properties upon biotreatments and after different bleaching stages. The results obtained warrant upscaling any of the biobleaching sequences for flax pulp as they provide sustainable flax fibre with a high cellulose content and brightness above 80% ISO. The use of xylanase pretreatment was found to efficiently remove HexA and enhance delignification by laccase.

Material development is necessary before all-optical signal processing (AOSP) can be realized. Traditional AOSP will require materials with a large magnitude of the real part of the third-order polarizability, while having a small imaginary magnitude of third-order polarizability. The aim of this thesis is to investigate the potential for polymethine dyes to be used for AOSP applications. The basic structure of a polymethine dye was synthetically modified in a variety of positions to observe the effects upon the linear and nonlinear optical properties. The modifications included variation in the terminal group, substituents in the polymethine bridge, and exchange of the counterion. The dyes were generally synthesized using the Vilsmeyer-Haack reaction to form simple polymethine precursors, and then complexity was added by performing the Knoevenagel condensation with various acceptors and the polymethine precursor. Ion metathesis was often employed to exchange the initial counterion for a counterion that provided increased solubility in common organic solvents. The third-order nonlinear optical polarizabilities were characterized by Dr. Joel Hales who used the open- and closed-aperture Z-scan technique at 1300 nm and non-degenerate two-photon absorption experiments to identify the position of the two-photon absorption bands.

<p>This thesis addresses the mechanisms involved in cellulosedegradation in general and Totally Chlorine Free (TCF) bleachingof pulp in particular. The thesis shows that the cellulosedegradation during high consistency ozone bleaching is explainedby free radical chain reactions.</p><p>By simulation, it has been shown that the number, weight andviscosity average of liner polymer chain length can be used tocalculate the number of random scissions in a linear polymer ofany molecular weight distribution, provided that there is acalibrated Mark-Houwink equation. A model describing partialdegradation of molecular weight distributions of linear polymersmeasured with viscometry was developed and verifiedexperimentally. The model predicts viscometric measurement ofchemical cellulose degradation by a rapidly reacting reagent tobe strongly dependent on cellulose accessibility.</p><p>The role of free radical reactions in cellulose degradationwas studied by varying the amount of ferrous ions and ozone addedto the cotton linters. The result was compared to the resultsobtained from cellulose of lower crystallinity (cellulose beads)by measuring average chain length. When a ferryl ion reacted withcotton linters in the presence of ozone, the very formation ofone glycosidic radical was more significant to degradation thanthe final step of forming one oxidised glycoside. The inefficientdegradation observed of the oxidation step is explainable by theamount of accessible glycosides being too small to influenceviscometry. The efficient degradation observed in associationwith the glycosidic radical formation is explained by initiationof free radical chain reactions that are propagated as long asthere is ozone in the system. As none of these phenomena werefound in the less crystalline cellulose, cellulose structureappears to be important for how free radical mediated cellulosedegradation develops.</p><p>The theory of free radical chain reactions coupled withdiffusion suggests a concentric expansion of the chain reactionsoutwards from the initial site of radical formation duringozonation of carbohydrates. This was confirmed by demonstratingfree radical chain reactions spreading from a spot of initiationoutwards during ozonation of a filter paper, using a pH-indicatorto monitor acid formation. Furthermore, the interior and exteriorof cellulose fibres doped with initiator were shown to bepermeated by small holes after ozonation.</p><p>Ethylene glycol was shown to improve the selectivity duringozone bleaching of oxygen bleached kraft pulp at pH 3. Optimalconditions were obtained at pH 3 for 25 wt% ethylene glycol. Theinfluence of ethylene glycol on selectivity is explained by aproportion of the free radical chain reactions being carried bythe ethylene glycol instead of the cellulose during ozonebleaching. The observations were summarised in the form of amodel where the observed degradations for pulp, bleached pulp andcotton fibres during both ozone bleaching and ethylene glycolassisted ozone bleaching were shown to agree with each other.</p><p>From g-irradiation of ozonised aqueous solutions of alcohol,the rate constant of superoxide formation from the peroxylradical of methanol was estimated to be 10 s^-1. Rate constants of the reactions between ozone andalkylperoxyl radicals were determined to be around 10⁴M^-1s^-1. The possibility of the reaction betweenalkylperoxyl radicals and ozone contributing significantly tofree radical chain reactions during ozonation of carbohydratesand alcohols could therefore be ruled out.</p><p>Cellulose, degradation, free radical, ozone, selectivity,ethylene glycol, alcohol, bleaching, kraft pulp, cotton linters,delignification, fibre, fibril, crystallinity, ferryl ion, freeradical chain reactions, TCF, viscometry, molecular weightdistributions, random scissions.</p>

Both Notch and the Wnt pathways are key regulators in maintaining the homeostasis in the intestine. Defects on the key tumor suppressor adenomatous polyposis coli, APC a gene in the Wnt pathway is most frequently mutated in colorectal cancer. Previous studies have indicated that there is a crosstalk between these two pathways. We investigate if there is correlation by first using bioinformatics to find Lef1/Tcf sites in several of the Notch pathway gene promoters. Bioinformatically we found that a lot of the genes contained theses sites controlled by the APC's destruction target β-catenin. By using semi quantitative PCR and western blot we found that Hes 1, Hes 7, JAG 2, MAML 1, Notch 2, NUMB, NUMBL, RFNG and LFNG was downregulated in HT29 colon cancer cells carrying a vector containing wild type APC. All but JAG 2 contains at least one Lef1/Tcf site in their promoter region. The results were verified in HT29 cells transfected with siRNA against β-catenin. We also investigated what would happen to the Lef1/Tcf target gene program of the Wnt pathway, if the Notch pathway was inhibited with the gamma-secretase inhibitor DAPT. Results showed no downregulution of β-catenin or its target gene Cyclin D1.Taken together, these results demonstrate that the Wnt pathway can be placed upstream of the Notch pathway and regulates the latter through β-catenin and the Lef1/Tcf target gene program. However, preliminary results indicate that there is no regulation of APC/β-catenin by the Notch pathway.

Hhex transcription factor is expressed in multiple endoderm-derived tissues, like the liver, where it is essential for proper development. The pleiotropic effect of Hhex in the embryo and its dual role as a transcriptional repressor/activator suggest the presence of different interaction partners capable of modulating its activity and function. In the current study we identified two new Hhex protein interactors: SOX13 and c-Myc. We show that Hhex interacts directly with SOX13. By doing so, Hhex sequesters SOX13 from the SOX13•TCF1 complex, overturning SOX13-dependent repression of the Wnt pathway. On the other hand, Hhex induces proliferation of non-tumorigenic human fibroblast through a Myc-dependent mechanism. Hhex and c-Myc interact directly upregulating Cyclin D1, a c-Myc target gene involved in cell cycle progression and proliferation. Elevation of Cyclin D1 might be the final effector of Hhex capacity to regulate cell proliferation.