The CNS monoaminergic systems are considered as the head engine regulating neuropsychiatric functions and personality. Transcription factor AP-2 is known to be essential for the development of the brainstem including the monoaminergic nuclei, and has the ability to regulate many genes in the monoaminergic systems. The ability of transcription factors to regulate specific gene expression, has lately made them hot candidates as drug targets. In this thesis, results indicating a role of AP-2 in the molecular effects of the antidepressant drugs citalopram and phenelzine, are presented. A polymorphism in the second intron of the gene encoding AP-2ß has previously been associated with anxiety-related personality traits as estimated by the Karolinska Scales of Personality (KSP). In this thesis, results confirming this association, gained by using a larger material and several different personality scales, are presented. Furthermore, data is presented showing an association between the activity of platelet monoamine oxidase, a trait-dependent marker for personality, and the genotype of the AP-2ß intron 2 polymorphism. The functional importance of the AP-2ß intron 2 polymorphism has not yet been elucidated. Included in this thesis are results showing that the AP-2ß intron 2 polymorphism is not in linkage disequilibrium with the only other described polymorphism in the AP-2ß gene, i.e. in the AP-2ß promoter (-67 G/A). Introns have in several studies been shown to include binding sites for regulatory proteins, and thus, to be important in transcriptional regulation. Results are presented demonstrating that one human brain nuclear protein binds only to the long variant of the AP-2ß intron 2 polymorphism. If this protein is involved in the regulation of the AP-2ß gene, it would affect the expression levels of the AP-2ß protein. In general, this thesis further establishes the role of transcription factor AP-2 as a regulatory factor of importance for personality and monoaminergic functions.

Notch signaling is important for cell-cell signaling during development. Notch signaling is highly conserved across all metazoans and failure in Notch signaling is causative in many human diseases. In the Drosophila eye, activation of the Notch pathway requires Lqf (Drosophila Epsin)-dependent and Clathrin-dependent internalization of the Notch receptor ligands, Delta or Serrate, by the signal-sending cells. However, it is unclear why ligand must be internalized into the signal-sending cells to activate Notch signaling in the signal-receiving cells. Evidence suggests that in addition to Clathrin and Epsin, Auxilin is essential for signaling and is indirectly required for internalization of the Notch receptor ligand Delta. Auxilin functions in uncoating Clathrin-coated vesicles to maintain a pool of free Clathrin and Epsin in the cell. auxilin mutants were used as an entryway to identify previously unknown components of the Notch signaling pathway. An F1, FLP/FRT, EMS screen was performed and enhancers of an auxilin mutant rough eye defect were isolated. The enhancers ultimately formed one complementation group on the 2nd chromosome and fourteen complementation groups on the 3rd chromosome. Three of the 3rd chromosome complementation groups were each identified as Delta, lqf, or hsc70. A single allele was identified as faf. Delta and Epsin have known roles in signaling cells to activate Notch as described above. Hsc70 is an ATPase that functions with Auxilin to uncoat Clathrin-coated vesicles and Faf is a deubiquitinating enzyme that maintains levels of active Epsin in the cell. These results suggest I have isolated mutations in genes closely tied to Notch signaling or functioning directly with Auxilin. Mutations in two genes previously undescribed in Notch signaling in the developing Drosophila eye were also isolated from the screen and identified. The second chromosome complementation group was identified as α-adaptin. α-Adaptin is a subunit of the heterotetrameric Clathrin adaptor protein AP-2. One of the third chromosome complementation groups was identified as crumbs. Crumbs is an integral membrane protein that functions at adherens junctions and in establishing apical/basal polarity in cells. Characterizing roles for α-Adaptin and Crumbs during Notch signaling may elucidate the purpose for Delta internalization to activate Notch signaling. / text

Les plantes sont exposées, à chaque étape de leur cycle de vie, à différents stress abiotiques notamment la sécheresse, le froid et la salinité. Bien que ces différents types de stress aient des effets spécifiques pour chacun d'entre eux, ils ont tous la capacité d'induire un effet commun soit le déficit hydrique qui affecte la croissance et le développement des plantes. Certaines plantes s'adaptent au déficit hydrique en modifiant leurs métabolismes ainsi que l'expression de leurs gènes afin de synchroniser leur développement avec les conditions de stress. Cette modification se traduit par la synthèse de nouvelles protéines, particulièrement régulée par le déficit hydrique, notamment les protéines de type Ap2/EREBP. Chez Arabidopsis thaliana, ces protéines forment une famille de facteurs de transcription de 145 membres. Certains membres jouent des rôles essentiels de régulateur dans plusieurs processus de développement tandis que d'autres sont impliqués dans les mécanismes de réponses des plantes aux stress. Chez les céréales, très peu de gènes Ap2/EREBP avec des fonctions connues ont été identifiés. Ce projet de recherche vise à identifier cette famille de gènes chez le blé hexaploid (Triticum aestivum L.). Le choix de ce type de blé comme modèle d'étude réside dans sa capacité de tolérance au gel et sa grande variabilité de réponses aux stress abiotiques. Le blé est la céréale la plus importante au monde puisqu'il représente environ 73% de la production céréalière mondiale. Il est cultivé dans divers types d'environnements climatiques, de productions et de fermes. De plus, il représente la principale source d'énergie alimentaire, d'emploi et de revenu dans plusieurs pays en voie de développement. Le blé agronomique est plus nutritif et économique qu'Arabidopsis. Cependant, le choix entre les deux espèces est influencé par la taille et la complexité de leurs génomes. Le premier chapitre décrit l'identification, la phylogénie et la localisation chromosomique des gènes Ap2/EREBP du blé. Une approche génomique combinant une recherche de bases de données de séquences suivie d'une analyse bioinformatique, un criblage de banques d'ADNc et des amplifications par réaction de polymérisation en chaîne a permis d'identifier 107 ADNc du blé qui codent potentiellement pour des facteurs de transcription de type Ap2/EREBP. Leur localisation chromosomique montre que ces gènes sont dispersés sur tout le génome du blé. Cependant, quelques gènes de cette famille, appelés CBFs (C-repeat Binding Factors), sont regroupés principalement sur le long bras du groupe de chromosomes 5 et ont été associés directement à des déterminants génétiques (QTLs) contrôlant les réponses aux stress et divers aspects du développement floral. Le deuxième chapitre présente la caractérisation de la sous-famille de gènes CBFs. L'analyse phylogénétique a indiqué que les espèces de blé contiennent au moins 23 gènes différents de type CBF. Chez les Poaceae, les CBFs sont classés dans 10 groupes qui partagent une origine phylogénétique commune et des caractéristiques structurales semblables. Six de ces groupes (3C, 3D, 4A, 4B, 4C et 4D) sont trouvés seulement chez les Pooideae, suggérant ainsi qu'ils représentent les mécanismes de réponse de CBF qui ont évolué récemment pendant la colonisation des habitats tempérés. Les études menées sur le profil d'expression de ces gènes démontrent que 5 des groupes spécifiques aux Pooideae montrent une expression constitutive élevée et une expression inductible par les basses températures chez le cultivar d'hiver. Par contre à des températures modérées, la régulation de l'expression varie en fonction de la période de la journée. L'expression inductible et non héritée au sein des groupes de CBF a possiblement joué un rôle prédominant dans l'aptitude des cultivars d'hiver à tolérer les basses températures et elle est probablement à la base de la variabilité génétique de la tolérance au gel chez les Pooideae. Le troisième article décrit l'identification de deux gènes chez le blé, ICE1-like (Inducteur de l'expression de CBF 1) codant pour un facteur de transcription qui régule l'expression des gènes CBF. TaICE87 et TaICE41 codent pour un activateur de transcription de type MYC-like bHLH. TaICE87 et TaICE41 se lient spécifiquement à différentes séquences de reconnaissance MYC du promoteur de TaCBFIVd-B9. TaICE87 and TaICE41 sont constitutivement exprimés chez le blé et leur surexpression chez Arabidopsis mène à l'augmentation du niveau d'expression des gènes AtCOR et AtCBF3 et de la tolérance des plantes au gel. Ces résultats suggèrent que TaICE87 and TaICE41 sont des orthologues fonctionnels d'AtICE1 et peuvent réguler la transcription d'AtCBF3. La structure complexe des éléments MYC au niveau des promoteurs CBF et la différence d'affinité entre TalCE87 et TalCE41 pour les éléments MYC suggèrent que ces 2 protéines peuvent différentiellement activer CBF chez le blé. En résumé, le clonage de la famille de gènes Ap2/EREBP chez le blé a permis d'identifier et de caractériser les membres CBF régulés par les basses températures. De plus, les résultats de cette étude démontrent que l'expression des gènes CBF est amplifiée chez les Pooideae, suggérant ainsi un rôle plausible dans la tolérance au froid des cultivars d'hiver. L'analyse des gènes CBFs pourrait nous conduire à améliorer la tolérance au gel chez le blé, et par conséquent faciliter l'établissement de nouvelles stratégies visant à améliorer la productivité des céréales et leur adaptation aux changements climatiques. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Blé, Acclimatation au froid, Tolérance au gel, Facteurs de transcription, Ap2/EREBP.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 5 uppsatser.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 225-248). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Nef, an accessory protein of HIV-1, is a critical determinant of viral pathogenicity. The pathogenic effects of Nef are in large part dependent on its ability to decrease the amount of CD4 on the surface of infected cells. Early studies suggested that Nef induces downregulation by linking the cytosolic tail of CD4 to components of the host-cell protein-trafficking machinery. However, the specific sorting pathway that Nef uses to modulate CD4 expression remained uncertain. According to one model, Nef was thought to interfere with the transport of newly synthesized CD4 from the TGN to the cell-surface. Another model claimed that Nef facilitated the removal of CD4 from the plasma membrane. The primary goal of this thesis was to determine which of these models was correct. To accomplish this objective, a novel Nef-CD4 system was developed in Drosophila S2 cells. Nef was not only able to downregulate human CD4 in S2 cells, but it did so in a manner that was phenotypically indistinguishable from its activity in human cells. An RNAi screen targeting protein-trafficking genes in S2 cells revealed a requirement for clathrin and the clathrin-associated, plasma membrane-localized AP-2 complex in the Nef-mediated downregulation of CD4. In contrast, depletion of the related AP-1 and AP-3 complexes, which direct transport from the TGN and endosomes, had no effect. The requirement for AP-2 was subsequently confirmed in a human cell line. Yeast three-hybrid and GST pull-down assays were then used to demonstrate a robust, direct interaction between Nef and AP-2. This interaction was found to depend on a [D/E]xxxL[L/I]-type dileucine motif, located in the C-terminal loop of Nef, that is essential for CD4 downregulation. While mapping the binding site of AP-2 on Nef, a second determinant of interaction in the C-terminal loop was identified. Mutation of this motif, which conforms to a consensus [D/E]D diacidic sequence, prevented Nef from binding to AP-2 and down-regulating CD4. However, the same mutations did not affect the ability of Nef to interact with either AP-1 or AP-3, providing further evidence that these complexes are not required for the modulation of CD4 expression. Additional experiments indicated that the Nef diacidic motif most likely binds to a basic patch on AP-2 α-adaptin that is not present in the homologous AP-1 γ and AP-3 δ subunits. As with the Nef diluecine and diacidic motifs, the α-adaptin basic patch was shown to be necessary for CD4 downregulation. Moreover, all three of these motifs were needed for the cooperative assembly of a CD4-Nef-AP-2 tripartite complex, which was observed here for the first time using a yeast four-hybrid system. The data in this thesis uniformly support an endocytic model of Nef-mediated CD4 downregulation. Indeed, there is now strong evidence that Nef simultaneously binds CD4 and AP-2, thereby connecting the receptor to the cellular endocytic machinery and promoting its rapid internalization from the plasma membrane. In addition, the identification of novel motifs required for this process has provided new insights on endocytosis, and may facilitate the development of pharmacological inhibitors of Nef function.

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Made available in DSpace on 2014-07-29T15:01:40Z (GMT). No. of bitstreams: 1 Viniciu Fagundes Barbara em PDF.pdf: 2483097 bytes, checksum: 5b34f64b65562b6c494035ae3e2630ee (MD5) Previous issue date: 2006-12-12 / The current investigation presents a characterization of the hydraulics and water quality of the medium course of the Araguari River, State of Amapá, Brazil, which has an approximate extension of 120 km. The Araguari River is the main water resource of the State of Amapá, located in the domains of the Amazon (the greatest tropical forest of the planet) in a region with great hydropower potential. In the studied river stretch, there is a hydropower generation station called Coaracy Nunes (UHECN), the first barrage build in the Amazon for electrical generation. A model for DO and BOD has been built with the help of the QUAL2E modeling system, developed by the United States Environmental Protection Agency. For this purpose, during one and a half year, field trips have been conducted with the intent of characterizing spacially and seasonally the hydraulics, kinetics and water quality of the Araguari River. The modeling stages involved model construction, sensitivity analisys and calibration. After model calibration, three hypothetic cenarios were simulated: (1) the introduction of a new barrage upstream of the UHECN; (2) municipal untreated wastewater inflow from Porto Grande and Ferreira Gomes into the waterbody, including populational growth, during the high flow period; (3) the same cenario as before but during the low flow period. The carbonaceous organic matter present in water hás shown to decompose, in average, in the first five days of incubation. The values obtained for the BOD first order reaction coefficient layed between 0,13 d-1 and 0,90 d-1. The reaeration coefficient, K2, estimated based on hydraulic characteristics, layed between 0,01 d-1 and 1,40 d-1. The results of the sample collections in the river stretch has shown that, amongst the parameters measured, the following were mostly altered: pH, electric conductivity, total solids, suspended solids, fecal coliforms, ammonium, nitrate, BOD and DO. Fecal coliforms, pH, nitrate and BOD were in disagreement with limits defined by Brazilian Legislation on water quality (CONAMA 357/2005). The river impoundment has shown not to significantly alter the DO and BOD concentrations, however, the same statement can not be made for other water characteristics. The water characteristics mostly altered by the UHECN were: hydraulic (flow, velocity, breadth, cross-sectional area) and water quality (pH, electric conductivity, water temperature, total solids, suspended solids, fecal coliforms, nitrate, chloride, BOD and DO). The simulated scenario with municipal untreated wastewater inflow from Porto Grande and Ferreira Gomes (including population growth) into the waterbody during low flow period showed the most critical results in terms of the water quality. The worst simulated situation happened in the dry period in which the river flows decreased significantly and so did the river dilution capacity. In this period the greatest OD depletion occurred and achieved concentrations bellow 4.0 mg/L, in disagreement with Brazilian Water Quality Legislation (CONAMA 357/2005). / A presente investigação apresenta uma caracterização hidráulica e da qualidade da água do médio curso do Rio Araguari (AP), em uma extensão de aproximadamente 120 km. O Rio Araguari, principal corpo hídrico do Estado do Amapá, está situado dentro dos domínios da Amazônia (a maior floresta tropical do planeta), em uma região com grande potencial hidrelétrico. No trecho em estudo, encontra-se implantada a Usina Hidrelétrica de Coaracy Nunes (UHECN), a primeira barragem edificada na Amazônia com fins de geração de energia elétrica. Foi desenvolvido um modelo de oxigênio dissolvido (OD) e de demanda bioquímica de oxigênio (DBO) com o auxílio do sistema de modelagem QUAL2E, desenvolvido pela Agência de Proteção Ambiental dos Estados Unidos (USEPA). Para isso, procedeu-se, durante um ano e meio, com expedições de campo que objetivaram caracterizar, espacialmente e sazonalmente, os parâmetros hidráulicos, cinéticos e de qualidade da água do Rio Araguari. As etapas da modelagem envolveram a construção, análise de sensibilidade e a calibração do modelo. Posteriormente efetuou-se, com o modelo calibrado, a simulação de três cenários hipotéticos na bacia: (1) a introdução de uma nova barragem a montante da UHECN; (2) o descarte de efluentes in natura oriundos dos municípios de Porto Grande e Ferreira Gomes (com crescimento populacional) nas águas do corpo hídrico (período de alta vazão); e (3) o mesmo cenário anterior, porém para a condição de baixas vazões. A matéria orgânica carbonácea presente na água desse rio decompõe-se, em média, nos primeiros cinco dias de incubação. Os valores obtidos experimentalmente para o coeficiente de deoxigenação, K1, ficaram compreendidos entre 0,13 d-1 e 0,90 d-1. O coeficiente de reaeração, K2, obtido com base nas características hidráulicas, ficaram compreendidos entre 0,01 d-1 a 1,40 d-1. Os resultados das campanhas amostrais mostraram que os parâmetros de qualidade da água que se apresentaram mais alterados no trecho em estudo foram: potencial hidrogeniônico, condutividade, sólidos totais, sólidos suspensos, coliformes termotolerantes, amônia, nitrato, demanda bioquímica de oxigênio e oxigênio dissolvido. Quando comparados com a Resolução 357/2005 do Conselho Nacional de Meio Ambiente (CONAMA), os parâmetros que mais infringiram esse instrumento jurídico foram: potencial hidrogeniônico, coliformes termotolerantes, nitrato e demanda bioquímica de oxigênio. A nova barragem não interfere significativamente as concentrações de OD e DBO do Rio Araguari, não se podendo fazer a mesma afirmação para os demais parâmetros de qualidade da água. As características que mais se alteraram devido à obra da UHECN foram: hidráulicas (vazão, velocidade, largura e área da seção transversal), e de qualidade da água (potencial hidrogeniônico, condutividade, temperatura da água, sólidos totais, sólidos suspensos, coliformes termotolerantes, nitrato, cloreto, demanda bioquímica de oxigênio e oxigênio dissolvido). O cenário simulado com o descarte nas águas do manancial de efluentes in natura dos municípios de Porto Grande e Ferreira Gomes, com o crescimento populacional, no período das baixas vazões, foi o mais desfavorável dentre os cenários investigados com relação à qualidade da água. A pior situação aconteceu na época das estiagens, em que as vazões do curso d água diminuem consideravelmente e, consequentemente, sua capacidade de diluição também. Neste período, ocorreu uma maior depleção do oxigênio dissolvido presente na água, que alcançou concentrações abaixo de 4,00 mg/L, vindo a infringir a Resolução 357/2005 do CONAMA.

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