Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 102-123).

Made available in DSpace on 2015-03-26T13:32:16Z (GMT). No. of bitstreams: 1 texto completo.pdf: 862787 bytes, checksum: b23a777918d630895ac6bf3d64e985c8 (MD5) Previous issue date: 2012-07-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In recent years Geostatistics has been widely used in the field of precision agriculture, this is due to being a tool to analyze the spatial variability exists in the area of agricultural production, enabling assess the need for creation of subareas or zones where they are handlings performed differently. One focus of precision agriculture is to identify management zones within the field based on the variability, and through Geostatistics is possible to produce thematic maps that assist in the establishment of management zones through adjustments of models of spatial dependence. The objective of this study was to analyze the influence of models of spatial dependence in the definition of thematic maps of management zones, using different models and different semivariogram sampling grids. To conduct the study, we analyzed simulated data generated in SAS software, where he held various structures of spatial dependence (DE) and different sampling grids (grid), with different sizes and densities of points, with a total of 9 data set simulated. To analyze the spatial dependence structure experimental semivariograms were used and adjusted three theoretical models to experimental semivariogram: exponential, spherical and Gaussian for each data set under study, according to the parameters set forth by autotune software ArcGis 9.3. Were adjusted 27 different semivariogram models using the method of ordinary kriging and through cross-validation technique checked the fit of the models semivariográficos. Thematic maps management zones were generated from the technique of kriging, and with the aid of software Idrisi was performed to compare the maps using Kappa Index of agreement, which was tested by testing their significance Z. From the analyzes, it was concluded that the model of spatial dependence does not influence the definition of thematic maps of management zones. / Nos últimos anos a Geoestatística vem sendo amplamente utilizada na área de agricultura de precisão, isso se deve ao fato de ser uma ferramenta que permite analisar a variabilidade espacial existente na área de produção agrícola, possibilitando avaliar a necessidade de criação de subáreas ou zonas onde serão realizados manejos de forma diferenciada. Um dos focos da agricultura de precisão é a identificação de zonas de manejo dentro do campo baseadas na variabilidade existente, e por meio da Geoestatística é possível produzir os mapas temáticos que auxiliam no estabelecimento das zonas de manejo através de ajustes de modelos de dependência espacial. Assim, o objetivo geral deste estudo foi analisar a influência de modelos de dependência espacial na definição de mapas temáticos de zonas de manejo, usando diferentes modelos de semivariogramas e diferentes grades de amostragem. Para a realização do estudo foram analisados dados simulados gerados no software SAS, onde se considerou diferentes estruturas de dependência espacial (DE) e diferentes grades de amostragens (grid), com diferentes tamanhos e densidades de pontos, sendo um total de 9 conjunto de dados simulados. Para a análise da estrutura de dependência espacial foram utilizados semivariogramas experimentais e ajustados três modelos teóricos ao semivariograma experimental: exponencial, esférico e gaussiano, para cada conjunto de dados em estudo, conforme os parâmetros estipulados pelo auto-ajuste do software ArcGis 9.3. Foram ajustados 27 diferentes modelos de semivariogramas através do método da krigagem ordinária e através da técnica de validação-cruzada verificados os ajustes dos modelos semivariográficos. Mapas temáticos de zonas de manejo foram gerados a partir da técnica de krigagem ordinária, e com o auxilio do software Idrisi foi realizada a comparação dos mapas através do Índice Kappa de concordância, o qual foi testado sua significância através do teste Z. A partir das análises realizadas, concluindo-se que o modelo de dependência espacial não influencia na definição de mapas temáticos de zonas de manejo.

Made available in DSpace on 2014-06-11T19:31:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-12Bitstream added on 2014-06-13T21:02:16Z : No. of bitstreams: 1 guirro_ecbp_dr_jabo.pdf: 1021752 bytes, checksum: 2f91e133459fe9dddd2a2939344fae32 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A dor interfere na homeostase e dificulta a realização de diversos procedimentos em eqüinos. Os opióides agonistas mu são analgésicos potentes, porém podem induzir efeitos adversos como aumento da atividade locomotora espontânea, enquanto que agonistas de receptores kappa, como o U50-488H, produzem analgesia e alterações comportamentais mínimas em eqüinos. A crotalfina é um agonista kappa que produz analgesia potente em ratos. Assim, dezoito eqüinos Puro Sangue Árabe foram utilizados para se avaliar os efeitos comportamentais, clínicos e antinociceptivos promovidos pela injeção intravenosa de dose única de crotalfina (3,8ng/kg) em comparação com morfina (0,1mg/kg), U50-488H (160mg/kg), fenilbutazona (4,4mg/kg) ou NaCl 0,9%. A crotafina não alterou a atividade locomotora espontânea, a taxa de micção e/ou defecação, a freqüência cardíaca e a motilidade intestinal; houve apenas discretas mudanças comportamentais, sedação leve e aumento sutil e transitório de temperatura retal e de freqüência respiratória. Em pele incisionada ocorreu efeito antinocicetivo por até seis horas e efeito anti-hipernociceptivo por até três dias em região isquiática, mas em pele íntegra e em região escapular incisionada o efeito antinociceptivo não foi significativo. Portanto, a administração intravenosa de dose única de crotalfina (3,8ng/kg), em eqüinos, não gera alterações clínicas e/ou comportamentais severas e promove efeito antinociceptivo por até seis horas e inibe a instalação de estados hipernociceptivos por até três dias, em modelo incisional de dor inflamatória realizado em região isquiática, enquanto que a crotalfina em pele íntegra e em região escapular incisionada não promove efeito antinociceptivo relevante. / Pain unbalances in homeostase and makes difficult a lot of procedures in horses. The mu-opioids agonists are powerful analgesics powerful, however can induce adverse effect as increase of the spontaneous locomotor activity, while kappa agonists, like U50-488H, produce analgesia and minimal behavioral changes in horses. Crotalfine is a kappa opioid that promote potent analgesia in rats. Thus, eighteen Arabian horses were used to evaluate behavioral, clinical and antinociceptive effects promoted by only intravenous injection of crotalfina (3,8ng/kg) in comparison with morphine (0,1mg/kg), U50-488H (160mg/kg), phenylbutazone (4,4mg/kg) or NaCl 0.9%. Crotalfine didn’t change spontaneous locomotor activity, rate to urine and defecate, heart rate and intestinal motility; were observed just discrete behavioral changes, light sedation and subtle and transitory increase of body temperature and respiratory rate. At incisioned skin was verified antinociceptive effect for up to six hours and anti-hypernociceptive effect occurred from six hours until three days in isquiatic region, but in complete skin and incisioned scapular region the antinociceptive effect was not significant. Therefore, only intravenous administration of crotalfina (3,8ng/kg), in horses, does not generate severe clinical and behavioral changes and promotes antinociceptive effect for up to six hours and inhibits installation of hypernociceptive states for up to three days in incisional model of inflammatory pain at isquiatic region, whereas crotalfine does not promote relevant antinociceptive effect in complete skin and in incisioned scapular region.

Orientador: Rosiane Lopes da Cunha / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-10T02:52:06Z (GMT). No. of bitstreams: 1 Takeuchi_KatiuchiaPereira_D.pdf: 2512071 bytes, checksum: bf144af04917964fdd59c0d4a482b84e (MD5) Previous issue date: 2008 / Resumo: A avaliação do processo de gelificação ácida de caseinato de sódio (CS) induzida por glucona-d-lactona (GDL) foi realizada em diferentes taxas e com ampla faixa de concentração de proteína (2-6% p/p). A cinética de acidificação e gelificação foi avaliada desde o pH 6,7 até o ponto isoelétrico das caseínas através da medida de pH e de propriedades mecânicas obtidas em compressão uniaxial (tensão e deformação na ruptura). A formação da rede do gel foi mais influenciada pelas interações eletrostáticas do que pelas diferentes taxas de acidificação, principalmente em pH próximo ao pI das caseínas. Além disso, interações hidrofóbicas e ligações de hidrogênio também estiveram envolvidas na estabilização da estrutura da rede, promovendo géis mais fortes. Também foi avaliado o processo de gelificação de proteínas do leite em pH 6,7 em sistemas contendo carragena. Neste caso, a ?-carragena foi adicionada em concentração de 0,3 a 0,8% (p/p) em misturas contendo caseinato de sódio (2 a 8% p/p), isolado protéico de soro (0,5 a 7% p/p) ou sacarose (5 a 30% p/p). Estes sistemas foram estudados a partir de ensaios reológicos em cisalhamento oscilatório, propriedades mecânicas e microestrutura. A temperatura de início da gelificação ou do desenvolvimento de estrutura (Ts) aumentou com a concentração de carragena em sistemas puros, enquanto que a presença de sacarose ou isolado protéico de soro promoveu um aumento da Ts e a adição de caseinato de sódio não modificou esta temperatura, em relação aos géis puros. Após a fomação do gel, um aumento da concentração de carragena levou a géis mais elásticos, rígidos, deformáveis e firmes, sendo que a adição de sacarose exerceu pouco efeito nas propriedades reológicas destes sistemas. A adição de isolado protéico de soro enfraqueceu a rede do gel em baixas concentrações (até 3%), mas houve formação de uma rede mista em maiores concentrações, sem demonstração de sinergismo entre os biopolímeros. A mistura de carragena e caseinato de sódio mostrou sinergia até 5% (p/p) desta proteína e para maiores concentrações, ocorreu o enfraquecimento da rede, diminuição da rigidez e firmeza do gel, provavelmente relacionada à micro-separação de fases, observada por microscopia confocal. Os espectros mecânicos mostraram que a maioria destes sistemas mistos apresentou comportamento de gel fraco, devido ao efeito simultâneo das interações físicas e incompatibilidade termodinâmica. No entanto, géis fortes foram observados na maior e menor concentração de carragena e caseinato de sódio, respectivamente, indicando a importância da interação eletrostática entre estes dois biopolímeros. O aumento da concentração de carragena e de proteínas promoveu um aumento da heterogeneidade da microestrutura do gel e a intensidade de interações repulsivas entre os biopolímeros e a formação da rede do gel, dominado pela carragena, afetou de maneira complexa as propriedades físicas destes sistemas / Abstract: Evaluation of acid-induced sodium caseinate (CS) gelation promoted by glucono-d- lactone (GDL) was performed at different acidification rates with several protein concentrations (2-6% w/w). The kinetics of acidification and gelation were followed from pH 6.7 to the isoelectric point of casein by evaluation of the pH and mechanical properties using uniaxial compression measurements (stress and strain at rupture). Gel network formation was more influenced by electrostatic interactions than different acidification rates, showing the contribution of rearrangements of bonds to strengthening the network, mainly at steady-state pH close to pI of caseins. Besides the hydrophobic interactions and hydrogen bonds were also important forces involved in structure stabilization, leading to stronger gels. Moreover, evaluation of gelation process of milk proteins at pH 6.7 in systems containing ?-carrageenan. In this study, the ?-carrageenan was added at concentration from 0.3 to 0.8% (w/w) into mixtures containing sodium caseinate (2 a 8% w/w), whey protein isolate (0.5 a 7% w/w) or sucrose (5 a 30% w/w). This systems was analysed using rheological measurements under oscillatory shear, mechanical properties and microstructure. The temperature at which structure development began or initial of gelation (Ts) augmented with increasing carrageenan concentration for pure systems, but sucrose or whey protein isolate addition promoted an increase of Ts and sodium caseinate did not affect this temperature, in relation to pure gel. After gel formation, increasing carrageenan concentration promoted more elastic, stronger, deformable and firmer, as well as sucrose addition showed a little effect to decrease elastic character of gel. Whey protein addition weakened gel network at lower concentrations (up to 3% w/w), but at higher concentration was observed a mixed gel network entanglement of carrageenan and whey protein, without sinergism between this biopolymers. Mixed gels of carrageenan and sodium caseinate showed synergism up to 5% (w/w/) of this protein and increasing concentration led to weaken the gel network, decreasing the rigidity and the firmness of gel, probably related to micro-phase separation, observed by confocal microscopy. Mechanical spectra showed that most of mixed systems presented weak gel behaviour, due to simultaneous effect of physical interactions and thermodynamic interactions. However, stronger gels were formed at higher and lower concentration of carrageenan and sodium caseinate, respectively, which indicate the relevance of electrostatic interactions between these biopolymers. Increasing carrageenan and proteins concentrations led to greater microstructure heterogeneity and increased repulsive interactions between biopolymers and thus gel formation, dominated by carrageenan, affected in a complex way the physical properties of these systems / Doutorado / Doutor em Engenharia de Alimentos

Os objetivos gerais do presente estudo foram avaliar o efeito do polimorfismo genético da kappa-caseína, da raça e da estação do ano sobre as características físico-químicas (acidez, pH e crioscopia), composição (gordura, lactose, sólidos totais, contagem de células somáticas, uréia, proteína bruta, proteína verdadeira, nitrogênio não protéico e nitrogênio não caseinoso) e estabilidade do leite. Foram selecionados 11 rebanhos leiteiros comerciais, sendo cinco deles da raça Holandesa e seis da raça Girolanda, dos quais foram amostradas em média 122 vacas em lactação por rebanho, totalizando 1350 vacas amostradas em três períodos: 2 no período seco e 1 no período chuvoso. As vacas selecionadas foram analisadas quanto a composição e propriedades físico-químicas do leite, assim como para a determinação do polimorfismo de kappa-caseína. O presente trabalho foi dividido em dois estudos, no primeiro avaliou-se os efeitos da raça e estação do ano sobre características físico-químicas, composição e estabilidade do leite. Foi observado efeito de raça sobre acidez titulável, pH, lactose, uréia e estabilidade do leite. O efeito de sazonalidade mostrou-se significativo sobre pH, crioscopia, teores de lactose, uréia, proteína bruta, sólidos totais, contagem de células somáticas, proteína verdadeira, caseína, nitrogênio não protéico e estabilidade térmica do leite. Em animais da raça Girolanda, foram observados no período seco maiores teores de sólido totais. O ponto crioscópico do leite sofreu efeito do período de coleta somente em animais da raça Holandesa, onde valores mais altos foram observados durante o período seco. Os teores de lactose apresentaram maiores médias no período seco em animais da raça Holandesa. Os teores de uréia sofreram efeito de raça e período de coleta estudado. A proteína bruta do leite sofreu efeito dos fatores raça e período de coleta, sendo que vacas da raça Holandesa e Girolanda apresentam maiores teores no período seco. A concentração de caseína do leite sofreu efeito significativo do período de coleta. A estabilidade térmica do leite sofreu influência da raça, pois o leite de vacas da raça Holandesa mostraram-se mais estáveis do que os da raça Girolanda. A sazonalidade é um fator determinante para a estabilidade do leite, pois o leite mostrou-se mais estável no período chuvoso que em período seco. No segundo estudo foram avaliados o polimorfismo genético da kappa-caseína em vacas Holandesas e Girolandas e o efeito deste sobre características físico-químicas, de composição e estabilidade térmica do leite. A freqüência do alelo A foi maior do que a do alelo B no que diz respeito ao gene da kappa-caseína. Com relação às características físico-químicas do leite, não houve efeito do polimorfismo do gene da kappa-caseína sobre teores de gordura, sólidos totais, lactose, contagem de células somáticas e uréia do leite. Não foram observados efeitos do polimorfismo genético do gene para kappa-caseína sobre a composição protéica do leite, sendo que teores de proteína bruta, nitrogênio não protéico, nitrogênio não caseinoso e proteína verdadeira não sofrem influência. A estabilidade do leite frente à prova do álcool não sofreu influencia do polimorfismo genético de kappa-caseína / The objective of this study were to determine the effects of kappa-casein gene polymorphisms, breed and season on physical-chemical properties (acidity, pH and cryoscopy), composition (fat, lactose, total solids, somatic cells count, urea, crude protein, true protein, non protein nitrogen and non casein nitrogen) and stability of milk. For this aim 11 dairy herds were selected, six of them composed of Girolando cows and five from Holstein cows, in average milk samples were taken of 112 cows from each herd, collected three times: twice in dry season and once on rainy season. Each cow were analyzed for milk composition, physico-chemical properties, and to determine its kappa-casein polymorphism. This study was divided in two, the first one analyzed possible breed and season effects over milk physico-chemical characteristics, composition and stability. Breed effect was observed over acidity, pH, lactose, urea and milk stability. Season effects was significant for pH, cryoscopy, lactose, urea, CP, total solids, SCC, TP, casein, EqNPN and heat milk stability. In cows Girolando it was observed highest total solids in dry season. Milk cryoscopy had season effects only on Holstein cows, with highest results in dry season. Lactose concentration was greatest in Holstein cow in dry season. Urea concentration showed breed and season effect on this study. Crude protein was affected by breed and season, in dry season the concentration were highest for Holstein and Girolanda. Casein milk concentration demonstrated season effect. Heat milk stability showed breed stability, milk from Holstein cow were more stable that milk from Girolanda cows. Season is a determinant factor for milk stability, milk showed more stable in rainy season than during dry period. The second study analyzed: kappa-casein gene polymorphisms in Holstein and Girolando cows and its effects over milk physico-chemical characteristics, milk composition and milk heat stability. Kappa-casein allele A had a higher frequency, than allele B, in Girolando and Holstein cows in comparison with other polymorphisms. No effect of kappa-casein polymorphism was observed on milk phisico-chemical characteristics and on milk fat, total solids, lactose, SCC, and milk urea. There were no difference for milk protein composition (EqNPN, NCNC, TP, casein and EqNCN) for kappa-casein polymorphism. Milk alcohol stability did not showed effect of gene polymorphism

L'addiction est une affection chronique emmaillée de rechutes, caractérisée principalement par des comportements compulsifs de recherche et de consommation de drogue. Il s'agit d'une pathologie grave et fréquente. Les sujets qui parviennent à se désengager de ces compulsions sont considérés comme abstinents. Les études épidémiologiques montrent que l'abstinence, notamment pour les opiacés est fortement associée à une prévalence accrue de la dépression. Le but de mon travail de thèse était d'aborder spécifiquement la cooccurrence des troubles dépressifs et addictifs. J'ai participé à l'élaboration d'un modèle murin de cette comorbidité, et nous nous sommes focalisés sur l'addiction aux opiacés, en particulier l'héroïne. Suite à un traitement chronique par l'héroïne et au cours de l'abstinence paraissent progressivement des comportements apparentés à la dépression. Ce traitement chronique à l'héroïne modifie le fonctionnement du système sérotoninergique et l'activité du récepteur opioïde kappa (Article publié: Distinct mu, delta, and kappa opioid receptor mechanisms underlie low sociability and depressive-­like behaviors during heroin abstinence). Les déficits comportementaux observés peuvent être prévenus et reversés par un traitement antidépresseur ciblant le system sérotoninergique ou en inhibant l'activité du récepteur opioïde kappa avec un antagoniste, dans des portions similaires (Manuscript submis: Kappa opioid receptor antagonism prevents and reverses heroin abstinence-­induced social deficit with similar efficacy compared to chronic antidepressant treatment). Notre étude nous permet de proposer que le récepteur opioïde kappa constitue un acteur majeur à l'interface de l'addiction et de la dépression(Revue publié: The kappa opioid receptor: from addiction to depression, and back). / Addiction is a chronic, relapsing disorder and is mainly described as compulsive craving and consumption of a drug in spite of adverse consequences. Individuals who have achieved to refrain from such compulsive behaviour are considered abstinent, but present symptoms reminiscent of depression. Epidemiological studies report that abstinence, particularly from opiates, strongly associates with higher prevalence of depression. Aim of my thesis was to specifically address the co-­occurrence of opiate addiction and major depression in preclinical research. Thus, I have contributed to develop a mouse model of opiate abstinence, and in particular extend our model of morphine abstinence to heroin. Following exposure to escalating doses of heroin, abstinent mice progressively exhibit a depressive-­like phenotype, revealed by low sociability., and show altered serotonergic and kappa opioid receptor signaling (Published article: Distinct mu, delta and kappa opioid receptor mechanisms underlie low sociability and depressive-­like behaviors during heroin abstinence).Importantly, these behavioural deficits can be both prevented and reversed by antidepressant treatment targeting serotonergic signaling, or inhibiting the activity of the kappa opioid receptor withan antagonist, with similar efficacy (Submitted article: Kappa opioid receptor antagonism prevents and reverses heroin abstinence-­induced social deficit with similar efficacy compared to chronic antidepressant treatment). Altogether, our results allow us to propose the kappa opioid receptor is a major player at the interface of addiction and depression (Published review: The kappa opioid receptor: from addiction to depression, and back).

En premier lieu, en utilisant des inhibiteurs pharmacologiques du NF-[kappa]B, nous avons constaté que l'inhibition du facteur de transcription NF-[kappa]B chez ces cellules diminue de beaucoup l'expression génique et la sécrétion de diverses cytokines et chimiokines (TNF-[alpha], IL-8 ou CXCL8, Mip-1[alpha]/[bêta] induites par des stimuli tels que TNF-[alpha] ou LPS. Nous montrons ensuite que le complexe IKK (IKK[alpha], IKK[bêta], et IKK[gamma]) est aussi partiellement localisé dans le noyau, alors que les kinases reliées à IKK (IKK[epsilon] et TBK-1) sont cytoplasmiques; la kinase NIK, quant à elle, est strictement nucléaire. Suite à une activation des neutrophiles, IKK[bêta] et IKK[gamma] deviennent transitoirement phosphorylées dans le cytoplasme et le noyau, alors qu'IKK[alpha] disparaît temporairement de ces deux compartiments cellulaires d'une manière qui semble dépendante de IKK[bêta]. Ces réponses s'accompagnent, dans les deux compartiments, de la dégradation d'I[kappa]B[alpha] et de la phosphorylation du RelA sur la sérine 536. Bien que les deux protéines puissent être des substrats de IKK, l'inhibition de ce dernier empêche la dégradation d'I[kappa]B[alpha], tandis que le niveau de phosphorylation du RelA est essentiellement inchangé. Nous apportons enfin une preuve que des isoformes de IKK nucléaires s'associent à la chromatine suivant l'activation des neutrophiles, ce qui suggère un rôle potentiel dans la régulation de gènes. Deuxièmement, nous rapportons que les neutrophiles expriment la MAP3K, TAK1, ainsi que ses partenaires associés, TAB1/2, dans le cytoplasme et le noyau. La kinase TAK1 est associée de façon constitutive aux protéines TAB1 et TAB2, ainsi qu'au complexe IKK[alpha]/[bêta] dans les neutrophiles au repos. Le niveau d'interaction de ces complexes demeure inchangé suite au traitement des neutrophiles avec le TNF-[alpha] ou le LPS. La kinase TAK1 devient rapidement et transitoirement activée suite à une stimulation des cellules avec le TNF-[alpha] ou le LPS. L'inhibition de l'activité kinase de TAK1 avec un inhibiteur hautement sélectif (5z-7-oxozeaenol) a empêché la phosphorylation d'IKK[alpha]/[bêta], de RelA, et la dégradation de I[kappa]B[alpha] dans les fractions cytoplasmiques et nucléaires, ainsi que la liaison à l'ADN du NF-[kappa]B dans des neutrophiles activés.En conséquence, l'expression et la sécrétion de cytokines inflammatoires induites par le TNF-[alpha] ou le LPS ont été profondément altérées suivant une inhibition de TAK1.En revanche, la phosphorylation de IKK[gamma] induite par le LPS n'a pas été affectée par l'inhibition de TAK1. Finalement, nos résultats indiquent que l'activation du NF-[kappa]B et les réponses cellulaires dépendantes du NF-[kappa]B sont indépendantes des ROS endogènes dans les neutrophiles humains primaires ou dans la lignée promyélocytaire PLB-985, qui peut être différenciée en granulocytes et se comporte comme les neutrophiles. Parallèlement, nous avons optimisé les conditions de transfection des PLB-985 différenciées, ce qui nous a permis de montrer pour la première fois l'activation de promoteurs [kappa]B-dépendants chez des granulocytes humains. Ces travaux rendent par ailleurs possibles les études portant sur l'activation des promoteurs chez les granulocytes. Dans leur ensemble, ces observations démontrent l'importance du NF-[kappa]B dans la génération inductible de cytokines et chimiokines par les neutrophiles. Il s'agit de la première étude qui montre la présence et l'activation (phosphorylation) du complexe IKK et la phosphorylation des protéines NF-[kappa]B/Rel dans les neutrophiles humains. Plus important encore, nos résultats dévoilent un mode d'activation de la cascade de signalisation IKK/I[kappa]B/NF-[kappa]B dans le noyau de cellules primaires. Nos données établissent également le rôle central de TAK1 dans le contrôle de la cascade de signalisation IKK/I[kappa]B/NF-[kappa]B cytoplasmique et nucléaire dans les neutrophiles primaires humains, ce qui pourrait représenter une cible prometteuse pour une intervention thérapeutique considérant le rôle critique des neutrophiles dans plusieurs conditions inflammatoires.

BACKGROUND:Detection of B cell clonality is useful for assisting in the diagnosis of B cell lymphomas. Clonality assessment can be accomplished through evaluation of KAPPA and LAMBDA light chain expression. Currently, only slide based methods are available for the majority of patient biopsies and do not detect light chain protein or mRNA in many B-cell lymphomas. Herein we evaluated a new method, known as colorimetric in situ hybridization (CISH), with improved sensitivity and multiplexing capacity, for its usefulness in clonality detection in mature B cell malignancies.METHODS:The KAPPA and LAMBDA ISH was performed on a Ventana Benchmark XT utilizing two color chromogenetic detection. The probes comprised 2 haptenated riboprobes each approximately 500 base pairs long directed against the conserved regions of either KAPPA or LAMBDA mRNA. The dual colors consisted of silver deposition (black) for KAPPA light chain and a novel (pink) chromogen for LAMBDA light chain. Following optimization, CISH allowed visualization of mRNA in benign B cells in reactive tissues including germinal center, mantle zone, and post-germinal center cells. We then identified 79 cases of B cell lymphoma with formalin-fixed paraffin-embedded (FFPE) biopsies including: follicular (36 cases), mantle cell (6 cases), marginal zone (12 cases), lymphoplasmacytic (6 cases), small lymphocytic (4 cases), and diffuse large B cell (15 cases), which were selected on the basis of either prior flow cytometry or immunohistochemistry (IHC) results to serve as the predicate, "gold standard," comparator.RESULTS:39/79 (49.4%) cases were classified as KAPPA and 29/79 (36.7%) as LAMBDA light chain restricted / while 9/79 (11.3%) cases were classified as indeterminate. Of the 70 cases with KAPPA or LAMBDA light chain restricted CISH, 69/70 (98.6%) were concordant with the reference method, while 1/70 (1.4%) was discordant.CONCLUSIONS:Optimized CISH detected lower levels of mRNA than can be visualized with current slide based methods, making clonality assessment in FFPE biopsies possible for mature B cell neoplasms. In this preliminary study, CISH was highly accurate compared to flow cytometry or IHC. CISH offers the possibility of wider applicability of light chain ISH and is likely to become a useful diagnostic tool.Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1430491067123856

Atherosclerosis is a chronic inflammatory disease of arterial blood vessels, characterized by deposition of lipoproteins in the arterial wall. Atherosclerotic plaques form preferentially in distinct regions of the vasculature such as branch points, curvatures and bifurcations, suggesting that local hemodynamic forces may contribute to disease susceptibility. Shear stress imparted on endothelial cells (ECs) by the flowing blood has been shown to modulate gene expression and remodelling of the artery. In this thesis, an in vitro model was established to recreate the contrasting environments found in atherosclerosis-prone and atherosclerosis-resistant regions of the vasculature to demonstrate a direct causal-relationship between shear stress and expression of endothelial nitric oxide synthase (eNOS) and p65 in ECs. In vitro assessment of cell shape and expression patterns of these anti- and atherogenic genes demonstrated that shear stress can induce cell morphology and gene expression patterns that are similar to ECs in atherosclerosis-prone and atherosclerosis-resistant regions of the mouse vasculature. Regulation of eNOS transcription by shear stress was demonstrated using a transgenic mouse model and in vitro heterogeneous nuclear RNA (hnRNA) quantification. Similar to ECs in atherosclerosis-prone regions, epithelial cells lining the small intestine lumen express high levels of p65. To investigate the effects of p65 expression levels on constitutive and tumour necrosis factor α (TNFα)-induced nuclear factor-κB (NF-κB) signalling, p65 expression was suppressed in HeLa cells by RNA interference. Lower p65 expression resulted in reduced TNFα-induced expression of NF-κB target genes, including many subunits of inhibitor of nuclear factor κB (IκB), demonstrating modulation of NF-κB priming by p65 expression levels. Suppression of p65 also affected constitutive expression levels of IκB, and resulted in re-setting of the NF-κB/IκB equilibrium. Experiments using inhibitors of canonical NF-κB signalling found that basal expression of NF-κB components is independent of nuclear factor κB kinase β (IKKβ) activity and proteasome-mediated degradation of IκBα. Together, these studies elucidate the mechanism of flow-mediated gene regulation and the effect of resulting changes in p65 expression on NF-κB signalling.

Les neutrophiles représentent une composante fondamentale du système immunitaire. En effet, il est bien connu qu'ils sont essentiels à la défense de l'organisme contre les agressions microbiennes. En plus de leurs fonctions anti-microbiennes classiques, les neutrophiles, via la sécrétion de cytokines (incluant les chimiokines), jouent un rôle prépondérant dans l'initiation et la modulation de la réponse immunitaire. Lors d'une infection ou d'une blessure, les neutrophiles agissent à titre de premier répondant et migrent rapidement au site de l'aggression. La production rapide de cytokines par les neutrophiles permet le recrutement additionnel de neutrophiles au site inflammatoire et celui d'autres populations leucocytaires, tout en modulant les fonctions de ces dernières. La génération de cytokines par les neutrophiles est induite par une vaste gamme de stimuli: agonistes d'origine microbienne, facteurs chimiotactiques, cytokines, chimiokines et facteurs de croissance. Ces signaux inflammatoires induisent l'activation de nombreuses voies de signalisation menant ultimement à l'activation de facteurs transcriptionnels. L'expression des gènes de cytokines inflammatoires doit être finement régulée afin qu'il y ait une production adéquate de ces médiateurs. Elle repose, en grande partie, sur le recrutement de facteurs transcriptionnels capables de se lier à des séquencescibles sur les promoteurs de ces gènes.Les travaux présentés dans cette thèse portent sur l'identification des facteurs de transcription impliqués, ainsi que des sentiers de signalisation en amont, dans l'expression de gènes encodant des cytokines chez le neutrophile humain. Dans le premier chapitre de la section résultats, nous avons montré que l'expression génique des cytokines dépend fortement de l'activation du facteur de transcription NF-[kappa]B. Celui-ci contrôle l'expression inductible de tous les gènes étudiés. De plus, nous avons montré que la génération de ces cytokines chez les neutrophiles dépend fortement de l'activation des voies p38 MAP kinases (aux niveaux transcriptionnel et traductionnel) et MEK/ERK (au niveau traductionnel seulement). Dans le deuxième chapitre, nous avons étudié l'expression et la localisation des différents facteurs de transcription de la famille C/EBP, de même que l'activation de C/EBP[bêta] via sa phosphorylation en Thr 235 chez les neutrophiles humains. Grâce à la surexpression d'un répresseur des C/EBP (A-C/EBP2N3T) chez les cellules PLB-985 granulocytiques, nous avons montré l'importance de la famille de C/EBP dans la production de MIP-1[alpha], de MIP-1[bêta] et d'IL-8 chez les neutrophiles humains.Les facteurs C/EBP ne semblent toutefois pas impliqués dans la production de TNF-[alpha] par ces cellules. La surexpression d'un mutant de C/EBP[bêta] (C/EBP[bêta] T235A) chez ces mêmes cellules nous a par ailleurs permis de déterminer que la phosphorylation de cet acide aminé est essentielle à la production des chimiokines MIP-1[alpha], MIP-1[bêta] et IL-8. Nous démontrons enfin une association constitutive de C/EBP[bêta] et de C/EBP[epsilon] avec le promoteur de l'IL-8 chez les neutrophiles primaires humains. Un des éléments déclencheurs de la transcription de ce gène serait probablement l'induction de la phosphorylation de C/EBP[bêta] sur les promoteurs géniques. Enfin, dans le troisième chapitre de la section résultats, nous explorons le rôle de la MAPK p38 dans la production de cytokines chez les neutrophiles. Nous y identifions quelques-uns de ses substrats impliqués dans la transcription et la traduction de cytokines inflammatoires. Ainsi, nous avons determiné que la MAPK p38 participe à l'activation des facteurs de transcription C/EBP[bêta] et CREB, probablement via l'activation des kinases MSK-1 et RSK. Nous avons également établi l'impact de la phosphorylation de la Ser 133 de CREB sur la production de cytokines chez les neutrophiles humains. Finalement, nous avons documenté l'activation de la kinase MNK1 par la MAPK p38, et son rôle dans la régulation traductionnelle des cytokines. L'ensemble de ces travaux a permis d'identifier des molécules-clés dans la génération de cytokines, plus particulièrement de chimiokines, chez le neutrophile humain. Nos travaux ont donc contribué de manière substantielle à nos connaissances des mécanismes transcriptionnels et traductionnels gouvernant l'expression de gènes précoces chez le neutrophile. Nos résultats permettront en outre l'identification de cibles thérapeutiques potentielles qui pourraient servir à atténuer l'inflammation chronique observée dans de nombreuses pathologies où les neutrophiles prédominent.