A minoria dos pacientes em processo de remodelamento pulmonar por bronquiolite obliterante (BO) responde a corticosteróides. Nos propusemos assim, a avaliar a importância da tolerância gerada pela imunização via nasal pelo colágeno tipo V (ctV) como uma opção no tratamento da BO. Através da análise morfométrica, mensuramos as dimensões, a densidade de colágeno e infiltrado celular no eixo pré-acinar visando o entendimento na patogênese da BO. Aplicamos essa metodologia a três protocolos: primeiro, o estabelecimento do modelo da BO em camundongos BALB/c. Segundo, a tolerância preventiva a BO. Terceiro, comparar os tratamentos prednisona vs tolerância após a BO já estabelecida. Oito semanas após uma única instilação de HNO3-nasal, as mudanças pulmonares foram caracterizadas pela distorção do lúmen, perda da barreira epitelial, redução ou total obliteração do lúmen do bronquíolo terminal e aumento do espessamento da parede. Modelo da BO: A densidade do infiltrado celular total mostrou valores significantes quando comparados os pulmões BO vs salina e controle (P = 0,001 para ambos), com maior evidência a densidade macrófagos nos pulmões controle vs BO e salina (P = 0,01 e P = 0,04, respectivamente). Tolerância preventiva: A densidade de CD3+ mostrou diminuição significante quando comparado ao estágio intermediário da doença nos pulmões BO vs BO+ctV e controle (P = 0,001 e P = 0,002, respectivamente). Houve também diminuição estatística da densidade de células CD20+ quando comparados os pulmões BO vs ctv+BO, BO+ctV, e controle (P = 0,008, P = 0,004 e P = 0,001). Prednisona vs tolerância: A densidade total de células diminuiu drasticamente quando comparados os pulmões BO vs BO+ctV e controle (P = 0,003 e P = 0,001, respectivamente). As células CD3+ mostraram diminuição quando comparados os pulmões BO vs BO+pr, BO+ctV e controle (P = 0,03, P = 0,03 e P = 0,05, respectivamente). Houve também diminuição das células CD20+ e CD4+ quando comparados os pulmões BO vs BO+ctV e controle (P = 0, 006 e P = 0,004, respectivamente) e (P = 0,001 para ambos). A histoarquitetura e a densidade de células dos pulmões BO+ctV se assemelharam ao pulmões controle. A tolerância pelo ctV comparada com o prednisona, mostrou marcante diminuição da deposição de colágeno e infiltrado celular no eixo pré-acinar. Nossos resultados indicam que o ctV pode atuar como um supressor da resposta imune. Concluímos, portanto, que a morfometria foi um método apropriado para relatar o espectro de mudanças histológicas no modelo de BO proposto. / A minority of patients with remodeling process of lungs following bronquiolite obliterante (BO) responds to corticosteroids. So, we sought to validate the importance of type V collagen (tVc) tolerance from immunization as option in BO model treatment. Througt of morphometric analyses, we have mensured for the dimensions, the collagen and cell infiltration density on pre-acinar axis, target the understand of BO pathogenesis. We applied for tree protocols: First, the establishment of BO model on BALB/c mice. Second, preventive tolerance to BO. Third, prednisone treatment vs tolerance, after BO established. Eight weeks after a single HNO3- nasal instillation, lung changes were characterized by lumen distortion, epithelial layer folding, reduction or total obliteration of terminal bronchiole lumen, and wall thickness increase. The morphological changes coincide with the measurement difference in the study for the tree protocols established. BO model: The total density of immune cells showed stasticaly significance when was compared BO vs saline and control lungs (P = 0.001 for both), more evidence to macrophage on control vs BO and saline lungs (P = 0.01 and P = 0.04, respectevely). Preventive tolerance: The CD3+ density showed a decreased statiscaly significance in lower BO vs BO+tVC and control lungs (P = 0.001 and P = 0.002, respectevely). Also the CD20+ density was decreased when was compared BO vs tVc+BO, BO+tVc and control (P = 0.008, P = 0.004 and P = 0.001). Prednisone vs tolerance: The total density of immune cells was decreased drastically when was compared BO vs BO+tVc and control lungs (P = 0.003 and P = 0.001, respetevely). These cells were CD3+ when was compared BO vs BO+pr, BO+tVc and control lungs (P = 0.03, P = 0.03 and P = 0.05, respectevely); Also CD20+ cells and CD4+ were decreased when was compared BO vs BO+tVc and conmtrol lungs (P = 0.006 and P = 0.004, respectevely) and (P = 0.001, for both). The histoarchiteture from BO+tVc and immune cells as simmilar to control lungs. The tolerance with tVc when was compared to prednisona, showed us a decreased of collagen and immune cell density in pre-acinar axis. Our results indicating that tVc may acts as a supressor in inflammatory process in bronchiolar remodeling. We conclued that method morphometric was effective for related us the spectrum of histologic changes in BO model propose. Ours results indicating that induction of tVc acts in suppression of the immune response. We conclude that morphometric analise was a aproprieated for related the spectrum of histologic changes for propose BO model.

Introdução: As células-tronco são células indiferenciadas que apresentam capacidade de auto renovação, ou seja, são capazes de se multiplicar, mantendo seu estado indiferenciado, o que proporciona uma reposição ativa de sua população de maneira constante nos tecidos; e, possui também, a capacidade de se diferenciar em diversos tipos celulares. Desta forma, acredita-se que células tronco presentes nos diferentes tecidos tenham papel regenerativo quando estes sofrem uma lesão ou injúria. Atualmente tem aumentado o número de estudos que envolvem a utilização de células tronco mesenquimais de tecido adiposo (ADSCs) na medicina regenerativa e curativa, estando o avanço desta terapia ligado à identificação de mecanismos e de moléculas que controlam e mediam a diferenciação de uma linhagem específica. Entre estas moléculas está o colágeno, proteína estrutural responsável pelas propriedades mecânicas, forma e organização tecidual. Dentre os 28 tipos de colágeno, o tipo V (Col V) e o XI são considerados nucleadores da fibrilogênese e modulam a adesão e proliferação celular. Além disso, o Col V é altamente expresso em tecido embrionário, sugerindo que ele possa atuar na interação célula-matriz no remodelamento e reparo de tecidos lesados, como a cartilagem. Dentre as doenças com acometimento articular, a osteoartrite (OA) é considerada a artropatia mais comum e, ainda, sem tratamento eficaz, culminando, em casos avançados, em intervenção cirúrgica. Estudos recentes mostram que as ADSCs seriam uma alternativa no restabelecimento do tecido cartilaginoso. Por esta razão a proposta do estudo foi avaliar a resposta das ADSCs, frente ao estímulo com Col V in vitro, e se o transplante autólogo dessas células poderia apresentar um efeito na regeneração da cartilagem articular na OA induzida em coelhos. Métodos: Foram cultivadas ADSCs, derivadas do tecido adiposo de coelhos (CEUA 123/14), estimuladas com Col V, para a avaliação da síntese dos principais componentes da matriz cartilaginosa: proteoglicanos e colágeno II. A preservação do fenótipo celular frente ao estímulo com Col V foi avaliada através da expressão do colágeno dos tipos I, II, III, CD34 e vimentina e dos genes COL2A1, ACAN e POU5F1. Os animais (n=24) foram submetidos à indução de OA, por meniscectomia parcial, e divididos nos grupos: OA (n=8), sem tratamento; OA/ADSCs (n=8), tratado com injeções mensais de ADSCs, e OA/ADSCs/V (n=8), tratado com injeções mensais de ADSCs, estimuladas previamente com Col V. As articulações foram coletadas após 22 semanas, descalcificadas e coradas com Hematoxilina e Eosina para histomorfometria da celularidade e espessura da cartilagem, perda de proteoglicanos pela Safranina O/Fast green e imunomarcação para colágeno II e Fas-L, usando o software Image Pro-Plus 6.0®. Resultados: As células estimuladas com Col V apresentaram-se negativas para colágeno I, III e CD34 e positivas para vimentina, colágeno total e proteoglicanos in vitro. Ainda, foi obtido um aumento significativo da expressão de colágeno II e dos genes COL2A1 e ACAN e a expressão de POU5F1 não foi significativa após estímulo. A análise morfológica da cartilagem indicou aumento da quantidade de condrócitos, da espessura da cartilagem e diminuição da perda de proteoglicanos nos grupos OA/ADSCs/V e OA/ADSCs, em relação ao grupo OA. Também foi observado um aumento da quantidade de colágeno II e diminuição de condrócitos apoptóticos no grupo OA/ADSCs/V. Conclusão: O Col V atua como mediador da condrogênese in vitro, estimulando colágeno II e proteoglicanos, além de aumentar a expressão dos genes COL2A1 e ACAN. A terapia com ADSCs estimuladas com Col V atenuou significativamente o processo osteoartrítico em coelhos, sugerindo uma nova perspectiva para o tratamento da OA / Introduction: Stem cells are undifferentiated cells that are capable of self-renewal, that is, they are capable of multiplying maintaining their undifferentiated state, which provides an active replacement of their population in a constant way in the tissues; stem cells also have the ability to differentiate into several cell types. Thus, it is believed that stem cells present in tissues have a regenerative role when they suffer an injury. The number of studies involving the use of adipose-derived stem cells (ADSCs) in regenerative medicine has increased and the progress of this therapy is link to the identification of mechanisms and molecules that control and mediate the specific lineage\'s differentiation. Collagen is among these molecules and it is a structural protein responsible for the mechanical properties, shape and tissue organization. Among 28 types of collagen, type V (Col V) and XI are considered nucleators of fibrillogenesis and they modulate cell adhesion and cell proliferation. In addition, Col V is highly expressed in embryonic tissue, suggesting that it may act on cell-matrix interaction in remodeling and repair of damaged tissues such as cartilage. Osteoarthritis (OA) is the most common arthropathy among the diseases with joint involvement and, it has no effective treatment that results in surgical intervention in advanced cases. Recent studies show that ADSCs would be an alternative in restoring cartilaginous tissue. Therefore, the aim of this study was to evaluate the response of ADSCs to the Col V stimulus in vitro and the effect of these autologous cells on the regeneration of articular cartilage of rabbits with OA. Methods: ADSCs from rabbit (CEUA 123/14) were cultured with Col V to evaluate the synthesis of the main components of the cartilaginous tissue as proteoglycans, collagen type II. The preservation of the cellular phenotype was evaluated through the collagen I, II, III, CD34 and vimentin expression and COL2A1, ACAN and POU5F1 genes. Rabbits (n=24) were submitted to OA induction though partial meniscectomy and divided into the following groups: OA (n=8), without treatment; OA/ADSCs (n=8), treated with monthly injections of ADSCs and OA/ADSCs/V (n=8), monthly injections of ADSCs previously treated with Col V. Joints were collected after 22 weeks, decalcified and stained with H&E for cellular histomorphometry and cartilage thickness. Safranin O/fast green staining was used for proteoglycan evaluation and immunostaining for collagen type II and Fas-L expression using Image Pro Plus 6.0 software. Results: ADSCs stimulated with Col V were negative for collagen I, III and CD34 and positive for vimentin, total collagen and proteoglycans in vitro. Furthermore, a significant increase in the expression of collagen II, COL2A1 and, ACAN genes was obtained, but the POU5F1 gene expression was not significant after stimulation. Morphological analysis of cartilage indicated increased in the number of chondrocytes, cartilage thickness, and decrease in loss of proteoglycans in the OA/ADSCs/V and OA/ADSCs groups, compared to the OA group. In addition, an increase in the amount of collagen II and decrease of apoptotic chondrocytes in the OA/ADSCs/V group was observed. Conclusion: Col V acts as a mediator of chondrogenesis in vitro stimulating collagen II, proteoglycans and COL2A1, ACAN genes expression. Therapy with Col V-stimulated ADSCs significantly attenuate the osteoarthritic process in rabbits, suggesting a new perspective for the treatment of OA

<p>Einstein's theory of general relativity is a cornerstone in the process of gaining increased understanding about problems of gravitational nature. It can be applied to problems on the huge length scales of cosmology and as far as we know it does not break down before the Planck scale is approached. Irrespective of scale, a perturbative approach is often a very useful way to reduce the Einstein system to manageable complexity and size.</p><p>The projects included in this thesis can be divided into three subcategories. In the first category the keyword is photon-photon scattering. General relativity predicts that scattering can take place on a flat background due to the curvature of space-time caused by the photons themselves. The coupling equations and cross-section are found and a comparison with the corresponding quantum field theoretical results is done to leading order. Moreover, photon-photon scattering due to exchange of virtual electron-positron pairs is considered as an effective field theory in terms of the Heisenberg-Euler Lagrangian resulting in a possible setup for experimental detection of this phenomenon using microwave cavities. The second category of projects is related to cosmology. Here linear perturbations around a flat FRW universe with a cosmological constant are considered and the corresponding temperature variations of the cosmic microwave background radiation are found. Furthermore, cosmological models of Bianchi type V are investigated using a method based on the invariant scheme for classification of metrics by Karlhede. The final category is slowly rotating stars. Here the problem of matching a perfect fluid interior of Petrov type D to an exterior axisymmetric vacuum solution is treated perturbatively up to second order in the rotational parameter.</p>

Einstein's theory of general relativity is a cornerstone in the process of gaining increased understanding about problems of gravitational nature. It can be applied to problems on the huge length scales of cosmology and as far as we know it does not break down before the Planck scale is approached. Irrespective of scale, a perturbative approach is often a very useful way to reduce the Einstein system to manageable complexity and size. The projects included in this thesis can be divided into three subcategories. In the first category the keyword is photon-photon scattering. General relativity predicts that scattering can take place on a flat background due to the curvature of space-time caused by the photons themselves. The coupling equations and cross-section are found and a comparison with the corresponding quantum field theoretical results is done to leading order. Moreover, photon-photon scattering due to exchange of virtual electron-positron pairs is considered as an effective field theory in terms of the Heisenberg-Euler Lagrangian resulting in a possible setup for experimental detection of this phenomenon using microwave cavities. The second category of projects is related to cosmology. Here linear perturbations around a flat FRW universe with a cosmological constant are considered and the corresponding temperature variations of the cosmic microwave background radiation are found. Furthermore, cosmological models of Bianchi type V are investigated using a method based on the invariant scheme for classification of metrics by Karlhede. The final category is slowly rotating stars. Here the problem of matching a perfect fluid interior of Petrov type D to an exterior axisymmetric vacuum solution is treated perturbatively up to second order in the rotational parameter.

A minoria dos pacientes em processo de remodelamento pulmonar por bronquiolite obliterante (BO) responde a corticosteróides. Nos propusemos assim, a avaliar a importância da tolerância gerada pela imunização via nasal pelo colágeno tipo V (ctV) como uma opção no tratamento da BO. Através da análise morfométrica, mensuramos as dimensões, a densidade de colágeno e infiltrado celular no eixo pré-acinar visando o entendimento na patogênese da BO. Aplicamos essa metodologia a três protocolos: primeiro, o estabelecimento do modelo da BO em camundongos BALB/c. Segundo, a tolerância preventiva a BO. Terceiro, comparar os tratamentos prednisona vs tolerância após a BO já estabelecida. Oito semanas após uma única instilação de HNO3-nasal, as mudanças pulmonares foram caracterizadas pela distorção do lúmen, perda da barreira epitelial, redução ou total obliteração do lúmen do bronquíolo terminal e aumento do espessamento da parede. Modelo da BO: A densidade do infiltrado celular total mostrou valores significantes quando comparados os pulmões BO vs salina e controle (P = 0,001 para ambos), com maior evidência a densidade macrófagos nos pulmões controle vs BO e salina (P = 0,01 e P = 0,04, respectivamente). Tolerância preventiva: A densidade de CD3+ mostrou diminuição significante quando comparado ao estágio intermediário da doença nos pulmões BO vs BO+ctV e controle (P = 0,001 e P = 0,002, respectivamente). Houve também diminuição estatística da densidade de células CD20+ quando comparados os pulmões BO vs ctv+BO, BO+ctV, e controle (P = 0,008, P = 0,004 e P = 0,001). Prednisona vs tolerância: A densidade total de células diminuiu drasticamente quando comparados os pulmões BO vs BO+ctV e controle (P = 0,003 e P = 0,001, respectivamente). As células CD3+ mostraram diminuição quando comparados os pulmões BO vs BO+pr, BO+ctV e controle (P = 0,03, P = 0,03 e P = 0,05, respectivamente). Houve também diminuição das células CD20+ e CD4+ quando comparados os pulmões BO vs BO+ctV e controle (P = 0, 006 e P = 0,004, respectivamente) e (P = 0,001 para ambos). A histoarquitetura e a densidade de células dos pulmões BO+ctV se assemelharam ao pulmões controle. A tolerância pelo ctV comparada com o prednisona, mostrou marcante diminuição da deposição de colágeno e infiltrado celular no eixo pré-acinar. Nossos resultados indicam que o ctV pode atuar como um supressor da resposta imune. Concluímos, portanto, que a morfometria foi um método apropriado para relatar o espectro de mudanças histológicas no modelo de BO proposto. / A minority of patients with remodeling process of lungs following bronquiolite obliterante (BO) responds to corticosteroids. So, we sought to validate the importance of type V collagen (tVc) tolerance from immunization as option in BO model treatment. Througt of morphometric analyses, we have mensured for the dimensions, the collagen and cell infiltration density on pre-acinar axis, target the understand of BO pathogenesis. We applied for tree protocols: First, the establishment of BO model on BALB/c mice. Second, preventive tolerance to BO. Third, prednisone treatment vs tolerance, after BO established. Eight weeks after a single HNO3- nasal instillation, lung changes were characterized by lumen distortion, epithelial layer folding, reduction or total obliteration of terminal bronchiole lumen, and wall thickness increase. The morphological changes coincide with the measurement difference in the study for the tree protocols established. BO model: The total density of immune cells showed stasticaly significance when was compared BO vs saline and control lungs (P = 0.001 for both), more evidence to macrophage on control vs BO and saline lungs (P = 0.01 and P = 0.04, respectevely). Preventive tolerance: The CD3+ density showed a decreased statiscaly significance in lower BO vs BO+tVC and control lungs (P = 0.001 and P = 0.002, respectevely). Also the CD20+ density was decreased when was compared BO vs tVc+BO, BO+tVc and control (P = 0.008, P = 0.004 and P = 0.001). Prednisone vs tolerance: The total density of immune cells was decreased drastically when was compared BO vs BO+tVc and control lungs (P = 0.003 and P = 0.001, respetevely). These cells were CD3+ when was compared BO vs BO+pr, BO+tVc and control lungs (P = 0.03, P = 0.03 and P = 0.05, respectevely); Also CD20+ cells and CD4+ were decreased when was compared BO vs BO+tVc and conmtrol lungs (P = 0.006 and P = 0.004, respectevely) and (P = 0.001, for both). The histoarchiteture from BO+tVc and immune cells as simmilar to control lungs. The tolerance with tVc when was compared to prednisona, showed us a decreased of collagen and immune cell density in pre-acinar axis. Our results indicating that tVc may acts as a supressor in inflammatory process in bronchiolar remodeling. We conclued that method morphometric was effective for related us the spectrum of histologic changes in BO model propose. Ours results indicating that induction of tVc acts in suppression of the immune response. We conclude that morphometric analise was a aproprieated for related the spectrum of histologic changes for propose BO model.

Orientador: Mário Tyago Murakami / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T10:58:02Z (GMT). No. of bitstreams: 1 Polo_CarlaCristina_M.pdf: 4286137 bytes, checksum: cf12a2b67d90255468d1906d719759a3 (MD5) Previous issue date: 2012 / Resumo: As miosinas pertencem a uma família de proteínas motoras que através da hidrólise de ATP são capazes de se movimentarem pelas fibras de actina. Sua estrutura é dividida em três domínios principais: motor, responsável pela hidrólise do ATP; regulador, envolvido na ligação de cadeias leves de calmodulina e a cauda, que tem papel essencial na mediação de interações com cargas celulares, como organelas, ácidos nucleicos e outras proteínas. O organismo modelo para insetos, Drosophila melanogaster, possui 11 miosinas pertencentes a 8 classes sendo que informações funcionais e bioquímicas são escassas, e estruturais, inexistentes. Apesar dos grandes avanços obtidos nos estudos de miosinas humanas, as miosinas de inseto ainda são pouco caracterizadas não havendo estudos comparativos, como por exemplo, para a determinação de conservação de parceiros moleculares entre os diferentes filos da classificação de Lineu. Neste contexto, foram selecionados os seguintes domínios de inseto para caracterização molecular: quinase da miosina III, cauda globular da miosina V e FERM da miosina XV. A partir do cDNA da pupa e adulto do inseto foram amplificados os fragmentos de genes de interesse e clonados em vetor de clonagem, pGEM T-easy, e posteriormente de expressão, pET28a e pET28aSUMO. Após testes em diversas condições no sistema bacteriano, as construções cauda globular da miosina V (GLOB_2 e GT-f) e domínio FERM-f da miosina XV foram obtidos na fração solúvel e com rendimento suficiente para estudos estruturais. O protocolo de expressão em larga escala assim como de purificação foram estabelecidos para cada uma dessas construções. Estudos biofísicos por aSEC, DLS, CD e SAXS foram realizados para a proteína GT-f da miosina V, mostrando a proteína monomérica com alto conteúdo de hélices-alfa corroborando as análises in silico e com as caudas globulares de miosinas ortólogas já descritas. Além disso, sua termoestabilidade foi avaliada com Tm de 46 ºC e que sua estabilidade é alterada pela adição de alguns ligantes, mas não pela força iônica como verificado para a cauda globular da miosina Va humana. A estrutura a baixa resolução da GT-f foi determinada pelos experimentos de SAXS, mostrou uma proteína monomérica com forma alongada (Dmax =100 Å). Para a construção FERM-f, apesar do sucesso alcançado no protocolo de expressão e purificação, a amostra permaneceu polidispersa e com uma estrutura random coil, inviabilizado futuros experimentos. Acredita-se que a ausência do domínio MyTH4, gerou a perda de estabilidade do domínio FERM-f e será necessária a clonagem dos dois domínios fusionados para garantir a estabilidade estrutural e funcional / Abstract: Myosins belong to a motor protein superfamily that uses the ATP hydrolysis to move along actin filaments. Its structural architecture comprises three main domains: motor, responsible for ATP hydrolysis; regulator, which binds calmodulin light chains; and tail, responsible for cellular cargoes binding. Drosophila melanogaster, an insect model, has 11 myosins belonging to 8 different myosin classes; however functional and biochemical data are scanty, and, structural, absent. Although the great advances on investigating human and yeast myosins with the elucidation of molecular partners and molecular mechanisms involved in signaling, there are no comparative studies among different phyla. Therefore, in order to gain insights into insect myosins, in silico analysis were performed and some domains like kinase (myosin III), globular tail (myosin V), and FERM (myosin XV) were selected for biophysical and structural studies. From pupa and adult cDNAs, the target genes were amplified and cloned into pGEM- T-easy, pET28a and pET28aSUMO vectors. After expressions tests, the myosin V globular tail constructs, GLOB_2 and GT-f, and myosin XV FERM-f were obtained in soluble fraction and in sufficient amounts for structural studies. aSEC, DLS, CD e SAXS biophysical studies were performed to myosin V GT-f, showing that the protein is monomeric with predominance of alpha-helix in accordance with the in silico atomic model. The stability of GT-f was assessed with a Tm of 46 ºC and some molecules are able to increase the stability, while the ionic strength has no difference like was observed for the human globular tail of myosin Va. The low resolution structure determined by SAXS experiments confirmed the monomeric state and its elongated molecular shape (Dmax = 100 Å). The FERM-f construct, although the purification and expression protocol were standardized, the sample remained polydisperse with a random coil structure, disabling other biophysical experiments. It is supposed that the absence of the MyTH4 domain in the FERM-f domain results in loss of stability, and cloning and expression of fused domains will be necessary to guarantee structural and functional stability / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Le Syndrome d’Ehlers-Danlos classique (EDSc) est une maladie génétique du tissu conjonctif lié, dans 90% des cas, à des mutations sur les gènes du collagène V (COLV). Elle se caractérise par une peau fine, vulnérable et hyper-extensible, des défauts de cicatrisation et une hypermobilité articulaire. Afin d’établir un lien entre le génotype et le tableau clinique des patients, j’ai étudié l’impact de mutations identifiées sur le N-propeptide de la chaine α1 de COLV, particulièrement la mutation S254L associée à un syndrome chevauchant EDSC/UCMD (Dystrophie Musculaire Congénitale d’Ullrich), sur la maturation de COLV, la production et l’organisation de la matrice extracellulaire (MEC), et sur l’intégrité et la fonction des fibroblastes du derme.J’ai ainsi montré que seule la mutation S254L abolit le clivage BMP-1 du N-propeptide, étape importante de la maturation de COLV. Le COLV muté est normalement sécrété dans la MEC qui va toutefois présenter une organisation anarchique des fibres de collagène et du réseau de collagène VI. À l’aide de tests de blessure sur couche cellulaire, j’ai observé que les fibroblastes EDSc (fb/EDSc) et S254L (fb/SL) ont un niveau de cicatrisation inférieur à celui observé chez les fibroblastes sains (fb/WT). Ce défaut de recouvrement est associé à des défauts de migration et à un plus faible niveau de prolifération. De plus, les fb/SL ont une surface d’adhésion inférieure à celle des fb/WT. J’ai également observé une augmentation du flux autophagique chez les fb/EDSc et fb/SL par rapport aux fb/WT. L’autophagie des fb/SL, contrairement au fb/EDSc, n’est pas associée à un stress du réticulum endoplasmique mais à la mitophagie, un mécanisme permettant la dégradation spécifique des mitochondries qui sont altérées chez les fb/SL.Mon étude portant sur les mutations du N-propeptide a ainsi permis de caractériser certains des processus biologiques associés à la pathogénèse de l’EDSc et ceux spécifiquement liée au syndrome chevauchant EDS/UCMD. / Classic Ehlers-Danlos syndrome is a heritable connective tissue disorder, characterized by fragile and hyperextensible skin, and joint hypermobility. 90% of patients harboured a mutation in the COL5A1 gene, which encode the pro α1(V) chain of the collagen V, that could assemble with the pro α2(V) chain to form the predominant isoform of collagen V, the heterotrimer α1(V) ₂ α2(V). This isoform is expressed in bones, skin and tendons and is able to control collagen fibrillogenesis. Here we described for the first time a mutation, localized in the BMP-1 cleavage site on the α1(V) chain in a patient exhibiting muscular contractures and weakness, an abnormal skin and problems with nerve conductions, which could remind an UCMD pathology, which is normally due to mutations in the collagen VI molecule. We first showed that this new mutation totally abolished the maturation of the pro α1(V) chain by the BMP-1 enzyme but didn’t affect the secretion of the collagen V in the ECM. Analysis of fibroblasts from this patient showed that the collagen V is secreted but that the collagen fibrils are not correctly organized in the ECM of the cells. Our analysis also revealed that fibroblasts migration is affected, compared to wild-type cells and that number, area and length of vinculin-containing focal adhesion are reduced. Interestingly, there is an increase of the autophagic flux in those cells, associated with mitophagy but without ER stress. These data highlight that a mutation in the COL5A1 gene could leads to a new pathology in which skin, muscle and nerves are affected.

Le syndrome de chylomicronémie multifactorielle (MCS) est une maladie complexe au cours de laquelle les valeurs de triglycérides (TG) dépassent 10 mmol/L. Le MCS se manifeste à l'âge adulte et a une prévalence d’environ 1 adulte sur 600 au Québec. Deux conditions doivent être réunies pour développer cette maladie : une composante génétique (oligogénique ou polygénique) ainsi que la présence de facteurs de risque reliés au style de vie (une alimentation riche en gras et en sucres raffinés, une consommation excessive d'alcool, un diabète non contrôlé ou l'obésité). Le MCS est une condition de santé grave, puisqu’il augmente considérablement le risque de pancréatites aigües et peut doubler le risque de maladies cardiovasculaires. Actuellement, il n’y a pas d’étude d’intervention nutritionnelle, réalisée dans cette population, qui permette de connaitre l’approche nutritionnelle la plus bénéfique. Ce mémoire présente les résultats d’une étude croisée randomisée dont l’objectif était d’évaluer l’impact d’une diète faible en lipides et d’une diète faible en glucides sur le profil lipidique à jeun et postprandial chez des patients atteints de MCS en fonction de la présence d’un variant rare à l’état hétérozygote du gène de la lipoprotéine lipase (LPL). Les résultats de cette étude suggèrent qu’une diète faible en lipides permettrait une diminution plus importante des TG chez les sujets porteurs d’un variant rare à l’état hétérozygote de la LPL et pourrait ultimement contribuer à réduire le risque de pancréatite aigüe sur le long terme. / Multifactorial chylomicronemia syndrome (MCS) is a complex disease in which triglyceride (TG) values exceed 10 mmol/L. MCS occurs in adulthood and has a prevalence of approximately 1 in 600 adults in Quebec. Two conditions must be met to develop this disease: a genetic component (oligogenic or polygenic) as well as the presence of lifestyle risk factors (a diet high in fat and refined sugars, excessive alcohol consumption, uncontrolled diabetes or obesity). MCS is a serious health condition, as it significantly increases the risk of acute pancreatitis and can double the risk of cardiovascular disease. Currently, there are no nutritional intervention studies conducted in this population to determine the most beneficial nutritional approach. This thesis presents the results of a randomized crossover study whose objective was to evaluate the impact of a low-fat diet and a low-carbohydrate diet on the fasting and postprandial lipid profile in patients with SCD according to the presence of a rare heterozygous lipoprotein lipase (LPL) gene variant. The results of this study suggest that a low-fat diet would result in a greater reduction in TGs in subjects with a rare heterozygous variant of LPL and may ultimately help reduce the risk of acute pancreatitis in the long term.

The D3 dopamine receptor (D3R) belongs to D2-class of dopamine receptors (DARs) and is involved in emotion, movement, and reward. D3R dysfunction has been reported in some neuropsychiatric disorders such as addiction, cognitive deficits, depression, schizophrenia, and Parkinson’s disease. Genetic studies have shown two polymorphic variants of the D3R gene resulting from substitution of serine to glycine at position nine of the amino terminus. Isoform 5 of adenylyl cyclase (AC5) is one of the nine transmembrane bound ACs in the brain and myocardium. Previous studies in rats have shown that AC5 is expressed in the striatum, nucleus accumbens and olfactory tubercle and at lower levels in islands of Calleja, where the D3R is also expressed. Previous studies showed that although D2R and D4R inhibit ACs activity in different cell types, inhibition of ACs by D3R is weak and often undetectable. It has been shown that D3R selectively inhibits AC5 activity in human embryonic kidney 293 (HEK293) cells co-transfected with D3R and AC5. Co-expression of D3R and AC5 in brain regions which are major coordinators of normal and pathological movement, and the selective inhibition of AC5 activity by D3R raise the possibility of a functional link between AC5 and D3R in the modulation of signal transduction and trafficking. I hypothesized that AC5 plays a unique role in modulation of D3R trafficking and signaling pathways through interaction between D3R and AC5. Herein, I demonstrated an interaction between D3R and AC5 in vivo and in vitro using reciprocal co-immunoprecipitation/immunoblotting (co-IP/IB) assays. Interestingly, DA may facilitate the formation of protein complex between D3R and AC5 in vitro. Radio ligand binding assays revealed that heterodimerization of D3R polymorphic variants with AC5 does not change ligand binding affinity and expression of the D3R. Furthermore, taking advantages of GloSensor assays, selective inhibition of AC5 activity by D3Ser9 and D3Gly9 has been shown following activation by DA and quinpirole. Using ELISA studies showed that AC5 promotes cell surface expression and total expression of D3Ser9 and D3Gly9. Moreover, ELISA results suggested that AC5 facilitates DA-induced D3Ser9 endocytosis in dynamin and β-arrestin 2 dependent process, while having no effect on D3Gly9 polymorphic variant. The results also revealed that AC5 attenuates heterologous (PKC-induced) internalization of D3Ser9, while it does not have any effect on D3Gly9 heterologous internalization. My results also displayed a complex formation between D3R, AC5 and, β-arrestin 2 under basal and DA stimulation conditions, which emphasize the role of β-arrestin 2 in D3R signal transduction. Overall, a new regulatory mechanism for D3R has been suggested. My results suggested that complex formation between both D3R polymorphic variants with AC5 can regulate signaling and trafficking properties of D3R without changing the binding affinity of the receptor. These data will be meaningful for understanding of diseases and developing treatment strategies.

Indiana University-Purdue University Indianapolis (IUPUI) / Rationale: Tissue remodeling and complement activation are asthma hallmarks. Type V collagen [col(V)], a cryptic antigen, becomes exposed during lung remodeling. IL-17 is key to anti-col(V) immunity, and regulates complement activation. We have reported that col(V)-induced tolerance down regulates IL-17 and prevents immune-mediated lung diseases. Objectives: Determine a role for anti-col(V) immunity in asthma. Methods: Serum anti-col(V) antibodies were measured in asthma patients, and immunohistochemistry utilized to detect interstitial col(V) in fatal asthma. Balb/c mice were tolerized with col(V) prior to sensitization with ovalbumin (OVA), and subsequent OVA intranasal challenge. Airway hyper-responsiveness (AHR) to methacholine was measured; and RT-PCR utilized to determine local Il17 transcripts. Bronchoalveolar lavage levels of C3a¸ C5a and OVA-specific IgE were measured; and immunohistochemistry utilized to detect expression of complement regulatory proteins, expression, CD46/Crry and CD55, in lung tissue. Results: Compared to normal subjects, anti-col(V) antibodies were increased in asthmatics; and interstitial col(V) was over expressed in fatal asthma. OVA-induced AHR up regulated anti-col(V) antibodies systemically, and increased OVA-specific IgE and C3a in BAL, and parenchymal Il17 transcripts. Col(V)-induced tolerance abrogated AHR, down regulated OVA-induced T cell proliferation, as well as total and OVA-specific IgE, C3a, IL-17 expression and tracheal smooth muscle contraction. Crry/CD46 and CD55, key to preventing complement activation, were down regulated on goblet cells in murine allergic airway disease. Conclusions: Anti-col(V) immunity correlates with asthma pathogenesis, and col(V)-induced tolerance may be a novel therapeutic for asthma. Decreased expression of Crry/CD46 and CD55 on goblet cells may in part account for complement activation in asthma.