Pythiosis is a disease caused by the oomycete Pythium insidiosum that affects horses, cattle, sheep, cats and humans, and occurs in tropical, subtropical and temperate regions. In horses the lesions are characterized by ulcerative eosinophilic granulomas. Most drugs are ineffective against this pathogen and immunotherapy has shown promise in the regression of the disease, even without the complete elucidation of the immune mechanisms involved. The purinergic system via adenine nucleotides (ATP and ADP) and its derivated nucleoside (adenosine), plays an important role in modulating the immune and inflammatory responses. Once released by cells, nucleotides interact with specific receptors and their extracellular concentrations are controlled by a group of ecto-enzymes, among which are ENTPDase (ecto-nucleoside triphosphate diphosphohydrolase) and ADA (adenosine deaminase). Taking into account the importance of immunotherapy in the treatment of pythiosis and a possible involvement of purinergic signaling in the immune response, the activities of E-NTPDase and E-ADA were evaluated in lymphocytes of rabbits with experimental pythiosis. For disease induction, zoospores were inoculated, subcutaneously, in the lateral region of the torax of each rabbit. These animals were evaluated weekly and the nodular area (cm2) developed was determined after 28 days of inoculation. Animals with experimental pythiosis, confirmed by enzyme immunoassay and the development of characteristic lesions, were treated with immunotherapy, whereas the ratio between the enzymatic activity in lymphopcytes and the consequent triggered immune response was investigated. Animals that did not develop lesions and those from the control group showed the same pattern of ectoenzymatic activity. E-NTPDase activity in lymphocytes of rabbits with experimental pythiosis was significantly higher (p<0,001) in relation to ATP hydrolysis (about 100%) and may be related to decreased (p<0,05) serum ATP (54,04%), when compared to the control group. After immunotherapeutic treatment, the activity of E-NTPDase showed similar values to those observed on the day of inoculation. Moreover, rabbits with experimental pythiosis showed a significant decrease (p<0,01) in the activity of E-ADA (82,36%), and this would lead to a significant increase in the extracellular concentration of adenosine (2,51 times), in relation to the control group (p<0,01). After immunotherapy, this group of animals showed a significant increase in the activity of E-ADA (78,62%) (p<0,01). Through this study, it can be observed that an increased activity of E-ADA, during pythiosis, leads to extracellular regulation of ATP and adenosine concentration. As a result, low levels of extracellular ATP could activate P2Y receptors, while high levels of extracellular adenosine may activate A2A and/or A2B receptors, triggering a TH2 response, responsible for the tissue damage generated by infection. After immunotherapy, it can be observed an inversion in the behaviour of the enzymatic activities, stimulating a TH1 response in the host. The switch from a TH2 response, responsible for the injuries that occur on pythiosis, to a TH1 response, is the most accepted hypothesis to explain the healing properties of immunotherapy. Thus, it is suggested the involvement of purinergic system in the pattern of immune response that occurs during pythiosis and after immunotherapy. / A pitiose é uma doença causada pelo oomiceto Pythium insidiosum que acomete equinos, bovinos, ovinos, felinos e seres humanos, e ocorre em regiões tropicais, subtropicais e temperadas. Em equinos as lesões são caracterizadas por granulomas eosinofílicos ulcerados. A maioria das drogas antifúngicas é ineficaz contra este patógeno e a imunoterapia tem se mostrado promissora na regressão da doença, mesmo sem a elucidação completa dos mecanismos imunes envolvidos. O sistema purinérgico, através dos nucleotídeos de adenina (ATP e ADP) e seu derivado nucleosídeo (adenosina), desempenha um importante papel na modulação da resposta imune e inflamatória. Uma vez liberados pelas células, os nucleotídeos interagem com receptores específicos e suas concentrações extracelulares são controladas por um grupo de ecto-enzimas, entre as quais estão a E-NTPDase (Ecto-Nucleosídeo Trifosfato Difosfoidrolase) e ADA (adenosina desaminase). Levando-se em conta a importância da imunoterapia no tratamento da pitiose e uma possível participação da sinalização purinérgica na resposta imune, foram avaliadas as atividades das ecto-enzimas NTPDase e ADA em linfócitos de coelhos com pitiose experimental. Para a indução da doença, zoósporos foram inoculados por via subcutânea, na região lateral do tórax, de cada coelho. Os coelhos inoculados foram avaliados semanalmente e a área nodular (cm2) desenvolvida foi determinada após 28 dias da inoculação. Os animais com pitiose experimental, confirmada por teste imunoenzimático e pelo desenvolvimento de lesão característica, foram tratados com imunoterápico, sendo que a relação entre as atividades enzimáticas nos linfócitos e o tipo de resposta imune foi investigada. Os animais que não desenvolveram lesão e os animais do grupo controle mostraram o mesmo padrão de atividade das ectoenzimas. A atividade da E-NTPDase nos linfócitos dos coelhos com pitiose experimental mostrou-se significativamente maior (p<0,001) em relação à hidrólise de ATP (em cerca de 100%), podendo estar relacionada à diminuição (p<0,05) da concentração sérica de ATP (54,04%), quando comparado ao grupo controle. Após o tratamento com o imunoterápico, a atividade da E-NTPDase apresentou valores semelhantes aqueles observados no dia da inoculação. Por outro lado, os coelhos com pitiose experimental apresentaram uma diminuição significativa (p<0,01) na atividade da E-ADA (82,36%), o que levaria a um importante aumento na concentração extracelular de adenosina (2,51 vezes) em relação ao grupo controle (p<0,01). Após a imunoterapia, este grupo de animais apresentou um aumento significativo na atividade da E-ADA (78,62%) (p<0,01). Através deste estudo, podese observar que a atividade aumentada da E-NTPDase e a atividade diminuída da E-ADA, durante a pitiose, levam à regulação da concentração do ATP e da adenosina no meio extracelular. Em conseqüência, baixos níveis extracelulares de ATP poderiam ativar receptores P2Y, enquanto, altos níveis de adenosina poderiam ativar receptores A2A e/ou A2B desencadeando uma resposta TH2, responsável pelos danos teciduais gerados na infecção. Com a imunoterapia, pode-se observar uma inversão no comportamento das atividades enzimáticas, estimulando uma resposta TH1 no hospedeiro. A mudança de uma resposta TH2, responsável pelas lesões que ocorrem na pitiose, para um padrão de resposta TH1, é hipótese mais aceita para explicar as propriedades curativas da imunoterapia. Dessa forma, sugere-se a participação do sistema purinérgico no padrão de resposta imune que ocorre durante a pitiose e após a imunoterapia.

Rodd är en sport som kräver hög kondition då de flesta av kroppens stora muskler används och kroppens alla tre energisystem är inblandade. Därmed har kroppen ett stort behov av energi vid rodd.    Roddmaskin är en utrustning som används av roddare under vintersäsongen för att utveckla prestationen och göra fysiologiska tester.    Syftet med projektet var att undersöka hur takt (drag per minut) och styrka (1RM marklyft) påverkade, sluttiden (s) vid 1000 m rodd på roddergometrar. Studien utfördes på 9 manliga deltagare över 18 år och sambanden studerades med linjär regressionsanalys. Hypotesen var att lägre takt leder till högre kraft i draget och högre takt leder till lägre kraft. Resultaten visade inget samband mellan takt, högre vikt i marklyft och sluttid där en lägre takt och högre medeleffekt gav en lägre sluttid, p och r2 värdet för sambandet mellan tid och takt var 0.109 för r2och p=0.385, p= 0.210 och r2 värdet 0.214 för sambandet mellan tid och marklyft, för sambandet mellan tid och medeleffekt var r2 värdet 0.75 och p = 0.0025, vilket tyder på ett starkt samband.    Det är troligen större fördel om man drar med större kraft på roddmaskin än om man skulle ro på vatten och resultaten är därför inte direkt överförbara till rodd på vatten. Vidare antyder resultaten att det kan vara en fördel om roddarna tränar mer styrka då mer styrka leder till kraftigare drag, vilket i sin tur leder till en lägre tid. / Rowing is a sport dependent on high endurance capacity, where most of the large muscles in the body are used, the three energy systems are involved and the body is in need of a large amount of energy. Concept 2 rowing ergometers is a sport of itself but is performed mostly during the winter season for outdoor rowers to see the performance and do physiological tests. The purpose of this project was to see if there is any correlation between stroke rate, average power, 1RM deadlift and finishing time on 1000 m rowing machine performance, for 9 adult male rowers. The correlation was studied with linear regression analysis.    The hypothesis was that lower rate leads to higher mean power in each stroke and higher stroke rate leads to lower power in each stroke.    The results showed no correlation between stroke rate, heavier weight lifted in deadlift and finishing time. Where a lower stroke rate and higher mean effect lead to a lower finishing time, p and r2 value for the correlation between time and stroke rate was 0.109 for r2 and p=0.385, p=0.210 and r2 value 0.214 for the correlation between time and deadlift, for the correlation between time and mean effect, the r2 value was 0.75 and p=0.0025, which indicate a strong correlation.        It is more benefit if one row with higher force on the rowing machine than rowing on the water. Further, the results show that it is an advantage if the rowers train more strength, because more power leads to a more powerful stroke and lower time.

Le transporteur mitochondrial de nucléotides adényliques (Ancp), localisé dans la membrane interne mitochondriale, catalyse l'échange ADP/ATP entre le cytoplasme et la matrice mitochondriale. Il lie deux classes d'inhibiteurs naturels avec une grande spécificité et une haute affinité. Ces deux types d'inhibiteurs, BA et CATR, stabilisent Ancp dans deux conformations distinctes impliquées dans le transport des nucléotides. La compréhension des changements conformationnels subits par Ancp est essentielle pour décrire précisément le mécanisme d'échange des nucléotides. La structure atomique du transporteur de bœuf a montré que les hélices transmembranaires 1, 3 et 5 sont coudées par la présence de prolines qui pourraient donc être impliquées dans les changements conformationnels associés au transport.Dans la première partie de ce manuscrit, ces prolines ont été mutées en alanine ou en leucine et les conséquences de ces mutations ont été étudiées au niveau de la cellule (phénotype, morphologie, contenu en protéines) et des mitochondries en examinant le transport lui-même ainsi que toutes les fonctions mitochondriales (respiration, contenu en protéines, importation des protéines, morphologie…). Il peut-être conclu de ces études que ces prolines jouent un rôle dans le transport mais également dans la biogenèse mitochondriale (import d'Ancp, équilibre fusion/fission mitochondriale).Dans la deuxième partie, ont été étudiées des mutations qui stabilisent Ancp dans la conformation BA ou CATR. L'objectif était d'obtenir des formes stables du transporteur représentant des états intermédiaires du transport pour en étudier la structure atomique par cristallographie. Les résultats préliminaires sont prometteurs. / The mitochondrial ADP/ATP carrier (Ancp), located in the inner mitochondrial membrane, catalyzes the ADP/ATP exchange between the cytoplasm and the mitochondrial membrane. Two classes of natural inhibitors can bind to the carrier with high specificity and affinity. These two families of inhibitors, BA and CATR, stabilize Ancp in two different conformations, which are involved in the nucleotide transport. Understanding the conformational changes undergone by Ancp is essential to describe precisely the nucleotide exchange mechanism. The atomic structure of the Beef Ancp unveiled kinks in transmembrane helices 1, 3 and 5 induced by the prolines, which therefore could be involved in the conformational changes associated with the nucleotide transport. In the first part of this manuscript, the three prolines were mutated into alanine or leucine and the results of these mutations were studied at the level of the cell (phenotype, morphology, protein content) and of the mitochondria by examining the transport itself and various mitochondria functions (respiration, protein content, protein import, morphology...). It can be concluded from these studies that these prolines play a key role in the transport but also in mitochondria biogenesis (Ancp import, mitochondrial fusion/fission balance).In the second part were studied mutations that stabilize Ancp in BA or CATR conformation. The goal was to obtain stable forms of Ancp that would correspond to intermediate steps of the transport to study their atomic structure by crystallography. The preliminary results are promising.

L'obésité est une maladie multifactorielle qui constitue un facteur de risque de nombreuses pathologies, notamment les maladies cardiovasculaires, le diabète et les maladies neurodégénératives. Des études épidémiologiques récentes suggèrent un effet obésogène des contaminants environnementaux, mais peu d'informations sont disponibles sur leur effet potentiel sur le métabolisme des lipoprotéines hépatiques. L'objectif de cette étude était de déterminer l'effet de polluants environnementaux de la famille des hydrocarbures aromatiques polycycliques (HAP) sur trois récepteurs des lipoprotéines, le récepteur des LDL (LDL-R), le lipolysis-stimulated lipoprotein receptor (LSR) et le scavenger receptor B1 (SR-B1) ainsi que sur les ATP binding cassette transporter A1 (ABCA1) et G1 (ABCG1) en utilisant des modèles cellulaires et/ou animaux. Les études par immunoblots et immunofluorescence in vitro ont révélé que l'exposition de cellules Hepa1-6 au B[a]P diminue de manière significative les taux de protéine LSR, LDL-R et ABCA1, alors qu’aucune modification significative du taux et de l’activité du LSR n’a été observée lors d’une exposition au pyrène ou au phénanthrène. L’analyse en temps réel par PCR et les études avec la lactacystine ont révélé que cet effet était dû principalement à une augmentation de la dégradation par le protéasome plutôt qu’à une diminution de la transcription ou à une augmentation de la dégradation lysosomale. En outre, les ligand-blots ont révélé que les lipoprotéines exposées au B[a]P avaient une affinité réduite pour le LSR ou le LDL-R. Des souris C57Bl/6RJ ont été traitées par le B[a]P (0,5 mg / kg, i.p) toutes les 48 h pendant 15 jours. Le gain de poids observé est accompagné d’une augmentation des taux de triglycérides et de cholestérol plasmatiques, du taux de cholestérol hépatique, et d’une diminution du taux de LDL-R et ABCA1 chez animaux traités au B[a]P par rapport aux témoins. Les corrélations observées entre les taux de LSR et de LDL-R hépatiques chez les souris contrôle ne sont plus observées chez les souris traitées au B[a]P, ce qui suggère un dérèglement du métabolisme des lipoprotéines hépatiques. Ces résultats suggèrent que la prise de poids induite par le B[a]P est peut-être liée à son action inhibitrice sur le LSR et le LDL-R, ainsi que sur l’ABCA1 et le métabolisme des lipoprotéines hépatiques, ce qui conduit à un statut lipidique modifié chez les souris traitées au B[a]P, donnant ainsi un nouvel éclairage sur les mécanismes sous-jacents de la contribution des polluants tels que le B[a]P à la perturbation de l'homéostasie lipidique, susceptible de contribuer à la dyslipidémie associée à l'obésité / Obesity is a multifactorial disorder that represents a significant risk factor for many pathologies including cardiovascular diseases, diabetes and neurodegenerative diseases. Recent epidemiological studies suggest potential obesogenic effects of environmental contaminants, but little information is available on their potential effect on hepatic lipoprotein metabolism. The objective of this study was to determine the effect of the common environmental pollutants, belonging to polycyclic aromatic hydrocarbon (HAP) on three lipoprotein receptors, the LDL-receptor (LDL-R), the scavenger receptor B1 (SRB1) and the lipolysis-stimulated lipoprotein receptor (LSR) as well as the ATP binding cassette transporters A1 (ABCA1) and G1 (ABCG1) using cell and/or animal models. Immunoblot and immunofluorescence in vitro studies revealed that exposure of Hepa1-6 to benzo[a]pyrene (B[a]P) significantly decreased LSR, LDL-R and ABCA1 protein levels, whereas no significant changes in protein levels and LSR activity where observed upon cell treatment with pyrene or phenanthrene. Real-time PCR analysis, lactacystin and chloroquine studies revealed that this effect was due primarily to increased proteasome-mediated degradation rather than to decreased transcription or to increased lysosomal degradation. Furthermore, ligand blots revealed that lipoproteins exposed to B[a]P displayed markedly decreased binding to LSR or LDL-R. C57Bl/6RJ mice were treated with B[a]P (0.5 mg/kg, i.p) every 48 h for 15 days. The increased weight gain observed was accompanied by increased plasma triglycerides and cholesterol levels, increased liver cholesterol content, and decreased LDL-R, ABCA1, ABCG1 and SR-B1 protein levels in B[a]P-treated animals as compared to controls. Correlations observed between hepatic LSR and LDL-R levels in control mice were no longer observed in B[a]P treated mice, suggesting a potential dysregulation of hepatic lipoprotein metabolism.Taken together, these results suggest that B[a]P-induced weight gain may be due its inhibitory action on LSR and LDL-R, as well as ABCA1 and lipoprotein metabolism in the liver, which leads to the modified lipid status in B[a]P-treated mice, thus providing new insight into mechanisms underlying the involvement of pollutants such as B[a]P in the disruption of lipid homeostasis, potentially contributing to dyslipidemia associated with obesity

Lors d'une infection virale, l'hôte déclenche une réponse rapide, la rémonse immunitaire innée, dont l'interféron (IFN) de type I est la cytokine centrale. Des motifs moléculaires associés aux micro-organismes (MAMP) sont déteectés par de nombreux récepteurs dédiés, dont les récepteurs cytoplasmiques de type RIG-I (RLR) identifiés à partir de 2004. Les RLR, au nombre de trois, RIG-I, MAD5 et LGP2, sont les ARN-hélicases composées de deux ou trois types de domaines : deux domaines CARD, resposables du recrutement de la cascade de signalisation, un domaine C-terminal CTD, site de liaison initial de l'ARN viral, et le domaine central hélicase, site secondaire de liaison àl'ARN et possédant également une activité enzymatiques ATP-dépendante. RIG-I est impliqué dans la détection de plusieurs virus dont ceux de l'ordre des mononegavirales (virus de la rage, de la rougeole, Ebola). Ce récepteur reconnait des ARN viraux possédant une région double brin adjacente à une extrémité 5'-triphosphate. Les nombreuses études menées n'ont cepnedant pas encore permis de dégager un mécanisme complet et cohérent de l'activation de RIG-I. Notre objectif était donc d'apporter des réponses molécualires quant au mécanismes d'activation de RIG-I. Dans un premier temps, l'élucidation de la structure de la protéine entière RIG-I de canard, par l'équipe de Stephen Cusack, leur a permis d'identifier une conformation auto-réprimée de la protéine. En l'absence d'ARN le domaine CARD2 interagit avec le sous domaine Hel2i du domaine hélicase. Nous avons apporté une preuve fonctionelle de cette observation. Les mutations F540 A/D, du résidu situé dans le sous domaine Hel2i, inhibent l'interaction CARD2-Hel2i et produisent des mutant constituvement actifs. A l'opposé, les mutations correspondantes dans le domaine CARD2 rendent RIG-I inactif. L'interaction CARD2-Hel2i semble donc impliquer une double auto-répression via (i) le masquage du site de liaison à l'ARN du sous-domaine Hel2i, et (ii) le masquage de résidus du domaine CARD2 impliqués dans le recrutement d'intermédiaires requis pour la transduction du signal. Par ailleurs, l'étude de mutants impliqués dans la liaison et l'hydrolyse de l'ATP nous a permis de proposer un nouveau rôle régulateur pour cette activité enzymatique. Dans un deuxième temsp, l'étude de la nécessité de l'oligomérisation de RIG-I pour l'activation de la réponse IFN a été menée. Eva Kowalinski, en thèse dans l'équipe de Stephen Cusack, n'observe la formation de dimères de RIG-I, in vitro, qu'en présence d'un ARN synthétique possédant deux extrémités 5'-triphosphate. Nous avons complété cette observation avec des analyse montrant que des ARN synthéttiques leader incapables d'induire la dimérisation de RIG-I in viro, activent néanmoins ce récepteur in cellula. Par ailleurs, nim'utilisation de la technique de co-immunoprécipitation, ni celle du test de complémentation basé sur la luciférase Gaussia, avec ou sans activiation par un ARN ou une infection virale, n'ont permis d'observer d'oligomérisation de RIG-I. L'auto-association de RIG-I ne semble donc pas être indispensable pour son activation. / Vertebrate are permanently threatened by infections that they manage to counteract using a dedicated system. The innate immunity allows a rapid response against viral infection, mainly through the type I interferon (IFN) production. Dedicated receptors detect microbe-associated molecular patterns (MAMPs), and among them the RIG-likereceptors (RLRs), RIG-I, MDA5 and LGP2, can sense viral RNA into the cytoplasm. RLRs are compossed of two or three different domains : two N-terminal CARDs domains are resposible for signal transduction, a C-terminal domains is the first RNA binding site, and a central helicase domainis the second RNA binding site and possesses an ATP-dependent activity. RIG-I is important for sensing of several mononegavirales, such as rabies, measle and Ebola viruses, and recongnized 5'-triphosphorylated double stranded RNA. Despite intensive studies, a full and comprehensive model of the mechanismof RIG-I activation is still lacking. Our aim was to clarify the first molecular steps of RIG-I activation. First, Stephen Cusack's team elucidated the structure of the full lenght duck RIG-I protein and identified the principle of RIG-I auto-repressed conformation. In absence of ligand RNA, CARD2 domain interacts with Hel2i subdomain of helicase domain. We confirmed this conformation with functional evidence. Mutaions F540A/D, in Hel2i subdomain, inhibits CARD2:Hel2i interaction and renders RIG-I constitutively active. In contrast, the corresponding mutations in the Hel2i contacting site of CARD2 domain produce inactive mutants. Thus CARDS;Hel2i interactio induces an auto-repressed state through a deual masking of both Hel2i RNA binding site and CARD2 residus necessary for signal transduction. Moreover, study of mutants involved in ATP binding and hydrolysis reealed a portential unsuspected regulatory role for the ATP-dependent enzymatic activity of RIG-I. Second, we studied the necessity of RIG-I oligomerization for RIG-I activation. Eva Kowalinski, PhD student in Stephen Cusack's team, observed RIG-I dimers in vitro, only in presence of synthetic RNA with two 5'triphosphorylated ends. We complete this observation with functional assays showing that synthetic leader RNA incapable to induce RIG-I oligomerization in vitro, did activate RIG-I in cellula. Moreover, we did not observe RIG-I oligomerization using either co-immunoprecipitation or Gaussia Luciferase-based-protein complementation assay, after activation with cognate RNA or viral infection. Altogether our results indicate that the self-oligomerization og RIG-I is either dispensable or very transient for signal transduction.

Le récepteur hépatique aux lipoprotéines LSR est impliqué dans la clairance des lipoprotéines riches en triglycérides telles que les résidus de chylomicrons pendant la phase postprandiale. La réduction de l’expression du LSR chez la souris (LSR+/-) est associée à une dyslipidémie et une lipémie postprandiale élevée. Afin de mieux comprendre la régulation de la distribution des lipides alimentaires, nous avons cherché quels étaient les facteurs pouvant affecter le niveau protéique de LSR. La leptine, hormone sécrétée par le tissu adipeux et connue pour son action d’hormone de satiété au niveau du système nerveux central, a été démontrée dans cette thèse comme modulant l’expression de LSR par la régulation de la transcription du gène lsr. La leptine est impliquée dans la régulation de la lipogénèse à travers SREBP-1. Grâce à l’utilisation d’un extrait de Garcinia cambogia contenant un inhibiteur de l’ATP citrate lyase, nous avons démontré une interaction importante entre les enzymes lipogéniques, l’expression de LSR et d’autres récepteurs lipoprotéiques, afin de maintenir un équilibre entre la synthèse de lipides endogènes et l’apport alimentaire de lipides exogènes. Soumises à un régime hyperlipidique, les souris sauvages montrent une diminution de l’expression des enzymes lipogéniques hépatiques, aggravée chez les souris LSR+/-. Ces résultats indiquent qu’il existe un mécanisme de maintien de l’équilibre entre la lipogénèse (synthèse endogène de lipides), la lipolyse (utilisation lipidique comme substrat énergétique) et le stockage de lipides à travers une forte interaction entre les enzymes lipogéniques et LSR. / The hepatic lipoprotein receptor LSR is involved in the clearance of triglyceride-rich lipoproteins including chylomicrons remnants during the post-prandial phase. Reduced LSR protein expression in mice (LSR+/-) is associated with dyslipidemia and increased postprandial lipemia; these mice exhibit increased weight gain with aging or when placed under a high-fat diet. In order to better understand the regulation of the distribution of dietary lipids, we looked for factors that could regulate LSR protein levels. Leptin is a hormone secreted by the adipose tissue that is a centrally-acting satiety factor, and was demonstrated to modulate LSR mRNA and protein expression through the modulation of transcription of the gene lsr. Leptin has been reported be involved in the control of lipogenesis through SREBP-1c. Using Garcinia cambogia extract containing an inhibitor of ATP citrate lyase, we demonstrated that there is an important link between lipogenic enzymes and LSR protein levels and with other lipoprotein receptors that provides the means to maintain a balance between endogenous lipid synthesis and dietary intake of exogenous lipids. When exogenous lipid intake is increased in the form of a high-fat diet, mice exhibited a decrease in hepatic lipogenic enzymes expression, but a deficiency of LSR led to increased lipid content in the peripheral tissues. These results suggest the presence of mechanisms for the maintenance for the balance between lipogenesis (de novo endogenous lipid synthesis), lipolysis (lipids used as energy substrate), and lipid storage through an important link between lipogenic enzymes and LSR.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2016-05-24T21:28:26Z No. of bitstreams: 1 Flavia Regina Greiffo.pdf: 886904 bytes, checksum: 4aa27a44bc94c9d06427df1b649e9b9e (MD5) / Made available in DSpace on 2016-05-24T21:28:26Z (GMT). No. of bitstreams: 1 Flavia Regina Greiffo.pdf: 886904 bytes, checksum: 4aa27a44bc94c9d06427df1b649e9b9e (MD5) Previous issue date: 2015-03-02 / In the last years, a growing number of studies have demonstrated that the aerobic physical training (APT), performed in the proper intensity, duration and frequency, display several benefits to asthmatic patients, including improvement in quality of life, reduction in the levels of anxiety and depression, improvements in minute ventilation, reduction in the number of crisis, reduction in the levels of exhaled nitric oxide, beyond reduced daily needing of corticosteroids. More recently, was demonstrated for the first time that APT presents a direct anti-inflammatory effect on the airways of asthmatic patient, since that APT reduced the number of eosinophils in the induced sputum of these patients. However, several questions and hypothesis were raised up about the possible mechanism involved in these anti-inflammatory effects of APT for asthma. In this way, a growing number of experimental studies using models of asthma have been published, revealing possible mechanism involved of action APT for asthma. On the other hand, until this moment no study evaluated the effects of APT on the via of purinergic receptors, a family of receptors that present a central role in the physiopathology of asthma. Therefore, the present study aim is to evaluate if the anti-inflammatory effects of APT for asthma is mediate, at least partially, by the inhibition of expression of purinergic receptor P2X7, as well as the extracellular adenosine triphosphate (ATP). Thus, using 40 C57Bl/6 mice, divided in 4 experimental groups (Control, n = 10), (Exercise, n = 10), (Asthma, n = 10) and (Asthma + Exercise, n = 10). The experimental model of asthma (chronic pulmonary allergic inflammation) was induced through the intra-peritoneal injection of ovalbumin solution on days 0, 14, 21 and 42 followed by inhalation of 1%, 3% and 5% ovalbumin, 3x/week, and beginning on 21st day until the 53rd day of experimental protocol. To evaluate the therapeutic effects of APT on the inflammation and on the via of purinergic receptors, the low intensity APT was performed 5x/week, 60 min/session, begining on 27th day of experimental protocol until the 53rd day, when the airways inflammation is already established. Twenty-four hours after the last inhalation and training session, mice were anesthetized, tracheotomized, canuled and the bronchoalveolar lavage was collected and analyzed for the ATP levels and also the total number of cells as well as the differential number of cells. The analysis of hyperresponsiveness was performed. The histological analysis was performed to evaluate and quantify the number of lymphocytes and eosinophils in the airways wall, and also the airway remodeling. The expression of purinergic receptor P2X7 in the lung tissue homogenate was performed using western blotting and also the immunohistochemistry technique. / Nos últimos anos um crescente número de estudos tem demonstrado que o treinamento físico aeróbio (TFA), quando realizado de maneira adequada em termos de intensidade, duração e freqüência, apresenta inúmeros benefícios para o paciente asmático, incluindo melhora na qualidade de vida, diminuição dos níveis de ansiedade e depressão, melhora da ventilação minuto, diminuição do número de crises, diminuição dos níveis de óxido nítrico exalado, além de diminuir a necessidade diária de corticoesteróides. Recentemente, foi demonstrado pela primeira vez que o TFA apresenta um efeito antiinflamatório direto sobre as vias aéreas do paciente asmático, uma vez que o TFA diminuiu o número de eosinófilos no escarro induzido desses pacientes. Entretanto, inúmeras perguntas e hipóteses foram levantadas sobre os possíveis mecanismos envolvidos nesse efeito antiinflamatório do TFA na asma. Nesse sentido, um crescente número de estudos com animais de experimentação em modelos de asma tem sido publicado, os quais têm revelado possíveis vias de atuação do TFA na asma. Entretanto, até o momento não existem publicações sobre os efeitos do TFA sobre a via dos receptores purinérgicos, os quais têm um papel central na fisiopatologia da asma. Portanto, o presente estudo teve com objetivo avaliar se os efeitos antiinflamatórios do TFA na asma é mediado, pelo menos em parte, pela inibição da expressão dos receptores purinérgicos, assim como pela diminuição dos níveis do principal ativador dos receptores purinérgicos, a adenosina trifosfato (ATP) extracelular. Para isso, foram utilizados 40 camundongos C57Bl/6 divididos em 4 grupos experimentais (n=10 / cada) Controle, Exercício, Asma e Asma + Exercício. O modelo de asma (inflamação pulmonar alérgica crônica) foi induzido por meio de injeção intra-peritoneal com ovalbumina nos dias 0, 14, 28 e 42 seguidos por inalação com solução de ovalbumina (1, 3 e 5%) três vezes por semana, a partir do dia 21 até o dia 53 do protocolo experimental. Com o intuito de avaliar o efeito terapêutico do TFA sobre a inflamação e sobre a via dos receptores purinérgicos, o TFA de baixa intensidade foi realizado 5x/semana, 60 minutos/sessão, iniciando no dia 27 do protocolo experimental até o dia 53, quando a inflamação das vias aéreas já estava estabelecida. Vinte e quatro horas após a última inalação e sessão de treinamento, os animais foram anestesiados, traqueostomizados, canulados e o lavado broncoalveolar foi coletado e analisado para os níveis de ATP e também para o número de células totais e contagem diferencial. A avaliação da expressão do receptor purinérgico P2X7 foi realizada no homogenato do tecido pulmonar por meio da técnica de western blotting, e também nos cortes histológicos por meio da técnica de imunohistoquímica. Através da técnica de histologia foi quantificado o número de linfócitos e eosinófilos nas vias aéreas, assim como o remodelamento brônquico. A hiperresponsividade brônquica das vias aéreas foi avaliada através do sistema Buxco. Os resultados obtidos neste trabalho corroboram com a literatura, uma vez que o TFA reduz a inflamação crônica das vias aéreas. Nossos resultados também sugerem que o TFA atua na via dos receptores purinégicos diminuindo a inflamação pulmonar.

Purinerge P2X3-Rezeptoren spielen eine bedeutende Rolle in der Vermittlung chronischer Schmerzen, welche ein führendes Problem des Gesundheitswesens mit vielen sozioökonomischen Konsequenzen darstellen. Die Tatsache, dass P2X3-Rezeptoren fast ausschließlich von nozizeptiven Neuronen exprimiert werden, macht sie trotz ihres besonderen Desensitisierungsverhaltens zu vielversprechenden Angriffspunkten zukünftiger Schmerztherapien, beispielsweise mithilfe kompetitiver Antagonisten an diesen Rezeptoren. Zur Analyse der Wechselwirkungen zwischen Agonist und kompetitivem Antagonist wird meist der Schild-Plot benutzt. Jedoch ist dieser im Falle der sehr schnell desensitisierenden P2X3-Rezeptoren ungeeignet, da die Vorbedingung eines stabilen Gleichgewichts zwischen Agonist und Antagonist aufgrund der Desensitisierung nicht erfüllt ist. Ziel der vorliegenden Arbeit war es, eine neue Methode zur Analyse der Interaktion kompetitiver Antagonisten mit ihrer Bindungsstelle am Beispiel des P2X3-Rezeptors zu entwickeln und so für die Antagonistenbindung bedeutende Aminosäuren der Bindungsstelle zu identifizieren. Mittels der Patch-Clamp-Technik wurden die Effekte der Antagonisten A-317491, TNP-ATP und PPADS auf die vom P2X1,3-Rezeptor-selektiven Agonisten α,β-MeATP induzierten Ströme am P2X3-Wildtyp-Rezeptor und an fünf Rezeptormutanten mit veränderter Ligandenbindungsstelle untersucht. Alle Rezeptoren wurden in HEK293-Zellen exprimiert. Anhand der gemessenen Daten wurde ein Hidden Markov Model (HMM) erstellt, welches die sequentiellen Übergänge des Rezeptors von geschlossen zu offen und desensitisiert in An- und Abwesenheit des Antagonisten miteinander kombiniert. Die am P2X3-Rezeptor induzierten Ströme konnten mithilfe dieses Modells korrekt gefittet und die für die Antagonistenbindung wichtigen Aminosäuren innerhalb der Bindungsstelle bestimmt werden. Als Resultat dieser Arbeit konnte außerdem gezeigt werden, dass das HMM eine geeignete Methode zur Analyse der Wirkung kompetitiver Antagonisten an schnell desensitisierenden Rezeptoren darstellt. Die untersuchten Antagonisten A-317491 und TNP-ATP haben einen kompetitiven Wirkmechanismus, während PPADS eine pseudoirreversible Blockade verursacht.

1 Abstract It has been estimated that the mammalian mitochondrial proteome consists of ~1500 distinct proteins and approximately one quarter of them is still not fully characterized. One of these proteins is TMEM70, protein involved in the biogenesis of the eukaryotic F1Fo-ATP synthase. TMEM70 mutations cause isolated deficiency of ATP synthase often resulting in a fatal neonatal mitochondrial encephalocardiomyopathies in patients. To understand the molecular mechanism of TMEM70 action, we generated constitutive Tmem70 knockout mice, which led to embryonic lethal phenotype with disturbed ATP synthase biogenesis. Subsequently generated inducible Tmem70 mouse knockout was lethal by the week 8 post induction. It exhibited primarily impaired liver function, which contrasts with the predominantly cardiologic phenotype at disease onset in humans. Liver mitochondria revealed formation of labile ATP synthase subcomplexes lacking subunit c. Thus, in case of TMEM70 deficiency c-oligomer was not incorporated into ATP synthase, which led to critical impairment of mitochondrial energy provision, analogous to TMEM70 dysfunction in humans. In TMEM70 deficient models, the ATP synthase deficiency reached the 'threshold' for its pathologic presentation, which we quantified at 30 %. We observed compensatory increases in the...

With the rapid development of power systems, locating and classifying faults is critical to the continuity and reliability of the transmission system. In this thesis, a traveling-wave based technique for fault location and classification on high voltage and extremely high-voltage transmission lines is proposed. The traveling-wave based protection has the advantage of fast response and not being affected by power swing and CTs saturation. In this thesis, the transient characteristics of single line to ground fault (which can be divided into solid fault and arcing fault) and lightning disturbance are extracted by using Clarke transformation and wavelet transformation. The differences among recorded traveling wave arrival times are used to calculate the fault location, and the wavelet energy at different frequency bands is utilized to distinguish between lightning and different kinds of fault. A criterion is proposed according to the energy ratio. The proposed scheme can identify different faults correctly and quickly. In addition, the influence of busbar capacitance, current transformer and coupling capacitor voltage transformer are considered. The simulation of a transmission system has been made in ATP/EMTP, and the calculations have been made in MATLAB. / Med den snabba utvecklingen av kraftsystem är lokalisering och klassificering av fel avgörande för kontinuiteten och tillförlitligheten hos överföringssystem. I denna avhandling föreslås en vågrörelse-baserad teknik för fellokalisering och klassificering av kraftledningar för högspänning och extremt hög spänning. Vågrörelsebaserat skydd har fördelen av snabb respons och att det inte påverkas av kraft fluktuationer och strömtransformsmättnad. I denna avhandling tas momentana egenskaperna av jord till ledningsfel (vilket kan delas in i stumt jordfel och ljusbågefel) och blixtstörning fram med hjälp av Clarke transformation och wavelet transformation. Skillnaderna mellan de uppmätta vågrörelsernas ankomsttider används för att beräkna fellokalisering och wavelet energin vid olika frekvensband, vilket används för att skilja mellan blixt och olika sorters fel. Ett kriterium föreslås enligt energiförhållandet. Det föreslagna systemet kan identifiera olika sorters fel korrekt och snabbt. Dessutom övervägs påverkan av strömskenans kapacitans, strömtransformator och kopplingskondensatorspänningsomvandlare. Simuleringen av transmissionssystem har gjorts med ATP/EMTP, och beräkningarna är gjorda med MATLAB.