Submitted by PPG Odontologia (odontologia-pg@pucrs.br) on 2018-06-04T18:05:30Z No. of bitstreams: 1 NATALIA_PRADELLA_CIGNACHI_TES.pdf: 6840858 bytes, checksum: 8e5f3ae6a98395bf09135b6770903f35 (MD5) / Rejected by Sheila Dias (sheila.dias@pucrs.br), reason: Devolvido devido ? falta da capa institucional no arquivo em PDF. on 2018-06-14T14:34:13Z (GMT) / Submitted by PPG Odontologia (odontologia-pg@pucrs.br) on 2018-06-19T13:28:41Z No. of bitstreams: 1 NAT?LIA_PREDELLA_CIGNACHI_TES.pdf: 11102267 bytes, checksum: 121fbf4d2b9ae1c4a36e21d23ab3140d (MD5) / Approved for entry into archive by Sheila Dias (sheila.dias@pucrs.br) on 2018-06-27T14:32:32Z (GMT) No. of bitstreams: 1 NAT?LIA_PREDELLA_CIGNACHI_TES.pdf: 11102267 bytes, checksum: 121fbf4d2b9ae1c4a36e21d23ab3140d (MD5) / Made available in DSpace on 2018-06-27T14:43:00Z (GMT). No. of bitstreams: 1 NAT?LIA_PREDELLA_CIGNACHI_TES.pdf: 11102267 bytes, checksum: 121fbf4d2b9ae1c4a36e21d23ab3140d (MD5) Previous issue date: 2018-01-25 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / This thesis encompasses two parts: firstly, we compared the bone healing in female and male mice, after induction of type 1 diabetes (T1D); secondly, the bone regeneration was evaluated in a menopause model induced by bilateral ovariectomy (OVX), with or without T1D induction. For the part I, the animals (female and male) were initially assigned into two groups, namely control or T1D (elicited by streptozotocin; STZ). In the part II, the females were divided into four experimental groups: sham-operated or OVX, with or without STZ T1D induction. After T1D induction, a monocortical femoral defect was created. In either parts of the present study, we evaluated the effects of supplementation with vitamin D3 and/or insulin. In the second part, the effects of estrogen replacement were also analyzed. Following 21 days of bone defect creation, the animals were euthanized; the femurs and blood were collected for posterior analysis. Both T1D females and males presented a reduction in body weight gain, associated with hyperglycemia. There were no changes in the serum levels of the pro-inflammatory cytokines [interleukin-1? (IL-1?), tumor necrosis factor (TNF) and interferon-? (IFN-?)] in all the evaluated groups. T1D mice of both sexes presented a delayed bone regeneration, according to the histological and micro-CT assessment. The supplementation with vitamin D3 restored the bone healing in female and male T1D mice, reaching values close to controls. The insulin therapy improved the bone remodeling in T1D mice of both sexes, but the effects of this hormone were superior in males. The evaluation of osteoclast activity did not reveal significant differences among the experimental groups. Real time PCR revealed slight differences in the mRNA expression of two transcription factors related to osteoblast differentiation, namely runx2 and osterix, as measured in the area into the bone defect. A higher upregulation of both factors was seen in T1D males treated with vitamin D3. Conversely, vitamin D3-treated T1D females displayed an upregulation of insulin-like growth factor 1 (IGF-1), further indication sex-related differences for the treatments. Besides the experimental protocols described for the 12 part I of this thesis, in the part II, we also evaluated some behavioral locomotor parameters and serum levels of calcium and alkaline phosphatase. OVX animals presented increased body weight gain, accompanied by uterus atrophy. Otherwise, T1D induction elicited a reduction of body weight gain, which was more pronounced in OVX-T1D animals. Serum levels of alkaline phosphatase were divergent in the non-diabetic and T1D OVX animals. Calcium or cytokine levels were similar in all the experimental groups. The sham-operated T1D, the non-diabetic OVX and the OVX-T1D groups presented a delayed bone regeneration, as indicated by histological and micro-CT analysis. Estrogen replacement improved the bone healing in all OVX groups. There was a trend toward an upregulation of IGF-1 mRNA in non-diabetic OVX animals, which was not mirrored in OVX-T1D mice. Locomotor parameters remained unaltered, except by a general reduction of rearing numbers in T1D animals. / A presente tese est? dividida em duas partes: na parte I, comparou-se a regenera??o ?ssea em camundongos f?meas e machos, com diabetes do tipo 1 (T1D). Na parte II, foi avaliada a regenera??o ?ssea no modelo de menopausa experimental induzido por ovariectomia (OVX), com ou sem T1D. Na parte I, os animais (machos e f?meas) foram divididos em dois grandes grupos: controle e T1D (induzido por estreptozotocina; STZ). Na parte II, os animais foram divididos em quatro grandes grupos: f?meas falso-operadas e OVX, com ou sem T1D. Ap?s a indu??o do T1D, foi criado um defeito ?sseo monocortical no f?mur. Nas duas partes do trabalho, foram avaliados os efeitos da suplementa??o com vitamina D3 e/ou insulina. Na segunda parte, tamb?m se avaliou o efeito da reposi??o hormonal com estradiol. Decorridos 21 dias do procedimento da cria??o do defeito, os animais foram eutanasiados; o f?mur e o sangue foram coletados para an?lises posteriores. Tanto as f?meas, quanto os machos T1D, apresentaram uma redu??o do ganho de peso corporal, associado ? hiperglicemia. N?o houve altera??es nos n?veis s?ricos das citocinas pr?-inflamat?rias interleucina-1? (IL-1?), fator de necrose tumoral (TNF) ou interferon-? (IFN?). Os animais T1D, de ambos os sexos, apresentaram um comprometimento na regenera??o ?ssea, como demonstrado pelas an?lises histol?gicas e de micro-CT. A suplementa??o com vitamina D3 reestabeleceu a regenera??o ?ssea em f?meas e machos T1D, apresentando valores pr?ximos aos encontrados nos animais do grupo controle. A terapia com insulina melhorou a remodela??o ?ssea nas f?meas e machos T1D; por?m, os efeitos foram mais pronunciados nos machos. A avalia??o da atividade osteocl?stica n?o revelou diferen?as significativas entre os grupos experimentais, ap?s a indu??o de T1D, em machos ou f?meas. Os resultados do PCR em tempo real para runx2 e osterix (dois fatores de transcri??o relacionados aos osteoblastos), no tecido ?sseo, n?o demonstraram nenhuma diferen?a significativa, exceto por um aumento nos n?veis de RNAm dos dois fatores nos camundongos machos T1D, que receberam suplementa??o com vitamina 10 D3. Por outro lado, f?meas T1D que receberam vitamina D3 apresentaram um aumento na express?o dos n?veis de RNAm para o fator de crescimento semelhante ? insulina do tipo 1 (IGF-1), quando comparado com os machos que tiveram o mesmo tratamento, sugerindo assim, diferen?as relacionadas ao sexo. Al?m das an?lises j? mencionadas anteriormente, na parte II da tese, par?metros comportamentais e n?veis s?ricos de c?lcio e fosfatase alcalina tamb?m foram analisados. Os resultados da segunda parte do trabalho demonstraram que os animais submetidos a OVX tiveram um aumento do peso corporal, com atrofia uterina. Em contrapartida, quando foi induzido T1D, houve uma diminui??o do peso corporal mais acentuada no grupo OVX que no grupo falso-operado. Os animais falso-operados e OVX T1D apresentaram hiperglicemia, confirmando o desenvolvimento do diabetes. Os n?veis s?ricos de fosfatase alcalina foram divergentes entre os grupos n?o-diab?ticos OVX e OVX T1D. N?o houve varia??es dos n?veis de c?lcio. Os animais falso-operados T1D, n?o diab?ticos OVX e OVX T1D apresentaram preju?zos similares na regenera??o ?ssea, como observado pelas an?lises histol?gicas e imagens de micro-CT. A reposi??o com estradiol melhorou a cicatriza??o ?ssea nos animais n?o-diab?ticos OVX e OVX T1D. Houve uma tend?ncia ao aumento nos n?veis de RNAm de IGF-1 no grupo OVX, o que n?o foi observado quando da associa??o de T1D e menopausa. N?o foram observadas diferen?as na atividade locomotora dos animais com T1D e/ou OVX, a n?o ser por uma diminui??o no n?mero de rearings no grupo falso-operado T1D, independente do tratamento com vitamina D3, insulina e estradiol, de forma isolada ou em associa??o.

A maciez da carne bovina é o resultado do processo de proteólise miofibrilar influenciado pelas calpaínas (CAPN), enzimas ativadas pelo cálcio. A calpastatina (CAST) constitui um regulador das calpaínas, atuando como substrato e degradando-se pela ação da própria calpaína. A suplementação com vitamina D3 na dieta dos animais tem alterado positivamente a maciez da carne, por obter maior absorção e deposição de cálcio nos músculos. Tendo em vista a importância desses genes para a maciez da carne em bovinos e o metabolismo de vitamina D3, o objetivo deste estudo foi verificar a expressão gênica de 1-HYD, 24-HYD, CAPN1 e CAST (Isoformas). Foram utilizados 42 machos castrados da raça Nelore que foram divididos em 6 tratamentos (7 animais por tratamento). Os animais foram alojados em baias individuais e os tratamentos foram: T1) sem suplementação de Vitamina D3 + sem sombrite; T2) sem suplementação de Vitamina D3 + com sombrite filtração de 50% de UV; T3) com 2x106UI de vitamina D3 por dois dias consecutivos pré-abate + sem sombrite; T4) com 2x106UI de vitamina D3 por dois dias consecutivos pré-abate + com sombrite filtração de 50% de UV; T5) com 2x106UI de vitamina D3 por oito dias consecutivos pré-abate + sem sombrite; 6) com 2x106UI de vitamina D3 por oito dias consecutivos pré-abate + com sombrite filtração de 50% de UV. A expressão gênica foi analisada por PCR quantitativa em tempo real. O gene 1-HYD foi mais expresso nos tratamentos com 2 e 8 dias de suplementação com vitamina D3, independente da exposição diferenciada aos raios ultravioleta. O gene 24-HYD foi mais expresso no tratamento com 8 dias de suplementação pré-abate quando comparado ao tratamento controle, não diferindo do tratamento com suplementação por 2 dias consecutivos pré-abate. Portanto os resultados não corroboram com o mecanismo de regulação das atividades das enzimas modulada de forma oposta para regular os níveis circulantes de 1,25-di-hidroxi-vitamina D3 (1,25 D). Quando a concentração de 1,25 D atinge níveis elevados um mecanismo de regulação inativa a 1,25 D e promove a diminuição da expressão de 1-HYD. Assim pode-se inferir que neste estudo esses fatores não exerceram influência sobre o gene 1-HYD que teve um aumento da expressão. A única possibilidade de verificar o aumento da expressão do gene da enzima 1-HYD parece envolver um hiperparatireoidismo secundário decorrente de hipocalcemia em algum momento do experimento. A suplementação com vitamina D3 não exerceu influência na expressão de RNAm da CAPN1, isoforma I e isoforma total da CAST. A isoforma II do gene da calpastatina foi mais expressa no tratamento com 8 dias de suplementação pré-abate, onde parece que o aumento da expressão é devido a uma sinalização intracelular indicando que a calpastatina está sendo degradada pela ativação prolongada das calpaínas, em decorrência do aumento do influxo de cálcio. As análises de associação apontam para um possível descompasso entre abundância de RNAm e atividade da protease do sistema das calpaínas no processo de amaciamento da carne. / The tenderness of beef is the result of the process of myofibrillar proteolysis influenced by calpains (CAPN), enzymes activated by calcium. The calpastatin (CAST) is a regulator of calpains, acting as a substrate and degraded by the action of calpain itself. Supplementation with vitamin D3 in the diet has changed positively on meat tenderness, to obtain greater absorption and calcium deposition in muscle. Given the importance of these genes for meat tenderness in cattle and metabolism of vitamin D3, the objective of this study was to investigate the gene expression of 1-HYD, HYD-24, CAPN1, CAST (isoforms). We used 42 Nellore steers were divided into 6 treatments (seven animals per treatment). The animals were housed in individual pens and treatments were: T1) without vitamin D3 feeding + without shadow; T2) without vitamin D3 feeding + with black - 50% filtration of UV; T3) with 2x106UI of vitamin D3 for two days consecutive slaughter + without shadow; T4) with 2x106UI vitamin D3 for two days consecutive slaughter + with black - 50% filtration of UV; T5) with 2x106UI vitamin D3 for eight days consecutive slaughter + without shading; T6) 2x106UI with vitamin D3 for eight days consecutive slaughter + with black - 50% filtration of UV. Gene expression was analyzed by quantitative real-time. The 1-HYD gene was more expressed in the treatments with 2 and 8 days of supplementation with vitamin D3, independent of differential exposure to ultraviolet rays. The 24-HYD gene was more expressed in the treatment with 8 days of pre-slaughter supplementation compared to control treatment, the treatment did not differ with supplementation for 2 consecutive days pre-slaughter. These findings do not corroborate the mechanism for regulating the activities of enzymes modulated so diametrically opposite to regulate the circulating levels of 1,25- dihydroxy vitamin D3 (1,25 D). When there is an overproduction of 1,25 D regulation mechanism inactivates 1.25 D and promotes the reduction of the expression of 1-HYD. Thus we can infer that in this study, these factors did not influence the 1-HYD gene that had increased expression. The only way to verify the increase of gene expression of the enzyme 1-HYD seems to involve a secondary hyperparathyroidism due to hypocalcemia sometime in the experiment. Supplementation with vitamin D3 did not influence the mRNA expression of CAPN1, isoform I and isoform total of CAST. The isoform II of the calpastatin gene expression was higher in treatment with 8-day preslaughter supplementation, where it appears that increased expression is due to an intracellular signaling indicating that calpastatin is degraded by prolonged activation of calpain as a result of increased influx of calcium. The association analysis point to a possible mismatch between mRNA abundance and activity of the calpain system components in these physiological conditions.

No description available.

Der Embryonale Stammzelltest (EST) ist ein validierter In-vitro-Embryotoxizitätstest, der zur Untersuchung embryotoxischer Wirkungen von Chemikalien eingesetzt werden kann. Während des zehntägigen Differenzierungsassays differenzieren sich die pluripotenten murinen embryonalen Stammzellen (ES-Zellen) der Linie D3 in vitro in spontan kontrahierende Herzmuskelzellen. Dabei rekapitulieren sie Prozesse der frühen Embryogenese in vivo. Ein Zytotoxizitätsassay mit D3-Zellen und ausdifferenzierten, adulten 3T3-Maus-Fibroblasten dient der Ermittlung allgemeiner zytotoxischer Effekte und unterschiedlicher Sensitivitäten beider Zelllinien. Somit basiert der EST auf den beiden wichtigsten Mechanismen pränataler Toxizität, der Störung der Differenzierung und der Zytotoxizität. Ziel dieser Arbeit war es, mit Hilfe des EST das embryotoxische Potential der vier Chemikalien Trichostatin A (TSA), Methylazoxymethanolacetat (MAMac), Natriumdodecylsulfat (SDS) und Benzoesäure (BA) abzuschätzen. Dazu wurde mikroskopisch ermittelt, bei welcher Testsubstanzkonzentration in 50 % der während der In-vitro-Differenzierung gebildeten Embryonalkörperchen die Kardiomyozytendifferenzierung inhibiert wird (ID50). Außerdem wurde die halbmaximale Hemmkonzentration des Zellwachstums auf die beiden Zelllinien bestimmt (IC50D3 bzw. IC503T3). Als Erweiterung dieses konventionellen EST wurden mittels quantitativer Real Time-PCR an den Tagen 5, 7 und 10 der Differenzierung zusätzlich Genexpressionsanalysen etablierter herzmuskelspezifischer Markergene (Mesoderm Posterior 1, Tag 5; Myosin light chain 1, Tag 7 und 10) durchgeführt. Deren Expression korreliert in den ES-Zellen mit der embryonalen Herzdifferenzierung in vivo und kann zur Ermittlung der von der Prüfsubstanz hervorgerufenen halbmaximalen Hemmung der Genexpression in den Kardiomyozyten (IC50 Exp) herangezogen werden. Um letztlich embryotoxische Effekte in vivo auf Grundlage der ermittelten In-vitro-Daten abschätzen zu können, wurden die ermittelten Parameter mittels eines für den EST empirisch abgeleiteten mathematischen Prädiktionsmodells (PM) zur Klassifizierung der Testsubstanzen als nicht, schwach oder stark embryotoxisch herangezogen. Für jede der Substanzen waren die ermittelten Halbhemmkonzentrationen in den überwiegenden Fällen vergleichbar und führten unter Verwendung des PMs im konventionellen und im molekularen EST zu deren identischer Klassifizierung. TSA wurde als „stark embryotoxisch“ klassifiziert und beeinflusste insbesondere das Differenzierungspotential der ES-Zellen. Das als „schwach embryotoxisch“ klassifizierte SDS wirkte auf die D3-Zellen stärker differenzierungsinhibierend als zytotoxisch, hemmte jedoch das Wachstum der 3T3-Zellen bereits in deutlich niedrigeren Konzentrationen. MAMac und BA wurden als „nicht embryotoxisch“ klassifiziert. Bei ihnen stand die zytotoxische Wirkung deutlich im Vordergrund. Diese Prädiktionen stimmten mit In-vivo-Befunden überein, was von der Stabilität und der Brauchbarkeit der im konventionellen und molekularen EST ermittelten Parameter zeugte. Einzige Ausnahme war das als Entwicklungsneurotoxin in vivo bekannte MAMac. Da der EST auf mesodermaler Differenzierung basiert, können spezifische Effekte auf neuronale Entwicklungsprozesse offenbar nicht vollständig erfasst werden. Substanzkonzentrationen, die sich als differenzierungsinhibierend auf die morphologische Kardiomyozytendifferenzierung erwiesen haben, führten auch zu einer messbaren Repression der herzmuskelspezifischen Genexpression. Dabei erwies sich die IC50 Exp als ebenso sensitiv wie die konventionellen Parameter und als nutzbringende Ergänzung zu diesen, da sie bereits nach 5 bzw. 7 Tagen der In-vitro-Differenzierung eine mit dem mikroskopischen Parameter übereinstimmende Einschätzung des embryotoxischen Potentials der Chemikalien in vivo ermöglichte. Genexpressionsanalysen weiterer differenzierungsspezifischer Gene können zusätzlich zur Aufklärung zu Grunde liegender Mechanismen der Embryotoxizität von Testsubstanzen dienen. Somit kann der EST durch die Vorteile der Stammzelltechnologie und der Genexpressionsanalyse als neues prädiktives Screening-Instrument zur frühzeitigen Detektion embryotoxischer Substanzeffekte in der pharmazeutischen und chemischen Industrie genutzt werden. / The embryonic stem cell test (EST) represents a validated in vitro embryotoxicity test that can be utilised for investigations of embryotoxic effects of chemical substances. During the 10-day differentiation assay the pluripotent murine embryonic stem cells (ES cells) of the D3 line differentiate in vitro into spontaneously beating cardiac muscle cells that can be observed microscopically. Thereby, ES cells recapitulate processes of early embryogenesis in vivo. A cytotoxicity assay with D3 cells as well as differentiated, adult 3T3 mouse fibroblasts is used to determine general cytotoxic effects and to consider differences in the sensitivity of both cell lines. Hence the EST is based on the two most important mechanisms of prenatal toxicity, such as inhibition of differentiation and cytotoxicity. The aim of the presented work consisted in the evaluation of the embryotoxic potential of the four chemicals trichostatin A (TSA), methylazoxymethanolacetate (MAMac), sodium dodecyl sulfate (SDS) and benzoic acid (BA) by means of the EST. For this purpose the concentration of the test substance that causes an inhibition of cardiomyocyte differentiation in 50 % of the embryoid bodies which are formed during the in vitro differentiation (ID50-value) and the halfmaximal inhibiting concentration of cell proliferation of D3 and 3T3 cell lines (IC50D3 and IC503T3) were determined. As extension of this conventional EST, the effect of test substances was investigated at the molecular level by gene expression analyses of cardiac specific genes (Mesoderm Posterior 1, day 5; Myosin light chain 1, day 7 and 10). Their expression in ES cells correlates with the embryonic heart differentiation in vivo. Quantitative Real Time-PCR gene expression analysis was used to determine the halfmaximal inhibition of the cardiomyocyte gene expression (IC50 Exp) caused by the test compound. To predict embryotoxic effects in vivo from the determined in vitro data, these parameters were used for the classification of the test chemicals as non, weak or strong embryotoxic via a mathematical prediction model (PM). In the majority of cases comparable halfmaximal inhibiting concentrations were calculated in the conventional and molecular EST that resulted in the identical classification of the tested chemicals concerning their embryotoxic potential. TSA was estimated as “strongly embryotoxic” and affected particularly the differentiation potential of the ES cells. SDS was classified as “weakly embryotoxic” and acted by inhibiting the differentiation of D3 cells at concentrations lower than cytotoxic concentrations but already repressed the growth of the 3T3 cells in significantly lower ranges. As to MAMac and BA that were classified as “non-embryotoxic” the cytotoxic effects on both cell lines predominated. These predictions were consistent with in vivo findings that testifies the stability and the usefulness of the parameters used in the conventional and molecular EST. MAMac, which is known as a developmental neurotoxin in vivo, represented the single exception. Its misclassification as compared to in vivo data may originate from the limitations of the model system that is based on mesodermal differentiation. Thus, specific effects on neuronal developmental processes obviously cannot be detected completely. Gene expression analysis showed that test substance concentrations which were proved to be inhibiting on the morphological differentiation of cardiomyocytes caused a repression of cardiac-specific marker gene expression as well. Thereby, IC50 Exp-values proved to be just as sensitive as the conventional parameters and can provide valuable and supportive data. They allowed a prediction of the embryotoxic potential of the chemicals in vivo already at day 5 and day 7 of in vitro differentiation. Moreover, gene expression analysis of appropriate differentiation specific genes could be used to investigate mechanisms that are responsible for embryotoxic properties of the test compounds. Thus, the EST is considered to represent a new, predictive screening test especially in the pharmaceutical industry to detect the embryotoxic potential of chemical compounds early in the process of compound development.

No description available.

Les dyskinésies tardives (DT) sont des troubles moteurs associés à l’utilisation chronique des antagonistes des récepteurs dopaminergiques D2 tels que les antipsychotiques et le métoclopramide. Ces dyskinésies correspondent à une incoordination motrice portant préférentiellement sur la musculature oro-faciale. La gestion des DT s'est imposée comme défi de santé publique surtout en l’absence d’une alternative thérapeutique efficace et abordable. L’hypothèse classiquement avancée pour expliquer la physiopathologie des DT inhérente au traitement par les antipsychotiques s’articule autour de l’hypersensibilité des récepteurs dopaminergiques D2, cibles principales de ces molécules. Néanmoins, plusieurs données remettent la véracité de cette hypothèse en question. Hypothèse: nous proposons que le blocage chronique des récepteurs dopaminergiques soit effectivement responsable d’un phénomène d’hypersensibilisation mais contrairement à l’hypothèse classique, cette hypersensibilisation porterait sur des paramètres de la transmission dopaminergique autres que les récepteurs D2. De même nous postulons que cette hypersensibilisation se traduirait par des altérations des cascades signalétiques au niveau des cellules du striatum. Ces altérations aboutissent à des changements portant sur le récepteur nucléaire (Nur77), qui est hautement associé au système dopaminergique; l’induction de ces récepteurs déclencherait des cascades associées à la compensation ou à la genèse des DT. Matériels et méthodes: 23 femelles Cebus apella, réparties en 3 groupes: groupe halopéridol, groupe clozapine, et groupe contrôle, ont été exposées aux traitements respectifs pendant 6-36 mois. Après l’analyse comportementale, les animaux ont été décapités et leurs cerveaux isolés pour fin d’analyse. Hybridation in situ: nous avons fait appel à cette technique pour mesurer l’expression de l’ARNm de Nur77 et du neuropeptide enképhaline. Hybridation in situ double: nous avons exploités cette technique pour identifier les populations neuronales exprimant les récepteurs dopaminergiques D3 et localiser leur éventuelle induction. Autoradiographies des récepteurs dopaminergiques D1, D2 et D3 et autoradiographies des récepteurs i glutamatergiques mGluR5. Ces autoradiographies avaient pour objectif d’évaluer l’expression de ces différents récepteurs. Mutagenèse dirigée et transfection cellulaire: nous faisons appel à ces techniques pour reproduire le polymorphisme identifié au niveau de la région 3’UTR de l’ARNm Nur77 et évaluer l’impact que pourrait avoir ce polymorphisme sur la stabilité de l’ARNm Nur77 sinon sur l’expression de la protèine Nur77. Western Blot des kinases ERK 1 et 2: cette technique nous a servi comme moyen pour quantifier l’expression globale de ces kinases. Analyses statistiques: l’expression de l’ARNm Nur77 a été évaluée en utilisant l’analyse de la variance à un seul facteur (One way ANOVA). Nous avons procédé de la même façon pour mesurer l’expression des récepteurs D2, D3 et mGluR5. Résultats: le groupe des animaux traités par l’halopéridol montre une plus forte expression des récepteurs D3 par rapport aux sujets des autres groupes. Cette expression se produit au niveau des neurones de la voie directe. De plus, cette augmentation corrèle positivement avec la sévérité des DT. L’expression des récepteurs D2 et mGluR5 reste relativement inchangée entre les différents groupes, alors qu’un gradient d’expression a été observé pour le récepteur D1. Par ailleurs, Nur77 est induit par l’halopéridol, alors que son expression semble baisser chez les animaux traités par la clozapine. L’induction de l’expression de Nur77 par l’halopéridol est plus accrue chez les animaux non dyskinétiques. Les animaux traités par la clozapine démontrent une expression amoindrie de l’ARNm de Nur77 qui tend à être plus faible que l’expression de base. D’autre part, la présence du polymorphisme au niveau de la région 3’UTR semble affecter l’expression cellulaire de Nur77. Conclusion: ces résultats confortent notre hypothèse concernant l’existence d’un phénomène d’hypersensibilisation prenant place suite un traitement chronique par les antipsychotiques. Ce phénomène s’est traduit par une augmentation de l’expression des récepteurs D3 sans porter sur les récepteurs D2 tel que prôné classiquement. Cette hypersensibilisation des récepteurs D3 implique également l’existence d’un débalancement des voies striatales pouvant ainsi sous tendre l’apparition des DT. Ces résultats dévoilent ainsi un nouveau mécanisme qui pourrait contribuer à l’apparition des DT et pourraient permettre une meilleure gestion, nous l’espérons, des DT à l’échelle clinique. / Tardive dyskinesia (TD) is a potentially disabling and irreversible motor complication including all persistent, abnormal, involuntary movements, classicaly caused by the chronic therapy with typical antipsychotic drugs (haloperidol, fluphenazine). Atypical antipsychotic drugs like clozapine have been introduced because they showed little potential to induce TD, raising the hope to completely eradicate this complication. However, it has been later shown that these drugs have several serious metabolic side- effects and that some atypical molecules are as responsible as typical drugs for inducing TD. Besides, the typical drugs are still widely prescribed in a large spectrum of disorders. For all these reasons, TD still constitutes a major challenge for psychotic disorders treatments especially that the pathophysiology of TD remains elusive and therapeutics are difficult. Based on rodent experiments, it was proposed that dopamine D2 receptor hypersensitivity could be responsible for TD. However, this hypothesis lacks strong support in humans. We suggest, in this thesis, that TD is associated with the hypersensitivity of other receptors, than D2. To investigate the neurochemical basis of TD, we chronically exposed 23 adult capuchin monkeys to haloperidol (median 18.5 months, N=11) or clozapine (median 6 months, N=6). Six unmedicated animals were used as controls. Five haloperidol-treated animals developed mild TD movements, and no TD was observed in the clozapine group. Using receptor autoradiography, we measured dopamine D1, D2, D3 and mGluR5 receptor levels. We also examined the D3 receptor/preprotachykinin mRNA co-expression, and quantified enkephalin and Nur77 mRNA levels, in striatal sections. Unlike clozapine, haloperidol strongly induced dopamine D3 receptor binding sites in the anterior striatum, particularly in TD animals, and binding levels positively correlated with TD intensity. In contrast, D2 receptor binding was comparable to controls, and dopamine D1 receptor binding reduced in the anterior (haloperidol and clozapine) and posterior (clozapine) putamen. Preprotachykinin mRNA-labeled cell count was unaffected by either haloperidol or clozapine, enkephalin mRNA widely increased in all animals, but to a greater extent in TD-free animals. Nur77 mRNA levels in the caudate-putamen were strongly up regulated iv in animals exposed to haloperidol but were spared following clozapine treatment. Interestingly, within the haloperidol-treated group, TD-free animals showed higher Nur77 expression in putamen sub territories compared with dyskinetic animals. These results corroborate our hypersensitivity hypothesis, and indicate that an imbalance between the striatal pathways could contribute to the pathophysiology of TD.

A maciez da carne bovina é o resultado do processo de proteólise miofibrilar influenciado pelas calpaínas (CAPN), enzimas ativadas pelo cálcio. A calpastatina (CAST) constitui um regulador das calpaínas, atuando como substrato e degradando-se pela ação da própria calpaína. A suplementação com vitamina D3 na dieta dos animais tem alterado positivamente a maciez da carne, por obter maior absorção e deposição de cálcio nos músculos. Tendo em vista a importância desses genes para a maciez da carne em bovinos e o metabolismo de vitamina D3, o objetivo deste estudo foi verificar a expressão gênica de 1-HYD, 24-HYD, CAPN1 e CAST (Isoformas). Foram utilizados 42 machos castrados da raça Nelore que foram divididos em 6 tratamentos (7 animais por tratamento). Os animais foram alojados em baias individuais e os tratamentos foram: T1) sem suplementação de Vitamina D3 + sem sombrite; T2) sem suplementação de Vitamina D3 + com sombrite filtração de 50% de UV; T3) com 2x106UI de vitamina D3 por dois dias consecutivos pré-abate + sem sombrite; T4) com 2x106UI de vitamina D3 por dois dias consecutivos pré-abate + com sombrite filtração de 50% de UV; T5) com 2x106UI de vitamina D3 por oito dias consecutivos pré-abate + sem sombrite; 6) com 2x106UI de vitamina D3 por oito dias consecutivos pré-abate + com sombrite filtração de 50% de UV. A expressão gênica foi analisada por PCR quantitativa em tempo real. O gene 1-HYD foi mais expresso nos tratamentos com 2 e 8 dias de suplementação com vitamina D3, independente da exposição diferenciada aos raios ultravioleta. O gene 24-HYD foi mais expresso no tratamento com 8 dias de suplementação pré-abate quando comparado ao tratamento controle, não diferindo do tratamento com suplementação por 2 dias consecutivos pré-abate. Portanto os resultados não corroboram com o mecanismo de regulação das atividades das enzimas modulada de forma oposta para regular os níveis circulantes de 1,25-di-hidroxi-vitamina D3 (1,25 D). Quando a concentração de 1,25 D atinge níveis elevados um mecanismo de regulação inativa a 1,25 D e promove a diminuição da expressão de 1-HYD. Assim pode-se inferir que neste estudo esses fatores não exerceram influência sobre o gene 1-HYD que teve um aumento da expressão. A única possibilidade de verificar o aumento da expressão do gene da enzima 1-HYD parece envolver um hiperparatireoidismo secundário decorrente de hipocalcemia em algum momento do experimento. A suplementação com vitamina D3 não exerceu influência na expressão de RNAm da CAPN1, isoforma I e isoforma total da CAST. A isoforma II do gene da calpastatina foi mais expressa no tratamento com 8 dias de suplementação pré-abate, onde parece que o aumento da expressão é devido a uma sinalização intracelular indicando que a calpastatina está sendo degradada pela ativação prolongada das calpaínas, em decorrência do aumento do influxo de cálcio. As análises de associação apontam para um possível descompasso entre abundância de RNAm e atividade da protease do sistema das calpaínas no processo de amaciamento da carne. / The tenderness of beef is the result of the process of myofibrillar proteolysis influenced by calpains (CAPN), enzymes activated by calcium. The calpastatin (CAST) is a regulator of calpains, acting as a substrate and degraded by the action of calpain itself. Supplementation with vitamin D3 in the diet has changed positively on meat tenderness, to obtain greater absorption and calcium deposition in muscle. Given the importance of these genes for meat tenderness in cattle and metabolism of vitamin D3, the objective of this study was to investigate the gene expression of 1-HYD, HYD-24, CAPN1, CAST (isoforms). We used 42 Nellore steers were divided into 6 treatments (seven animals per treatment). The animals were housed in individual pens and treatments were: T1) without vitamin D3 feeding + without shadow; T2) without vitamin D3 feeding + with black - 50% filtration of UV; T3) with 2x106UI of vitamin D3 for two days consecutive slaughter + without shadow; T4) with 2x106UI vitamin D3 for two days consecutive slaughter + with black - 50% filtration of UV; T5) with 2x106UI vitamin D3 for eight days consecutive slaughter + without shading; T6) 2x106UI with vitamin D3 for eight days consecutive slaughter + with black - 50% filtration of UV. Gene expression was analyzed by quantitative real-time. The 1-HYD gene was more expressed in the treatments with 2 and 8 days of supplementation with vitamin D3, independent of differential exposure to ultraviolet rays. The 24-HYD gene was more expressed in the treatment with 8 days of pre-slaughter supplementation compared to control treatment, the treatment did not differ with supplementation for 2 consecutive days pre-slaughter. These findings do not corroborate the mechanism for regulating the activities of enzymes modulated so diametrically opposite to regulate the circulating levels of 1,25- dihydroxy vitamin D3 (1,25 D). When there is an overproduction of 1,25 D regulation mechanism inactivates 1.25 D and promotes the reduction of the expression of 1-HYD. Thus we can infer that in this study, these factors did not influence the 1-HYD gene that had increased expression. The only way to verify the increase of gene expression of the enzyme 1-HYD seems to involve a secondary hyperparathyroidism due to hypocalcemia sometime in the experiment. Supplementation with vitamin D3 did not influence the mRNA expression of CAPN1, isoform I and isoform total of CAST. The isoform II of the calpastatin gene expression was higher in treatment with 8-day preslaughter supplementation, where it appears that increased expression is due to an intracellular signaling indicating that calpastatin is degraded by prolonged activation of calpain as a result of increased influx of calcium. The association analysis point to a possible mismatch between mRNA abundance and activity of the calpain system components in these physiological conditions.

Sweden produced 215 kilotons of electronic waste in 2016. One way to reduce this amount is to repair electronic devices when they fail instead of replacing them. The company Mavenoid has a troubleshooting service in the form a web application to help troubleshoot machines like for example consumer electronics. The company desired a tool to visualize data about the usage of the troubleshooting models in their web app. The prospect was that the users who build the troubleshooting models would be able to analyse how their models are being used and how well they work with the tool. To implement the tool, a method with three phases was used. An initial preparation phase with requirements elicitation via a domain analysis and set-up of the development environment, a development phase with iterative development and requirements management with weekly meetings and an analysis phase with evaluation of the tool according to the requirements and the status of a pull request. The product met eight out of ten requirements and was of a sufficiently high quality that it could be integrated into the company’s system. The use of the library C3 resulted in a 21% increase of the web application’s build size. / Sverige producerade 2016 215 kiloton elektronikavfall. Ett sätt att minska denna mängd är att reparera elektronik när den fallerar istället för att byta ut den. Företaget Mavenoid erbjuder en tjänst i form av en React webapplikation för att underlätta felsökning av maskiner som t.ex. hemelektronik. Företaget eftersökte ett verktyg för att visualisera data om användningen av felsökningsmodellerna i sin webbapplikation. Förhoppningen var att de användare som bygger modellerna via det nya verktyget skulle kunna analysera hur deras modeller används och väl de fungerar. För att utveckla verktyget användes en metod med tre faser. En inledande förberedelsefas med kravinsamling via en domänundersökning och uppbyggnad av utvecklingsmiljö, en utvecklingsfas med iterativ utveckling och kravhantering med veckomöten och en analysfas med utvärdering av verktyget efter kraven och status på en pull-request. Produkten uppfyllde åtta utav tio av de insamlade kraven och var av tillräckligt hög kvalitet att integreras i företagets system. Användningen av biblioteket C3 medförde att webapplikationens transaktionsstorlek ökade med ca 21%

This paper analyses the sources of income measurement error in surveys with a unique dataset.We use the Austrian 2008-2011 waves of EU-SILC which provide individual information on wages,pensions and unemployment benefits from survey interviews and officially linked administrativerecords. Thus, we do not have to fall back on complex two-sample matching procedures likerelated studies. We empirically investigate four sources of measurement error, namely (i) socialdesirability, (ii) socio-demographic characteristics of the respondent, (iii) the survey design, and(iv) the presence of learning effects. We find strong evidence for a social desirability bias inincome reporting, while the presence of learning effects is mixed and depends on the income typeunder consideration. An Owen value decomposition reveals that social desirability is a majorexplanation of misreporting in wages and pensions, whereas socio-demographic characteristicsare most relevant for mismatches in unemployment benefits. / Series: INEQ Working Paper Series

B lymphopoiesis requires a network of transcription factors that orchestrate changes in gene expression amidst immunoglobulin gene rearrangement and periods of cell proliferation. Although proteins required for the function of this network have been identified, the precise mechanisms that coordinate these processes as hematopoietic progenitors differentiate into lineage-committed B cells remain unclear. Justy mice display a profound arrest of B cell development at the time of lineage commitment due to a point mutation that decreases expression of the protein Gon4-like. Previous studies suggested that Gon4-like functions to coordinate gene expression and cell division to determine cell fate, but the role of Gon4-like in B lymphopoiesis is largely unknown. Here we demonstrate that Gon4-like is required to regulate gene expression and cell cycle progression in B cell progenitors. Expression of genes required for B cell development is intact in Justy B cell progenitors, yet these cells fail to repress genes that promote the development of alternative lineages. In addition, Justy B cell progenitors are unable to upregulate genes that instruct cell cycle progression. Consistent with this, B cell progenitors from Justy mice show signs of impaired proliferation and undergo apoptosis despite containing elevated levels of activated STAT5, a transcription factor that promotes cell proliferation and survival. Genetic ablation of p53 or retroviral-mediated overexpression of pro-survival factors failed to rescue these defects. In contrast, overexpression of proteins that promote the G1/S transition of the cell cycle, including D-type cyclins, E2F2 and cyclin E, rescued pro-B cell development from Justy progenitors, an effect that was not observed upon overexpression of proteins that function during the S and G2M phases of the cell cycle. Further, overexpression of cyclin D3 led to partial restoration of gene repression in Justy pro-B cells. Notably, Gon4-like interacted with STAT5 when overexpressed in transformed cells, suggesting Gon4-like and STAT5 function together to activate expression of STAT5 target genes. Collectively, our data indicate that Gon4-like is required to coordinate gene repression and cell cycle progression during B lymphopoiesis.