Nous avons précédemment montré que l’activation du récepteur natriurétique de type C (NPR-C) par son agoniste spécifique, le C-ANP4-23, atténue l’augmentation de la prolifération des cellules du muscle lisse vasculaire (CMLV) induite par les peptides vasoactifs (Ang II, ET-1 et l’AVP). Puisque les CMLV provenant de rats spontanément hypertendus (SHR) montrent elles aussi un taux de prolifération plus élevé que leur contrôle, les CMLV de rats Wystar-Kyoto (WKY), nous avons entrepris cette étude dans le but de déterminer si C-ANP4-23 peut également diminuer le taux élevé de prolifération des CMLV de SHR et, le cas échéant déterminer les mécanismes responsables de cette réponse. Nos résultats montrent que le taux de prolifération des CMLV de SHR est significativement plus élevé que celui des CMLV de WKY et que la présence de C-ANP4-23 diminue de manière-dose dépendante le taux de prolifération des CMLV de SHR. En plus, l’expression des protéines de la phase G1 du cycle cellulaire, la cycline D1, la kinase dépendante des cyclines 2 (cdk2) et la forme phosphorylée de la protéine du rétinoblastome (pRb) est augmentée dans les CMLV de SHR comparativement aux CMLV de WKY et est atténué par C-ANP4-23. De plus, nos résultats montrent que les inhibiteurs du complexe cycline D1/cdk4 (NSC 625987) et cdk2 (NU2058) diminue le taux de prolifération élevé des CMLV de SHR. Les CMLV de SHR montrent également un taux de phosphorylation de ERK1/2 et d’AKT et est atténué par C-ANP4-23. De plus, le taux d’expression élevé des protéines cycline D1, cdk2 et pRb des CMLV de SHR est diminué par la toxine pertussis qui inactive la protéine Giα, le PD 98095, un inhibiteur de MEK de la voie des MAPK, du wortmannin, un inhibiteur de la PI3-K et finalement du losartan, un antagoniste du récepteur AT1. Ces résultats suggèrent que l’activation du récepteur NPR-C par C-ANP4-23 diminue le taux de prolifération élevé des CMLV de SHR par une régulation à la baisse des composantes du cycle cellulaire via l’inhibition de la protéine Giα et des voies signalétique MAP kinase/PI3-K. / We have previously shown that natriuretic peptide receptor-C (NPR-C) activation by C-ANP4-23 decreased the proliferation of vascular smooth muscle cells (VSMC) induced by vasoactive peptides (Ang II, ET-1 and AVP). Since, VSMC from SHR also exhibit an enhanced proliferation as compared to VSMC from WKY, we undertook the present study to investigate if C-ANP4-23 could also attenuate the enhanced proliferation of VSMC from SHR and to further explore the underlying mechanisms responsible for this response. The proliferation of VSMC from SHR was significantly increased as compared to VSMC from WKY as determined by [3H]thymidine incorporation and was attenuated by C-ANP4-23 in a concentration-dependent manner. Furthermore the expression of cyclin D1, cyclin-dependent kinase 2 (cdk2) and phosphorylated retinoblastoma protein (pRb) was enhanced in VSMC from SHR compared to WKY which was attenuated by C-ANP4-23. In addition, the inhibitor of cdk4/cyclinD1 (NSC 625987) and cdk2 (NU2058) also attenuated the enhanced proliferation of VSMC from SHR in a concentration-dependent manner. VSMC from SHR also exhibited the enhanced phosphorylation of ERK1/2 and AKT as compared to WKY which was attenuated by C-ANP4-23.. Furthermore, the enhanced expression of cyclin D1, cdk2 and pRb in VSMC from SHR were also attenuated by pertussis toxin that inactivates Giα protein, PD 98095, a MEK kinase inhibitor, wortmannin, PI3K inhibitor as well as by losartan, an AT1 receptor antagonist. These results suggest that NPR-C activation attenuates the enhanced proliferation of VSMC from SHR which may be attributed to Giα /MAP kinase/PI3K-mediated inhibition of the expression of cell cycle components.

L'endométriose concerne 8 à 10% des femmes en âge de procréer et est responsable de douleurs pelviennes chroniques et d'infertilité. Seule l'exérèse chirurgicale des lésions permet un traitement curatif de la maladie. Dans le cas de l'endométriose profonde avec atteinte rectale la chirurgie est extensive et associée à une morbidité significative. Les traitements médicaux reposent sur une hormonothérapie visant à bloquer la fonction ovarienne dont l'effet n'est que suspensif et transitoire. Il n'existe à ce jour aucun traitement ciblant les mécanismes à l'origine de la maladie. L'objectif de notre travail était d'explorer différents mécanismes potentiellement impliqués dans le développement de la maladie et d'identifier des molécules capables d'intervenir sur ces mécanismes. Dans un premier temps nous avons exploré le phénotype hyperprolifératif des cellules endométriosiques profondes et cherché un lien avec différentes voies métaboliques impliquées dans la prolifération cellulaire telles que le stress oxydant, la voie ERK et la voie Akt. Dans un deuxième temps, nous avons exploré le recrutement des cellules endométriales au sein de la cavité péritonéale au travers de l'interaction CXCR4-CXCL12. Des cultures cellulaires ont été réalisées à partir de prélèvements humains de nodules endométriosiques profonds, d'endomètre eutopique et d'endomètre sain. Des lames histologiques ont été préparées à partir de nodules endométriosiques profonds. Des prélèvements de liquide péritonéal de femmes endométriosiques et de témoins ont été congelés. La prolifération cellulaire a été étudiée par incorporation de thymidine tritiée. La production des FRO a été évaluée par spectrofluorimétrie. La voie ERK a été évaluée par western blot, ELISA et immunohistochimie. La voie Akt été évaluée par western blot et immunohistochimie. Nous avons montré un phénotype hyperprolifératif des cellules endométriosiques profondes en rapport avec une activation de la voie ERK par le biais du stress oxydant et à une activation de la voie Akt. Nous avons montré qu'un anti-oxydant (NAC), un inhibiteur de protéines kinases (A771726), un inhibiteur de Raf (sorafenib), un inhibiteur de mTOR (temsirolimus), un agoniste des cannabinoïdes (WIN 55212-2) et un anti-métabolite (5-FU) pouvaient contôler la prolifération des cellules endométriosiques profondes in vitro et la progression de nodules endométriosiques profonds implantés dans des souris Nudes. L'interaction CXCR4-CXCL12 a été étudiée par western blot, analyse de migration, cytométrie de flux et ELISA. Nous avons montré une attraction spécifique des cellules endométriosiques profondes sur-exprimant le CXCR4 par la chimiokine CXCL12 présente en quantité accrue dans le liquide péritonéal des femmes endométriosiques. En conclusion, nous avons montré que le traitement médical de l'endométriose pouvait être non hormonal et que le stress oxydant, la voie ERK et la voie Akt constituaient de nouvelles pistes thérapeutiques à évaluer dans le cadre d'essais cliniques. Nous avons également montré comment la modification constitutive des cellules de l'endomètre eutopique pouvait favoriser leur recrutement dans la cavité péritonéale.

La maladie rénale chronique (MRC) par ses conséquences organiques et psychologiques représente unenjeu majeur de santé publique. Sa physiopathologie reste mal connue, mais il est établi que toute atteinte rénale,quelle qu'en soit la cause, aboutit à une réduction du nombre de néphrons fonctionnels. Cette réductionnéphronique est responsable de processus adaptatifs complexes des néphrons sains restants pour maintenir unefonction rénale satisfaisante. Si la perte néphronique est suffisamment importante, le parenchyme rénal vas'altérer progressivement aboutissant au remplacement des néphrons sains par un tissu fibreux puis au déclin dela fonction rénale. Les mécanismes moléculaires impliqués, tant dans l'adaptation à la réduction néphronique,que dans la dégradation progressive du parenchyme rénal sont mal connus et les possibilités d'interventionsthérapeutiques limitées.La voie AKT/mTOR est une voie de signalisation intracellulaire ubiquitaire, très conservée, jouant unrôle central dans l'homéostasie cellulaire par sa fonction de régulation de la croissance, de l'apoptose et du cyclecellulaire. L'extraordinaire complexité de son mode de recrutement témoigne du rôle de carrefour de cette voie.Elle intègre des signaux multiples et très variés (facteurs de croissance, acides-aminés, niveau énergétiquecellulaire, disponibilité de l'oxygène) modulant l'anabolisme cellulaire. Notre compréhension du rôle de cettevoie dans la progression de la MRC est encore très limitée et les données sont peu nombreuses et controversées.Mon travail de thèse a consisté à évaluer, en utilisant un modèle expérimental de réduction néphroniquechez la souris, le rôle de la voie AKT/mTORC au cours de la progression des MRC.Nous résultats démontrent que AKT, et plus précisément AKT2, est une molécule essentielle àl'adaptation podocytaire aux contraintes imposées par la réduction néphronique. En appliquant plusieursmodèles de réduction néphronique à des souris génétiquement modifiées, nous avons pu établir la fonctioncruciale de cette isoforme. En effet, l'inactivation d'Akt2, soit systémique soit conditionnelle dans le podocyte,est responsable d'une hyporéactivité de la voie AKT podocytaire, d'un remodelage du cytosquelette, d'uneaugmentation de l'apoptose glomérulaire avec raréfaction podocytaire et de lésions de glomérulosclérose.Transposant nos données à la pathologie humaine, nous avons mis en évidence une activation podocytaired'AKT2 après transplantation rénale chez les patients présentant une altération importante de la fonction rénale.De façon marquante, la survenue d'une protéinurie en réponse à un traitement par sirolimus s'associait à uneperte de cette activation et à une augmentation de l'apoptose glomérulaire.Parallèlement, nous avons évalué le rôle de cette voie chez l'homme au cours d'une MRC trèsparticulière secondaire à la présence d'anticorps antiphospholipides. Cette néphropathie est caractérisée par laprésence de lésions vasculaires sévères prolifératives, hypertrophiques et progressivement obstructivesaboutissant à la destruction du parenchyme rénal. Jusqu'à maintenant, aucun lien formel n'avait été établi entre laprésence de ces anticorps et le développement des lésions vasculaires, et aucune thérapeutique n'était disponible.Nos résultats indiquent que ces anticorps sont directement pathogènes pour l'endothélium induisant l'activationde la voie AKT/mTORC. L'activation de cette voie stimule la prolifération des cellules endothéliales mais aussi ....

Resistance to cisplatin (CDDP)-based therapy is a major hurdle to the successful treatment of human ovarian cancer (OVCA) and the chemoresistant phenotype in OVCA cells is associated with Akt-attenuated, p53-mediated apoptosis. Pro-apoptotic functions of p53 involve both transcription-dependent and -independent signaling pathways and dysfunctional localization and/or inactivation of p53 contribute to the development of chemoresistance. PARC is a cytoplasmic protein regulating p53 subcellular localization and subsequent function. Little is known about the molecular mechanisms regulating PARC. Although PARC contains putative caspase-3 cleavage sites, and CDDP is known to induce the activation of caspases and calpains and induce proteasomal degradation of anti-apoptotic proteins, if and how PARC is regulated by CDDP in OVCA is unknown. Here we present evidence that CDDP promotes calpain-mediated PARC down-regulation, mitochondrial and nuclear p53 accumulation and apoptosis in chemosensitive but not resistant OVCA cells. Inhibition of Akt is required to sensitize chemoresistant cells to CDDP in a p53-dependent manner, an effect enhanced by PARC down-regulation. CDDP-induced PARC down-regulation is reversible by inhibitor of calpain but not of caspase-3 or the 26S proteasome. Furthermore, in vitro experiments confirm the ability of calpain in mediating Ca2+-dependent PARC down-regulation. The role of Ca2+ in PARC down-regulation was further confirmed as ionomycin induced PARC down-regulation in both chemosensitive and chemoresistant ovarian cancer cells. The data presented here implicates the regulation of p53 subcellular localization and apoptosis by PARC as a contributing factor in CDDP resistance in OVCA cells and Ca2+/calpain in PARC post-translational processing and chemosensitivity.

Thesis (M.Sc.). / Written for the Dept. of Human Genetics. Title from title page of PDF (viewed 2008/12/09). Includes bibliographical references.

Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2014-12-17T13:10:33Z No. of bitstreams: 2 dirceu_de_souza_melo.pdf: 1243588 bytes, checksum: e609b2371fe339ca91cc379149b3beca (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2014-12-17T13:13:48Z (GMT) No. of bitstreams: 2 dirceu_de_souza_melo.pdf: 1243588 bytes, checksum: e609b2371fe339ca91cc379149b3beca (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2014-12-17T13:13:44Z (GMT) No. of bitstreams: 2 dirceu_de_souza_melo.pdf: 1243588 bytes, checksum: e609b2371fe339ca91cc379149b3beca (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2014-12-17T13:14:26Z (GMT) No. of bitstreams: 2 dirceu_de_souza_melo.pdf: 1243588 bytes, checksum: e609b2371fe339ca91cc379149b3beca (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) / Made available in DSpace on 2014-12-17T13:14:27Z (GMT). No. of bitstreams: 2 dirceu_de_souza_melo.pdf: 1243588 bytes, checksum: e609b2371fe339ca91cc379149b3beca (MD5) license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Previous issue date: 2013 / Evid?ncias recentes sugerem que a restri??o cal?rica intensa (RCI) (>40%) exerce efeitos ben?ficos sobre o cora??o de ratos. No entanto, a maioria destes estudos avaliaram os efeitos da RCI em cora??es de ratos que iniciaram esta restri??o j? na idade adulta. Neste trabalho, investigamos as conseq??ncias de uma restri??o cal?rica de 50% desde o nascimento sobre a fun??o e morfologia card?aca de ratos adultos e avaliamos os poss?veis mecanismos envolvidos nestas adapta??es. Desde o nascimento at? a idade de 90 dias ratos RC50 tiveram sua alimenta??o restrita a 50% do consumo do grupo ad libitum (AL). Durante o per?odo de 90 dias os animais tiveram sua ingest?o alimentar, peso corporal e press?o arterial monitorados. Ap?s este per?odo, foi realizado um teste de capacidade aer?bica m?xima para avaliar indiretamente a fun??o cardiovascular global. Quarenta e oito horas ap?s este teste os animais foram eutanasiados, o sangue foi coletado para an?lise do hemat?crito, bioqu?mica s?rica e estresse oxidativo e o m?sculo s?leo retirado para an?lise do estresse oxidativo. A t?bia foi retirada para aferi??o do comprimento e f?gado, ba?o, supra-renais, test?culos e gordura viceral removidos para aferi??o do peso. O cora??o foi retirado e o ?ndice de desenvolvimento de tens?o m?xima (+dT/dt) e m?nima (-dT/dt) do mioc?rdio foram analisados pela prepara??o de cora??o isolado. Tamb?m foi realizada an?lise do estresse oxidativo card?aco e o di?metro, n?mero e densidade de cardiomi?citos, assim como os n?veis de fibrose card?aca foram obtidos atrav?s da an?lise histol?gica. Mi?citos ventriculares foram isolados para avalia??o do transiente de Ca2+ em microscopia confocal e os n?veis de fosforila??o da Akt e express?o da SERCA2 foram avaliados pela t?cnica de Western blot. Comparado ao grupo AL, os animais RC50 apresentaram menor peso corporal e de ?rg?os, menor comprimento da t?bia, menor glicemia, maior colesterol HDL, menor press?o arterial, menor estresse oxidativo card?aco, maior desempenho aer?bio e da fun??o card?aca, como mostrado pelo aumento ?dT/dt. Apesar do menor di?metro dos cardiomi?citos, ratos RC50 apresentaram aumento na rela??o cora??o/peso corporal, aumento do n?mero e densidade dos cardiomi?citos, e n?veis semelhantes de fibrose card?aca em compara??o aos animais AL. Os n?veis de fosforila??o da Akt foram superiores nos cardiomi?citos dos animais RC50 e n?o houve diferen?as significativas no transiente de Ca2+ e na express?o de SERCA2 entre os cardiomi?citos dos animais RC50 e AL. Em conjunto, estas observa??es revelaram efeitos positivos de uma RCI de 50% desde o nascimento sobre a fun??o, estrutura card?aca e vias de sinaliza??o da sobreviv?ncia de cardiomi?citos em ratos adultos. / Disserta??o (Mestrado) ? Programa Multic?ntrico de P?s-gradua??o em Ci?ncias Fisiol?gicas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2013. / ABSTRACT There has been growing evidence suggesting that a severe caloric restriction (SCR) (above 40%) leads to beneficial effects on heart rats. However, most of the reports are focused on the effects of SCR on hearts of rats which started this restriction at adulthood. In this work, we investigated the consequences of a 50% caloric restriction since birth on cardiac morphology and function in adult rats and evaluated the possible mechanisms involved in these adaptations. From birth up to the age of 90 days CR50 rats were fed restricted at 50% of the Ad Libitum group (AL). During the period of 90 days the animals had their food intake, body weight and blood pressure monitored. After this period, a maximal aerobic test was performed in order to indirectly evaluate the global cardiovascular function. Forty-eight hours after this test the animals were euthanized the blood collected for analysis of hematocrit, serum biochemistry and oxidative stress and soleus muscle collected for oxidative stress analysis. The tibia was removed for measurement of length and liver, spleen, adrenals, testes and visceral fat were removed for measurement of weight. The heart was removed and the index of velocity of myocardial contraction (+dT/dt) and relaxation (-dT/dt) was analyzed by isolated heart preparation. We also carried out analysis of cardiac oxidative stress and cardiomyocyte diameter, number, density and myocardium collagen content were obtained through histological analysis. Ventricular myocytes were isolated by standard methods in order to evaluate phosphorylated AKT levels and the expression of SERCA2a was assessed by Western blot. Compared to AL, CR50 animals had lower body and organs weight, lower tibia length, lower blood glucose, higher HDL cholesterol, lower blood pressure, lower oxidative stress in the heart and blood, increased aerobic performance and cardiac function, as shown by increased ? dT/dt. Despite the smaller cardiomyocyte diameter, CR50 rats presented increased heart/body weight ratio, increased cardiomyocyte density and number, and similar levels of myocardium collagen content compared to AL rats. AKT was hyperphosphorylated in cardiomyocytes from CR50 rats and no significant differences in Ca2+ transient and SERCA2 levels were found between cardiomyocytes of CR50 and AL rats. Collectively, these observations revealed beneficial effects of a 50% caloric restriction from birth on cadiac function, structure and signaling pathways for survival of adult rat cardimyocytes.

Administrative Justice in the CSR in the Years 1918 - 1938 within European context. For my thesis I chose as a topic 'Administrative Justice in the CSR in the Years 1918 - 1938 within European Context', as administrative justice including the protection against illegal interventions of executive power is topical for any democratic state at any time. The purpose of administrative justice is reviewing administrative acts of public administration namely by independent courts. I decided to target my thesis on the period of so-called first republic thus on the period from 28 October 1918 i.e. from establishing the independent Czechoslovak state to 30 September 1938, i.e. the signing of the Munich agreement. After establishing the independent Czechoslovak state The Act on the Supreme Administrative Court and on Solving Competence Litigations was one of the first acts passed by the National Czechoslovak Committee. This year i.e. in 2018 it has been 100 years since constituting administrative justice in the Czechoslovak Republic. However, when the Czechoslovak Republic came into being administrative justice was not formed from scratch, but by the above mentioned Act on Administrative Justice and Solving Competence Litigations Austrian administrative justice was adopted. During the first republic, however,...

Made available in DSpace on 2016-06-02T19:23:02Z (GMT). No. of bitstreams: 1 6406.pdf: 2384873 bytes, checksum: 540107ae0b5b377c4dd745fed9717832 (MD5) Previous issue date: 2014-07-12 / Universidade Federal de Minas Gerais / Menopause is associated with higher risks of metabolic changes that may compromise women s life quality. Glicemia is regulated by the liver which is responsible for glucose storage at postprandial and for glucose efflux in a fastened state. The absence or the reduction of stradiol levels cause glucose intolerance and deregulated insulin output in bloodstream, setting of the insulin resistance process (RI). Hepatic glucose regulation is directly related to the accurate control of gene expression which encodes different isoforms of oxidation proteins and glucose input proteins. Studies suggest that Resistance Training (TR) prevents RI on ovariectomized (Ovx) rats liver. However there are few molecular events that support TR. Objective: To investigate the Ovx and TR effects over protein and gene expression of biomarkers associated with insulin signalization and glucose oxidation in rats liver. Methods: Adults Sprague-Dawley were divided into 4 groups (n=6 each group): Sedentary Sham-surgical (Sham-Sed); Sedentary-Ovx (Ovx-Sed); (Sham-Tr) and (Ovx-Tr). Tr protocol included 1.1 m vertical climbing with tied weight to the rats tail. Each session consisted of 4 to 9 climbing and 2 minutes of resting between the exercises. Training was performed 3 times a week during 10 weeks. Gene expression was analysed using real time quantitative PCR and protein assays by Western Blotting technic. Results: GLUT2 gene and protein expression and PGC-1α gene expression increased significantly; and p-Akt Ser473 protein expression decreased in Ovx group. TR promoted a greater increase of PGC-1α gene expression and further repair of GLUT2 gene and protein expression and p-Akt Ser473 protein expression. Conclusions: The results show the ovariectomy promotes overexpression of molecular markers that induced RI. These findings suggest that TR may play an important role on the RI prevention in Ovx animals through gene and protein expression repairment of the glycolytic metabolism signalling molecules. / A menopausa está associada ao risco aumentado de diversas alterações metabólicas que podem comprometer a qualidade de vida. A glicemia é regulada pelo fígado o qual é responsável pelo armazenamento de glicose no período pós-prandial e pelo efluxo da glicose no jejum. A ausência ou redução dos níveis de estradiol provocam liberação desregulada de insulina na circulação sanguínea e intolerância à glicose, desencadeando o processo de resistência à insulina (RI). A regulação dos níveis de glicose hepática está diretamente relacionada ao controle preciso da expressão dos genes que codificam as diferentes isoformas de proteínas de oxidação e captação de glicose. O treinamento resistido (TR) pode prevenir a RI no fígado de ratas ovariectomizadas (Ovx). No entanto ainda há poucos eventos moleculares que fundamentam o TR no modelo experimental de menopausa. Objetivo: investigar os efeitos da Ovx e do TR sobre a expressão gênica e proteica de biomarcadores relacionados à sinalização da insulina e oxidação da glicose no fígado de ratas. Métodos: Ratas Sprague Dawley adultas foram divididas em 4 grupos (n = 6 por grupo): sham operado sedentário (Sham-Sed), Ovx sedentário (Ovx-Sed), Sham-Tr e Ovx-Tr. O protocolo TR exigiu dos animais a escalada vertical de 1,1 m com pesos atados as suas caudas. Cada sessão consistiu de 4-9 escaladas, com intervalo de 2 minutos entre as escaladas, realizados 3 vezes por semana durante 10 semanas. A análise da expressão gênica foi realizada por PCR-RT pelo método ΔΔCt e as análises proteicas pela técnica de Western Blotting. Resultados: Aumentou significativamente expressão gênica e proteica de GLUT2 e gênica de PGC-1α, também a diminuição da quantificação proteica de Akt-p(Ser473) no grupo ovariectomizados sedentário. A diminuição da expressão gênica e proteica de GLUT2, o aumento da expressão gênica de PGC-1α e proteica de Akt-p Ser473 nos grupos treinados em relação ao grupo controle e ovariectomizados. Conclusão: Os resultados demonstram que a ovariectomia promove a superexpressão de sinalizadores moleculares que induz a RI e o TR foi capaz de promover a restauração desta sinalização. Estes achados sugerem que o TR exerce efeitos notórios na modulação dos sinalizadores que podem induzir a RI em animais Ovx, por meio da restauração da expressão gênica e proteica das moléculas que sinalizam o metabolismo glicolítico.

Undersökningens syfte var att ta reda på vilka motiv och målsättningar en förskollärare hade inför en naturvetenskaplig aktivitet, samt hur dessa kom till uttryck i aktiviteten.  Undersökningen baserades på en kvalitativ ansats. Inför aktiviteten intervjuades en förskollärare. Syftet med intervjun var att synliggöra de motiv och målsättningar som fanns inför aktiviteten. Intervjun dokumenterades med hjälp av ljudupptagning och papper och penna. I den aktivitet vi observerade deltog två barn och en förskollärare. För att kunna synliggöra hur motiv och målsättningar kom till uttryck under aktiviteten videofilmades aktiviteten. Resultatet av vår undersökning visade att barns intresse var centralt i de motiv som låg till grund för aktiviteten. Resultatet visade också att förskollärarens sätt att rikta barnens uppmärksamhet mot olika fenomen var avgörande på vilket sätt barnen erövrade kunskap.

Tumor suppressor genes play a role as a growth regulator and a gatekeeper of a cell. Their inactivation is often detected in malignant tumors. Identification of novel tumor suppressor gene candidates may help to further understand tumorigenesis and aid in the discovery of a new treatment leading toward cure of cancer. This PhD research project aimed to understand functional significance of a novel tumor suppressor gene candidate, myosin IC (MYO1C) and to identify potential interaction(s) of the MYO1C protein with key components of the signaling pathways involving in cancer development. In an experimental rat model for endometrial carcinoma (EC), detailed molecular genetic analysis of a candidate tumor suppressor region located distal to the tumor protein 53 (Tp53) suggested the myosin IC gene (Myo1c) as the best potential target for deletion of the genetic material. The question arising was whether and how MYO1C could function as a tumor suppressor gene. By using qPCR, Western blot or immunohistochemistry analyses, we examined MYO1C protein level in panels of well-stratified human colorectal cancer (CRC) and EC respectively. We found that MYO1C was significantly down-regulated in these cancer materials and that for the EC panel, the observed down-regulation of MYO1C correlated with tumor stage, where tumors at more advanced stages had less expression of MYO1C. In cell transfection experiments, we found that over-expression of MYO1C significantly decreased cell proliferation, and silencing MYO1C with siRNA increased cell viability. Additionally, knockdown of MYO1C impaired the ability of cells to migrate, spread and adhere to the surface. Recent published studies suggested a potential interplay between MYO1C and the phosphoinositide 3-kinase (PI3K)/AKT pathway. To examine this hypothesis, we analyzed the expression and/or activation of components of the PI3K/AKT and RAS/ERK signaling pathways in vivo in CRC samples, and in vitro in cells transfected with the MYO1C gene expression construct or MYO1C-targeted siRNA. To identify other potential pathways/ mechanisms through which MYO1C may exert its tumor suppressor activity, we additionally performed new sets of MYO1C-siRNA knockdown experiments. At different time points post transfection, we performed microarray global gene expression experiments followed by bioinformatics analysis of the data. Altogether, the results suggested an early PI3K/AKT response to altered MYO1C expression. We additionally identified several cancer-related genes/pathways with late response to MYO1C knockdown. All things considered, the identification of MYO1C-expression impact on cell proliferation, migration, and adhesion in combination with its interplay between several cancer-related genes and signaling pathways provide further evidence for the initial hypothesis of a tumor suppressor activity of MYO1C. / Cellular, Molecular and Functional Characterization of the Tumor Suppressor Candidate MYO1C