An appropriate selection of isolated oocytes with respect to the level of folliculogenesis is one of the ways to improve current methods. For this reason, this disertation thesis was focused on research of changes in distribution of cortical granules in different subpopulations of porcine oocytes with different meiotic competence during folliculogenesis. Oocytes from small (to less than or equal to 5mm) and medium (5-9 mm) follicles, collected from selected ovaries divided according to the phase of estral cycle, were used for the experiments. The oocytes were stained with PNA conjugated with fluorescent probe fluorescein isothiocyanate. A laser confocal microscope was used for visualization of the cortical granules. The experiment revealed a significant decrease of cortical granules in peripheral region of the ooplasm with increasing meiotic competence of oocytes during maturation in vitro. It also showed that oocytes with different meiotic competence differ in the ability to undergo normal fertilization and that fertilization in vitro in oocytes with lower meiotic competence is associated to higher incidence of polyspermic oocytes. However, disruption during cortical reaction as the cause for higher incidence of polyspermy could not be confirmed.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-10-10T17:18:18Z No. of bitstreams: 1 2011 - Talita de Santana Matos.pdf: 652723 bytes, checksum: 16c30eb83b6db70231ab44534e188e6c (MD5) / Made available in DSpace on 2016-10-10T17:18:18Z (GMT). No. of bitstreams: 1 2011 - Talita de Santana Matos.pdf: 652723 bytes, checksum: 16c30eb83b6db70231ab44534e188e6c (MD5) Previous issue date: 2011-10-31 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / A study was conducted to evaluate the agronomic efficiency of nitrogen fertilizer with slow-release urea, measuring their losses by volatilization of NH3-N, N2O emission and recovery of fertilizer nitrogen applied as top dressing compared with commercioal urea to corn crop. All experiments were conducted at Embrapa Agrobiology. Firstly, the experiments were performed under controlled conditions in a greenhouse using soil layer of 0-10 cm of a haplic planosol. In the first experiment, the plastic trays were used as experimental units for evaluation of losses due to volatilization chambers with the aid of semi-open static free (SALE). Commercial urea was applied (UC), urea + KCl (UK), humic acid + urea (UH), urea + zeolitic sandstone (UZ) and urea + gypsum (UG) in two conditions of pH (5.4 and 6,5). Secondly, the other experiment was conducted using plastic pots containing the same soil samples as experimental units where they were planted three plants of Brachiaria decumbens. In this case, fertilizers were enriched with 15N, in two pH conditions too (pH 5,4 and 6,5). Treatments UZ and UK were more efficient in retaining N in the soil than the UC, with smaller losses through volatilization of NH3-N, and 20,2 and 15,8% on condition of lime and 22 and 17,2% when Liming did not occur, respectively. The UK fertilizer and UG showed overall increase of about 149 and 146% on dry biomass production at the end of the cycle on condition of pH 5,4. At pH 6,5 UG fertilizer showed yield increases of 149,3%. The accumulation of N in the plant and was 279,2 and 270,3 mg N.vaso-1 when no lime was applied to the UK and UG treatments, respectively, and the limed, 207.4 and 200,6 mg N. vaso-1 for the treatments UG and UH, respectively. Treatments UZ and UK had the highest recovery of applied N by plants, with values of 65,5 and 61,9% without lime, 60,2 and 45,7% with lime, respectively. Thirdly, other experiment was conducted at the experimental field at Embrapa Agrobiology in order to quantify the PNV, N2O emissions and efficiency of nitrogen fertilizer use (NFUE) slow-release by a corn crop on N balance Treatments consisted of field application of nitrogen fertilizers in coverage along the rows and a control treatment. Emissions of N2O were evaluated using static chambers closed. Was used fertilizers enriched with 15N in little plots for the assessment of NFUE. The treatments UZ and UK reduced the losses of N-NH3 by volatilization in approximately 18 and 14%, respectively. These losses corresponded to 32,3 and 35,7% of total N applied to soil. For N2O emissions the UK treatment showed the largest emission of N2O losses reaching values of 2.02 kgN.ha-1. The highest yield of grain were obtained by treatments that UG and UZ had a better response of grain production reaching values of 9666 and 9940 kg ha-1 respectively. To NFUE treatment UZ showed the highest values of N recovered reaching 67% of the total N applied system soil plant. / O trabalho objetivou avaliar a efici?ncia agron?mica de fertilizantes nitrogenados de libera??o lenta baseados no uso da ureia, quantificando suas perdas por volatiliza??o de NNH3 (PNV), emiss?o de N2O e a recupera??o do N-fertilizante aplicado em cobertura em compara??o com a ureia comercial na cultura de milho. Todos os esperimentos foram conduzidos na Embrapa Agrobiologia. Os primeiros experimentos foram realizados em condi??es controladas em casa de vegeta??o, utilizando solo da camada de 0-10 cm de um Planossolo H?plico. No primeiro experimento foram usadas bandejas pl?sticas como unidades experimentais para avalia??o das perdas por volatiliza??o com aux?lio de c?maras semi-aberta livre est?tica (SALE). Aplicou-se ureia comercial (UC), ureia + KCl (UK), ureia + ?cido h?mico (UH), ureia +arenito zeol?tico (UZ) e ureia +gesso agr?cola (UG) em duas condi??es de pH (5,4 e 6,5). O outro experimento utilizou vasos pl?sticos contendo amostras do mesmo solo como unidades experimentais onde foram plantados 3 plantas de Brachiaria decumbens. Neste caso os fertilizantes foram enriquecidos com 15N, tamb?m em duas condi??es de pH (pH 5,4 e 6,5). Os tratamentos UZ e UK foram mais eficientes na reten??o do N no solo do que a UC, apresentando menores perdas por volatiliza??o de N-NH3 de 20,2 e 15,8% sob condi??o de calagem e 22 e 17,2% quando sem calagem, respectivamente. Os fertilizantes UK e UG apresentaram aumento total de aproximadamente 149 e 146% na produ??o de biomassa seca ao final do ciclo da cultura em condi??o de pH 5,4. Em pH 6,5 o fertilizante UG apresentou aumento de rendimento de 149,3%. O ac?mulo de N na planta foi de 279,2 e 270,3 mg N.vaso-1 quando n?o foi aplicado calagem para os tratamentos UK e UG, respectivamente e quando com calagem, 207,4 e 200,6 mg N.vaso-1 para os tratamentos UG e UH, respectivamente. Os tratamentos UZ e UK apresentaram maior recupera??o pelas plantas do N aplicado, com valores de 65,5 e 61,9% sem calagem e 60,2 e 45,7% com calagem, respectivamente. O outro experimento foi realizado no campo na ?rea experimental da Embrapa Agrobiologia com objetivo de quantificar as perdas por volatilza??o de am?nio, as emiss?es de N2O e a efici?ncia do uso de fertilizantes nitrogenados (EUFN) de libera??o lenta pela cultura de milho, no balan?o de N. Os tratamentos consistiram da aplica??o no campo em cobertura dos fertilizantes nitrogenados ao lado da linha de plantio e um tratamento controle. As emiss?es de N2O foram avaliadas utilizando-se c?maras est?ticas fechadas. Foi utilizado fertilizantes enriquecidos com 15N em microparcelas para a avalia??o da EUFN. Os tratamentos UZ e UK reduziram as perdas de N-NH3 por volatiliza??o em aproximadamente 18 e 14%, respectivamente. Estas perdas corresponderam a 32,3 e 35,7% do total de N aplicado no solo. Para as emiss?es de N2O o tratamento UK foi o que apresentou maiores perdas por emiss?o de N2O atingindo valores de 2,02 kg N.ha-1. As maiores produtividades de gr?o foram obtidas pelos tratamentos UG e UZ que apresentaram melhor resposta de produ??o de gr?os atingindo valores de 9.666 e 9.940 kg.ha-1, respectivamente. Para EUFN o tratamento UZ apresentou o maior valor de N recuperado chegando a 67% do total do N aplicado no sistema solo-planta.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-07-03T12:22:22Z No. of bitstreams: 1 SloanyGuedesMedeiros_DISSERT.pdf: 4950272 bytes, checksum: da30052a82bfbac8284e64d77d53e6f5 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-07-05T15:57:09Z (GMT) No. of bitstreams: 1 SloanyGuedesMedeiros_DISSERT.pdf: 4950272 bytes, checksum: da30052a82bfbac8284e64d77d53e6f5 (MD5) / Made available in DSpace on 2017-07-05T15:57:09Z (GMT). No. of bitstreams: 1 SloanyGuedesMedeiros_DISSERT.pdf: 4950272 bytes, checksum: da30052a82bfbac8284e64d77d53e6f5 (MD5) Previous issue date: 2016-08-25 / Comp?sitos de matriz met?lica de n?quel refor?ados por dispers?o de part?culas de NbC e WC s?o materiais promissores por apresentarem elevados n?veis de resist?ncia mec?nica e de resist?ncia ao desgaste. O objetivo principal deste trabalho foi avaliar a microdureza, microestrutura e o comportamento dilatom?trico dos comp?sitos Ni-NbC e dos Ni-WC. Os comp?sitos foram obtidos atrav?s da t?cnica de metalurgia do p? utilizando a moagem de alta energia para proporcionar a dispers?o da fase dura de NbC e WC na matriz de n?quel. O moinho utilizado foi do tipo attritor, com velocidade de 850 rpm durante 2 horas, via ?mido. Os p?s dos comp?sitos foram preparados nas composi??es de 5%, 10%, 15% em massa de NbC e mesmas composi??es para o refor?ado com WC, estes foram granulados utilizando a t?cnica de granula??o em tambor. P?s granulados e n?o-granulados foram compactados a frio utilizando press?o de 600 MPa em matriz uniaxial. A densidade dos compactados ? verde e dos sinterizados foi calculada pelo m?todo geom?trico. Testes de microdureza e an?lise microestrutural foram realizados nas amostras sinterizadas. Durante os ensaios em dilat?metro foram utilizadas as seguintes condi??es: (i) 25?C a 1200?C; (ii) 25?C a 1300?C, ambos os par?metros com patamar isot?rmico de 1 h e taxa de aquecimento de 10?C/min; (iii) 25?C a 1200?C com patamar isot?rmico de 2 h e a mesma taxa de aquecimento. Os resultados mostraram que as amostras sinterizadas de n?quel sem adi??o de refor?os apresentaram maior densifica??o e maiores valores de retra??o quando preparadas a partir da granula??o em tambor. As imagens por MEV das misturas mo?das de Ni-NbC comprovaram certa homogeneiza??o dos p?s, assim como a incorpora??o mec?nica do refor?o cer?mico na matriz de n?quel, entretanto os resultados para Ni-WC n?o indicaram homogeneidade e pouca incorpora??o mec?nica. As amostras sinterizadas de Ni-NbC para todas as composi??es apresentaram maiores valores de densidade na condi??o de sinteriza??o (iii), enquanto que as de Ni-WC obtiveram os menores valores de densidade relativa. Com rela??o ?s imagens obtidas por MEV, os comp?sitos sinterizados com 5% de carbetos foram as que apresentam melhor aspecto microestrutural quanto ? porosidade. Os comp?sitos Ni-NbC10% e Ni-WC15% apresentaram-se com microestrutura mais porosa e com o aparecimento de ?clusters?. A inser??o de NbC na matriz de n?quel apresentou maiores resultados de microdureza do que com a inser??o de WC, entretanto os comp?sitos Ni-WC apresentaram maior homogeneidade nos resultados com desvios padr?es similares para todas as composi??es. / Nickel matrix composites reinforced by dispersed NbC or WC particles are promising materials due to high mechanical strength and wear resistance. The objective of this study was to evaluate the dilatometric behavior, microstructure and microhardness of Ni-NbC and Ni- WC composites. The composites were obtained by powder metallurgy using high energy milling to provide the dispersion of the hard NbC or WC particles in the nickel matrix. An attritor with speed 850 rpm was used to wet mill and mix the powder mixtures for 2 hours. The powders were prepared with 5%, 10% or 15% (wt.%) of NbC or WC, and subsequently drum granulated. Both granulated and non-granulated powders were uniaxially cold pressed under 600 MPa. The density of green and sintered samples was measured by the geometric method. Microhardness tests and microstructural analyses were performed on the sintered samples. For dilatometric tests, the following conditions were employed: (i) 25?C to 1200?C; (ii) 25?C to 1300?C, both parameters with isothermal step of 1 h and heating rate of 10 ?C/min; (iii) 25 ? C to 1200 ? C with isothermal step of 2 h and the same heating rate. The results showed that the sintered nickel samples without addition of reinforcements depicted major densification and high retraction when drum granulated. The SEM images of milled mixtures of Ni-NbC confirmed a certain degree of homogenization and the mechanical incorporation of the ceramic particles in the matrix On the other hand, the results for Ni-WC did indicate non-homogeneity and little mechanical incorporation. The sintered samples of all compositions of Ni-NbC showed higher values of density under sintering conditions (iii), whereas Ni-WC depicted the lowest relative density values. With respect to the images obtained by SEM, the sintered composite with 5% of carbides presented better microstructural characteristics regarding porosity. The Ni-NbC10% and Ni-WC15% composites presented porous microstructure and clusters. The addition of NbC in nickel matrix showed higher microhardness results than the one with WC, although Ni-WC composites showed higher homogeneity.

Made available in DSpace on 2014-12-17T14:07:20Z (GMT). No. of bitstreams: 1 MariaRPF_TESE.pdf: 4797522 bytes, checksum: 8d9626466b01e2425f6ea6036398adcf (MD5) Previous issue date: 2014-01-24 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Carbide reinforced metallic alloys potentially improve some important mechanical properties required for the overall use of important engineering materials such as steel and nickel. Nevertheless, improved performance is achieved not only by composition enhancement but also by adequate processing techniques, such as novel sintering methods in the case of powder metallurgy. The method minimizes energy losses in addition to providing uniform heating during sintering. Thus, the general objective of this study was to evaluate the density, hardness, flexural strength, dilatometric behavior and to analyze the microstructure of metal matrix composites based nickel with addition of carbides of tantalum and / or niobium when sintered in a conventional furnace and Plasma assisted debinding and sintering (PADS). Initially, were defineds best parameters of granulation, screening and mixing procedure. After, mixtures of carbonyl Ni and 5%, 10% and 15 wt.% NbC and TaC were prepared in a Y-type mixer under wet conditions during 60 minutes. The mixtures were then dried and granulated using 1.5 wt. % paraffin diluted in hexane. Granulates were cold pressed under 600 MPa. Paraffin was then removed from the pressed pellets during a pre-sintering process carried out in a tubular furnace at 500 ?C during 30 min. The heating rate was 3 ?C/min. The pellets were then sintered using either a plasma assisted reactor or a conventional resistive tubular furnace. For both methods, the heating rate was set to 8 ?C/min up to 1150 ?C. The holding time was 60 minutes. The microstructure of the sintered samples was evaluated by SEM. Brinell hardness tests were also carried out. The results revealed that higher density and higher hardness values were observed in the plasma-assisted sintered samples. Hardness increased with the concentration of carbides in the Ni-matrix. The flexural strength also increased by adding the carbides. The decline was larger for the sample with addition of 5% 5% TaC and NbC. In general, compositions containing added carbide 10% showed less porous and more uniform distribution of carbides in the nickel matrix microstructural appearance. Thus, both added carbide and plasma sintering improved density, hardness, flexural strength and microstructural appearance of the composites / Ligas met?licas refor?adas por carbetos melhoram potencialmente algumas propriedades mec?nicas necess?rias para a utiliza??o de importantes materiais de engenharia, tais como o a?o e o n?quel. No entanto, o desempenho ? conseguido n?o apenas pela melhoria de composi??o, mas tamb?m por t?cnicas de processamento adequadas, tais como, novos m?todos de sinteriza??o, no caso da metalurgia do p?. Desta forma, o objetivo do presente trabalho foi avaliar a dureza, resist?ncia ? flex?o, comportamento dilatom?trico e analisar a microestrutura de comp?sitos de matriz met?lica base n?quel com adi??o de carbetos de t?ntalo e/ou ni?bio quando sinterizados em forno tubular convencional e Plasma assisted debinding sintering (PADS). Os carbetos (5%, 10% e 15% em massa) foram misturados ao p? de n?quel carbonila via ?mido com aux?lio do misturador Y adaptado durante 1h. Ap?s secagem, as misturas foram submetidas ao processo denominado de granula??o em tambor. Utilizou-se 1,5% de parafina (% massa) dilu?da em hexano. Os p?s granulados foram compactados a frio utilizando press?o de 600 MPa. Antes da sinteriza??o a uma taxa de 8 ?C/min com patamar 1h na temperatura de 1150?C tanto em forno tubular quanto em reator PADS, as amostras foram pr?-sinterizadas em forno tubular para extra??o da parafina a uma taxa de 3 ?C/min com patamar 30 min em 500 ?C. A dureza avaliada foi a Brinell e a an?lise microestrutural por MEV. Os resultados mostraram que as amostras sinterizadas assistidas por plasma apresentaram dureza superior ?quelas sinterizadas em forno convencional. As imagens por MEV comprovaram esses maiores valores de durezas, uma vez que a matriz apresentou-se mais densificada. Com rela??o ? adi??o dos carbetos, a dureza aumentou com o aumento da concentra??o dos mesmos. A resist?ncia ? flex?o tamb?m aumentou ao adicionar os carbetos. A retra??o foi maior para a amostra com adi??o de 5% de TaC e 5% NbC. De maneira geral, as composi??es com adi??o de 10% de carbetos apresentaram um aspecto microestrutural menos poroso e com uma distribui??o mais uniforme dos mesmos na matriz de n?quel. Assim, tanto a adi??o de carbetos quanto a sinteriza??o com aux?lio do plasma melhoraram a dureza e o aspecto microestrutural dos comp?sitos

Eukaryotic initiation factor 4E (eIF4E) is a key part of initiation and regulation of translation in human cells. Three members of human eIF4E proteins have been characterized: eIF4E1, eIF4E2 and eIF4E3. Cellular stress causes translation initiation inhibition followed by disassembly of the polysomes, those processes are accompanied by the assembly of cytoplasmic RNA granules, called stress granules (SG). Stress granules are dynamic structures whose composition may vary depending on the cell type and the stress stimulus. In this study, human cells were subjected to the following stress conditions: high temperature (HS), sodium arsenite (AS) or hypoxia. Using fluorescence microscopy, pairs of human translational initiation factors from the 4E protein family were visualized and their localization to SG was assessed with one GFP- 4E incorporated in the stable cell line and the other one detected endogenously. Here we show eIF4E1 being a part of all the SGs, both in HS and AS conditions. Next, the eIF4E1 and eIF4E3 proteins together form more SGs than proteins eIF4E1, respectively eIF4E3, with eIF4E2. And last, that the presence of the particular 4E protein has no effect on the composition of SGs. Furthermore, selected groups of proteins were assessed for their potential to localize to the SGs under HS...

Das plastidäre Genexpressionssystem höherer Pflanzen ist hochkomplex und differiert beträchtlich von dem prokaryotischer Vorfahren. Jeder einzelne Schritt der RNA-Prozessierung und Translation ist abhängig von nukleär kodierten, posttranslational in den Chloroplasten importierten Proteinen, insbesondere RNA-Bindeproteinen, wie den chloroplastidären Ribonukleoproteinen (cpRNP). Ein wichtiger und im Fokus dieser Arbeit stehender Vertreter ist CP29A, welcher ein breites Bindespektrum aufweist und in vivo mit einer Vielzahl von Transkripten interagiert. Frühere Studien belegen dennoch, dass ein Knockout von CP29A unter Standardkultivierungsbedingungen nicht in einem makroskopisch vom Wildtyp differierenden Phänotyp resultiert. Unter Kältestress hingegen ist CP29A für die Entwicklung photosynthetisch aktiver Chloroplasten essenziell. Zur tiefergehenden Charakterisierung der molekularen Funktion von CP29A wurde in der vorliegenden Arbeit eine genomweite Transkriptomanalyse der cp29a-Mutante durchgeführt. Es konnte erstmals gezeigt werden, dass CP29A bereits unter Standardkultivierungsbedingungen das Spleißen vieler plastidärer Introns unterstützt. Kälteinduziert weist das phänotypisch auffällige Gewebe der cp29a-Mutante eine globale Beeinträchtigung der Genexpression sowie massive Defekte der plastidären mRNA-Prozessierung auf. Da die Funktionalität von Proteinen substanziell von ihrer Lokalisation abhängig ist, wurden antikörperbasierte mikroskopische Lokalisationsstudien durchgeführt. Diese dokumentieren, dass CP29A kältestressinduziert zu dynamischen Granula lokalisiert, welche separiert von den plastidären Nukleoiden vorliegen und mit einem häufig in stressinduzierten Granula detektierten Protein kolokalisieren. / The plastid gene expression system in higher plants is highly complex and differs significantly from the gene expression system of the prokaryotic ancestors. Each individual step of RNA-processing and translation is dependent on nuclear-encoded RNA-binding proteins, such as chloroplast ribonucleoproteins (cpRNP), which are imported post-translationally into the chloroplast. An important representative is CP29A, which has a broad binding spectrum and interacts with a large number of transcripts in vivo. Earlier studies show that, while a knockout of CP29A under standard-cultivation-conditions does not result in a macroscopically different phenotype, under cold stress conditions CP29A is essential for the development of photosynthetically active chloroplasts. For a more detailed characterization of the molecular function of CP29A, a genome-wide transcriptome analysis of the cp29a mutant was carried out. It could be shown for the first time that CP29A already supports the splicing of many chloroplast introns under standard-cultivation-conditions. Cold-induced the phenotypically noticeable mutant tissue shows a global impairment of gene expression and massive defects in plastid mRNA processing. Since the functionality of proteins is substantially dependent on their localization, antibody-based microscopic localization studies were carried out. They reveal that cold stress-induced CP29A localizes to dynamic granules, which are separate from the plastid nucleoids and colocalize with a protein commonly detected in stress-induced granules.

The trafficking of insulin secretory granules(SGs) of pancreatic b-cells is a tightly controlled complex network. Increasing evidence indicates that the cortical actin cytoskeleton modulates the mobility and exocytosis of SGs,yet the mechanisms anchoring SGs to the cytoskeleton is not completely understood.It has been shown by Ort et al.(2000,2001) that the cytoplasmic tail of an intrinsic membrane protein of the SGs named ICA512/IA-2 binds the PDZ domain of b2-syntrophin,which in turn binds to the F-actin-binding protein utrophin. These data also indicate that stimulation of SG exocytosis affects the phosphorylation of b2-syntrophin,hence altering its binding to ICA512.Therefore a model was proposed whereby SGs are anchored to the actin cytoskeleton through the ICA512/b2-syntrophin complex, whose dynamics are regulated by phosphorylation.To test this model GFP-b2-syntrophin stable INS-1 cell clones were generated.GFP-b2-syntrophin expression and localization pattern were similar to those of the endogenous protein. Electron microscopy showed that in GFP-b2-syntrophin INS-1 cells the number of SGs with a pear-like shape was increased relative to control cells. Insulin content and stimulated secretion were increased in three GFP-â2-syntrophin INS-1 cell clones,compared to non-transfected INS-1 cells and INS-1 cells expressing GFP. These increments correlated with the different expression levels of GFP-b2-syntrophin in the three GFP-b2-syntrophin INS-1 cell clones. These findings support the hypothesis that b2-syntrophin regulates the trafficking and exocytosis of SGs by modulating their tethering to the actin cytoskeleton.In order to confirm the proposed model, the phosphorylation of b2-syntrophin was investigated in more detail. Similar to endogenous b2-syntrophin,GFP-b2-syntrophin underwent Ca2+-dependent and okadaic acid-sensitive dephosphorylation upon stimulation of insulin secretion. Stimulation-dependent dephosphorylation was confirmed by immunoprecipitation of 32P-labeled GFP-b2-syntrophin.Mass spectrometry of immunoprecipitated GFP-b2-syntrophin allowed the identification of four serine-phosphorylation sites (S75,S90,S213,S373) that could affect the binding to ICA512.Mutants,in which all four phosphoserines, were replaced by either asp or ala to mimic(S/D) or prevent(S/A) phosphorylation were expressed in INS-1 cells. All S/D mutants retained a cortical localization,but by immunoblotting the pattern of the S75D allele differed from wild type and all other S/D alleles.Conversely, all S/A alleles were diffused cytosolically, except S213A,which was still restricted to the cortex. Finally, pull down assays showed increased binding of ICA512 to the S75A and S90D alleles compared to wild type b2-syntrophin,while the opposite was observed with the S75D and S90A mutants.Additionally,both the S75 and the S213 allele conform a consensus for phosphorylation by Cdk5,which is known to modulate insulin secretion. The phosphorylation of GFP-b2-syntrophin and particularly the S75 allele by Cdk5 was exhibited with pharmacological inhibitors,by in vitro phosphorylation and by RNAi. Taken together, these findings are consistent with the model by which phosphorylation of b2-syntrophin modulates the tethering of SGs to the cytoskeleton, and thereby their mobility and exocytosis. Specifically, the data of this thesis suggest that Cdk5-dependent phosphorylation of the S75 site of GFP-b2-syntrophin facilitates insulin secretion by reducing the interaction of b2-syntrophin with ICA512,thereby decreasing the actin cytoskeleton constrain on SG mobility. This process could occur in combination with the phosphatase-dependent dephosphorylation of b2-syntrophin at phosphosites other than S75. / Der Transport Insulin-gefüllter sekretorische Granula(SG) ist ein streng kontrollierter komplexer Prozess.Es gibt vermehrt Beweise,dass das kortikale Actinzytoskelett die Ausschüttung der SGs beeinflusst.Bisher ist der Mechanismus der Verankerung von SGs am Zytoskelett noch nicht vollständig aufgeklärt.Ort et al.(2000,2001) haben gezeigt,daß der zytosoplasmatische Teil des trans-membranen SG-Proteins ICA512 mit der PDZ-Domäne von b2-Syntrophin interagiert.Dieses Protein bindet das F-Actin-Bindeprotein Utrophin.Die Ergebnisse zeigen außerdem,daß durch Stimulation der SG-Exozytose der Phosphorilierungsstatus von b2-Syntrophin beeinflusst wird,woraus ein verändertes Bindungsvermögen zu ICA512 resultiert.Es wurde ein Funktionsmodel vorgestellt,in dem sich SGs durch die Interaktion des ICA512/b2-Syntrophin Komplexes an das Actinzytoskelett binden.Dabei wird die Bindedynamik durch Phosphorilierung reguliert.Um dieses Model zu etablieren,wurden stabile GFP-b2-Syntrophin produzierende INS-1-Zellklone erzeugt.Die zelluläre Lokalisation und das Expressionsmuster von GFP-b2-Syntrophin stimmen mit dem des endogenen Proteins überein.Elektronenmikroskopie zeigte eine größe Anzahl oval-verformter SGs in GFP-b2-Syntrophin INS-1-Zellen im Vergleich zu Kontrollzellen.Verglichen mit nicht-transfizierten INS-1 Zellen waren in drei GFP-b2-Syntrophin INS-1-Zellklonen der Insulingehalt der Zellen und die stimulierte Insulinsekretion erhöht.Die Werte korrelierten mit den unterschiedlichen GFP-b2-Syntrophin Expressionsmengen der Klone.Diese Ergebnisse untermauern die Hypothese,daß b2-Syntrophin den Transport und die Sekretion der SGs durch Modulation ihres Bindevermögens an Actin reguliert.Um das postulierte Model genauer zu prüfen,wurde die Phosphorilierung von b2-Syntrophin detaillierter untersucht.Das GFP-Protein wurde,ähnlich dem endogenen b2-Syntrophin,durch Stimulation der Insulinausschüttung dephosphoriliert.Diese Dephosphorilierung ist Ca2+-abhängig und Okadeinsäuresensitiv.Die stimulationsabhängige Dephosphorilierung wurde durch Immunoprezipitation von 32P-markiertem GFP-b2-Syntrophin bestätigt.Massenspektrometrie des präzipitierten Proteins ermöglichte die Identifikation von vier Serin-Phosphorilierungsstellen(S75,S90,S213,S373),welche die Bindung zu ICA512 beeinflussen könnten.Mutanten,in denen die vier Phosphoserine durch Asp beziehungsweise Ala ersetzt wurden,um entweder eine Phosphorilierung(S/D) oder Dephosphorilierung(S/A) nachzuahmen,wurden in INS-1-Zellen exprimiert.Alle S/D Mutanten blieben kortikal lokalisiert.Das Expressionsmuster des S75D Allels unterschied sich jedoch von denen des Wild-Typs(wt).Im Gegensatz dazu waren alle S/A Allele zytosolisch verteilt.Eine Ausnahme bildete S213A,das an der Zellkortex lokalisiert blieb.Im Vergleich zu wt b2-Syntrophin zeigten PullDown-Assays eine erhöhte Bindung von ICA512 zu den S75A und S90D Allelen.Das Gegenteil konnte für die S75D und S90A Mutanten nachgewiesen werden.S75,S90 und S213 sind in einer Konsensussequenz für Cdk5-Phosphorilierung enthalten.Diese Kinase kann die Insulinsekretion regulieren.Die Phosphorilierung von b2-Syntrophin,insbesondere des S75 Allels durch Cdk5 wurde durch pharmakologische Inhibitoren,in vitro-Phosphorilierung und RNAi demonstriert.Zusammenfassend stimmen diese Erkenntnisse mit dem Model überein,daß die Phosphorilierung von b2-Syntrophin die Vernetzung von SGs mit Actin und dadurch deren Mobilität und Exozytose moduliert.Im Speziellen postulieren die Ergebnisse dieser Arbeit eine Cdk5-abhängige Phosphorilierung der S75 Stelle des b2-Syntrophins.Durch eine verminderte Interaktion von b2-Syntrophin und ICA512 erleichtert diese Mutante vermutlich die Insulinsekretion,da der Einfluss des Actinzytoskeletts auf die Granulamobilität vermindert ist.Dieser Prozess ereignet sich möglicherweise in Kombination mit einer Dephosphorilierung des b2-Syntrophins.in Kombination mit einer Dephosphorilierung des b2-Syntrophins.

The trafficking of insulin secretory granules(SGs) of pancreatic b-cells is a tightly controlled complex network. Increasing evidence indicates that the cortical actin cytoskeleton modulates the mobility and exocytosis of SGs,yet the mechanisms anchoring SGs to the cytoskeleton is not completely understood.It has been shown by Ort et al.(2000,2001) that the cytoplasmic tail of an intrinsic membrane protein of the SGs named ICA512/IA-2 binds the PDZ domain of b2-syntrophin,which in turn binds to the F-actin-binding protein utrophin. These data also indicate that stimulation of SG exocytosis affects the phosphorylation of b2-syntrophin,hence altering its binding to ICA512.Therefore a model was proposed whereby SGs are anchored to the actin cytoskeleton through the ICA512/b2-syntrophin complex, whose dynamics are regulated by phosphorylation.To test this model GFP-b2-syntrophin stable INS-1 cell clones were generated.GFP-b2-syntrophin expression and localization pattern were similar to those of the endogenous protein. Electron microscopy showed that in GFP-b2-syntrophin INS-1 cells the number of SGs with a pear-like shape was increased relative to control cells. Insulin content and stimulated secretion were increased in three GFP-â2-syntrophin INS-1 cell clones,compared to non-transfected INS-1 cells and INS-1 cells expressing GFP. These increments correlated with the different expression levels of GFP-b2-syntrophin in the three GFP-b2-syntrophin INS-1 cell clones. These findings support the hypothesis that b2-syntrophin regulates the trafficking and exocytosis of SGs by modulating their tethering to the actin cytoskeleton.In order to confirm the proposed model, the phosphorylation of b2-syntrophin was investigated in more detail. Similar to endogenous b2-syntrophin,GFP-b2-syntrophin underwent Ca2+-dependent and okadaic acid-sensitive dephosphorylation upon stimulation of insulin secretion. Stimulation-dependent dephosphorylation was confirmed by immunoprecipitation of 32P-labeled GFP-b2-syntrophin.Mass spectrometry of immunoprecipitated GFP-b2-syntrophin allowed the identification of four serine-phosphorylation sites (S75,S90,S213,S373) that could affect the binding to ICA512.Mutants,in which all four phosphoserines, were replaced by either asp or ala to mimic(S/D) or prevent(S/A) phosphorylation were expressed in INS-1 cells. All S/D mutants retained a cortical localization,but by immunoblotting the pattern of the S75D allele differed from wild type and all other S/D alleles.Conversely, all S/A alleles were diffused cytosolically, except S213A,which was still restricted to the cortex. Finally, pull down assays showed increased binding of ICA512 to the S75A and S90D alleles compared to wild type b2-syntrophin,while the opposite was observed with the S75D and S90A mutants.Additionally,both the S75 and the S213 allele conform a consensus for phosphorylation by Cdk5,which is known to modulate insulin secretion. The phosphorylation of GFP-b2-syntrophin and particularly the S75 allele by Cdk5 was exhibited with pharmacological inhibitors,by in vitro phosphorylation and by RNAi. Taken together, these findings are consistent with the model by which phosphorylation of b2-syntrophin modulates the tethering of SGs to the cytoskeleton, and thereby their mobility and exocytosis. Specifically, the data of this thesis suggest that Cdk5-dependent phosphorylation of the S75 site of GFP-b2-syntrophin facilitates insulin secretion by reducing the interaction of b2-syntrophin with ICA512,thereby decreasing the actin cytoskeleton constrain on SG mobility. This process could occur in combination with the phosphatase-dependent dephosphorylation of b2-syntrophin at phosphosites other than S75. / Der Transport Insulin-gefüllter sekretorische Granula(SG) ist ein streng kontrollierter komplexer Prozess.Es gibt vermehrt Beweise,dass das kortikale Actinzytoskelett die Ausschüttung der SGs beeinflusst.Bisher ist der Mechanismus der Verankerung von SGs am Zytoskelett noch nicht vollständig aufgeklärt.Ort et al.(2000,2001) haben gezeigt,daß der zytosoplasmatische Teil des trans-membranen SG-Proteins ICA512 mit der PDZ-Domäne von b2-Syntrophin interagiert.Dieses Protein bindet das F-Actin-Bindeprotein Utrophin.Die Ergebnisse zeigen außerdem,daß durch Stimulation der SG-Exozytose der Phosphorilierungsstatus von b2-Syntrophin beeinflusst wird,woraus ein verändertes Bindungsvermögen zu ICA512 resultiert.Es wurde ein Funktionsmodel vorgestellt,in dem sich SGs durch die Interaktion des ICA512/b2-Syntrophin Komplexes an das Actinzytoskelett binden.Dabei wird die Bindedynamik durch Phosphorilierung reguliert.Um dieses Model zu etablieren,wurden stabile GFP-b2-Syntrophin produzierende INS-1-Zellklone erzeugt.Die zelluläre Lokalisation und das Expressionsmuster von GFP-b2-Syntrophin stimmen mit dem des endogenen Proteins überein.Elektronenmikroskopie zeigte eine größe Anzahl oval-verformter SGs in GFP-b2-Syntrophin INS-1-Zellen im Vergleich zu Kontrollzellen.Verglichen mit nicht-transfizierten INS-1 Zellen waren in drei GFP-b2-Syntrophin INS-1-Zellklonen der Insulingehalt der Zellen und die stimulierte Insulinsekretion erhöht.Die Werte korrelierten mit den unterschiedlichen GFP-b2-Syntrophin Expressionsmengen der Klone.Diese Ergebnisse untermauern die Hypothese,daß b2-Syntrophin den Transport und die Sekretion der SGs durch Modulation ihres Bindevermögens an Actin reguliert.Um das postulierte Model genauer zu prüfen,wurde die Phosphorilierung von b2-Syntrophin detaillierter untersucht.Das GFP-Protein wurde,ähnlich dem endogenen b2-Syntrophin,durch Stimulation der Insulinausschüttung dephosphoriliert.Diese Dephosphorilierung ist Ca2+-abhängig und Okadeinsäuresensitiv.Die stimulationsabhängige Dephosphorilierung wurde durch Immunoprezipitation von 32P-markiertem GFP-b2-Syntrophin bestätigt.Massenspektrometrie des präzipitierten Proteins ermöglichte die Identifikation von vier Serin-Phosphorilierungsstellen(S75,S90,S213,S373),welche die Bindung zu ICA512 beeinflussen könnten.Mutanten,in denen die vier Phosphoserine durch Asp beziehungsweise Ala ersetzt wurden,um entweder eine Phosphorilierung(S/D) oder Dephosphorilierung(S/A) nachzuahmen,wurden in INS-1-Zellen exprimiert.Alle S/D Mutanten blieben kortikal lokalisiert.Das Expressionsmuster des S75D Allels unterschied sich jedoch von denen des Wild-Typs(wt).Im Gegensatz dazu waren alle S/A Allele zytosolisch verteilt.Eine Ausnahme bildete S213A,das an der Zellkortex lokalisiert blieb.Im Vergleich zu wt b2-Syntrophin zeigten PullDown-Assays eine erhöhte Bindung von ICA512 zu den S75A und S90D Allelen.Das Gegenteil konnte für die S75D und S90A Mutanten nachgewiesen werden.S75,S90 und S213 sind in einer Konsensussequenz für Cdk5-Phosphorilierung enthalten.Diese Kinase kann die Insulinsekretion regulieren.Die Phosphorilierung von b2-Syntrophin,insbesondere des S75 Allels durch Cdk5 wurde durch pharmakologische Inhibitoren,in vitro-Phosphorilierung und RNAi demonstriert.Zusammenfassend stimmen diese Erkenntnisse mit dem Model überein,daß die Phosphorilierung von b2-Syntrophin die Vernetzung von SGs mit Actin und dadurch deren Mobilität und Exozytose moduliert.Im Speziellen postulieren die Ergebnisse dieser Arbeit eine Cdk5-abhängige Phosphorilierung der S75 Stelle des b2-Syntrophins.Durch eine verminderte Interaktion von b2-Syntrophin und ICA512 erleichtert diese Mutante vermutlich die Insulinsekretion,da der Einfluss des Actinzytoskeletts auf die Granulamobilität vermindert ist.Dieser Prozess ereignet sich möglicherweise in Kombination mit einer Dephosphorilierung des b2-Syntrophins.in Kombination mit einer Dephosphorilierung des b2-Syntrophins.

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