Pflanzen erkennen potentielle Pathogene anhand von konservierten Mikroben-assoziierten molekularen Mustern (MAMPs) welche sie über membranlokalisierte Rezeptoren wahrnehmen. Der durch diese Rezeptoren aktivierte Signalweg spielt eine wesentliche Rolle in der pflanzlichen angeborenen Immunität. Das Binden eines MAMPs an die oberflächenexponierten Ektodomänen der Rezeptoren führt typischerweise dazu, dass diese homo- oder heteromere Komplexe bilden. Diese Komplexe können aus rezeptorartigen Kinasen (RLKs), rezeptorartigen Proteinen (RLPs) sowie aus rezeptorartigen zytoplasmatischen Kinasen (RLCKs), welche keine extrazelluläre Domäne zur Ligandenbindung besitzen, bestehen. Der Fokus dieser Arbeit liegt auf einem möglichen heteromeren Signalkomplex der unteranderem aus der lysinhaltigen-Motiv (LysM) RLK CERK1 besteht. CERK1 spielt eine Rolle in der durch Chitin induzierten Signaltransduktion und Abwehrantwort in Arabidopsis. In einer vorangegangenen Hefe-Zwei-Hybrid-Analyse wurde die RLCK CLR1 als möglicher Interaktor der CERK1 Kinasedomäne identifiziert. Vergleichende Sequenzanalysen zeigen, dass die Aminosäuresequenz von CLR1 eine hohe Homologie zu den Sequenzen der Kinasedomänen anderer Arabidopsis LysM-RLKs aufweist. Dies könnte möglicherweise für die Funktion des Proteins eine Rolle spielen. Die auf TAIR10 annotierte CLR1 Sequenz scheint falsch annotiert worden zu sein, da das eigentliche Protein laut Analysen in dieser Arbeit wahrscheinlich erst 23 Aminosäuren Richtung C-Terminus beginnt, wodurch dann ein mögliches N-Myristoylierungsmotiv exponiert wird. In vitro wird CLR1 direkt von der CERK1 Kinasedomäne phosphoryliert. CLR1 Fusionsproteine wurden in stabil transgenen Arabidopsis-Pflanzen CERK1-abhängig durch Chitin phosphoryliert. Unabhängig von der möglichen N-terminalen Myristoylierung scheint CLR1 sowohl in vitro also auch in vivo ein Phosphorylierungssubstrat von CERK1 darzustellen. Mikrosomale Fraktionierungen und Analysen zur subzellulären Lokalisation in transgenen Pflanzen zeigten dass die Mehrheit der CLR1 Proteine löslich ist, wobei auch eine kleine Subpopulation von CLR1 membrangebunden in Pflanzenzellen vorliegt. Drei unabhängige T DNA Insertionslinien wurden isoliert und im Hinblick auf die Weiterleitung Chitin-induzierter Signale und Immunität gegen pilzliche und bakterielle Schädlinge getestet. Die clr1 T-DNA Linien wiesen eine verringerte ROS Produktion, MAPK Aktivierung und Expression von Abwehrgenen auf, was eine Rolle für CLR1 im Chitin-induzierten Signalweg bestätigt. Dabei hing die Ausprägung des Phänotyps von der Position der T-DNA ab. clr1 Pflanzen waren nicht in der Resistenz gegen pilzliche Schädlinge beeinträchtigt, wohingegen sie eine leicht erhöhte Anfälligkeit gegenüber bakterieller Infektionen zeigten. Da der CLR1 Promotor erhöhte Aktivität in Hydathoden zeigt, könnte CLR1 darin involviert sein selektiv das Eintreten von Pathogenen über diese konstitutiv geöffneten Öffnungen einzugrenzen.

Doctor of Philosophy / Department of Agronomy / Tesfaye Tesso / Sweet Sorghum [Sorghum bicolor (L.) Moench] is a type of cultivated sorghum grown primarily for its sugar-rich stalks. Because of its high fermentable sugar content, the crop is widely recognized as an alternative feedstock source for bio-fuel production. The extent to which stalk sugar accumulation occurs may be determined by several factors including the sink size. Grain is the most important sink in sorghum and other grain crops. Three experiments were conducted in this study to determine the extent to which the grain sink can reduce sugar accumulation in the stalks, to test and validate a genetic system that allows development of sterile sweet sorghum hybrids, and to assess the potential of sugar-rich hybrids to overcome stalk rot diseases. The first experiment, based on 22 sweet sorghum genotypes, was undertaken to study the effect of eliminating the grain sink (removing the head prior to anthesis) on stalk juice yield, sugar accumulation, and biomass. The data showed that the grain sink had a significant effect on all traits measured. Elimination of the grain sink significantly increased oBrix % (17.8%), dry biomass (27.8%), juice yield (23.9%), and total sugar yield (43.5%). The second experiment was aimed at validating the role of A3 genetic male sterility system for producing sterile sweet sorghum hybrids. Ten sweet sorghum pollinator lines of variable sugar content were selected among the entries included in the previous experiment. The lines were crossed to four A1 and A3 cytoplasmic male sterile (CMS) lines using a Design II mating scheme. The A3 females did not have effective restorers so that the hybrids were expected to be sterile. The parental lines and corresponding hybrids were evaluated for biomass production, oBrix, juice and sugar yield using a randomized complete block design. All A3 hybrids were sterile and did not produce seed when heads were covered prior to pollination. The effect of grain sink represented by the A1 vs. A3 CMS were highly significant for Brix%, biomass, juice, and sugar yield. Comparison of parents vs. crosses component was highly significant, indicating marked heterosis effect for the traits. Both general (GCA) and specific (SCA) combining ability effects were also significant for all traits, indicating the role of both additive and dominance genetic effects in the inheritance of the characters. Earlier studies have shown positive relationships between stalk sugar concentration and stalk rot disease resistance in sorghum. Thus, the objective of the third experiment was to study the effects of the CMS mediated differential accumulation of stalk sugar on severity of charcoal rot disease caused by Macrophomina phaseolina. The experiment provided an opportunity to test the effect of variable stalk sugar in the same genetic backgrounds. The data indicated that hybrids produced from A3 cytoplasm were more resistant to charcoal rot (7.1cm lesion length) compared to those produced from the A1 hybrids (9.5 cm lesion length). The enhanced resistance of hybrids with higher sugar yield could have significant agronomic advantage in sugar based bio-fuel feedstock production.

Cytoplasmic dynein is a molecular motor that moves cargos along microtubules. Dynein, together with its large co-factor dynactin, is responsible for the vast majority of traffic towards the centre of the cell. The largest subunit of the dynein complex is called the dynein heavy chain (DHC). The DHC includes a C-terminal motor domain, which converts ATP hydrolysis into mechanical force, an N-terminal tail domain, and a flexible linker domain to join the two together. An intermediate chain (DIC) and light intermediate chain (DLIC) bind directly to the DHC tail, while light chains (DLCs) bind to the DIC. This tail complex is important for both cargo binding as well as homodimerisation of the DHC, which is necessary for processive movement. Previous studies suggest that the DLCs play an important role in homodimerisation, but it remains unclear how else the DHCs are held together. Using S. cerevisiae as a model system, I co-expressed all four dynein subunits and purified functional dynein motors. In this background, I found that truncating the DHC to include only the first 1004 residues (out of the total 4092) eliminates the motor domain as well as the flexible linker domain, while preserving binding to the DIC, DLIC and DLC. However, truncating just another 50 residues off of the C-terminus led to a loss of all accessory subunits. I developed a protocol for expressing and purifying large quantities of the 1004 residue construct, thus I provide the first description of a recombinant dynein tail domain. Using negative stain electron microscopy (EM), I also present the first 3D structural information for the tail region of the cytoplasmic dynein motor. I then describe a construct including only the first 557 residues of the DHC, which dimerises despite not being able to bind any of the other subunits. I present a crystal structure of this smaller DHC fragment, which shows that the N-terminal 180 residues of the DHC constitute an intricate dimerisation domain made up of a β-sheet sandwiched between α-helices. Not only is this the first crystal structure of any part of the DHC N-terminus, but it reveals a previously undocumented dimerisation domain within the DHC itself. Furthermore, information garnered from this crystal structure allowed for interpretation of a recent cryo-EM structure of a triple complex containing the dynein tail, dynactin and the cargo adaptor BICD2 (TDB) that was solved by my colleagues in the Carter group. Only by docking the DHC N-terminus crystal structure within the TDB EM density did it become clear that the N-terminus of the DHC is responsible for the majority of the contacts the dynein tail makes with both dynactin and BICD2. Therefore the work that I present here sheds new light on the unexpected importance of the DHC N-terminus and allows two important conclusions to be made. First, the N-terminal 180 residues of the DHC constitute a dimerisation domain of its own. Second, the next ~400 residues of the DHC form a domain that plays a key role in the complex interface between dynein, dynactin and BICD2.

Os efeitos maternos e, principalmente, os componentes citoplasmáticos devido sua herança matrilinear, podem influenciar de forma permanente na seleção e no potencial de expressão das características de importância econômica dos bovinos. Como objetivo deste estudo, estimou-se o impacto da inclusão do efeito genético da linhagem citoplasmática sobre estimativas de componente de (co)variância, parâmetros genéticos e sua influência sobre a predição dos valores genéticos para características de crescimento, reprodução e eficiência alimentar de animais da raça Nelore. Para traçar a genealogia, tomando como base a linha materna de cada animal, utilizou-se o software LinMat e arquivo de pedigree contendo 496.190 animais do rebanho de bovinos Nelore pertencentes à Agropecuária CFM e 8.635 animais compondo o arquivo provenientes dos experimentos para eficiência alimentar. Os componentes de (co)variância e parâmetros genéticos foram estimados com base nos registros para peso ao nascimento (PN), peso ao desmame (PD), ganho de peso ajustado para 345 dias de idade (GPSOB), perímetro escrotal aos 18 meses (PE), conformação (CONF), precocidade (PREC), musculosidade (MUSC), ingestão de matéria seca (IMS), consumo residual (CAR), taxa de conversão alimentar (CA) e ganho médio diário (GMD). As análises de componentes de variância foram realizadas no programa computacional BLUPF90 de forma uni-característica com modelo animal pelo método da máxima verossimilhança restrita (REML), e por inferência bayesiana para os indicadores de eficiência alimentar, considerando a linhagem citoplasmática como efeito aleatório. Os resultados obtidos revelam que as características analisadas podem ser utilizadas como critério de seleção pela variabilidade genética e potencial de transmissão. A inclusão do efeito de linhagem citoplasmática foi significativo para PN, CONF, PREC e MUSC. Ao longo prazo, a seleção de linhagens citoplasmáticas com maior efeito genético pode promover o avanço genético para as características de interesse econômico. / Maternal effects and its inheritance have been suggested to have a permanent influence on selection and expression potential traits in cattle. The objectives of this study were to estimate the impact of the inclusion of the cytoplasmic lineage genetic effect on component estimates of (co) variance, genetic parameters and evaluated the influence of this effect on the prediction of breeding values for growth, reproductive and feed efficiency traits of Nellore cattle. Pedigree data from 496,190 Nellore animals belonging to the Agropecuaria CFM and 8,635 animals from feed efficiency traits composing the pedigree file that was used to trace the genealogy, based on the maternal line of each animal by LinMat software. The records for birth weight (BW), weaning weight (WW), weight gain adjusted to 345 days of age (PWG), scrotal circumference at 18 months (SC), conformation (CONF), precocity (PREC), muscularity (MUSC), dry matter intake (DMI), residual feed intake (RFI), feed conversion rate (FCR) and average daily gain (ADG) were evaluated. Analyses were performed by computer program BLUPF90 with an animal model by the method of restricted maximum likelihood (REML) and Bayesian approach to feed efficiency indicators, considering cytoplasmic lineage as a random effect. The results show that the traits analyzed can be used as selection criteria by the genetic variability and the potential for heritability. The inclusion of cytoplasmic lineage effect was significant for BW, CONF, PREC and MUSC. In long-term, the selection of cytoplasmic lines with greater genetic effect may promote genetic progress for economic interest traits.

Le virus de l’hépatite B (VHB) utilise la machinerie transcriptionnelle nucléaire pour sa réplication. Le génome viral est transporté de la périphérie cellulaire à l’enveloppe nucléaire. Généralement, ce transport intracytoplasmique rétrograde est facilité par le réseau de Mt via l’utilisation du complexe moteur de la dynéine. Nous avons montré que le transport des capsides du VHB dépend des Mt, ce qui permet l’adressage des capsides aux complexes du pore nucléaire (NPC) ; lequel est requis pour l’étape de libération du génome de la capside dans le noyau.Dans cette étude, nous avons utilisé des capsides provenant de virus récupérés dans du surnageant de HepG2.2.15, qui contiennent le génome mature partiellement double brin (capsides matures), et des capsides exprimées chez E.coli. Ces dernières sont utilisées telles quelles, capsides E.coli contenant de l’ARN, ou bien sont utilisées pour préparer des capsides vides. Après microinjection dans des ovocytes de Xenopus laevis, nous avons observé que les capsides vides et les capsides matures sont transloquées aux NPC avec une cinétique similaire. Les capsides contenant de l’ARN ne sont pas identifiées aux NPCs ce qui implique que le transport des deux autres types de capsides est actif. Cela a été confirmé par la pré-injection d’anticorps anti tubuline qui neutralisent le transport assuré par les Mt.L’attachement spécifique des capsides matures et vides aux Mt a été confirmé en utilisant des Mt polymérisés in vitro, nous avons montré que cette interaction nécessitait des protéines cytosoliques. En utilisant des expériences de coïmmunoprécipitation et de cosédimentation nous avons identifié une chaîne légère de la dynéine (DynLL1 membre de la famille Lc8) comme partenaire des capsides. Dans les expériences de microinjection, la comicroinjection d’un excès de DynLL1 avec les capsides inhibe leur transport vers les NPCs, indiquant que DynLL1 est impliquée dans le transport actif des capsides.DynLL2 qui n’interagit pas avec les capsides diffère de DynLL1 de seulement six acides aminés. Par mutagénèse dirigée de DynLL1, nous avons montré l’implication de deux acides aminés dans l’interaction directe avec les capsides. Ces deux acides aminés sont présents à la surface du dimère de DynLL1 et absents dans le sillon résultant de la dimérisation de DynLL1, sillon impliqué dans l’interaction avec la DynIC. Nous avons partiellement reconstitué le complexe DynIC, DynLL1 et capsides vides qui doit en partie refléter la situation in vivo. / Hepatitis B virus (HBV) needs the nuclear transcription machinery for replication. The virus thus depends on the transport of its genome from the cell periphery to the nuclear envelope. In general this retrograde intracytoplasmic trafficking is facilitated along Mt (MT) using motor protein complexes of the dynein family. As we showed earlier HBV capsid transport also depends upon intact MT in order to allow their arrival at the nuclear pores, which in turn is required for genome liberation from the capsid.In the analysis we used virus-derived HBV capsids obtained from the supernatant of HepG2.2.15, which contain the mature partially double-stranded DNA genome (mature capsids) and capsids expressed in E. coli. The latter were applied in two forms: as unspecific E. coli RNA- containing capsids and as empty capsids. Upon microinjection into Xenopus laevis oocytes we observed that mature and empty capsids were translocated to the nuclear pores with a similar kinetic. RNA-containing capsids failed to arrive at the pores implying that transport of the two other capsid types was active. Active translocation was confirmed by pre-injecting anti tubulin antibodies which interfere with MT-mediated translocation.In vitro reconstitution assays confirmed the specific attachment of mature and empty capsids to MTs and showed the need of further cytosolic proteins. Using pull-down and co-sedimentation experiments we identified one dynein light chain (DYNLL1, member of the Lc8 family) as interaction partner of the capsids. Injecting an excess of recombinant DYNLL1 with empty capsids into Xenopus laevis oocytes inhibited capsid transport to the nuclear pores indicating that DYNLL1 was only functional interaction partner implied in active transport.DNYLL2 did not interact with the capsids although differing from DYNLL1 by just six amino acids. Site directed mutagenesis of DYNLL1 revealed that two amino acids were critical for a direct interaction with the capsids. Both localized at the exterior of the DYNLL1 dimer and not in the groove of DYNLL1, which interacts with the dynein intermediate chain. Accordingly we could reconstitute a complex consisting of empty capsids, DYNLL1 and dynein intermediate chain as it should be in the in vivo situation.

Les adénovirus (AdV), comme d'autres virus à réplication nucléaire, ont besoin d’arriver jusqu’aunoyau cellulaire afin de libérer leur génome. Pour ce faire, les particules des AdV contenant l’ADNviral sont transportées jusqu’au complexe du pore nucléaire (NPC), via le centre d’organisation desmicrotubules, par un mécanisme encore mal compris qui implique l’exportine cellulaire CRM1. Lacapside des AdV dépasse la taille limite d’entrée dans le noyau, et par conséquent, elle doit êtredésassemblée au niveau du NPC. Le mécanisme d’import de molécules d’ADN n’est pas un processusphysiologique. Pour cela, les AdV doivent détourner la machinerie cellulaire afin d’importer leurgénome dans le noyau. Le NPC est un complexe de protéines appelées nucléoporines. LaNup358/RanBP2, principal composant des filaments cytoplasmiques, sert de plateforme de liaison àdes karyopherines (e.g Importin-β, CRM1) et à la protéine GTPase Ran. Les karyopherinesreconnaissent des signaux spécifiques présents dans les cargos et facilitent leur transport d’unemanière très régulée dépendante de RanGTP. Nous avons constaté que l’import du génome AdV estmoins efficace en l’absence de Nup358. Dans ces conditions, nous avons observé que certaineskaryopherines deviennent limitantes pour l’import du génome viral, et identifié la région minimale deNup358 requise pour compenser ce défaut. D’autre part, nous avons confirmé l’implication de CRM1dans l’arrivé des particules virales au noyau et identifié un nouveau rôle de CRM1 dans ledésassemblage de la capside des AdV. Ces travaux contribuent à mieux connaître le mécanismed’entrée du génome AdV dans le noyau et donnent une idée de la façon dont les virus peuventcontourner la machinerie de transport cellulaire pour leur propre bénéfice. / Nuclear delivery of viral genomes is an essential step for nuclear replicating DNA viruses such asAdenovirus (AdV). AdV particles reach the nuclear pore complex (NPC) in the form of genomecontaining, partially disassembled capsids, through a poorly understood CRM1-dependent mechanism.These capsids exceed the NPC size limit and therefore, they must disassemble at the NPC to releasethe viral genome. Nuclear import of DNA cargos is not a physiological process. Consequently, AdVneed to divert the cellular transport machinery for nuclear genome delivery. The NPC is a multiproteincomplex consisting of nucleoporins (Nups). The Nup358/RanBP2 is the major component ofthe cytoplasmic filaments of the NPC and serves as binding platform for factors includingkaryopherins (i.e Importin-β, CRM1) and the small GTPase Ran. Selective transport of cargo throughthe NPC is mediated by karyopherins, which recognize specific signals within the cargos and facilitatetheir transport in a RanGTP-dependent regulated manner. We identified that Nup358-depleted cellsreduce nuclear import efficiency of the AdV genome. Indeed, we observed that karyopherins are ratelimitingfor AdV genome import under these conditions and we mapped the minimal region ofNup358 necessary to compensate the import defect. On the other hand, we could confirm therequirement of CRM1 in nuclear targeting of AdV capsids and identified and additional role inmediating AdV capsid disassembly. This work helps to understand the strategy used by AdV todeliver their genome and gives insight about how viruses hijack the cellular transport machinery fortheir own benefit.

Peptidyl-prolyl isomerization is an important post-translational modification of protein because proline is the only amino acid that can stably exist as cis and trans, while other amino acids are in the trans conformation in protein backbones. This makes prolyl isomerization a unique mechanism for cells to control many cellular processes. Isomerization is a rate-limiting process that requires a peptidyl-prolyl cis/trans isomerase (PPIase) to overcome the energy barrier between cis and trans isomeric forms. Pin1, a key PPIase in the cell, recognizes a phosphorylated Ser/Thr-Pro motif to catalyze peptidyl-prolyl isomerization in proteins. The significance of the phosphorylation-dependent Pin1 activity was recently highlighted for isomerization of ATR (ataxia telangiectasia- and Rad3-related). ATR, a PIKK protein kinase, plays a crucial role in DNA damage responses (DDR) by phosphorylating hundreds of proteins. ATR can form cis or trans isomers in the cytoplasm depending on Pin1 which isomerizes cis-ATR to trans-ATR. Trans-ATR functions primarily in the nucleus. The cis-ATR, containing an exposed BH3 domain, is anti-apoptotic at mitochondria by binding to tBid, preventing activation of pro-apoptotic Bax. Given the roles of apoptosis in many human diseases, particularly cancer, we propose that cytoplasmic cis-ATR enables cells to evade apoptosis, thus addicting cancer cells to cis-ATR formation for survival. But in normal DDR, a predominance of trans-ATR in the nucleus coordinates with a minimal level of cytoplasmic cis-ATR to promote DNA repair while preventing cell death; however, cells can die when DNA repair fails. Therefore, a delicate balance/equilibrium of the levels of cis- and trans-ATR is required to ensure the cellular homeostasis. In this review, we make a case that this anti-apoptotic role of cis-ATR supports oncogenesis, while Pin1 that drives the formation of trans-ATR suppresses tumor growth. We offer a potential, novel target that can be specifically targeted in cancer cells, without killing normal cells, to significantly reduce the adverse effects usually seen in cancer treatment. We also raise important issues regarding the roles of phosphorylation-dependent Pin1 isomerization of ATR in diseases and propose areas of future studies that would shed more understanding on this important cellular mechanism.

Type II 5'-deiodinase (D2) catalyzes the conversion of T4 to the transcriptionally active T3. When T4 levels are high, D2 activity levels are low. Conversely when T4 levels are low, D2 catalytic activity is high. Immunocytochemistry and biochemical data from cultured rat astrocytes revealed that physiological concentration of T4 and the non-transcriptionally active metabolite rT3, but not T3, initiates the budding of D2 containing endosomes and their subsequent translocation to the perinuclear space. Further analysis showed that this process required a polymerized actin cytoskeleton but not cellular transcription or translation; however the precise mechanism remained unknown. In this present investigation, we characterized the requirement of an unconventional myosin motor protein, myosin 5a, in the actin-based endocytosis of D2 containing vesicles. We developed an in vitro actin binding assay that exploited the T4 dependent binding of D2 containing vesicles to F-actin, and showed that D2p29:F-actin interactions are calcium, magnesium and ATP-dependent suggesting that a calmodulin (CaM) regulated myosin ATPase is required. Introduction of in vitro transcribed and translated vesicle-binding tail, which lacked the actin binding head, of myosin 5a to the in vitro actin binding assay created a dominant negative inhibitor of D2 binding to the actin cytoskeleton by competing with the native myosin 5a. A replication deficient adenoviral vector expressing the fusion protein of the 29 kDa substrate binding subunit of D2 with a green fluorescent protein reporter molecule enabled us to directly examine T4 dependent regulation of D2 in vitro as well as in living cells. Using immunoprecipitation we showed a T4 dependent association between the vesicle binding tail of myosin 5a and D2 containing vesicles. Biochemical analysis of the interaction of the myosin 5a tail with D2 containing vesicles revealed that the last 21 amino acids of myosin 5a were both necessary and sufficient for the attachment of D2 containing vesicles to the F-actin cytoskeleton. Using rapid acquisition time-lapse digital microscopy in p29GFP expressing rat astrocytes, we showed directed T4 dependent p29GFP movement from the plasma membrane to the perinuclear region. This hormone dependent vesicle movement was not observed in cells treated with T3 or no hormone. Time lapse motion studies allowed for the calculation of the velocity and of the distance traveled for individual fusion protein containing vesicles. The velocity for cells treated with T4 or rT3 was identical to that reported for vesicle-laden myosin 5a in mouse melanophores. In contrast cells treated with T3 or those receiving no hormone treatment had velocities similar to diffusion of proteins within the plasma membrane. Astrocytes constitutively expressing both p29GFP and dominant negative myosin 5a inhibitors failed to show hormone induced centripetal movement. These data demonstrate that myosin 5a is the molecular motor responsible for thyroid hormone dependent actin based endocytosis in astrocytes.

Orientador: Joaquim Mansano Garcia / Resumo: Análises morfológicas para seleção de oócitos podem subestimar os gametas femininos em relação ao seu potencial de desenvolvimento, pela subjetividade da técnica por identificar apenas alterações aparentes em microscopia de luz. Este método pode não ser capaz de avaliar por exemplo, a competência a nível molecular, nuclear e citoplasmática do oócito, como perfil de transcritos acumulados, organização de organelas e vias de apoptose ativas. Portanto, sob a hipótese que grânulos citoplasmáticos em oócitos não alteram a capacidade de desenvolvimento, a qualidade dos embriões gerados e o desenvolvimento embrionário e fetal, o objetivo do nosso estudo foi avaliar os efeitos de grânulos citoplasmáticos na produção in vitro de embriões. Para testar esta hipótese, utilizamos o modelo bovino em dois experimentos para avaliar grupos de oócitos com citoplasma homogêneo (controle) e oócitos que apresentam granulações no citoplasma (granular). No experimento 1, através de amostras de ovários de abatedouro, observamos que: i. Nas análises de perfil funcional do oócito, estruturas que possuem grânulos citoplasmáticos apresentam maiores níveis de caspase 3 ativa (indicador da cascata de apoptose), no entanto não demonstram nenhuma diferença em relação a atividade de junções GAP (comunicação oócito-células do cumulus), ii: Nas análises de expressão de genes marcadores de qualidade realizadas em oócitos, blastocistos e células do cumulus, apenas o transcrito ZAR1 foi diferencialmente expresso,... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Morphological analysis for oocyte selection may underestimate female gametes in regarding their developmental potential, due to the technique subjectivity for only identifies apparent changes in light microscopy. This method may not be able to evaluate for example the molecular, nuclear and cellular oocyte competence, such as transcript profile accumulations, organelles organization and active apoptosis pathways. Therefore, under the hypothesis that oocytes cytoplasmic granules do not alter the development capacity, embryos quality and embryonic and fetal development, the study objective was to evaluate cytoplasmic granules effects on in vitro embryo production. To test this hypothesis we used bovine model in two experiments to evaluate groups of homogeneous cytoplasm oocytes (control) and granular cytoplasm oocytes (granular). In experiment 1, through slaughterhouse ovarian samples we observed: i. In the analysis of oocyte functional profile structures that have cytoplasmic granules have higher active caspase 3 levels (apoptosis cascade indicator), but do not show any difference in GAP junction activity (oocyte-cumulus cell communication). ii. In the analysis of quality marker gene expression performed in oocytes, blastocysts and cumulus cells, only the ZAR1 transcript was differentially expressed and poorly detected in granular group oocytes and iii. In embryonic development rates (cleavage, blastocyst) and kinetics analysis we did not detect differences between the groups.... (Complete abstract click electronic access below) / Mestre

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21015号 / 医博第4361号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 渡邉 大, 教授 宮本 享 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM