No description available.

During meiosis eukaryotes produce four haploid gametes from a single diploid parental cell. In meiotic S-phase homologous chromosomes, which were inherited from maternal and paternal parents, are replicated. Homologous chromosomes then pair and undergo reciprocal crossover, which generates new mosaics of maternal and paternal sequences. Meiosis also involves two rounds of chromosome segregation, meaning that only one copy of each chromosome is finally packaged into the resulting haploid gametes. In this work I sought to genetically engineer two elements of meiosis, in order to generate tools which may be useful for plant breeding. The first project sought to generate a second division restitution (SDR) population, where the second meiotic division is skipped. This is created by crossing an SDR mutant, omission of second division1, which produces diploid pollen due to a defective meiosis-II, to a haploid inducer line, whose chromosomes are lost from the zygote post-fertilisation. This was intended to give rise to diploid plants possessing chromosomes from just the SDR parent. Importantly, the SDR parent used was heterozygous, meaning that SDR progeny should show mostly homozygous chromosomes, but with regions of residual heterozygosity, determined by crossover locations. This project succeeded in creating a small number of plants with the predicted SDR genotype, although a range of aberrant genotypes were also observed. I present several hypotheses that could account for the observed progeny genotypes. In a second project I attempted to direct meiotic recombination using DNA double strand breaks targeted to specific sites. This project used a spo11-1 mutant, which is unable to produce the endogenous meiotic DNA DSBs that normally mature into crossovers. Instead, TALFokI nucleases (TALENs) were expressed from meiotic promoters in order to generate exogenous DSBs at sites determined by the DNA binding specificity of the TAL repeat domains. The project succeeded in transforming TALENs into spo11-1 mutants and confirming their expression. However, this was not sufficient to recover the spo11-1 mutant infertility or direct crossovers. Potential reasons for this non-complementation are discussed, as well as their implications for control of meiotic recombination in plant genomes.

No description available.

The purpose of this research project was to elucidate the molecular interactions and detail the signaling pathways in obstetric pathologies. This work first seeks to understand inflammation related complications relevant to obstetrics. Prior research in our lab identified the implications of the receptor of advanced glycation end products (RAGE) during inflammatory response in the placenta. Current work identified the presence of DNA double-strand breaks (DNA-DSBs) in inflammation associated pregnancy complications of preeclampsia (PE) and preterm labor (PTL) and demonstrated the positive role of RAGE in repairing the damage. The confluent relevance of disrupted mitochondrial function and inflammation has been recognized in the etiology of numerous chronic diseases. Our current studies aim to understand the connections between energy metabolism and inflammation in pathologies of pregnancy complications. Previous research conducted in our laboratory has demonstrated the mediation of the Gas6/Axl pathway on the mechanistic target of rapamycin (mTOR), an important metabolic molecule. We observed the negative regulation of Gas6 treatment on the mTOR pathway and its negative effects on trophoblast cell invasion. In the current study looking at the aspect of energy regulation, we identified the activation of placental mTOR in gestational diabetes mellitus (GDM) and its decrease during PE and intrauterine growth restriction (IUGR). We further evaluated the regulation of mTOR on its downstream effector pyruvate kinase M2 (PKM2). We found that inhibition of mTOR decreased PKM2 activation; while PKM2 activation positively regulated trophoblastic invasion and rescued negative effects observed in our second-hand smoke IUGR murine model. Our work has opened a new direction of placental research, especially in pregnancy complications stemming from genomic instability. We also clarified details of mTOR and PKM2 meditated metabolic signaling that are crucial for future investigation on the dynamic metabolic regulation during pregnancy.

Les microtubules sont de longs polymères cylindriques de la protéine α, β tubuline, utilisés dans les cellules pour construire le cytosquelette, le fuseau mitotique et les axonèmes. Ces polymères creux sont cruciaux pour de nombreuses fonctions cellulaires, y compris le transport intracellulaire et la ségrégation chromosomique pendant la division cellulaire. Au fur et à mesure que les cellules se développent, se divisent et se différencient, les microtubules passent par un processus, appelé instabilité dynamique, ce qui signifie qu’ils basculent constamment entre les états de croissance et de rétrécissement. Cette caractéristique conservée et fondamentale des microtubules est étroitement régulée par des familles de protéines associées aux microtubules. Les protéines de kinésine-13 sont une famille de facteurs régulateurs de microtubules qui dépolymérisent catalytiquement les extrémités des microtubules. Cette thèse traite d’abord des concepts mécanistiques sur le cycle catalytique de la kinésine-13. Afin de mieux comprendre le mécanisme moléculaire par lequel les protéines de kinésine-13 induisent la dépolymérisation des microtubules, nous rapportons la structure cristalline d’un monomère de kinésine-13 catalytiquement actif (Kif2A) en complexe avec deux hétérodimères αβ-tubuline courbés dans un réseau tête-à-queue. Nous démontrons également l’importance du « cou » spécifique à la classe de kinésine-13 dans la dépolymérisation catalytique des microtubules. Ensuite, nous avons cherché à fournir la base moléculaire de l’hydrolyse tubuline-guanosine triphosphate (GTP) et son rôle dans la dynamique des microtubules. Dans le modèle que nous présentons ici, l’hydrolyse tubuline-GTP pourrait être déclenchée par les changements conformationnels induits par les protéines kinésine-13 ou par l’agent chimique stabilisant paclitaxel. Nous fournissons également des preuves biochimiques montrant que les changements conformationnels des dimères de tubuline précèdent le renouvellement de la tubuline-GTP, ce qui indique que ce processus est déclenché mécaniquement. Ensuite, nous avons identifié la kinésine de microtubule Kif2C comme une protéine associée à des modèles d’ADN imitant la rupture double brin (DSB) et à d’autres protéines de réparation DSB connues dans les extraits d’œufs de Xenope et les cellules de mammifères. Les cassures double brin d’ADN (DSB) sont un type majeur de lésions d’ADN ayant les effets les plus cytotoxiques. En raison de leurs graves impacts sur la survie cellulaire et la stabilité génomique, les DSB d’ADN sont liés à de nombreuses maladies humaines, y compris le cancer. Nous avons constaté que les activités PARP et ATM étaient toutes deux nécessaires pour le recrutement de Kif2C sur les sites de réparation de l’ADN. Kif2C knockout ou inhibition de son activité de dépolymérisation des microtubules a conduit à l’hypersensibilité des dommages à l’ADN et à une réduction de la réparation du DSB via la jonction terminale non homologue et la recombinaison homologue. Dans l’ensemble, notre modèle suggère que les protéines de kinésine-13 peuvent interagir avec les dimères de tubuline aux extrémités microtubules et modifier leurs conformations, moduler l’étendue des extrêmités tubuline-GTP dans les cellules et déclencher le désassemblage des microtubules. Ces deux modèles pourraient être des clés pour démêler les mécanismes impliqués dans le nouveau rôle de Kif2C dans la réparation de l’ADN DSB sans s’associer à des polymères de microtubules. / Microtubules are long, cylindrical polymers of the proteins α, β tubulin, used in cells to construct the cytoskeleton, the mitotic spindle and axonemes. These hollow polymers are crucial for many cellular functions including intracellular transport and chromosome segregation during cell division. As cells grow, divide, and differentiate, microtubules go through a process, called dynamic instability, which means they constantly switch between growth and shrinkage states. This conserved and fundamental feature of microtubules is tightly regulated by families of microtubule-associated proteins (MAPs). Kinesin-13 proteins are a family of microtubule regulatory factors that catalytically depolymerize microtubule ends. This thesis first discusses mechanistic insights into the catalytic cycle of kinesin-13. In order to better understand the molecular mechanism by which kinesin-13 proteins induce microtubule depolymerization, we report the crystal structure of a catalytically active kinesin-13 monomer (Kif2A) in complex with two bent αβ-tubulin heterodimers in a head-to-tail array. We also demonstrate the importance of the kinesin-13 class-specific “neck” in modulating Adenosine triphosphate (ATP) turnover and catalytic depolymerization of microtubules. Then, we aimed to provide the molecular basis for tubulin-Guanosine triphosphate (GTP) hydrolysis and its role in microtubule dynamics. Although it has been known for decades that tubulin-GTP turnover is linked to microtubule dynamics, its precise role in the process and how it is driven are now well understood. In the model we are presenting here, tubulin-GTP hydrolysis could be triggered via the conformational changes induced by kinesin-13 proteins or by the stabilizing chemical agent paclitaxel. We also provide biochemical evidence showing that conformational changes of tubulin dimers precedes the tubulin-GTP turnover, which indicates that this process is triggered mechanically. Next, we identified microtubule kinesin Kif2C as a protein associated with double strand break (DSB)-mimicking DNA templates and other known DSB repair proteins in Xenopus egg extracts and mammalian cells. DNA double strand breaks (DSBs) are a major type of DNA lesions with the most cytotoxic effects. Due to their sever impacts on cell survival and genomic stability, DNA DSBs are related to many human diseases including cancer. Here we found that PARP and ATM activities were both required for the recruitment of Kif2C to DNA repair sites. Kif2C knockdown/knockout or inhibition of its microtubule depolymerizing activity led to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both non-homologous end-joining (NHEJ) and homologous recombination (HR). Interestingly, genetic depletion of KIF2C, or inhibition of its microtubule depolymerase activity, reduced the mobility of DSBs, impaired the formation of DNA damage foci, and decreased the occurrence of foci fusion and resolution. Altogether, our findings shed light on the mechanisms involved in kinesin-13 catalyzed microtubule depolymerization. Our tubulin-GTP hydrolysis model suggests that kinesin-13 proteins may interact with tubulin dimers at microtubules ends and alter their conformations, modulate the extent of the GTP caps in cells and trigger microtubule disassembly. These two models could be keys to unravel the mechanisms involved in the novel role of Kif2C in DNA DSB repair without associating with microtubule polymers.