Spinal meningiomas (SM) comprise 5–10% of primary meningiomas and up to 30% of spinal intradural tumors. SMs are usually sporadic, but rarely, they can develop in association with genetic diseases like neurofibromatosis type 2 or schwannomatosis [2, 4, 6]. While the mutational landscape of intracranial meningiomas has been extensively studied [3, 5, 11, 14], our understanding of the molecular profile of SM remains incomplete. To date, genomic studies in SMs have been underpowered to make significant conclusions about the correlations between main genomic driver alterations and clinical features of these tumors. Here, we sought to assess the mutational profile of WHO grade 1 SM and to investigate the clinical characteristics that correlate with the genomic status.

Le canal calcique dépendant du voltage de type-T CaV3.2 joue un rôle important dans l’excitabilité neuronale et dans la perception de la douleur. Le canal CaV3.2 partage une grande homologie structurale et fonctionnelle avec les canaux NaV. Ces deux types de canaux sont activés par de faibles dépolarisations membranaires et possèdent des cinétiques de temps d’activation et d’inactivation plus rapides que les canaux CaV de type-L. Les structures cristallines à haute résolution des canaux bactériens NaVAb (Payandeh et al. 2011; Payandeh et al. 2012) et NaVRh (Zhang et al. 2012) suggèrent que l’hélice amphiphile S4S5 du domaine II peut être couplée avec les résidus de l’hélice S6 dans le domaine II ainsi qu’avec des résidus de l’hélice homologue dans le domaine adjacent, soit le domaine III, et ce, durant l’activation du canal. Pour déterminer les résidus fonctionnellement couplés, durant l’activation du canal CaV3.2, une analyse cyclique de doubles mutants a été effectuée par substitution en glycine et alanine des résidus clés entre l’hélice S4S5 du domaine II et le segment S6 des domaines II et III. Les propriétés biophysiques ont été mesurées à l’aide de la technique de « cut-open » sur les ovocytes. Les énergies d’activation ont été mesurées pour 47 mutations ponctuelles et pour 14 paires de mutants. De grandes énergies de couplage (ΔΔGinteract > 2 kcal mol-1) ont été observées pour 3 paires de mutants introduites dans les IIS4S5/IIS6 et IIS4S5/IIIS6. Aucun couplage significatif n’a été observé entre le IIS4S5 et le IVS6. Nos résultats semblent démontrer que les hélices S4S5 et S6 provenant de deux domaines voisins sont couplées durant l’activation du canal calcique de type-T CaV3.2. / Voltage-activated T-type calcium channel CaV3.2 plays an important role in neuronal excitability and in pain perception. CaV3.2 channel bears a strong structural and functional homology with voltage-dependent NaV channels. In particular, these channels are activated by relatively small depolarization and display faster activation and inactivation kinetics than the L-type CaV channel. High-resolution crystal structures of bacterial NaVAb (Payandeh et al. 2011; Payandeh et al. 2012) and NaVRh (Zhang et al. 2012) suggest that the amphiphilic helix S4S5 in Domain II may be coupled with S6 residues both in Domain II and in the adjacent Domain III during channel activation.To determine whether residues in the S4S5 helix of Domain II are functionally coupled with residues in the S6 helix in Domain II and Domain III during the voltage-dependent activation of CaV3.2, a double mutant cycle analysis was performed by introducing pairs of glycine and alanine residues in the S4S5 helix of Domain II and the S6 region of Domains II and III. Biophysical properties were measured with the cut-open oocyte technique. Activation gating was measured for 47 single mutants and 14 pairs of mutants. Strong coupling energies (ΔΔGinteract > 2 kcal mol-1) were reported for 3 pairs of mutants introduced in IIS4S5/IIS6 and IIS4S5/IIIS6. No significant coupling was observed between IIS4S5 and IVS6. Altogether, our results demonstrate that the S4S5 and S6 helices from neighboring domains are energetically coupled during the activation of the low voltage-gated T-type CaV3.2 channel.

Mitochondria are usually considered simply as the “powerhouses of the cell”, however in recent years it has become apparent that mitochondria are also of fundamental importance in programmed cell death (PCD), which refers to cell death resulting from a controlled, genetically defined pathway. In Arabidopsis, PCD induced by either heat shock or treatment with strong oxidants is found to be correlated with an early and irreversible change in mitochondrial morphology which manifests as an increase in the size of individual mitochondria. In addition, PCD causes a clustering of mitochondria and loss of motility. In this study, I have used two arginase negative mutant Arabidopsis lines (argah1-1 and argah2-1) which have elevated cellular NO concentrations to examine the effect of nitrosative stress on mitochondria undergoing PCD. Another three different Arabidopsis lines (mito-GFP/mTalin-mCherry, mito-GFP/MAP4-mCherry, mito- mCherry/EB1b-GFP) were used to visualize cytoskeletal elements alongside mitochondria to examine the mechanisms responsible for the mitochondrial morphology transition, clustering and motility inhibition. Results indicate that the elevated concentration of NO found in arginase negative mutants is not sufficient to induce PCD. There was no significant mitochondrial morphology or dynamic change detected between arginase negative mutants and wild type plants, with or without a heat shock. Disruption of either actin or microtubule (MT) cytoskeletal elements leads to the formation of mitochondrial clusters, although they showed different cluster morphology and sizes. Mitochondrial clusters were observed to be moving along the remaining actin cables after a mild heat treatment or cytoskeletal depolymerizing drug treatment. Intact microtubules or MT plus ends visualized with EB1b did not show any interaction with mitochondria under normal conditions. However, after a mild heat stress, EB1b appeared to be associated with clusters of enlarged, possibly swollen mitochondria.

<p>Obsessive-compulsive disorder (OCD) is a devastating illness that afflicts around 2% of the world's population with recurrent distressing thoughts (obsessions) and repetitive ritualistic behaviors (compulsions). While dysfunction at excitatory glutaminergic excitatory synapses leading to hyperactivity of the orbitofrontal cortex and head of the caudate - brain regions involved in reinforcement learning - are implicated in the pathology of OCD, clinical studies involving patients are unable to dissect the molecular mechanisms underlying this cortico-striatal circuitry defect. Since OCD is highly heritable, recent studies using mutant mouse models have shed light on the cellular pathology mediating OCD symptoms. These studies point toward a crucial role for deltaFosB, a persistent transcription factor that accumulates with chronic neuronal activity and is involved in various diseases of the striatum. Furthermore, elevated deltaFosB levels results in the transcriptional upregulation of Grin2b, which codes GluN2B, an N-methyl-D-aspartate glutamate receptor (NMDAR) subunit required for the formation and maintenance of silent synapses. Taken together, the current evidence indicates that deltaFosB-mediated expression of aberrant silent synapses in caudate medium spiny neurons (MSNs), in particular D1 dopamine-receptor expressing MSNs (D1 MSNs), mediates the defective cortico-striatal synaptic transmission that underlies compulsive behavior in OCD.</p> / Dissertation

Le canal calcique dépendant du voltage de type-T CaV3.2 joue un rôle important dans l’excitabilité neuronale et dans la perception de la douleur. Le canal CaV3.2 partage une grande homologie structurale et fonctionnelle avec les canaux NaV. Ces deux types de canaux sont activés par de faibles dépolarisations membranaires et possèdent des cinétiques de temps d’activation et d’inactivation plus rapides que les canaux CaV de type-L. Les structures cristallines à haute résolution des canaux bactériens NaVAb (Payandeh et al. 2011; Payandeh et al. 2012) et NaVRh (Zhang et al. 2012) suggèrent que l’hélice amphiphile S4S5 du domaine II peut être couplée avec les résidus de l’hélice S6 dans le domaine II ainsi qu’avec des résidus de l’hélice homologue dans le domaine adjacent, soit le domaine III, et ce, durant l’activation du canal. Pour déterminer les résidus fonctionnellement couplés, durant l’activation du canal CaV3.2, une analyse cyclique de doubles mutants a été effectuée par substitution en glycine et alanine des résidus clés entre l’hélice S4S5 du domaine II et le segment S6 des domaines II et III. Les propriétés biophysiques ont été mesurées à l’aide de la technique de « cut-open » sur les ovocytes. Les énergies d’activation ont été mesurées pour 47 mutations ponctuelles et pour 14 paires de mutants. De grandes énergies de couplage (ΔΔGinteract > 2 kcal mol-1) ont été observées pour 3 paires de mutants introduites dans les IIS4S5/IIS6 et IIS4S5/IIIS6. Aucun couplage significatif n’a été observé entre le IIS4S5 et le IVS6. Nos résultats semblent démontrer que les hélices S4S5 et S6 provenant de deux domaines voisins sont couplées durant l’activation du canal calcique de type-T CaV3.2. / Voltage-activated T-type calcium channel CaV3.2 plays an important role in neuronal excitability and in pain perception. CaV3.2 channel bears a strong structural and functional homology with voltage-dependent NaV channels. In particular, these channels are activated by relatively small depolarization and display faster activation and inactivation kinetics than the L-type CaV channel. High-resolution crystal structures of bacterial NaVAb (Payandeh et al. 2011; Payandeh et al. 2012) and NaVRh (Zhang et al. 2012) suggest that the amphiphilic helix S4S5 in Domain II may be coupled with S6 residues both in Domain II and in the adjacent Domain III during channel activation.To determine whether residues in the S4S5 helix of Domain II are functionally coupled with residues in the S6 helix in Domain II and Domain III during the voltage-dependent activation of CaV3.2, a double mutant cycle analysis was performed by introducing pairs of glycine and alanine residues in the S4S5 helix of Domain II and the S6 region of Domains II and III. Biophysical properties were measured with the cut-open oocyte technique. Activation gating was measured for 47 single mutants and 14 pairs of mutants. Strong coupling energies (ΔΔGinteract > 2 kcal mol-1) were reported for 3 pairs of mutants introduced in IIS4S5/IIS6 and IIS4S5/IIIS6. No significant coupling was observed between IIS4S5 and IVS6. Altogether, our results demonstrate that the S4S5 and S6 helices from neighboring domains are energetically coupled during the activation of the low voltage-gated T-type CaV3.2 channel.

New drug discoveries and new approaches towards diagnosis and treatment have improved cancer therapeutics remarkably. One of the most influential and effective discoveries in the field of cancer therapeutics was antimetabolites, such as the antifolates. The interest in antifolates increased as some of the antifolates showed responses in cancers, such as mesothelioma, leukemia, and breast cancers. When pemetrexed (PTX) was discovered, our laboratory had established that the primary mechanism of action of pemetrexed is to inhibit thymidylate 22 synthase (TS) (E. Taylor et al., 1992). Preclinical studies have shown that PTX has a broad range of antitumor activity in human and murine models of cancer (Adjei, 2000; Adjei, 2004; S. Chattopadhyay, Moran, & Goldman, 2007; Miller et al., 2000). Accordingly, in February 2004, the FDA issued first-line treatment approval for pemetrexed in malignant pleural mesothelioma and in 2008 for first line treatment for locally advanced or metastatic NSCLC (reviewed in (Rollins & Lindley, 2005). As an antifolate this level of therapeutic activity of PTX against lung cancers was surprising and atypical (Hazarika, White, Johnson, & Pazdur, 2004). This led us to the question whether the effects of pemetrexed on other folate-dependent targets could explain the clinical activity of the drug. Our lab showed that, in addition to inhibiting thymidylate synthase, PTX also inhibits aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate-dependent enzyme of de novo purine synthesis. Inhibition of AICART leads to massive accumulation of its substrate 5-amino-4-imidazolecarboxamide ribonucleotide (ZMP), causing activation of AMP-dependent kinase (AMPK), which ultimately leads to suppression of mTORC1 signaling, a central regulator of cell growth and proliferation. This secondary mechanism could explain the unusual activity of PTX against mesothelioma and lung cancers. The large proportion of lung cancers are either null or mutant for p53 function. Therefore, this thesis focused on defining what the role of p53 is in the PTX-mediated AMPK activation and mTORC1 inhibition and how the loss of p53 affects mTORC1 signaling. These two questions proved to be interlinked. Chapter 2 investigates this relationship in detail. We found that, upon loss of p53, mTORC1 signaling is enhanced to a significant degree in colon carcinoma and lung cancer cell lines. Clearly, this observation required explanation. We found that the major factors responsible for these differences in mTORC1 activity upon loss of p53 23 were lower levels of two p53 target genes Tuberin (TSC2) and sestrin2. Immunoprecipitation studies of mTORC1 complexes from p53 wt and p53 null cells revealed quite interesting differences in the components of the mTORC1 complex. Immunoprecipitates from p53 null cells had higher levels of mTOR and lower levels of TSC2 and PRAS40 bound to raptor. This suggested that, in comparision to p53 competent cells, p53 null cells have more mTORC1 complex with enhanced activity due to decreased interaction of TSC2 and PRAS40, both of which are inhibitors of mTORC1. These observations explained the higher mTORC1 in p53 null cells and laid the foundation for determining the role of p53 in PTX-activated AMPK and mTORC1 inhibition. In the experiments described in Chapter 3, we found that PTX-mediated AMPK activation inhibited mTORC1 regardless of the p53 status in colon carcinoma cells. This suggested that mTORC1 inhibition by PTX was either independent of p53 mediated negative regulation of mTORC1 or was somewhere bypassing it. Therefore, we compared the effects of PTX with the classic AMPK activator aminoimidazolecarboxamide ribonucleoside (AICAR). In spite of a common mechanism of AMPK activation, namely, expansion of cellular ZMP levels, signaling from AMPK activated by PTX or AICAR were quite different. PTX-activated AMPK phosphorylated the mTORC1 component Raptor but not tuberin (TSC2), whereas AICARactivated AMPK phosphorylated both the targets. This differential behavior of two AMPK activators was due to differential behavior of p53 under these two treatments. Both, AICAR and PTX treatment led to increase in p53 levels but the p53 that accumulated after AICAR treatment was transcriptionally active while the p53 that accumulated after PTX treatment was not. Transcription of p53 targets, including TSC2 and sestrin2, was activated in AICAR- but not in PTX-treated cells. In the absence of p53 function, TSC2 was deficient and mTORC1 activity 24 enhanced, but Raptor phosphorylation by AMPK following PTX was robust and independent of both p53 and TSC2. Therefore we concluded that p53 deficiency suppresses TSC2 and upregulates mTORC1, but AMPK-phosphorylation of Raptor after pemetrexed treatment was sufficient to suppress mTORC1, even in TSC2 deficiency. This suggested pemetrexed as a drug for treatment of Tuberous Sclerosis, a genetic disease caused by functional inactivity of TSC1 or TSC2 due to point mutations in these genes. Mutation of p53 is one of the most common genetic alterations in human cancers and tumors. Cancers that express mutant p53 tend to be more aggressive, resistant to chemotherapy and show worse prognosis then p53-null tumors (Elledge et al., 1993; Olivier et al., 2006). This tumor-promoting activity of mutant p53 has been correlated with acquired and novel transcriptional activities of mutant p53. It has been shown that mutp53 can activate the transcription of cell growth promoting genes, such as, NFκB2, PCNA, MDR1, Axl, EGFR, hTERT, and HSP70, which are not usually transcriptional targets of wt p53. Interestingly, we found that whereas DNA damaging drugs enhance the acquired oncogenic transcriptional activities of mutp53, PTX interferes with this transcription activation. We also found in Chapter 4 that PTX can limit or block the DNA damaging drug-mediated increment of transcriptional activation of mutp53. This suggests that blockade of transcriptional activation of mutp53 by pemetrexed may provide an additional therapeutic benefit in mutp53 bearing cancers. As discussed in Chapter Three, although pemetrexed (with TdR) increases the levels of p53 and its binding to the promoter of its target gene, p21, this p53 is transcriptionally inactive. In order to understand the mechanism of the pemetrexed-mediated transcriptional defect of wt p53, we studied the PTX-mediated signaling towards ATM and ATR and their effects on their substrates Chk2 and Chk1, respectively. These studies suggested that the difference between 25 signaling under AICAR treatment and PTX treatment was that, unlike PTX, AICAR treatment was leading to DNA damage, followed by Chk2 phosphorylation at Thr68. We found there were three major differences between AICAR and pemetrexed (+ TdR) mediated signaling: AICAR caused DNA damage, followed by ATM mediated phosphorylation of Chk2 at Thr68 and phosphorylation of p53 at Ser15 all of which lead to activation of p53 transcriptional activity, events which do not take place under PTX treatment. Studies aimed at understanding the effects of PTX on wt and mutp53 transcriptional activities are discussed in detail in Chapters Three and Four of this dissertation. Overall, we concluded that PTX interferes with the transcription activity of wild type as well as gain-of-function mutant p53. The blockade of DNA damaging agent-mediated enhancement of mutp53 transcription activity by PTX, suggests the clinical relevance of PTX in carcinomas with mutp53. We suggest that this could be one of the contributing factors in the effects of PTX against human lung cancers.

L’utilisation du cannabis peut précipiter la schizophrénie, en particulier chez les individus qui présentent une vulnérabilité génétique aux désordres mentaux. Des recherches humaines et animales indiquent que la neuréguline 1 (Nrg1) est un gène de susceptibilité à la schizophrénie. L’objectif de cette thèse est d’examiner si une modification du gène Nrg1 chez des souris mutante module les effets neuronaux et comportementaux des cannabinoïdes après traitement aiguë et chronique. De plus cette thèse examine les effets d'un pré-traitement au delta9-tétrahydrocannabinol, le principal composant psychotropique du cannabis, sur un modèle de flexibilité cognitive chez la souris. / Cannabis use may precipitate schizophrenia, especially in individuals who have a genetic vulnerability to the disorder. Human and animal researches indicate that neuregulin 1 (Nrg1) is a susceptibility gene for schizophrenia. This thesis aim at investigating if partial deletion of Nrg1 in mutant mice modulate the neuronal and behavioural effects cannabinoids after acute or chronic treatment. In addition, this thesis examine the effects of a pre-treatment with delta9-tetrahydrocannabinol, the main psychoactive constituent of cannabis, in a model of cognitive flexibility in the mice.

Mouse polyomavirus is a type species of Polyomaviridae family and serves as model for studying viral infection of human pathogenic polyomaviruses. Minor proteins of viral capsid have been found to be necessary for effective initiation of infection. In order to study their role in the early steps of infection we utilized the novel Cre-LoxP system for production of the viral mutant lacking both minor proteins. Virus produced this way was compared with virus produced by standard method and we found that both systems facilitate production of mutant virus with the comparable quality and quantity. The mutant virus contained reduced amount of viral DNA and formed virions with impaired stability. For further studies of intracellular virion trafficking we prepared virions with genomes modified by thymidine analogues 5- bromo-2'-deoxyuridine (BrdU) and 5-Ethynyl-2'-deoxyuridine (EdU) and optimized the methods for analogue detection. The viral genome become accessible for detection 4 hours post infection. For ultramicroscopic analysis of translocation of virus to the nucleus we used freeze substitution. All this methods will be utilized for detailed study of distinct steps in viral infection. Key words: Mouse polyomavirus, minor proteins,...

Genome transcription is much more widespread than has been traditionally thought because our view of a "gene" or "transcription unit" has changed dramatically over the past 4 to 5 years with the identification of many different non-coding ribonucleic acids. In the yeast, Saccharomyces cerevisiae, meiosis and sporulation are an important part of the life cycle and IME4 gene expression is required for these processes. IME4 sense transcript levels of expression are influenced by the level of its complementary non-coding antisense strand by mechanisms that are currently unknown. The a1-alpha2 heterodimer binding in the downstream 3' region of IME4 is one component required for repression of IME4 antisense transcription. However, this thesis shows that the general regulatory protein Reb1 is also required in this system. Reb1 involvement is most likely to create a nucleosome-free zone in the promoter region of the IME4 antisense strand therefore contributing to transcription.

O mutante redA de Dictyostelium discoideum, obtido por inativação gênica aleatória, tem crescimento aparentemente normal, porém seu ciclo de desenvolvimento não progride além do estágio de agregados compactos. Neste trabalho relatamos a caracterização deste mutante, cujo gene defeituoso codifica a enzima NADPH citocromo P450 redutase (NCPR). O principal papel desta enzima é transportar elétrons do NADPH para as várias isoformas do citocromo P450. Um cDNA de 2094 pb que codifica a NCPR de D. discoideum (DdNCPR) foi isolado através da varredura de uma biblioteca de cDNA com uma sonda derivada de um fragmento do gene inativado no mutante redA. A análise da seqüência de aminoácidos deduzida do cDNA DdNCPR revelou que esta codifica uma proteína de 631 aminoácidos com 31% de identidade e 50% de similaridade com a NCPR humana. Verificamos o acúmulo do mRNA da DdNCPR durante fase de crescimento mas durante as fases iniciais do desenvolvimento ocorre significativa diminuição em seus níveis até a formação dos agregados compactos onde o mRNA da NCPR não é detectável. Demonstramos que o gene que codifica a NCPR aparentemente está presente em uma única cópia no genoma de Dictyostelium. Ademais, a análise de outras linhagens mutantes nocautes do gene da NCPR confirmaram que a inativação deste gene está diretamente relacionada ao fenótipo exibido pelo mutante redA-. Contudo, é provável que um ou mais produtos gênicos possam complementar a ausência desta enzima, uma vez que nem a linhagem redA nem as outras linhagens nocautes do gene da NCPR apresentaram alteração na taxa de crescimento e, em algumas circunstâncias experimentais, não exibiram qualquer alteração no ciclo de desenvolvimento. Nossos resultados sugerem, ainda que o bloqueio do desenvolvimento eventualmente observado no mutante redA pode ser devido a um provável papel da NCPR no metabolismo de DIF-1 (fator indutor de diferenciação-1), que parece desempenhar um papel primordial no controle da diferenciação de células pré-talo e células pré-esporo durante o desenvolvimento de D. discoideum. / The Dictyostelium discoideum redA mutant, obtained by random gene inactivation, exhibits normal growth but has its developmental cycle impaired at tight mound stage. In this study we describe the characterization of this mutant whose defective gene encodes the enzyme NADPH cytochrome P450 reductase (NCPR). NCPR is known to play an essential role in the transfer of reducing equivalents from NADPH to various cytochrome P450 isoforms. We isolated a 2094 bp cDNA that encodes D. discoideum NCPR (DdNCPR) by screening a cDNA library using as probe the mutated gene fragment rescued from redA cells. Analysis of the deduced aminoacid sequence of DdNCPR cDNA shows that it encodes a 631 aminoacid protein with 31% of identity and 50% of similarity with human NCPR. Northern blot analysis showed that DdNCPR mRNA levels is maximum during growth phase and decreases at early stages of the development. After slug stage this mRNA is not detectable. D. discoideum has a single copy of NCPR gene and, as shown by analysis of other NCPR knockout mutants, inactivation of this gene is strongly correlated to the redA phenotype. However, redA, as well as the other NCPR knockout strains, do have growth alterations and in some circumstances they do not show the described developmental defects. Thus, it is possible that one or more proteins be able to compensate for the lack of NCPR in these mutants. Our results also suggest that the redA developmental phenotype might play a role of NCPR on the metabolism of DIF-1, a prime candidate for controlling prestalk and prespore cell differentiation during D. discoideum development.