Carvacrol is a phenol monoterpene found in aromatic plants specially in Lamiaceae family, which has been evaluated in an experimental model of breast cancer. However, any proposed mechanism based on its antitumor effect has not been reported. In our previous study, carvacrol showed a protective effect on 7,12-dimethylbenz[α]anthracene- (DMBA-) induced breast cancer in female rats. The main objective in this research was to evaluate by using in silico study the carvacrol on HER2, PI3Kα, mTOR, hERα, PR, and EGFR receptors involved in breast cancer progression by docking analysis, molecular dynamic, and drug-likeness evaluation. A multilevel computational study to evaluate the antitumor potential of carvacrol focusing on the main targets involved in the breast cancer was carried out. The in silico study starts with protein-ligand docking of carvacrol followed by ligand pathway calculations, molecular dynamic simulations, and molecular mechanics energies combined with the Poisson–Boltzmann (MM/PBSA) calculation of the free energy of binding for carvacrol. As result, the in silico study led to the identification of carvacrol with strong binding affinity on mTOR receptor. Additionally, in silico drug-likeness index for carvacrol showed a good predicted therapeutic profile of druggability. Our findings suggest that mTOR signaling pathway could be responsible for its preventive effect in the breast cancer. / Revisión por pares

Le développement dentaire est essentiellement étudié chez la souris, modèle mammifère le plus commun en biologie. Cependant, contrairement à la majorité des mammifères, les souris ne remplacent pas leurs dents. Ainsi, les mécanismes impliqués dans le remplacement dentaire mammalien sont encore inconnus. Au cours de cette thèse, nous nous sommes intéressés au développement et remplacement dentaire mammalien en utilisant le lapin Oryctolagus cuniculus comme modèle d’étude. Le lapin étant déjà séquencé, utilisé en recherche biomédicale avec une période de gestation courte et remplaçant ses dents, il semblait être un modèle pertinent en odontologie. Le lapin était un modèle méconnu du point de vue du développement dentaire, nous avons donc d’abord réalisé une étude histo-morphologique afin de caractériser la mise en place des dents déciduales et permanentes. Des reconstructions 3D des tissus mous ont été réalisés à différents stades embryonnaires afin d’obtenir une chronologie du développement et remplacement dentaire. Cette chronologie commence aux premières observations morphologiques de l’initiation du développement des premières dents jusqu’à la minéralisation des dernières dents à se développer.Puis, suite à l’identification dans la bibliographie de gènes candidats potentiellement impliqués dans le remplacement dentaire, nous avons étudiés les profils d’expressions de ces gènes afin de mieux comprendre la régulation spatio-temporelle du remplacement dentaire chez le lapin. Nous avons ensuite replacé nos résultats sur le développement chez le lapin dans un contexte évolutif. Ainsi, nous avons réalisé une étude d’anatomie comparée chez les lagomorphes actuels et quelques fossiles afin d’identifier des variations morphologiques dentaires au cours de leur histoire évolutive. Nous nous sommes particulièrement intéressé à la mise en place des cuspides au cours de l’odontogénèse ainsi qu’aux variations de la surface occlusale des dents supérieure tout au long de la vie des lapins et autres lagomorphes. En comparant les variations au cours de l’évolution avec celles observées lors de l’ontogénie dentaire chez le lapin nous avons identifié des processus d’hétérochronies du développement. Nous avons ainsi montré que la molaire actuelle du lapin suit un processus de péramorphose, donc de surdéveloppement, en comparaison aux lagomorphes fossiles. Le lapin est ainsi un modèle animal prometteur en biologie du développement et en évolution afin de mieux comprendre la mise en place du remplacement dentaire mammalien ainsi que les variations de forme dentaire au cours de l’évolution des mammifères. / Tooth development is essentially studied in mice, the favorite mammalian model in biology. However, mice do not replace their teeth on contrary to numerous mammals. So, the mechanisms involved in mammalian tooth replacement are still unknown. In this thesis, we focused on mammalian dental development and replacement using the European rabbit Oryctolagus cuniculus as animal model. The European rabbit is already sequenced, used in biomedical field, has a short gestation time and replaced its teeth, so rabbit seemed to be a relevant model in dental research.Rabbit dental development was not defined, so we first performed a histo-morphological study to characterize the development of deciduous and permanent teeth. 3D soft tissue reconstructions were performed at different embryonic stages to obtain a chronology of tooth development and replacement. This chronology begins with the first morphological observations of the initiation of the development of the first tooth until the mineralization of the last tooth to develop.Then, we identified in the bibliography candidate genes potentially involved in dental replacement. We studied the expression profiles of these genes in order to better understand the spatio-temporal regulation of tooth replacement in rabbits.We then returned our results to rabbit development in an evolutionary context. Thus, we performed a comparative anatomy study in the current lagomorphs and some fossils in order to identify dental morphological variations during their evolutionary history. We were particularly interested in the setting of cusps during odontogenesis as well as in the variations of the occlusal surface of the upper cheek teeth throughout the life of rabbits and other lagomorphs. Comparing changes during evolution with those observed during dental ontogeny in rabbits allow us to identify heterochronous processes of development. We have shown that the current molar rabbit follows a process of peramorphosis, so an overdevelopment compared to fossil lagomorphs.The rabbit is thus a promising animal model in developmental and evolutionary biology to better understand the implementation of mammalian tooth replacement and tooth shape variations during mammalian evolution.

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To form complex organisms characterized by different tissues with specialized functions, cells must acquire distinct identities during development. Yet, all the cells of an organism are equipped with the same genomic information. Elucidating the mechanisms that regulate the determination of a cell identity, i.e. the cell-fate commitment, is a main purpose in developmental biology. Numerous studies focused on genes that are activated or repressed at each stage of differentiation, identifying several key regulators of development. However, this approach ignores the transcript variability derived from alternative splicing, the transcriptional process by which different gene coding segments, i.e. exons, are combined giving rise to multiple transcripts and proteins from the same gene. With the advent of novel sequencing technologies, it is becoming clear that alternative splicing is widespread in higher organisms, regulates several processes and presents tissue- and cell-specificity. In mammals, the brain shows the highest degree of alternative splicing, with neurons expressing a high variety of splice variants. In this project I investigated whether and how alternative splicing could regulate cell-fate determination in the context of the embryonic development of the mouse neocortex, a highly complex structure presenting several different neuronal subtypes generated at specific time points. For this purpose, I analyzed transcriptome data of cells of the neurogenic lineage isolated from the developing mouse neocortex at subsequent stages of differentiation. I showed that the expression pattern of the proteins regulating splicing, i.e. the splicing factors, changes during neocortical development. By employing several bioinformatic tools, I described the splicing profile that characterizes each differentiation stage and, for the first time, I identified the splicing events that mark cell-fate commitment to a neurogenic identity. Alternative splicing mostly involved genes with a role in nervous system development, cell growth and signaling, mainly leading to the production of alternative protein isoforms. Splicing choices taken during the neurogenic commitment were kept throughout neurogenesis. Thus, exons that start to be included during cell-fate determination are always included in post-mitotic neurons. Exons gained during neurogenic commitment were characterized by strong features in their upstream intron, presented a general short length with an overrepresentation of microexons in the 3-27 nucleotides length range and showed an enrichment for binding motifs of the neural splicing factor nSR100. In vivo manipulation in the embryonic mouse neocortex highlighted isoform-specific effects on neocortical development, strongly suggesting a causal relationship between alternative splicing choices and cell-fate commitment. Moreover, the higher cell-specificity offered by the present dataset, compared to similar studies, allowed a better understanding of previously identified splicing events that characterize the nervous system and the relationships between neural-specific splicing factors.:Table of Contents Abstract I Zusammenfassung III Table of Contents V List of Figures VII List of Tables IX Abbreviations X Gene abbreviations XII 1 Introduction 1 1.1 Neurogenesis during embryonic development 2 1.1.1 Formation and patterning of the neural tube 2 1.1.2 Neural progenitors in the dorsal telencephalon 6 1.1.3 Neurogenesis 8 1.1.4 Regulation of neurogenesis 10 1.1.5 A novel tool to investigate cell-fate determination in the central nervous system: the Btg2RFP/Tubb3GFP mouse line 13 1.2 Alternative splicing: an additional level of genomic regulation 15 1.2.1 The splicing reaction 16 1.2.2 What makes splicing alternative? 18 1.2.3 Regulation of alternative splicing 19 1.2.4 The challenge to detect splicing 23 1.2.5 New sequencing technologies reveal a high transcriptome complexity 29 1.2.6 Splicing in nervous system development 31 1.2.7 Aims of the project 36 2 Materials and methods 38 2.1 Materials 38 2.1.1 Bacteria, cells, mouse strains 38 2.1.2 Vector 38 2.1.3 Primers 38 2.1.4 Chemicals and buffers 41 2.1.5 Antibodies 42 2.1.6 Kits and enzymes 42 2.2 Methods 43 2.2.1 Animal experiments 43 2.2.2 Molecular biology 44 2.2.3 Immunohistochemistry 46 2.2.4 Bioinformatics 47 3 Results 53 3.1 Splicing factors are differentially expressed during neurogenic commitment and neurogenesis 53 3.2 Detection of alternative splicing 55 3.2.1 Isoform-switching 55 3.2.2 Exon usage and splicing events 57 3.3 Validation 62 3.3.1 The isoform switching method has a poor validation rate 62 3.3.2 Analysis at the exon level has a high rate of validation 65 3.4 Pattern and representation of splicing events 67 3.4.1 Splicing choices during neurogenic commitment define the splicing profiles of neurons 67 3.4.2 Splicing events: microexon inclusion characterizes neurogenic commitment 69 3.5 Alternative splicing changes the protein output of genes involved in neurogenesis 75 3.5.1 Spliced genes are involved in neurogenesis and signaling 75 3.5.2 Impact of alternative splicing on the proteome 77 3.6 Splicing regulation: neural exon features and splicing factor binding 79 3.6.1 Included neural exons are short and preceded by strong exon-definition features 79 3.6.2 Early included exons are enriched for nSR100 binding sites 85 3.7 The Btg2RFP/Tubb3GFP mouse line outperforms previous models for the study of cell-type-specific splicing in the brain 88 3.8 In vivo manipulation of splice variants 90 4 Discussion 94 4.1 The combination of different bioinformatic approaches allows an accurate identification of splicing events at the exon-level 95 4.2 Splicing choices during neurogenic commitment establish a neural signature characterized by microexon inclusion 97 4.3 Splicing during neocortical development leads to the generation of alternative protein isoforms in genes involved in neurogenesis and signaling 98 4.4 Neural exons are short and present strong features facilitating inclusion 101 4.5 Neural exons are prevalently regulated by nSR100 during neurogenic commitment 102 4.6 In vivo overexpression of splice variants highlights isoform-specific functions in neurogenic commitment 105 4.7 Concluding remarks and future perspectives 108 5 Supplementary figures 110 6 References 118 Acknowledgments 137 Anlange I 138 Anlange II 139

During evolution, the mammalian cerebral cortex has undergone a considerable increase in size and complexity. The emergence of the cortical structure begins during embryonic development when neural stem cells initially undergo proliferative division to expand their pool and then switch to neurogenic division, generating differentiating progenitors that will give rise to neurons. Although the intrinsic molecular mechanisms instructing the switch from proliferative to neurogenic division have been well-studied, most work to date has focused on gene expression. However, as a consequence of transcriptional and post-transcriptional regulation, different transcripts can arise from a single gene. In particular, the process of alternative splicing occurs at a high frequency in the nervous system and can lead to changes in protein output regardless of gene expression. In the past years, the role of post- transcriptional mechanisms in neuronal maturation and function have been extensively investigated, mostly focusing on the function of specific isoforms or RNA binding proteins. Yet, the role of alternative splicing in generating transcript and protein diversity during neurogenic commitment is still unknown. Therefore, I used a combination of different RNA sequencing technologies and bioinformatic tools to reveal the transcript and protein diversity of proliferating progenitors, differentiating progenitors, and neurons isolated from double reporter mouse line. I identified widespread isoform diversity and many novel transcripts amongst expressed genes in the developing cortex. To date, this analysis represents the most comprehensive characterization of full-length transcript diversity at different stages of the neurogenic lineage in the developing mouse cortex. The resulting transcriptome annotation was used to quantify changes in exon inclusion across cells of the neurogenic lineage and identified alternative splicing events potentially involved in neurogenic commitment. These alternative splicing events were enriched in the coding sequence of isoforms, indicating that they might be relevant for protein diversity generation in the developing cortex. During neurogenesis, alternative splicing events were less enriched in regions that could disrupt or strongly affect protein structure and function, such as transmembrane regions, active sites, and domains. Interestingly, my results indicated that alternative splicing enables increased functional diversity by modulating protein-protein interaction sites and signaling properties of proteins. Still, further studies are required to delineate the causal relationship between these alternative splicing choices and cell-fate commitment. Applying a similar approach to other mammalian species, including humans, has the potential to uncover species-specific innovations and conserved features that underlie evolutionary cortex expansion. Moreover, understanding the function of isoforms during neural development could provide important insights into the molecular mechanisms involved in the onset of neurodevelopmental disorders. Therefore, the higher cell-specificity offered by the present dataset, compared to similar studies, allowed not only a better understanding of transcript and protein diversity generated by alternative splicing in the nervous system and highlighted potential functional consequences, but also shed light on the advantages of applying such strategy to address different biological questions.

La plasticité synaptique activité-dépendante forme la base physiologique de l’apprentissage et de la mémoire dépendants de l’hippocampe. Le rôle joué par les différents sous-types d’interneurones dans l’apprentissage et la mémoire hippocampiques reste inconnu, mais repose probablement sur des mécanismes de la plasticité spécifique aux synapses de certains sous-types d’interneurones. Les synapses excitatrices établies sur les interneurones de l’oriens-alveus dans l’aire CA1 exhibent une forme persistante de potentialisation à long terme induite par la stimulation chimique des récepteurs métabotropiques du glutamate de type 1 (mGluR1) [mGluR1-mediated chemical late long-term potentiation (cL-LTPmGluR1)]. Le présent projet de recherche avait pour objectifs d’identifier les sous-types d’interneurones de l’oriens-alveus exprimant la cL-LTPmGluR1 et d’examiner les mécanismes d’induction et d’expression de celle-ci. Nous avons déterminé que la stimulation répétée des mGluR1 induit de la cL-LTPmGluR1 aux synapses excitatrices établies sur le sous-type d’interneurones exprimant le peptide somatostatine (SOM-INs). Des enregistrements électrophysiologiques couplés à des inhibiteurs pharmacologiques et à un knock-out fonctionnel de mammalian target of rapamycin complexe 1 (mTORC1) ont montré que l’induction de la cL-LTPmGluR1 (qui consiste en trois applications de l’agoniste des mGluR1/5, le (S)-3,5-dihydroxyphénylglycine (DHPG) en présence de l’antagoniste des récepteurs métabotropiques du glutamate de type 5 (mGluR5), le 2-méthyl-6-(phényléthynyl)-pyridine (MPEP)) des SOM-INs requiert les voies de signalisation des mGluR1, de extracellular signal-regulated protein kinase (ERK) et de mTORC1. L’ensemble de nos résultats montre qu’une forme persistante de plasticité synaptique sous-tendue par mTORC1 est induite par la stimulation répétée des mGluR1 dans les interneurones hippocampiques exprimant le peptide somatostatine. La connaissance des mécanismes sous-tendant la cL-LTPmGluR1, couplée à l’utilisation de modèles animal in vivo, rendront maintenant possible le blocage de la cL-LTPmGluR1 dans les SOM-INs et l’examen de son rôle dans l’apprentissage et la mémoire dépendants de l’hippocampe. / Hippocampus-dependent learning and memory are mediated by activity-dependent synaptic plasticity. The role that different subtypes of interneurons play in hippocampal learning and memory remains largely unknown, but likely relies on cell type-specific plasticity mechanisms at interneuron synapses. Excitatory synapses onto CA1 oriens-alveus interneurons show persistent long-term potentiation induced by chemical stimulation of metabotropic glutamate receptor 1 (mGluR1) [mGluR1-mediated chemical late long-term potentiation (cL-LTPmGluR1)]. The objectives of this project were to identify the oriens-alveus interneuron subtypes expressing cL-LTPmGluR1 and examine its induction and expression mechanisms. We determined that repeated mGluR1 stimulation induces cL-LTPmGluR1 at excitatory synapses onto the somatostatin-expressing interneuron subtype (SOM-INs). Electrophysiological recordings coupled to pharmacological inhibitors and a functional knock-out of mammalian target of rapamycin complex 1 (mTORC1) showed that SOM-INs cL-LTPmGluR1 induction (which consisted of three applications of the mGluR1/5 agonist (S)-3,5-dihydroxyphenylglycine (DHPG) in the presence of metabotropic glutamate receptor 5 (mGluR5) antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP)) requires mGluR1, extracellular signal-regulated protein kinase (ERK) and mTORC1 signaling pathways. Collectively, our results show that persistent synaptic plasticity mediated by mTORC1 is induced by repeated mGluR1 stimulation in somatostatin-expressing hippocampal interneurons. Knowledge of cL-LTPmGluR1’s underlying mechanisms, coupled to in vivo models, will now make it possible to interfere with SOM-INs cL-LTPmGluR1 and examine its role in hippocampal-dependent learning and memory.

The development of safe and efficient gene delivery systems in pluripotent human embryonic stem cells (hESc) is essential to realising their full potential for basic and clinical research. The purpose of this study was to develop an efficient, non-integrating gene expression system in pluripotent hESc using human artificial chromosomes (HAC). Similar to endogenous chromosomes, HAC are capable of gene expression, replication and segregation during cell division. Unlike retroviral-mediated gene delivery vectors, HAC do not integrate into the host genome and can encompass large genomic regions for the delivery of multiple genes. Despite the advantages HAC offer, their use has been limited due to laborious cloning procedures and poor transfection efficiencies, and thus only studied in immortalised and tumour-derived human cell lines. In this study, the high transduction efficiency of herpes simplex virus type-1 (HSV-1) amplicons was utilised to overcome the described difficulties and delivered HAC vectors into pluripotent hESc. Analysis of stable hESc clones showed that de novo gene-expressing HAC were present at high frequencies ranging from 10-70% of metaphases analysed, without integrating into the genome. The established HAC contained an active centromere, and were stably maintained without integration or loss in the absence of selection for 90 days. Stable HAC-containing hESc clones retained their pluripotency as demonstrated by neuronal differentiation, in vitro germ layer and teratoma formation assays. HAC gene expression persisted, with some variation, post-differentiation in the various deriving cell types. This is the first report of successful de novo HAC formation in hESc for gene expression studies. These findings show potential for delivering high-capacity genomic constructs safely and efficiently into pluripotent cells for the purpose of genetic manipulation and ultimately patient-specific somatic gene therapy.

Topoisomerase 2α (Topo2α) is an essential protein with DNA decatenating enzymatic properties, indispensable for chromosome decatenation and segregation. It is a target for a plethora of antitumour drugs and Topo2α protein levels have been associated with the success of treatment, but also drug resistance and secondary malignancies. Although unique in its ability to resolve catenated chromosomes, the role of Topo2α in other steps of DNA metabolism, such as DNA replication elongation and termination have been elusive. A thorough understanding of the role of Topo2α in the cell will not only allow for increased insight into the mechanisms it is involved in, but it will also shed light on proteins and pathways that can act as back-up in its absence, and therefore hopefully expand the basis on which to improve treatment options. Through a synthetic lethal interaction (SLI) screen with an siRNA library targeting 200 DNA repair and signalling genes, Topo2α emerged as being synthetic lethal to Werner protein (WRN), a RecQ helicase involved in maintaining genome integrity mainly in S phase, and the loss of which leads to Werner Syndrome (WS), a segmental progeroid syndrome. The screen was performed in WRN deficient cells, with the initial aim to find proteins that act to buffer against loss of viability, which is the central idea in the concept of synthetic lethality in the absence of WRN. The screen revealed an SLI between WRN and Topo2α and although we were unable to fully validate this, it spurred the question of Topo2α’s role in DNA replication. The findings in this thesis suggest that Topo2α is not required for DNA elongation and timely completion of S phase, and that simultaneous loss of the closely related isoform Topo2β does not affect replication, suggesting that these proteins do not act in parallel back-up pathways during replication. Interestingly, cells accumulate in the polyploid fraction after both depletion and inhibition of Topo2α, albeit with different kinetics. The mechanistic basis of this phenotype remains to be understood through further research, but it is highly interesting as aneuplidity and polyploidy are implicated in the initial stages of tumour development.

This thesis is concerned with the topic of teaching histology at secondary schools. Histology is focused on the study of microscopic structure of tissue. By this it comprises the basic foundation stone for the studies of organs and organ systems not merely in humans, but also in other animals. The method of preparing the permanent histological slides, which has also been tested in practice, is thoroughly described in this thesis. The main goal of the practical part was to prepare a representative collection of the permanent histological sections through the organs of the house mouse (Mus muculus), to acquire its photo documentation as a base for an interactive histological atlas. House mouse (Mus musculus) was chosen as the model animal, because of its frequent utilization in various laboratory experiments and because this model mammal has the structure of tissues similar to human. The atlas consists of the photo documentation of histological sections through the following organs: lungs, skin, heart, thymus gland, spleen, epididymis, testicle, penis, spermatic sacs, striated muscle, heart muscle, smooth muscle, liver, tongue, pancreas, small intestine, large intestine, gall bladder, kidney, urinary bladder, urethra, cerebellum and hippocampus. The sections are stained with application of...