A recente explosão do conhecimento da genética molecular e o avanço da indústria da biotecnologia requerem que o público compreenda muitos conceitos da genética para a tomada de decisões sobre a pertinência do uso dessas novas ferramentas. Durante os últimos 30 anos a literatura educacional produziu conceitos e teorias para lidar com estas dificuldades, mas a maioria dos professores desconhece estas produções, principalmente porque os periódicos de referência são publicados em línguas estrangeiras (principalmente em inglês). Esta pesquisa-ação pretende preencher esta lacuna e foi baseada em testes que envolveram 283 estudantes de 12 a 15 anos de idade. Nela descrevemos as concepções dos estudantes sobre a localização e transmissão da informação genética antes e após a aplicação de uma seqüência didática elaborada especificamente para desenvolver estratégias metacognitivas de aprendizagem. As idéias dos estudantes foram colhidas por meio dos questionários e redações, nas quais os estudantes descrevem como imaginam ser o interior das células e dos gametas. Verificamos que as crianças do ensino fundamental possuem concepções semelhantes àquelas descritas para estudantes do ensino médio. A comparação das redações produzidas pelos estudantes antes e após a aplicação da seqüência didática permitiu verificar que o padrão mais comum de aprendizagem é sincrético, ou seja, as crianças tendem a distorcer as informações oferecidas pelo professor em virtude da existência de conhecimentos prévios. Por fim, a descrição e a documentação de seqüências didáticas planejadas a partir de conhecimentos produzidos na literatura educacional permitem o entendimento dos processos de transposição didática e a relação deste com a aprendizagem dos estudantes. / The recent knowledge explosion on the areas of genetics, molecular biology and biotechnology introduced many new concepts hard for common people to grasp in their decision-making processes. During the last 30 years or so the educational literature produced concepts and theories to cope with these difficulties but the vast majority of our elementary and highschool teachers remain unaware of them possibly because the available literature is written in foreign languages (mainly in English). The action-research here presented intends to fill this gap and was based on tests performed with 283 students aged 12 to 15. We describe their conceptions on the location and transmission of genetic information before and after the application of a didactic sequence specifically elaborated to develop metacognitive learning strategies. The students\' ideas were gathered by means of questionnaires and through essays describing how they imagine the interior of cells and germ-cells. We verified that children on basic educational level have conceptions very similar to those of students of middle-level education. The paired comparison of before and after essays suggests the existence of a common, syncretic learning standard. In plain language, the results indicate that previous informal knowledge of children tends to distort the formal information transmitted by their teachers. It is clear that the description and documentation of planned didactic sequences, available from the specialized literature, provide the understanding of the didactic transposition process and its relation with the students´ learning process.

Genome of S. pneumoniae contains only one copy of the gene coding eukaryotic type protein kinase StkP and corresponding phosphatase PhpP. These two enzymes form a functional signaling pair regulating cell division, which could be used in the future for the design of new bacteriostatic compounds. Not only kinase and phosphatase are important components of the system, but also other members of this pathway - specific substrates of these enzymes. The identification of the Ser/Thr phosphoproteom with a focus on the membrane fraction provided information not only about already known substrates such as LocZ, Jag and DivIVA but also about an unknown protein P15 with a molecular weight about 15 kDa. In this thesis the protein was identified as rhodanase (spr0595) by MS MALDI TOF. However, its subsequent deletion did not confirm it as a StkP/PhpP substrate. Therefore we investigated another substrate, protein FtsA, which has already been identified as a substrate of this kinase in a previous study (Beilharz et al., 2012). FtsA is an essential cell division protein that anchors FtsZ filaments into the membrane. Phosphorylation of this protein was detected on the Thr residue at position 404. Using phosphoablative substitution we found out, that Thr404 is indeed phosphorylated by protein kinase StkP, however, FtsA...

It is well known that tight coordination of cell division timing is essential for proper cell fate specification and tissue growth during metazoan development. However, how cell divisions are coordinated in vivo is largely unknown. In this thesis, a high- content screening was conducted to identify genes responsible for temporal coordination of cell division during Caenorhabditis elegans embryogenesis. A total of 822 genes were depleted using RNA interference (RNAi). The genes were prioritized based on their degree of conservation in human, as well as their roles in development. In addition to RNAi, an experimental pipeline was established, including 3D time-lapse imaging of an RNAi perturbed C. elegans embryos followed by automated lineaging, which allows systematic quantification of division timing of each individual cells up to approximately 350-cell stage. To identify genes with a significant reduction in the asynchrony of division between sister cells (ADS) upon perturbation, average division timings of each cell between at least two replicate perturbed embryos was compared against the average division timings of 92 wild type embryos. It was found that cell fate determinants were not only important for maintaining fate asymmetries, but are also imperative for establishing ADS regardless of cellular context. Hence, the results demonstrate that fate and temporal asymmetries share a common genetic architecture. The temporal coordination appears to facilitate cell migration during fate specification or tissue growth. Given the observation that perturbations of signalling pathways, especially Wnt and Notch pathways, are frequently associated with the ADS, it would be essential to map the exact signalling event that takes place at cellular level which is responsible for a given ADS or fate asymmetry. To this end, a miniMos transposon-meditated transgenic technique was adopted to insert a fusion between GFP and a promoter derived from individual components of the two signalling pathways into the C. elegans genome. The resulting insertion was crossed into a strain expressing lineaging markers, which allows automated lineaging and gene expression profiling to map the expression of each component with single cell resolution. A combination of cellular expression and cell-cell contact data would lead to a comprehensive map of each signalling event during C. elegans embryogenesis. Our preliminary results on cell-cell contacts and cellular expression validated the existing signalling events and identified potential novel ones that are associated with either ADS and/or fate asymmetry.

We describe the dynamic process of abdominal segment generation in the milkweed bug Oncopeltus fasciatus. We present detailed morphological measurements of the growing germband throughout segmentation. Our data are complemented by cell division profiles and expression patterns of key genes, including invected and even-skipped as markers for different stages of segment formation. We describe morphological and mechanistic changes in the growth zone and in nascent segments during the generation of individual segments and throughout segmentation, and examine the relative contribution of newly formed versus existing tissue to segment formation. Although abdominal segment addition is primarily generated through the rearrangement of a pool of undifferentiated cells, there is nonetheless proliferation in the posterior. By correlating proliferation with gene expression in the growth zone, we propose a model for growth zone dynamics during segmentation in which the growth zone is functionally subdivided into two distinct regions: a posterior region devoted to a slow rate of growth among undifferentiated cells, and an anterior region in which segmental differentiation is initiated and proliferation inhibited.

Phenotypic heterogeneity in terms of cell size, morphology, and metabolic status, which are believed to help the population survive under stress conditions, is known in mycobacterial populations. Such population heterogeneity had been observed in in vitro cultures, TB patients, and in animal models. Our laboratory had earlier shown that about 20-30% of the 15% septating cells of Mycobacterium smegmatis, Mycobacterium tuberculosis and Mycobacterium xenopi mid-log phase cultures divide by highly deviated asymmetric cell division (ACD), generating subpopulations of short cells and normal-sized/long cells. The remaining 70-80% of the septating cells divide by symmetric cell division (SCD) with 5-10% deviation of the division site constriction from the median. The proportion of short cells amounted to about 3-5% of the total population, while the remaining 97-98% of the population was constituted by normal-sized/long cells. This proportion of short cells has been found to be consistent and reproducible irrespective of culture media. Comparable proportion of short cells of tubercle bacilli has been found in the freshly diagnosed pulmonary tuberculosis patients’ sputum also. It indicated that such processes must be occurring in the tubercle bacillary population in the TB patients too and that the presence of short cells has some physiological relevance. Thus, ACD has been found to be one of the mechanisms that mycobacteria use to generate cell size heterogeneity in the population. However, there has not been any study on the mechanism that generate such subpopulation of short cells through ACD. Therefore, in the present study, we investigated the mechanism behind the ACD that generates short cells in the Mycobacterium smegmatis and Mycobacterium tuberculosis populations. The Chapter 1, which forms the Introduction to the thesis, gives an extensive literature survey on all the different areas of research in bacterial physiology that are linked by the present study. These areas of research include bacterial cell division process per se, the proteins involved in bacterial cell division, cell division in general in mycobacteria, different modes of cell division in mycobacteria, generation of cell size heterogeneity through symmetric and asymmetric cell division in mycobacteria, cell-cell communication in bacterial systems, and the characteristics and physiological significance of diadenosine polyphosphates from bacteria to humans. The complete account of the research in these areas that are linked in the present study thus justifies the introduction to the results that are given in the ensuing chapters. The Chapter 2 forms the Materials and Methods used in the present study. Here a detailed description of the methods used for the cell division bioassay used to score for the proportion of cells undergoing symmetric and asymmetric divisions, the biochemical assays performed to find out the biochemical identity of the molecule, the isolation and fractionation methods for the ACD-IM from the concentrated culture supernatants (CS), and finally the mass spectrometric methods used for the elucidation of the structure of the molecule are given The Chapter 3 forms the first data chapter that presents results on the detection of the presence of the asymmetric cell division inducing molecule (ACD-IM) from the concentrated culture supernatant (CS) of Mycobacterium smegmatis and Mycobacterium tuberculosis, as inducing ACD in higher proportions of cells. Further, it shows that the levels of ACD-IM increase at late growth phases of 0.8 and 1.0 OD600 nm. The chapter is concluded with a discussion of the results. The Chapter 4 describes the biochemical analyses of the CS to find out the chemical nature of the ACD-IM. DNase I, snake venom phosphodiesterase (SVP), RNase A, lipase, and proteinase K were used to find whether exposure of CS of M. smegmatis and M. tuberculosis cells to these enzymes could abolish the ACD inducing activity of the molecule in the CS on the respective cells. These experiments showed that the ACD-IM was susceptible to DNase I and SVP, but not to RNase A, lipase, or proteinase K. However, proteinase K showed direct effect on the cells by decreasing the proportion of cells dividing by ACD, indicating the probable presence of the receptor to the molecule on the external cell surface. The data also shows the conservation of the molecule in M. smegmatis and M. tuberculosis by demonstrating that the CS of M. smegmatis could induce ACD in higher proportions of M. tuberculosis cells and vice versa. The structural identification of ACD-IM present in the concentrated CS of M. smegmatis and M. tuberculosis using LC-ESI-MS and MS-MS analyses as diadenosine hexaphosphate (Ap6A). The structure of the natural Ap6A molecule was confirmed using synthetic Ap6A molecule subjected to LC-ESI-MS and MS-MS analyses. Further, the ACD inducing activity of the synthetic Ap6A molecule, its susceptibility to DNase I and SVP, but not to RNase A, lipase and proteinase K were verified to establish that the structural, biochemical, and functional properties of the naturally occurring Ap6A in the CS of M. smegmatis and M. tuberculosis were identical to those of synthetic Ap6A. The chapter is concluded with a discussion of the results. The Chapter 5 presents the data on the genes involved in the degradation and synthesis of Ap6A in M. smegmatis and M. tuberculosis. The knockout of MSMEG_2936 gene of M. smegmatis resulted with significant increase in the ACD proportion. The quantitation of Ap6A in the CS obtained from this strain found significantly higher concentration than wild type. These results showed that MSMEG_2936 protein might catalyse the degradation of Ap6A in mycobacteria. The knockout of MSMEG_2932 gene of M. smegmatis resulted with significant reduction in the ACD proportion. The quantitation of Ap6A in the CS obtained from this strain found comparable to wild type. These results showed that MSMEG_2932 protein might not involve in the synthesis of Ap6A in mycobacteria. The chapter is concluded with a discussion of the results. Thus, the present study, for the first time, establishes the identity, structure, and function of Ap6A as the molecule that induces asymmetric cell division in mycobacteria.

Les cellules positionnent leur plan de division de manière précise et prévisible. En particulier au tout début de l’embryogenèse, la cellule-œuf suit un patron de clivage extrêmement reproductible, mais néanmoins sensible aux perturbations (manipulation de la forme de la cellule,…), ce qui suggère une plasticité intrinsèque du système. Au cours de ma thèse, je me suis intéressée aux signaux qui déterminent la position des plans de division embryonnaires, et à leur compétition. Dans un premier temps, j’ai développé un modèle pour prédire le positionnement du plan de division à partir de la forme de la cellule, et de la présence éventuelle de polarité maternelle à la membrane ou d’une distribution inhomogène de yolk/organelles dans le cytoplasme. Ce modèle est basé sur les forces de traction exercées par les microtubules des astres interphasiques sur le fuseau mitotique/noyau. Sous l’hypothèse que ces forces dépendent de la longueur des microtubules (dynéine dans le cytoplasme) et sont modulées par la polarité membranaire, il est alors possible de trouver la position d’équilibre du fuseau, qui détermine le futur plan de division. J’ai également reproduit les formes et réarrangements des cellules (blastomères) dans l’embryon après la division, à l’aide d’un programme (The Surface Evolver) qui minimise l’énergie de surface sous différentes contraintes : ici les volumes, tensions de surface et éventuels confinements. En bouclant la génération des formes des blastomères avec la prédiction de leurs divisions (les formes permettent de prédire la division, qui permet de générer les formes des cellules filles, etc…), j’ai pu reproduire de manière quantitative quatre patrons de clivage représentatifs (poisson-zèbre, xenope, oursin, ascidie), jusqu’au stade 8 à 16 cellules, in silico. J’ai également testé le modèle sur des expériences classiques de perturbation dans ces quatre systèmes (Hertwig, Hörstadius, ablation de la polarité,…), et reproduit les observations de la littérature. Cette première partie suggère que ces systèmes sont auto-organisés et que la détermination du plan de division dépend principalement d’un nombre restreint de signaux. Dans un second temps, j’ai cherché à caractériser la compétition entre les signaux de forme et de polarité maternelle chez l’embryon d’oursin, de manière quantitative. Ce projet comprend une part importante d’imagerie 3D (position des centrosomes et division, polarité, forme des blastomères), ainsi que des expériences visant à tester le rôle de la forme/taille des blastomères et de la polarité (séparation des blastomères, microchambres de différentes formes, inhibition de la polarité,…). Les résultats obtenus sont comparés aux prédictions du modèle, cette fois basées sur la forme imagée des blastomères. Ces résultats expérimentaux confirment les hypothèses de l’étude in silico, et permettent d’évaluer la robustesse du système biologique pour affiner le modèle. / Cells position their cleavage plane in a precise and predictable way. In particular, during the early embryogenesis, the cleavage pattern of the egg cell is extremely reproducible, yet sensitive to perturbation (shape manipulation,…), which suggests an intrinsic plasticity of the system. My PhD project is about the signals that determine the positions of the cleavage planes in the embryo, and their competition. First, I developed a model to predict division plane positioning from cell shape and possible additional cortical maternal polarity or inhomogeneous yolk/organelles distribution within the cytoplasm. This model is based on pulling forces exerted by interphase astral microtubules on the mitotic spindle/nucleus. Under the hypothesis that these forces depend on microtubule lengths (dynein in the cytoplasm), and are modulated by cortical polarity, it is then possible to find the equilibrium position of the spindle, that sets the future division plane. In addition, I reproduced the shapes and rearrangement of cells (blastomeres) within the embryo, with a program (The Surface Evolver) that minimizes surface energy under various constraints : here cell volumes, surface tensions and possible confinements. The modeling framework I used consisted in a loop between cell shape generation and division plane prediction (cell shape allows to predict cell division, that gives the daughter cells volumes and positions to generate the next cell shapes, and so on…). I could quantitatively reproduce four representative cleavage patterns (zebrafish, xenopus, sea urchin, ascidian), up to the 8 to 16-cell stage, in silico. I also tested the model on classic perturbation experiments in these four systems (Hertwig, Hörstadius, polarity ablation,…), and reproduced the observations of the literature. This first part suggests an auto-organization of these systems, and that the determination of the cleavage plane mainly depends on a limited number of signals. Second, I aimed at characterizing the competition between shape and maternal polarity cues, in a quantitative manner. This project comprises 3D imaging (positions of the centrosomes and division planes, polarity, blastomere shape), as well as experiments assessing the roles of blastomere shape/size and of polarity (blastomere separation, microchambers of different shapes, polarity inhibition,…). The results are compared to the predictions of the model, that now inputs the imaged blastomere shapes. These experimental results confirm the hypotheses of the in silico study, and allow assessing the robustness of the biological system to refine the model.

Regulation of penicillin-binding protein Pbp2a in Streptococcus pneumoniae Streptococcus pneumoniae is an extracellular human pathogen that encodes a unique eukaryotic-type Ser/Thr protein kinase StkP in its genome. This enzyme is involved in other cellular processes, such as cell division and cell wall synthesis, through phosphorylation with its substrates. A transmembrane protein MacP has been identified as a substrate of StkP. It is an activator of penicillin-binding protein PBP2a, which is involved in the synthesis of peptidoglycan with its transpeptidase and transglycosylase activities. We found that MacP is phosphorylated by the protein kinase StkP at positions T32 and T56. We confirmed that proteins MacP and PBP2a interact with each other and that phosphoablative and phosphomimetic mutations of the major phosphorylated residues of the MacP protein do not affect the interaction with PBP2a and do not fundamentally affect the function of MacP in vivo. Furthermore, we showed that the ∆macP mutation is synthethically lethal with the ∆pbp1a mutation, confirming that MacP is an activator of the PBP2a protein. MacP is located in the cell septum and interacts with a number of S. pneumoniae cell division proteins. Keywords: Streptococcus pneumoniae, cell division, MacP, PBP2a, phosphorylation

Notch est le récepteur d’une voie de communication intercellulaire, conservée au cours de l’évolution et impliquée dans de nombreux processus développementaux. Chez Drosophila melanogaster, la spécification et la division des précurseurs des organes sensoriels (SOPs) sont gouvernées par l’activation différentielle de la voie de signalisation Notch. Cette activation est dépendante de l’interaction entre le récepteur Notch et les ligands Delta/Serrate et LAG-2. Cette interaction favorise le clivage protéolytique du récepteur Notch puis la libération et la translocation du domaine intracellulaire dans le noyau de la cellule receveuse du signal. L’activation de Notch est étroitement régulée dans le temps et dans l’espace et est sous le contrôle du trafic intracellulaire. Toutefois, la localisation exacte de l’interaction entre le ligand et le récepteur demeure encore débattue.Précédemment, la protéine Stratum, prédite pour avoir un rôle de facteur d’échange nucléotidique (GEF), fut identifiée comme régulateur de la voie de signalisation Notch. Ici, nous montrons que la perte de Stratum induit des phénotypes Notch associés à une délocalisation du co-facteur de Notch, Sanpodo, au pôle apical des cellules et dans le réseau trans- golgien, avec Notch et Delta. De plus, nous montrons que Rab8 est délocalisée en absence de Stratum et la perte de Rab8 récapitule les phénotypes Notch observés dans le mutant strat. Ensemble, nous résultats indiquent que Stratum et Rab8 régulent la voie de signalisation Notch en contrôlant à la fois le tri et le transport polarisé de Notch, Sanpodo et Delta à la sortie de l’appareil de Golgi. / Notch is the receptor of an evolutionarily conserved intercellular communication pathway, involved in numerous developmental processes. In Drosophila melanogaster, the specification and the division of sensory organ precursors (SOPs) are governed by the differential activation of the Notch signalling pathway. This activation depends on the interaction between the Notch receptor and its ligands Delta/Serrate and LAG-2. This interaction induces the proteolytic cleavage of the Notch receptor, the release and the translocation of the intracellular domain in the nucleus of the signal-receiving cell. The Notch activation is tightly regulated in time and in space and is controlled by intracellular trafficking. However, the exact localisation of the interaction remains debated.Previously, Stratum, predicted to be a guanine exchange factor (GEF), was identified as a regulator of the Notch signalling pathway. Here we show that the loss of Stratum induces Notch phenotypes associated with a mislocalization of the Notch co- factor, Sanpodo, at the apical pole of cells and in the trans-golgi network, with Notch and Delta. Moreover we show that Rab8 is mislocalized in the absence of Stratum and the loss of Rab8 recapitules Notch phenotypes observed in the strat mutant. Together our results indicate that Stratum and Rab8 regulate the Notch signalling pathway by controlling both the sorting and the transport of Notch, Sanpodo and Delta at the exit of the Golgi apparatus.

Mammals receive more germline mutations from fathers than mothers. While the paternal bias in mutation has historically been attributed to errors in DNA replication during spermatogenesis, evidence suggests that in humans mutational mechanisms independent from cell division may play a more prominent role. Understanding how the ratio of paternal-to-maternal mutations, 𝛼, varies across animals differing in their gametogenic development, physiologies, and habitats can provide unique insights into the processes by which mutation arises in male and female germlines. To these ends, this thesis examines features of paternal mutation bias in dozens of amniote species using a combination of sequencing and evolutionary approaches. A direct way of measuring the strength of paternal mutation bias involves sequencing pedigrees of related individuals and detecting mutations arising in a single generation. In Chapter 2, we applied this approach to measure 𝛼 in olive baboons (Papio anubis) and humans. Strikingly, we estimated that in baboons 𝛼 = 4.5, similar to humans, despite baboons experiencing far fewer spermatogenic cell divisions than humans. A model of mutation based on cell division differences in the two species failed to explain this observation. Our results provide added evidence for non-replicative processes driving paternal bias in mutation and suggest that these causes are likely shared across mammals. In Chapter 3, we expanded our analysis to survey 𝛼 across 42 amniote species. We estimated 𝛼 from putatively neutral substitution rates of sex chromosomes and autosomes and found that in mammals, 𝛼 ranges up to 4 and correlates with generation times. In contrast, birds and snakes harbor a stable 𝛼 of roughly 2. These results are well predicted by modeling sex bias in mutation as a product of an early developmental phase when mutation occurs equally in both parents and a late phase after sexual differentiation when the male germline is more mutagenic. That the paternal mutation bias is widespread and occupies a narrow range of values suggests that it is caused by endogenous damage sources that are similar across species. Through a combination of pedigree sequencing and evolutionary techniques, this work demonstrates how a comparative approach across diverse taxa can shed light on the origins of sex-bias in germline mutation.

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