A hallmark of human behaviour is that we can either couple or decouple our thoughts, decision and motor plans from actions. Previous studies have reported evidence of gating of information between intention and action that can happen at different levels in the central nervous system (CNS) involving the motor cortex, subcortical structures such as the basal ganglia and even in the spinal cord. In my research I examine the extent of this gating and its modulation by task context. I will present results obtained by data collected from (a) neck muscles and neural recording from frontal eye field (FEF) in macaque monkeys and (b) putative motor units (MUs) from high density electrode arrays using surface EMG signals in human to delineate the type of information that leaks into muscles in the periphery when subjects are involved in preparing eye and hand movements, respectively, and its modulation by task context Overall, my results reveal that we can assess some aspects of central planning in the activity of motor units Further, the recruitment of these motor units depend on task context.

Neurogenin 2 (Ngn2) is a basic Helix-Loop-Helix (bHLH) transcription factor regulating differentiation and cell cycle exit in the developing brain. By transcriptional upregulation of a cascade of other bHLH factors, neural progenitor cells exit the cell cycle and differentiate towards a neuronal fate. Xenopus laevis Ngn2 (xNgn2) is a short-lived protein, targeted for degradation by the 26S proteasome. I have investigated the stability of Ngn2 mediated by post-translational modifications and structural disorder. Firstly I will describe work focused on ubiquitylation of xNgn2, targeting it for proteasomal degradation. xNgn2 is ubiquitylated on lysines, the recognized site of modification. I will discuss the role of lysines in ubiquitylation and stability of xNgn2. In addition to canonical ubiquitylation on lysines, I describe ubiquitylation of xNgn2 on non-canonical sites, namely its amino-terminal amino group, and cysteine, serine and threonine residues. I show that the ubiquitylation of cysteines in particular exhibits cell cycle dependence and is also observed in mammalian cell lines, resulting in cell cycle-dependent regulation of stability. I will then discuss whether phosphorylation, a regulator of xNgn2 activity, also affects xNgn2 stability. I will provide evidence of cell cycle-dependent phosphorylation of cyclin dependent kinase (cdk) consensus sites affecting the stability of xNgn2. Finally I describe studies on the folding properties of Ngn2 to assess their role in protein stability. xNgn2 associates with DNA and its heterodimeric binding partner xE12 and may interact directly with the cyclin-dependent kinase inhibitor Xic1. I will discuss the role of these interaction partners in xNgn2 stability. xNeuroD, a downstream target of xNgn2, is a related bHLH transcription factor which is stable. Here I describe domain swapping experiments between these two proteins highlighting regions conferring instability on the chimeric protein. Finally I will provide nuclear magnetic resonance (NMR) data looking at the effect of phosphorylation on protein structure in mouse Ngn2 (mNgn2).

Microbiota becomes one of the most important subjects in biological research and numerous studies revealed that microbiota plays a broad spectrum of essential roles in different organisms. This master thesis focuses on the bacterial part of microbiota contained in mucosal tissues of wild house mice (Mus musculus musculus). Male and female samples were collected by nasal and oral cavity lavages, vaginal mucosa lavages and from urine and stool. We aimed to detect quantitative, qualitative and sex-specific differences in bacterial populations between mucosal tissues with particular focus on bacterial cycling in vaginal mucosa during the estrous cycles. Bacterial abundances were estimated by qPCR whilst bacterial diversity was detected by targeted metagenomic sequencing of the hypervariable region of the 16S rRNA gene. Significant differences were detected in bacterial abundances and alpha diversity between particular mucosal tissues. Stool samples contained the highest number of bacteria, while samples from the nasal mucosa and urine contained low amount of bacteria. The highest alpha diversity was discovered in stool samples, the least alpha diversity was found in the urine. Mucosal tissues also varied based on the bacterial composition on the level of particular genera. Detailed analysis of estrous cycles...

Los volcanes son formaciones geológicas que resultan atractivas para los asentamientos humanos debido a la fertilidad de sus suelos y su interés turístico. Pero también son una fuente importante de contaminación natural, debido a los procesos geoquímicos que tienen lugar durante los periodos eruptivos y no eruptivos. Se estima que 44 millones de personas viven a menos de 10 km de un volcán activo y ya se ha demostrado el aumento de incidencia de patologías respiratorias en humanos que habitan estos ambientes. La presente Tesis Doctoral tiene como objetivo valorar los efectos neurotóxicos en el sistema nervioso central de ratones domésticos (Mus musculus) que habitan en ambientes volcánicos activos. La acumulación de mercurio en cerebro y médula espinal de individuos expuestos, la activación de células astrocitarias y microgliales y la producción de citocinas proinflamatorias son los principales resultados de este trabajo de los que se desprende que el sistema nervioso central está afectado por la exposición crónica a estos ambientes.

No description available.

The cells from the inner cell mass (ICM) of an early embryo have the potential to differentiate into all the different cell types present in an adult organism. Cells from the ICM can be isolated and cultured in vitro, becoming embryonic stem cells (ESCs). ESCs have several properties that make them unique: they are unspecialized, can self-renew indefinitely in culture, and given the appropriate cues can differentiate into cells from all three germ layers (ecto-, meso-, and endoderm), including the germline, both in vivo and in vitro. Induced pluripotent stem cells (iPSCs) can be generated from adult, terminally differentiated somatic cells by transient exogenous expression of four transcription factors (Oct4, Sox2, Klf4, and cMyc; OSKM) present normally in ESCs. It has been shown that iPSCs are equivalent to ESCs in terms of morphology, gene expression, epigenetic signatures, in vitro proliferation capacity, and in vitro and in vivo differentiation potential. However, unlike ESCs, iPSCs can be obtained from a specific individual without the need for embryos. This makes them a promising source of pluripotent cells for regenerative medicine, tissue engineering, drug discovery, and disease modelling; additionally, in livestock species such as the bovine, they also have applications in genetic selection, production of transgenic animals for agricultural and biomedical purposes, and species conservancy. Nevertheless, ESC and iPSC lines that meet all pluripotency criteria have, to date, only been successfully produced in mice, rats, humans, and non-human primates. In the first part of this dissertation, we attempted reprogramming of three types of bovine somatic cells: fetal fibroblasts (bFFs), adult fibroblasts (bAFs), and bone marrow-derived mesenchymal stem cells (bMSCs), using six different culture conditions adapted from recent work in mice and humans. Using basic mouse reprogramming conditions, we did not succeed in inducing formation of ESC-like colonies in bovine somatic cells. The combination of 2i/LIF plus ALK5 inhibitor II and ascorbic acid, induced formation of colony-like structures with flat morphology, that occasionally produced trophoblast-like structures. These trophoblast-like vesicles did not appear when an inhibitor of Rho-associated, coiled-coil containing protein kinase 1 (ROCK) was included in the medium. We screened for expression of exogenous OSKM vector with RT-PCR and found upregulation of OSKM vector 24h after Dox was added to the medium; however, expression was sharply decreased on day 2 after Dox induction, and was not detectable after day 3. In a separate experiment, we induced reprogramming of bFF and bAFs using medium supplemented with 50% of medium conditioned by co-culture with the bovine trophoblast CT1 line. These cells expressed both OCT4 and the OSKM vector 24h after Dox induction. However, similar to our previous observations, both markers decreased expression until no signal was detected after day 3. In summary, we were unable to produce fully reprogrammed bovine iPSCs using mouse and human protocols, and the exact cause of our lack of success is unclear. It is possible that a different method of transgene expression could play a role in reprogramming. However, these ideas would be driven by a rather empirical reasoning, extrapolating findings from other species, and not contributing in our understanding of the particular differences of pluripotecy in ungulates. Our inability to produce bovine iPSCs, combined with the only partial reprogramming observed by others, justifies the need for in depth study of bovine pluripotency mechanisms, before meaningful attempts to reprogram bovine somatic cells to plutipotency are made. Therefore, we focused on getting a better understanding of bovine nuclear reprogramming. This would allow us to rationally target the specific requirements of potential bovine pluripotent cells. Cell fusion is a process that involves fusion of the membrane of two or more cells to form a multinucleated cell. Fusion of a somatic cell to an ESC is known to induce expression of pluripotency markers in the somatic nucleus. In the second part of this dissertation, we hypothesized that fusion of bFFs to mouse ESCs (mESCs) would induce expression of pluripotency markers in the bFF nucleus. We first optimized a cell fusion protocol based on the use of polyethylene glycol (PEG), and obtained up to 11.02% of multinucleated cells in bFFs. Next, we established a method to specifically select for multinucleated cells originated from the fusion of mESCs with bFFs (heterokaryons), using indirect immunofluorescence. With this in place, flow cytometry was used to select 200 heterokaryons which were further analyzed using RNA-seq. We found changes in bovine gene expression patterns between bFFs and heterokaryons obtained 24h after fusion. Focusing on the bovine transcriptome, heterokaryons presented upregulation of early pluripotency markers OCT4 and KLF4, as well as hypoxia response genes, contrasted with downregulation of cell cycle inhibitors such as SST. The cytokine IL6, known to increase survival of early embryos in vitro, was upregulated in heterokaryons, although its role and mechanism of action is still unclear. This indicates that the heterokaryon cell fusion model recapitulates several of the events of early reprogramming, and can therefore be used for further study of pluripotency in the bovine. The cell fusion model presented here can be used as a tool to characterize early changes in bovine somatic nuclear reprogramming, and to study the effect of different reprogramming conditions on the bovine transcriptome. / Ph. D. / The cells of an early embryo have the potential to give rise to any cell type found in the adult body. When these cells are transferred to a culture dish and kept under the right conditions, they become Embryonic Stem Cells (ESCs), and they retain the same developmental potential as the original embryonic cells they were derived from. In 2006, researchers in Japan found that it is possible to “reprogram” the cells of an adult individual (for example, fibroblast skin cells taken from a biopsy) to an embryonic state, by forcing the cells to express extra copies of genes that are normally active in embryos. These reprogrammed cells are called induced Pluripotent Stem Cells (iPSCs), and similarly to ESCs, they also have the potential to produce any cell type found in an adult organism. Lines of iPSCs from livestock species have possible applications in agriculture, species conservancy, biomedical industry, and veterinary and human health. Unfortunately, for reasons that are to date not fully understood, the technology to produce iPSCs has, so far, only worked in mice, rats, humans, and non-human primates. We first attempted to produce bovine iPSCs by adapting methods and conditions used to derive iPSCs in mice and humans. We observed partial reprogramming of bovine cells, but were ultimately not able to produce true bovine iPSCs. This suggests that the bovine requires alternative/additional factors to induce reprogramming in adult cells. However, not knowing exactly what conditions or reagents will induce the reprogramming process in the cow, we decided to take a different approach. We focused on trying to understand how nuclear reprogramming works in the bovine. This would allow us to rationally target the specific requirements of potential bovine pluripotent cells. It is known that the fusion (“merging”) of an adult cell with a stem cell, causes the adult cell to change its gene expression pattern to resemble a stem cell. We therefore fused mouse ESCs with bovine fibroblasts, and observed changes in bovine gene expression pattern as early as 24 hours after fusion. The gene expression changes observed resemble those found during early reprogramming of human and mouse iPSCs, and are accompanied by silencing of fibroblast specific genes. This suggests that our cell fusion model recreates the changes that happen during reprogramming, and can therefore be used as a tool to better understand pluripotency in the cow. The cell fusion method described in this dissertation can in theory be adapted to other species; by fusing somatic cells from other species to mouse ESCs, this model can be used to find species specific relevant pluripotency genes.

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...

Les relations hôte-parasite sont complexes et soumettent les deux protagonistes à un ensemble de compromis aussi bien plastiques qu’évolutifs. D'un côté, bien que l’immunité soit indispensable pour la lutte contre les parasites, elle peut aussi causer de nombreux dégâts à l’hôte lors de la réponse et mener à des maladies inflammatoires. De l’autre côté, les parasites, bien que dotés de mécanismes d'évasion, vont être affectés par l'environnement inflammatoire de l'hôte. Cela pose la question des coûts et bénéfices que l'interaction entre ces deux protagonistes va avoir sur chacun d'eux lors de l'apparition de troubles inflammatoires chez l'hôte. Grâce à différentes approches expérimentales et bibliographiques nous avons pu montrer que l’immunopathologie est un trait qui perdure vraisemblablement grâce aux bénéfices de la réponse immunitaire dans la lutte contre les parasites. Par ailleurs, j’ai pu mettre en évidence qu’une inflammation altérait positivement les traits d’histoire de vie du parasite Heligmosomoides polygyrus aussi bien de façon plastique que lors de processus de sélection. Cependant, le parasite investit davantage dans l’immunomodulation et le camouflage lorsqu'il est confronté à cet environnement, laissant planer la question du coût de cette inflammation à long terme et sur plusieurs générations. / Host-parasite interactions are characterized by trade-offs that involve both plastic and microevolutionary responses. On one hand, while immunity is essential to fight parasites, it can also cause damage to the host, leading to autoimmunity and inflammatory diseases. On the other hand, parasites have to cope with the immune environnement provided by the host. This raises the question of the costs and benefits of the inflammatory response for the two partners of the interaction. With different experimental and literature-based approaches, I showed that immunopathology is a trait that likely persists because of the immediate benefits of the immune response in terms of protection against parasites. Furthermore, I was able to show that inflammation positively altered the life history traits of the gastrointestinal nematode Heligmosomoides polygyrus both plastically or after experimental evolution. However, the parasite invested more in immunomodulation and camouflage when facing an inflammatory environment, leaving open the question of the costs associated with an inflammatory environment over the entire lifespan of the parasite and/or across generations.

La naissance est l’une des étapes les plus délicates à laquelle les nouveau-nés mammifères doivent faire face. Le nouveau-né doit opérer des changements physiologiques et comportementaux pour s’adapter à l’environnement aérien, et l’un des premiers défis est d’ingérer du colostrum et du lait. Il est surprenant que la nature des stimuli et les mécanismes impliqués dans le déclenchement de la tétée soient encore mal connus, alors que la survie du nouveau-né est conditionnée par le succès de la première tétée. Par conséquent, cette étude a pour but de comprendre comment un nouveau-né immature et inexpérimenté réussit à s’orienter vers une tétine, à la saisir et à la téter de façon efficace? Cette question générale est posée chez la souris en focalisant sur : i) la nature des substrats chimiques utilisés par les souriceaux pour atteindre les tétines maternelles ; ii) la variation de la puissance attractive de ces substrats au cours du développement ; et iii) l’implication des effets de l’expérience dans l’établissement des réponses adaptatives précoces. Premièrement, nos résultats mettent en lumière que les odeurs mammaires de femelles allaitantes induisent plus d’approches et de saisies de la tétine chez les souriceaux que celles émanant de femelles non allaitantes. Deuxièmement, les odeurs de liquide amniotique et de lait déclenchent la première saisie orale de la tétine chez des souriceaux à la naissance, alors que les odeurs de salives maternelle et infantile n’induisent ce comportement qu’après une brève expérience de tétée. Troisièmement, les souriceaux âgés de 0, 2 et 6 jours postnatals (P), ayant eu une expérience de tétée, affichent une attraction sélective envers des odeurs de laits collectés en début de lactation plutôt qu’en fin de lactation, alors que les souriceaux plus âgés P15 ne montrent aucune réponse sélective envers ces odeurs. En résumé, certains substrats biologiques présents sur les tétines de femelles allaitantes sont immédiatement attractifs après la naissance, tandis que d’autres ont besoin d’être appris pour être réactogènes. Par conséquent, la réponse initiale de recherche de la tétine chez le souriceau est contrôlée par des processus d’apprentissage postnatal. A ce stade, l’implication de l’apprentissage prénatal et de processus prédisposés n’a pu être prouvée, bien qu’elle ne soit pas exclue. Ces résultats montrent des capacités d’apprentissage sophistiquées chez le souriceau nouveau-né / Birth is one of the most delicate periods mammalian infants have to deal with. Newborns have then to adapt physiologically and behaviorally to the aerial environment, and one of their first challenges is to ingest colostrum and milk. It is paradoxical that the survival of pups is conditioned by the success of this first suckling, and that we have so little understanding of the stimuli that underlie and promote it. Thus, the present work aims to contribute to answer how immature and naïve newborns do manage to orient to a nipple, to grasp it, and to suckle efficiently? This general issue will be addressed in the mouse in focusing on: i) the nature of the chemical substrates that newborn mice use to reach nipples; ii) whether the attractive potency of these substrates changes as a function of development; and iii) whether exposure effects underly the establishment of early adaptive responses? The results highlight that mammary odors of lactating females are more behaviorally active for newly born pups than those of non-lactating females. Secondly, amniotic and milk odors provoke the first nipple grasping in newborns right at birth, while maternal and pup salivary odors induce this behavior after short sucking experience. Thirdly, younger pups on postnatal day (P) 0, 2 and 6 (with sucking experience), display a selective orientation toward the odor of milk collected during early-lactation rather than to the odor of late-lactation milk, whereas older pups P15 do not exhibit any selective attraction to these odors. To summarize, some of the biological substrates which are present on a nursing mouse nipples are attractive immediately after birth, while some others need to be postnatally learned to be active. Thus, the initial nipple search response of mouse pups is controlled by processes involving postnatal learning. At this stage, the involvement of prenatal learning and of predisposed processes that do not depend on previous exposure effect are not conclusive, although not excluded. These results show highly sophisticated learning abilities in a newborn mammal

Die Bildung und Aufrechterhaltung von Blutgefäßnetzen ist essenziell für Entwicklung, Gewebewachstum, Homöostase und Regeneration. Gefäßnetze erweitern sich durch ein Gleichgewicht von Endothelzellmigration und -proliferation. Während die Angiogenese gut untersucht ist, ist die Bildung arterieller Gefäße weniger erforscht. Piale Kollateralgefäße, eine seltene Form vaskulärer Redundanz im zentralen Nervensystem, verbinden Hirnarterien und schützen bei Schlaganfällen, indem sie blockierte Arterien ersetzen. Ein besserer pialer Kollateralfluss führt zu besseren Ergebnissen, was die Bedeutung ihrer Bildung und des Umbaus bei Schlaganfällen verdeutlicht. Diese Arbeit untersucht die zellulären Mechanismen, die der Bildung und dem Remodeling pialer Kollateralen zugrunde liegen. Mit Lineage-Tracing und hochauflösender Bildgebung pialer Gefäße von Mäusen wird gezeigt, dass Kollateralen primär aus wandernden, arteriellen Zellen und zu einem geringeren Teil aus Plexus-Zellen entstehen. Ich identifiziere den Mechanismus der Mosaikbesiedlung, bei dem arterielle und plexusstammende Endothelzellen (ECs) in vorkollaterale Kapillaren rekrutiert werden, die sich arteriell umgestalten. Während der embryonalen Entwicklung erfolgt die Kollateralbildung durch Rekrutierung von ECs, während der Umbau nach einem Schlaganfall auf der Proliferation lokaler, arterieller ECs beruht. Ultrastrukturelle Analysen zeigen, dass Kollateral-ECs eine hohe Caveolardichte aufweisen, die nach einem Schlaganfall im Vergleich zu Arterien rasch abnimmt. Die Arbeit beschreibt verschiedene Prozesse, die die Bildung und den Umbau pialer Kollateralen fördern, und hebt die Bedeutung endothelialer Linien hervor. Diese Erkenntnisse betonen die Relevanz arteriellen Wachstums für die Wiederherstellung des Kreislaufs und den Bedarf an verbesserten Kollateraltherapien für Schlaganfälle. / The formation and maintenance of blood vessel networks are crucial for development, tissue growth, homeostasis, and regeneration. Vascular networks expand and remodel through a balance of endothelial cell migration and proliferation. While angiogenesis—the growth of new vessels from existing ones—is well-studied, arterial vessel formation remains less explored. Pial collateral vessels, a rare form of vascular redundancy in the central nervous system, connect cerebral arteries and provide protection during stroke by dilating to replace blocked arteries. Patients with better pial collateral flow show improved outcomes, underscoring the importance of understanding collateral formation and remodeling in stroke. This work investigates the cellular mechanisms underlying pial collateral formation and post-stroke remodeling. Using lineage tracing and high-resolution imaging of mouse pial vasculature, I show that pial collaterals primarily arise from migrating artery-derived cells, with a smaller contribution from plexus-derived cells. I identify a novel mechanism—mosaic colonization—where arterial and plexus endothelial cells (ECs) are recruited into pre-collateral capillaries, coinciding with arterialization. Embryonic collateral formation involves EC recruitment, while post-stroke remodeling relies on proliferation of local artery-derived ECs. Ultrastructural analysis reveals collateral ECs exhibit high caveolar density, which rapidly declines after stroke compared to arterial caveolae. Overall, this thesis delineates distinct processes driving pial collateral formation and remodeling, highlighting the key endothelial lineages. These findings emphasize the importance of arterial growth in restoring circulation and underscore the need for improved collateral therapeutics for stroke.