Recurrent processes are a general feature of living systems, from the cell cycle to circadian day-night rhythms to hibernation and flowering cycles. During development and life, numerous recurrent processes are controlled by genetic oscillators, a specific class of genetic regulatory networks that generates oscillations in the level of gene products. A vital mechanism controlled by genetic oscillators is the rhythmic and sequential segmentation of the elongating body axis of vertebrate embryos. During this process, a large collection of coupled genetic oscillators gives rise to spatio-temporal wave patterns of oscillating gene expression at tissue level, forming a dynamic prepattern for the precursors of the vertebrae. While such systems of genetic oscillators have been studied extensively over the past years, many fundamental questions about their collective behavior remain unanswered. In this thesis, we study the behavior and the properties of genetic oscillators from the single oscillator scale to the complex pattern forming system involved in vertebrate segmentation. Genetic oscillators are subject to fluctuations because of the stochastic nature of gene expression. To study the effects of noisy biochemical coupling on genetic oscillators, we propose a theory in which both the internal dynamics of the oscillators as well as the coupling process are inherently stochastic. We find that stochastic coupling of oscillators profoundly affects their precision and synchronization properties, key features for their viability as biological pacemakers. Moreover, stochasticity introduces phenomena not known from deterministic systems, such as stochastic switching between different modes of synchrony. During vertebrate segmentation, genetic oscillators play a key role in establishing a segmental prepattern on tissue scale. We study the spatio-temporal patterns of oscillating gene expression using a continuum theory of coupled phase oscillators. We investigate the effects of different biologically relevant factors such as delayed coupling due to complex signaling processes, local tissue growth, and tissue shortening on pattern formation and segmentation. We find that the decreasing tissue length induces a Doppler effect that contributes to the rate of segment formation in a hitherto unanticipated way. Comparison of our theoretical findings with experimental data reveals the occurrence of such a Doppler effect in vivo. To this end, we develop quantification methods for the spatio-temporal patterns of gene expression in developing zebrafish embryos. On a cellular level, tissues have a discrete structure. To study the interplay of cellular processes like cell division and random cell movement with pattern formation, we go beyond the coarse-grained continuum theories and develop a three-dimensional cell-based model of vertebrate segmentation, in which the dynamics of the segmenting tissue emerges from the collective behavior of individual cells. We show that this model is able to describe tissue formation and segmentation in a self-organized way. It provides the first step of theoretically describing pattern formation and tissue dynamics during vertebrate segmentation in a unified framework involving a three-dimensional tissue with cells as distinct mechanical entities. Finally, we study the synchronization dynamics of generic oscillator systems whose coupling is subject to phase shifts and time delays. Such phase shifts and time delays are induced by complex signaling processes as found, e.g., between genetic oscillators. We show how phase shifts and coupling delays can alter the synchronization dynamics while leaving the collective frequency of the synchronized oscillators invariant. We find that in globally coupled systems, fastest synchronization occurs for non-vanishing coupling delays while in spatially extended systems, fastest synchronization can occur on length scales larger than the coupling range, giving rise to novel synchronization scenarios. Beyond their potential relevance for biological systems, these results have implications for general oscillator systems, e.g., in physics and engineering. In summary, we use discrete and continuous theories of genetic oscillators to study their dynamic behavior, comparing our theoretical results to experimental data where available. We cover a wide range of different topics, contributing to the general understanding of genetic oscillators and synchronization and revealing a hitherto unknown mechanism regulating the timing of embryonic pattern formation.

No description available.

<p> Per- and polyfluoroalkyl substances (PFAS) are a class of persistent environmental contaminants that have become ubiquitous, resulting in widespread exposure among humans and wildlife. Amphibians are regularly exposed in the field, making them susceptible to sublethal effects of PFAS exposure. In amphibians exposed to PFAS, deleterious effects have been observed, including reduction in body condition measured using the scaled mass index (SMI) and degraded hepatic condition, among others. PFAS may dysregulate lipid metabolism by altering signaling cascades regulated by peroxisome proliferator activated receptors (PPAR), but whether changes in energy stores can explain changes in amphibian SMI and/or hepatic condition remain underexplored. Since lipids are a critical energy reserve for anurans, understanding whether lipid metabolism is being perturbed is critical. The central objective of this thesis was to investigate the effect of PFAS on lipid homeostasis in <em>Xenopus laevis </em>tadpoles within the context of a PPAR mechanism of action (MOA), considering apical, molecular, and lipidomic endpoints. I conducted three studies: (a) a study to characterize SMI and the relative expression of the hepatic xPPARα/β/γ during metamorphosis, (b) a pharmaceutical exposure to assess the <em>in vivo</em> effects of xPPARα/β/γ agonism on hepatic gene expression for select downstream targets (<em>apoa5, fabp1, acox1,​ pck1</em>), and (c) a chronic PFAS exposure to investigate the effects of environmentally relevant concentrations (PFOS, PFHxS, PFOA, PFHxA at 0.5 ppb; binary mixture of PFOS:PFHxS at 1 ppb) on lipid homeostasis through apical endpoints (mass, snout vent length, SMI, hepatic condition), relative hepatic gene expression, and Multiple Reaction Monitoring (MRM) profiling of the hepatic lipidome for changes in relative class abundance. In study (a), I identified SMI and hepatic expression of <em>xPPARα/β/γ</em> is dynamic during late metamorphosis, indicating the potential for heightened susceptibility. However, in study (b), pharmaceutical agonists had no effect on <em>X. laevis</em> at high doses. For study (c), I did not observe effects on a majority of apical endpoints, including SMI, but detected a significant sex-specific reduction in hepatic condition for male<em> X. laevis</em> tadpoles exposed to single-chemical perfluorosulfonic acid (PFSA) treatments. For gene expression, I observed a transient downregulation for apolipoprotein-V (<em>apoa5</em>) at Nieuwkoop and Faber (NF) stage 62 for <em>X. laevis</em> tadpoles exposed to single-chemical perfluorocarboxylic acid (PFCA) treatments. Lipid profiling detected transient dysregulation of predominantly membrane lipids in-response to short-chain PFAS treatments at NF 58. Overall, our findings indicate PFAS may exert toxicity during anuran metamorphosis through multiple mechanisms of action (MOA) with sex-specific and developmental-stage specific outcomes.</p>

No description available.

<p dir="ltr">Primary cilia are hair-like structures found on nearly all mammalian cell types, including cells in the developing and adult brain. Cilia establish a unique signaling compartment for cells. For example, a diverse set of receptors and signaling proteins localize within cilia to regulate many physiological and developmental pathways including the Hh pathway. Defects in cilia structure, protein localization, or cilia function lead to genetic disorders called ciliopathies, which present with various clinical features including several neurodevelopmental phenotypes and hyperphagia associated obesity. Despite their dysfunction being implicated in several disease states, understanding their roles in CNS development and signaling has proven challenging. I hypothesize that dynamic changes to ciliary protein composition contributes to this challenge and may reflect unrecognized diversity of CNS cilia. The proteins ARL13B and ADCY3 are established ciliary proteins in the brain and assessing their localization is often used in the field to visualize cilia. ARL13B is a regulatory GTPase important for regulating cilia structure, protein trafficking, and Hh signaling, while ADCY3 is a ciliary adenylyl cyclase thought to be involved in ciliary GPCR singaling. Here, I examine the ciliary localization of ARL13B and ADCY3 in the perinatal and adult mouse brain by defining changes in the proportion of cilia enriched for ARL13B and ADCY3 depending on brain region and age. Furthermore, I identify distinct lengths of cilia within specific brain regions of male and female mice. As mice age, ARL13B cilia become relatively rare in many brain regions, including the hypothalamic feeding centers, while ADCY3 becomes a prominent cilia marker. It is important to understand the endogenous localization patterns of these proteins throughout development and under different physiological conditions as these common cilia markers may be more dynamic than initially expected. Understanding regional and development associated cilia signatures and physiological condition cilia dynamic changes in the CNS may reveal molecular mechanisms associated with ciliopathy clinical features such as obesity.</p>

Transcription factors of the helix-loop-helix (HLH) gene family are widespread in the animal kingdom. Among them, members of HLH subfamilies such as ASCL, Neurogenin, NeuroD, COE, Atonal, Oligo, NSCL, Hairy/E(spl) and Hey (here referred to as neural HLH genes) have been shown to be fundamental for the development of the nervous system. They are expressed at different time periods of neuronal differentiation, from the specification of ectoderm towards a neural lineage, to the ultimate differentiation of neurons. Few HLH genes have been identified in the lamprey; however, considering the wide diversity of HLH gene subfamilies in metazoans, including vertebrates, it is very likely that lampreys possess a large repertoire of HLH genes in their genome. In the present study, the identification of several HLH genes in the lamprey genome, as well as the isolation and expression of different lamprey neural HLH genes is reported. As expected, a wide repertoire of HLH genes was identified in the sea lamprey (Petromyzon marinus) genome. On the other hand, the identification and expression analysis of different neural HLH genes of the ASCL, Neurogenin, COE and Hairy/E(spl) in the brook lamprey Lampetra planeri showed an overall conservation with other vertebrates, both at the sequence and expression pattern levels. In addition, novel features of the lamprey nervous system are revealed, such as the identification of possible new sensory cranial placodes in pharyngeal arches. Furthermore, these genes can serve as molecular markers for different cranial placodes and dorsal root ganglia (DRG), and their expression also highlights the presence of a ventricular zone in the brain and spinal cord, along with a complementary marginal zone. Finally, with the use of a Notch pathway inhibitor in developing L. planeri embryos, the regulation of expression of the isolated genes by the Notch signaling pathway was shown to be generally conserved between lampreys and gnathostomes in the spinal cord. This functional study also revealed that the lamprey spinal cord likely presents an independent developmental programme from the brain. All together, the present study shows that the analysis of neural HLH genes represents an excellent tool to understand the lamprey nervous system.

Tandem amino acid repeats, also known as homopolimeric tract or homopeptides, are very common features of eukaryotic genomes and are present in nearly one-fifth of human encoded proteins. These structures have attracted much interest in the early 1990s when a number of neurological diseases associated with repeat expansion mutations were discovered in humans. Despite their abundance in coding proteins, little is known about their functional consequences. Two scenarios have been proposed. In one, tandem amino acid repeat is considered a neutral structure generated by slippage event and eventually tolerated in protein as long as it does not disrupt the protein function. However, an increasing number of studies proposed that tandem amino acid repeats may be involved in important functional or structural roles. For instance, tandem amino acid repeats had been found to be especially abundant in transcription factors and developmental proteins, where they can potentially modulate protein-protein interaction, exert an effect on gene transcriptional activity, or act as spacer between different protein domains. In addition, several studies have linked changes in repeat size to modification in developmental processes. Despite the advancement made in the last decade, little is known about the selective forces that shape their evolution. The aim of this thesis has been to gain further insight onto the evolutionary dynamics of tandem amino acid repeats by studying the different types of mutations that occur in the amino acid component of the human proteome, by studying the relationship between variability and abundance of amino acid tandem with the evolutionary constraints operating on the proteins, and by studying their conservation and distribution across various vertebrate genomes in both coding and non-coding sequences. The integration of these approaches enabled us to outline an evolutionary model of these structures.

The amphibian species Xenopus laevis, along with mouse and chicken is a very important model system, used widely to dissect the molecular intricacies of various aspects of vertebrate development. Study with Xenopus has clear advantages in terms of various technical considerations including the ease of handling early stage of embryos and due to the remarkable documentation of several early molecular events during development. The concept of inductive interactions between various cell types during early development was first revealed by the studies performed in Xenopus, and among the various factors proposed for mesoderm induction, the members of transforming growth factor-β (TGF- β) superfamily have been considered to be the most probable candidates. About forty different members of the TGF-β superfamily have been cloned and characterized from various organisms. The superfamily members like activins and BMPs have been studied extensively with respect to their functional role during development. While BMPs were assigned as candidates for inducing ventral mesoderm, activins oppose the role of BMPs by inducing dorsal mesoderm. Studies that helped in delineating their roles were performed using three approaches that utilized the ligands, receptors or down stream signaling components (Smads). All the three components were studied with respect to their endogenous expression pattern and effects of ectopic expressions of the wild type or dominant negative mutants. These approaches led to the accumulation of evidences supporting the importance of these signaling molecules. All the above mentioned studies were only possible due to the cloning and characterization of cDNAs of the various proteins involved in the signaling pathway including the ligands. TGF-β2 and 5 are the two isoforms of TGF-β cloned from the amphibian system. We have earlier cloned and characterized the promoter for TGF-β5 gene, which suggested possible regulation of this factor by tissue specific transcription factors. Messenger RNA in situ hybridization analysis to study the TGF-β5-expression pattern during Xenopus development, showed spatial and temporal expression pattern. The expression was confined to specific regions that include notochord, somites, and tail bud among others, in the various stages analyzed. This suggested a possible role for TGF-β5 in organogenesis during the amphibian development. To better understand the role of TGF-β in Xenopus development, studies to examine the specific receptor expression pattern for this growth factor is very essential. With the lack of any reports on cloning of TGF-β receptors from this system, the aim of the present study was to isolate and characterize the receptors for TGF-β from Xenopus laevis. PCR cloning using degenerate primers based on the conserved kinase domains of this class of receptors, coupled to library screenings enabled the identification of two novel receptor cDNAs of the TGF-β receptor superfamily. Characterization of the isolated cDNAs suggested that one of them codes for a type II receptor for TGF-β. Further the cDNAs were found to be ubiquitously expressed during development, as judged by RT-PCR analysis. The cloned cDNAs can now be employed as tools, to study the expression pattern by means of mRNA in situ hybridization, on the various developmental stage embryos and to perform studies using antisense and dominant negative mRNA injection experiments in vivo. Such studies will greatly assist in delineating the role of TGF-β ligands and receptors during amphibian development.

La palourde japonaise, Venerupis philippinarum, a été introduite en France au début des années 70 à des fins aquacoles. Depuis 1987, d'importants épisodes de mortalité, causés par la bactérie pathogène Vibrio tapetis, touchent cette espèce le long des côtes françaises et européennes. Cette vibriose, appelée Maladie de l'Anneau Brun (MAB), est considérée comme une maladie d'eau froide. L'interaction tripartite entre V. philippinarum, V. tapetis et l'environnement a été explorée à travers le rôle du système enzymatique des phénoloxydases (POs) dans le but de mieux comprendre la réponse immunitaire de la palourde japonaise, la virulence de l'agent pathogène mais aussi l'impact de l'environnement et plus particulièrement de la température. Les POs sont des oxydoréductases impliquées dans la synthèse de la mélanine et de ses dérivés mais aussi dans les processus de reconnaissance du non-soi et d'encapsulation chez les invertébrés. Dans un premier temps, l'activité PO du sérum d'hémolymphe a été caractérisée d'un point de vue biochimique comme étant majoritairement de type laccase ; une activité minoritaire de type tyrosinase a également été identifiée. Des infections de palourdes par trois souches de V. tapetis, à différentes températures, ont mis en évidence une modulation de la réponse du système PO en fonction du temps et du compartiment étudiés. De façon générale, l'infection bactérienne s'est traduite par une augmentation de l'activité PO. Toutefois, le niveau basal d'activité PO est variable d'une population à une autre et cette variabilité semble traduire une susceptibilité différente à la MAB. L'augmentation de la température de 15 à 22°C a entraîné une augmentation des capacités immunitaires de la palourde japonaise. La températurea également eu un impact sur la pathogénicité de V. tapetis et ce, de façon différentielle selon les souches. L'inhibition de l'activité PO observée in vitro en présence de produits extracellulaires bactériens souligne la complexité de l'interaction entre V. philippinarum et V. tapetis. Ainsi, le suivi de l'activité PO constitue un biomarqueur pertinent des capacités immunitaires des invertébrés marins dans l'interaction tripartite hôte-pathogène-environnement.

L'attention scientifique s'est récemment portée sur l'identification des mécanismes proximaux sous-tendant les compromis évolutifs;tels que les compromis existant entre croissance/reproduction et longévité. La production d'espèces réactives de l'oxygène (ROS )a été suggérée comme un candidat potentiel ,de par sa liaison étroite au métabolisme énergétique (sous-­produits du fonctionnement mitochondrial) et son caractère inévitable. Si la production de ROS excède le niveau des défenses antioxydantes, une situation de stress oxydant va en résulter et a été associé au vieillissement . Puisque la mitochondrie n'est pas uniquement la centrale énergétique de la cellule mais aussi le principal producteur de ROS, cette thèse s'est attachée à clarifier les relations entre métabolisme énergétique , fonctionnement mitochondrial et stress oxydant ; avec des études concernant l'impact d'activités coûteuses en énergie (croissance, reproduction, thermogénèse) sur l'équilibre de la balance oxydative.