Formins are proteins involved in regulation and construction of actin filaments of eucaryotic organism. They parcipitate in regulating cytokinesis, polar tip growth, and thus participate in development of whole organisms. There are 2 classes of formins in Arabidopsis thaliana. Both classes include FH1 and FH2 domains (formin homology 1 a 2). Class I formins have N-terminal transmembrane domain, unlike class II formins. Some formins of class II have a N-terminal PTEN domain (Phosphatase and Tensin Homolog). Sequence analyses predicted membrane binding via phosphatase or C2 subdomain of PTEN. This thesis was focused on the formin AtFH14, specifically its PTEN domain. Based on predicted sequence, a DNA fragment encoding the PTEN domain was amplified, sequenced and cloned to destination vectors for YFP and EOS phusions. Marked protein was visualized by transient expression in Nicotiana benthamiana. Stably transformed Arabidopsis lines were prepared for stably expression of protein. The tagged protein was localized in cortical cytoplasm, cytoplasmatical strands, probably in nuclear membrane or perinuclear cytoplasm, as well as in peculiar "folicle-like" structures that might be due to binding of PTEN at the periphery of some membrane organelles. Also were seen filament structures, maybe caused by PTEN binding...

A growing body of evidence shows that presynaptic nerve terminals throughout the nervous system are vulnerable to a range of traumatic, toxic and disease-related neurodegenerative stimuli. The aim of this study was to further characterise this vulnerability by examining the response of mouse α-motor nerve terminals at the neuromuscular junction (NMJ) to hypoxia-reperfusion injury. To address this aim, a novel model system was generated in which ex vivo skeletal muscle preparations could be maintained in an hypoxic environment, at an O2 concentration below in vivo normoxic values (<0.25% O2), for 2hr followed by 2hr reperfusion (2H-2R). Using this model system combined with quantitative assessment of immunohistological preparations as well as some ultrastructural observations, I present evidence to show that α-motor nerve terminals are rapidly and selectively vulnerable to hypoxia-reperfusion injury with no apparent perturbations to postsynaptic endplates or muscle fibres. I show that the severity of α-motor nerve terminal pathology is age and muscle type/location dependent: in 8-12wk old mice, nerve terminals in fast-twitch lumbrical muscles are more vulnerable than predominantly slow-twitch transversus abdominis and triangularis sterni. In 5-6 week old mice however, there is an age dependent increase in vulnerability of α-motor nerve terminals from the predominantly slow-twitch muscles while the fast-twitch lumbricals remained unaffected by age. The functional, morphological and ultrastructural pathology observed in α-motor nerve terminals following 2H-2R is indicative of selective and ongoing nerve terminal disassembly but, occurs via a mechanism distinct from Wallerian degeneration, as the neuroprotective slow Wallerian degeneration (Wlds) gene did not protect nerve terminals from these pathological changes. I also provide provisional evidence to show that 1A/II muscle spindle afferents and γ-motor nerve terminals are more resistant to hypoxia-reperfusion injury compared with α-motor nerve terminals. In addition to this, I also report preliminary finding that indicate that the oxygen storing protein, neuroglobin, maybe expressed at the mouse NMJ and report the difficulties of using mice that express yellow fluorescent protein (YFP) in their neurons for repeat/live imaging studies. Overall, these data show that the model of hypoxia-reperfusion injury developed in this study is robust and repeatable, that it induces rapid, quantitative changes in α-motor nerve terminals and that it can be used to further examine the mechanisms regulating nerve terminal vulnerability in response to hypoxia-reperfusion injuries. These findings have clinical implications for the use of surgical tourniquets and in the aetiology of many neurodegenerative diseases and neuropathic sequelae where mechanisms relating to hypoxia and hypoxia-reperfusion injury have been implicated.

Les protéines fluorescentes (PF) homologues d’AvGFP (Green Fluorescent Protein de la méduse Aequorea victoria) sont des outils incontournables de l’imagerie des processus de la cellule vivante. Leurs performances conditionnent la précision de l’analyse quantitative des signaux de fluorescence. Le développement de nouvelles PF demande donc à la fois de parvenir à une forte brillance tout en contrôlant la réponse de la protéine aux variations des paramètres physico-chimiques de la cellule en fonction de la question biologique étudiée. A ce jour, les PF jaunes disponibles montrent une forte sensibilité au pH. Afin d’élaborer des mutants moins sensibles, deux approches ont été considérées : une première consiste à mieux appréhender l’incidence de la dynamique du réseau de liaisons hydrogène entourant le chromophore sur son équilibre acido-basique. La seconde vise à identifier les facteurs structuraux à l’origine de la brillance particulièrement élevée de nouvelles PF jaunes et jaune-vert provenant d’un ver marin, Branchiostoma lanceolatum.J’ai d’abord mis au point un algorithme recherchant l’ensemble des liaisons hydrogène présentes au sein d’une protéine et qui étudie leur dynamique au cours de simulations par dynamique moléculaire. Il permet leur agrégation en réseaux, l’identification des réseaux connectés à un atome d’intérêt ainsi que le suivi de leur dynamique. Pour validation, cet algorithme a été appliqué à la recherche des réseaux de liaisons hydrogène présents au sein de différents mutants d’AvGFP pour lesquels un transfert de proton à l'état excité a été étudié expérimentalement. Cet algorithme pourra également servir à comprendre de façon dynamique le mécanisme d’autres systèmes biologiques dont la fonction repose sur le transfert de protons.D’autre part, j’ai résolu la structure de la protéine fluorescente jaune naturelle lanYFP de Branchiostoma lanceolatum, particulièrement brillante mais à la structure quaternaire tétramérique. Cette protéine a été rendue monomérique par évolution dirigée, ce qui a donné la protéine mNeonGreen à la fluorescence jaune-vert, protéine désormais étalon dans cette gamme spectrale, et dont j’ai également résolu la structure. Mon étude a permis de rationaliser a postériori l’ensemble des mutations introduites au cours de l’évolution. Enfin, j’ai réalisé une étude du dégât d’irradiation spécifique des rayons X permettant de comprendre le changement remarquable de couleur observé sur les cristaux de mNeonGreen après collecte de données de diffraction.L’ensemble des résultats obtenus au cours de ma thèse permet de proposer un cadre de compréhension à la fois théorique et expérimental des déterminants contrôlant les propriétés de fluorescence des PF jaunes. / Fluorescent Proteins (FPs) homologous to AvGFP (Green Fluorescent Protein from the jellyfish Aequoria victoria) are versatile tools used in live cell imaging. The amount of information that can be derived from the fluorescence signals depends on the spectroscopic performances of the FP. The development of new FPs should focus on both brightness increase and control of the protein response to physicochemical parameter variations within the cell. Current yellow FPs exhibit a strong sensitivity to pH. In order to engineer less sensitive variants, two complementary approaches have been used: the first one consists in studying the influence of the hydrogen bond network dynamics around the chromophore on its protonation state. In the second one, I have sought to identify the structural determinants of the particularly high brightness of newly discovered yellow FPs from a sea worm, Branchiostoma lanceolatum.First, I wrote an algorithm that can identify all hydrogen bonds within a protein and analyse their dynamics along molecular dynamics simulations. It allows for their clustering in networks, the identification of networks connected to a given atom and the monitoring of their dynamics. The method was validated by using the algorithm on various AvGFP mutants for which excited state proton transfer has been experimentally studied. This algorithm should also be useful for the study of other biological systems whose function is based on proton transfer.Besides, I solved the structure of the natural yellow FP lanYFP from Branchiostoma lanceolatum, which is particularly bright, but presents a tetrameric arrangement. This protein was monomerized by directed evolution, which led to the yellow-green FP mNeonGreen, now a benchmark in this spectral range. I also solved the structure of mNeonGreen, which allowed me to rationalize a posteriori the mutations that have been introduced during the evolution process. Finally, I performed a specific radiation damage study in order to explain the remarkable change in colour of mNeonGreen crystals upon X-ray data collection. Altogether, the results of my PhD work provides a theoretical and experimental framework of the determinants that drive the fluorescence properties of yellow FPs.

Formins are proteins facilitating formation of actin filaments. They affect structure of cytoskeleton and participate in cytokinesis and tip growth. There are 2 classes of formins in Arabidopsis thaliana, which include FH1 and FH2 (Formin Homology 1 and 2) domain. Formins of the class I have usually a transmembrane domain on N-terminus. Due to this fact they can interact with membranes. Some formins from the class II include PTEN domain (Phosphatase and Tensin Homolog) derived from sequences of PTEN proteins which has lost the function of phosphatase. It is assumed this domain can bind on a membrane via the phosphatase section or C2 domain. This thesis was focused on the formin AtFH13 from the class II in Arabidopsis thaliana and on its PTEN domain. There were analyzed differences between mutants and wild-types in length of roots in seedlings and in size of seeds and seed coats, and observed the effect of dexamethasone on the length of roots on AtFH13. PTEN domain of the formin was isolated from cDNA, cloned to a vector and fused with YFP. The tagged protein was visualized by the method of transient expression in epidermal cells in the leaves of Nicotiana benthamiana. No big differences were observed between plants mutant in the gene AtFH13 and wild-type in choice parameters. Dexamethasone did't influence...

Techniques for conditional gene or protein expression are important tools in developmental biology and in the analysis of physiology and disease. On the protein level, the tunable and reversible expression of proteins can be achieved by the fusion of the protein of interest to a destabilizing domain (DD). In the absence of its specific ligand (Shield-1), the protein is degraded by the proteasome. The DD-Shield system has proven to be an excellent tool to regulate the expression of proteins of interests in mammalian systems but has not been applied in teleosts like the medaka. We present the application of the DD-Shield technique in transgenic medaka and show the ubiquitous conditional expression throughout life. Shield-1 administration to the water leads to concentration-dependent induction of a YFP reporter gene in various organs and in spermatogonia at the cellular level.

Techniques for conditional gene or protein expression are important tools in developmental biology and in the analysis of physiology and disease. On the protein level, the tunable and reversible expression of proteins can be achieved by the fusion of the protein of interest to a destabilizing domain (DD). In the absence of its specific ligand (Shield-1), the protein is degraded by the proteasome. The DD-Shield system has proven to be an excellent tool to regulate the expression of proteins of interests in mammalian systems but has not been applied in teleosts like the medaka. We present the application of the DD-Shield technique in transgenic medaka and show the ubiquitous conditional expression throughout life. Shield-1 administration to the water leads to concentration-dependent induction of a YFP reporter gene in various organs and in spermatogonia at the cellular level.

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