Possibilities of remote sensing in grassland vegetation and management interventions monitoring in the Giant Mountains Abstract The aim of this thesis was to evaluate suitability of WorldView-2 imagery for grassland associations classification in the model area of Giant Mountains. The classification was based both on the legend compiled by a botanist, and on the legend of Natura 2000. In order to eliminate the effects of other types of land cover on the classification accuracy, a mask of grasslands was created. Using discriminant analysis, the significance of spectral bands of WorldView-2, as well as signifikance of selected vegetation indices and components from Principal Component Analysis (PCA) - to distinguish particular classes of grassland vegetation were evaluated. Based on the results of discriminant analysis, classifications using neural networks method and also maximum likelihood method were performed in ENVI 4.7 version software. The results of the both method were compared Key words: remote sensing, meadows association, classification, Giant mountains, WorldView 2

The purpose of the diploma thesis is the analysis of alpine timberline shifts in the Giant Mountains between 18th and 21st century. The altitudinal position of alpine timberline is a sensitive indicator which reflects human impacts as well as the climatic changes. Methodical approach included the reconstruction of alpine timberlines from the old maps (published in 1765, 1851-52, 1879, 1906) and their comparison with the newer data, which were evaluated from a series of aerial photographs dated 1936 and 2002 (actualized 2005; data provided by supervisor). Data obtained from the old maps and aerial photographs (1936) were compared and verified with old photos and postcards from Giant Mountains. The average altitude of alpine timberline increased during the study period. The increase in elevation of alpine timberline is significant between 19th and first half of 20th century, e. g. in period when the human influence in Giant Mountains decreased. It is concluded that position of alpine timberline in Giant Mountains depended on human influence. Key words: tree line, alpine timberline, human influence, Giant Mountains

Avalanches are important natural processes which shape the character of large mountainous areas. The reconstruction of their history has been performed on avalanche tracks in the Důl Bílého Labe Valley, Krkonoše Mts. through application of dendrogeomorphological methods. In total 101 trees from the area of 5 avalanche tracks were cored or cross-sectioned, which helped to identify 1253 growth disturbances (abrupt growth suppressions or releases, increases in tree-ring eccentricity, traumatic resin ducts, reaction wood, callus or determination of the tree death). The analysis spanning period from the 1859 led to the identification of 40 avalanche events, which prolong and complement written evidences of avalanche falls (avalanche cadastre). However, because of limitations of dendrogeomorphology (especially impossibility of dating of small events), these results have character of only minimal number of events. Strong influence of monthly snow melting and snow accumulation on initiation of events was pointed out through the statistical analysis of snowpack changes in relation to the reconstructed avalanche activity. Two main possible natural reasons for avalanche activity initiation were identified - vast spring snow melting and loading with fresh snow. The analysis demonstrates the potential of...

Au cours de ces travaux de thèse, nous avons étudié par microscopie magnétique à émission d’électrons balistiques (BEMM) les propriétés de magnétotransport d’électrons chauds de la vanne de spin Fe/Au/Fe épitaxiée sur GaAs(001). Dans ces expériences, la pointe d’un microscope à effet tunnel (STM) injecte localement un courant d’électrons chauds à la surface de la vanne de spin. La mesure sous champ magnétique du courant d’électrons balistiques collecté à l’arrière de l’échantillon donne accès aux propriétés locales de magnétoconductance de l’échantillon. Nous avons dans un premier temps étudié les propriétés de magnétotransport de vannes de spin planaires. Les mesures BEMM démontrent un magnétocourant d’électrons chauds pouvant atteindre 500 % à température ambiante. Ces forts effets de magnétoconductance ne sont que très faiblement dépendants des épaisseurs des électrodes de fer et ne peuvent donc être dus à l’asymétrie en spin de la longueur d’atténuation des électrons chauds dans les couches de fer. Dans cette structure épitaxiée, la polarisation en spin du faisceau d’électrons chauds s’acquiert principalement aux interfaces via des effets de structure électronique. L’électron traversant les couches minces métalliques se propage comme un état de Bloch. Sa transmission aux différentes interfaces se fait en conservant d’une part la composante transverse k║ du vecteur d’onde électronique, et d’autre part, la symétrie de la fonction d’onde. Au-dessus de la barrière Schottky, les électrons chauds sont collectés dans la vallée Г du GaAs se projetant à l’interface dans la direction k║=0. Dans cette direction k║=0, la conservation de la symétrie de la fonction d’onde à l’interface Fe/Au conduit au filtrage des états de Bloch de symétrie Δ1 du fer. Ces états de symétrie Δ1, totalement polarisés en spin, sont responsables des forts magnétocourants d’électrons chauds observés. Cette analyse est confirmée expérimentalement par l’observation d’une corrélation entre amplitude du magnétocourant et masse effective du substrat semiconducteur. En augmentant la masse effective du semiconducteur, on ouvre le collimateur filtrant le courant d’électrons chauds autour de la direction k║=0, et le magnétocourant diminue sans modifier la vanne de spin. Dans un second temps, tirant partie de la résolution latérale du microscope et de sa sensibilité au magnétisme, des microstructures de fer préparées sous ultra-vide par évaporation à travers un masque (méthode du nanostencil) ont été étudiées. Dans ces structures, la modulation du courant collecté par la structure locale en domaines magnétiques a permis la réalisation d’images magnétiques avec une haute résolution spatiale. Les contrastes observés sur ces microstructures sont en excellent accord avec les images BEMM calculées à partir de simulations micromagnétiques ouvrant la voie à une microscopie magnétique quantitative à forte sensibilité et résolution latérale nanométrique. / During this thesis work, we studied by ballistic electron magnetic microscopy (BEMM) the hot electron magnetotransport properties of epitaxial Fe/Au/Fe/GaAs(001) heterostructures. In these experiments, hot electrons are injected from an STM tip through the metallic base. The measurement of the ballistic electron current collected at the back of the substrate under magnetic field gives access to the local magnetoconductance properties of the sample. The first part of this work consists in the study of a planar heterostructures. BEMM measurements on epitaxial Fe/Au/Fe/GaAs(001) samples demonstrate hot electron magnetocurrent as high as 500% at room temperature. This high magnetocurrent value is observed to be almost independent of the Fe layers thickness, and thus can not be explained by the spin asymmetry of the electron attenuation length in the iron layers. In this epitaxial heterostructure, the hot electron beam is mainly spin-polarized at the interfaces due to band structure effects. In the metallic thin films, electrons propagate as Bloch states. The electron wave function transmission at the interfaces should satisfy two selection rules: the transverse momentum (k║) of the electron wave vector and the symmetry of the electron wave function should be conserved. Above the Schottky barrier height, hot-electrons are collected in the Г valley of GaAs selecting thus only electrons with a transverse momentum (k║) close to zero. Among these k|| ≈ 0 states, conservation of the electron wave-function symmetry at the Fe/Au epitaxial interfaces additionally selects electrons with the Δ1 symmetry. These Δ1 states are fully spin-polarized and are responsible for the observed high magnetocurrent in these heterostructures. This analysis is experimentally confirmed by the observation of a correlation between the magnetocurrent value and the semiconductor effective mass. By increasing the semiconductor effective mass, we open the collimator which filters the electronic states around k║=0 and the magnetocurrent value decreases. To take advantage of the lateral resolution of the microscope and of its high sensitivity to magnetism, the second part of this work was devoted to the study of sub-micrometric iron structures prepared under UHV by evaporation through a nanostencil. In these structures, the modulation of the collected current by the local magnetic domain structure in the Fe dots allows magnetic imaging with a high spatial resolution. The experimental magnetocontrasts observed on these sub-micrometric Fe dots are in excellent agreement with BEMM current maps calculated from micromagnetic simulation results. This opens the way to a quantitative magnetic microscopy with high contrast and nanometric lateral resolution.

La Neuropathie à Axones Géants (NAG) est une maladie neurodégénérative rare et fatale caractérisée par une détérioration du système nerveux central et périphérique, impliquant les fonctions motrices et sensorielles. La détérioration massive du système nerveux est accompagnée d'une désorganisation générale des Filaments Intermédiaires ce qui la différencie de nombreuses maladies neurodégénératives où seuls les neurofilaments(NFs) sont affectés. La protéine déficiente, la gigaxonine, est la sous-unité d'une ubiquitine ligase E3, responsable de la reconnaissance spécifique des substrats MAP1B, MAP1S et TBCB, seuls connus à ce jour.Dans le but d'étudier le rôle de la gigaxonine sur la survie neuronale, la désorganisation du cytosquelette et d'avoir un modèle animal suffisamment fort pour envisager des tests thérapeutiques, j'ai caractérisé un modèle murin de NAG. Pour ce faire, j'ai réalisé une étude comportementale des fonctions motrices et sensorielles ainsi qu'une étude histopathologique. Les souris NAG (129/SvJ) développent un phénotype moteur modéré dès 60 semaines alors que les souris NAG (C57BL/6) présentent un phénotype sensoriel dès 60 semaines. Les données histopathologiques ne présentent pas de mort neuronale mais les NFs sont sévèrement altérés. Les NFs sont plus abondant, leur diamètre est augmenté et leur orientation hétérogène, comme c'est observé chez les patients NAG.Nos résultats montrent que l'absence de gigaxonine induit un phénotype moteur et sensoriel modéré mais par contre reproduit la désorganisation massive des NFs observée chez les patients. Ce modèle va nous permettred'étudier le rôle de la gigaxonine, une ligase E3, sur l'organisation des NFs et ainsi comprendre les processus pathologiques impliqués dans d'autres maladies neurodégénératives caractérisée par une accumulation des NFs et un dysfonctionnement du système ubiquitine-protéasome comme les maladies d'Azheimer, de Parkinson etd'huntington ou la sclérose latérale amyotrophique. / Giant Axonal Neuropathy (GAN) is a rare and fatale neurodegenerative disorder characterized by a deterioration of the peripheral and central nervous system. The broad deterioration of the nervous system is accompanied with a general disorganization of the Intermediate Filaments which makes it different from other neurodegenerative disorders wherein only neurofilaments (NFs) are affected. The defective protein, gigaxonin, is the substrate adaptator of an E3 ubiquitin ligase, in charge of the specific recognition of MAP1B, MAP1S and TBCB. In order to study the role of gigaxonin on neuronal survival, the cytoskeleton disorganization and to have a relevant GAN animal model to evaluate efficacy of GAN treatments, I have characterized a GAN mouse model. I did a motor and sensory behavioural study and an histopathologic study. The GAN mice (129/SvJ) shown mild motordeficits starting at 60 weeks of age while sensory deficits were evidenced in C57BL/6 GAN mice. No apparent neurodegeneration was evidenced in GAN mice, but dysregulation of NFs was massive. NFs were more abundant, they shown the abnormal increased diameter and misorientation that are characteristics of the human pathology. Our results show that gigaxonin depletion induces mild motor and sensory deficits but recapitulates the severe NFs dysregulation seen in patients. Our model will allow us to study the role of the gigaxonin-E3 ligase in organizing NFs and understand the pathological processes engaged in other neurodegenerative disorders characterized by accumulation of NFs and dysfunction of the Ubiquitin Proteasome System, such as Amyotrophic Lateral Sclerosis, Huntington's, Alzheimer's and Parkinson's diseases.

Les virus sont présents dans tous les écosystèmes, et sont les entités les plus abondantes dans le milieu marin. Bien que nous associons systématiquement virus aux maladies, la plupart d'entre eux coexistent cependant en équilibre avec leur hôte. Les virus sont associés à tous les règnes de la vie, même les virus qui affectent d'autres virus(virophages). La définition aujourd'hui d'un virus chez les virologues, c'est qu'un virus est un parasite génétique qui utilise des systèmes cellulaires pour sa propre réplication. Les hôtes les plus couramment utilisés par les virus que nous avons étudiés sont principalement des protozoaires. Ainsi, les Amoebozoa font l'objet de nombreuses études et sont utilisés pour isoler de nouvelles espèces intracellulaires( virus, bactéries). Ces espèces ont évolué de manière à résister aux effets consécutifs à la phagocytose ou à l'ingestion dans des vacuoles, et restent viable dans le cytoplasme de l'amibe, et ont le potentiel de se multiplier dans les parasites. Dans cette étude, nous avons dans un premier temps étudier les diverses interactions existantes entre virus Acanthamoeba polypaghaga Mimivirus(APMV) et des bactéries au sein de l'amibe. Pour cela, nous avons choisi un système original basé sur la co-culture de l'APMV, soit seul ou en combinaison avec deux autres microorganismes isolés individuellement à partir de l'amibe. Il s'agit d'une bactérie intracellulaire stricte(BABL1) et le virophage de APMV (Sputnik). Cela nous a permis de mettre en évidence, d'une part la capacité du virophage à moduler la virulence d'APMV tout en révélant, d'autre part, la bataille qui a eu lieu entre eux au cours de l'infection de l'hôte. dans un deuxième temps, nous avons examiné l'activité virucide des biocides couramment utilisés en pratique clinique pour la désinfection des équipements hospitaliers. APMV et Marseillevirus montrent une grande résitance aux biocides chimiques, en particulier l'alcool. Seule la température de 75°C et le glutaraldéhyde ont réussi à réduire les titres d'APMV et Marseillevirus à des niveaux indétectables. Après dessiccation ou exposition aux rayonnements ultraviolets, APMV et marseillevirus ont démontré leur stabilité durable. Précédent le pré-traitement des échantillons de l'environnement par l'éthanol à 70°C, a permis la disparition des contaminants bactériens sans réduire la charge virale, permettant leur isolement sur amibe, sans avoir besoin d'utiliser des antibiotiques, qui peuvent avoir un effet délétère su les amibes. / In this study, we first examined the various interactions taking place between the virus Acanthamoeba polyphaga Mimivirus (APMV) and bacteria within the amoeba. We chose an original system based on a co-culture of APMV either alone or in combination with two other organisms isolated from amoeba, i.e a strict intracellular bacterium (BABL1) and the virophage of APMV (Sputnik). This allowed us to highlight, on the one hand, the possibility to modulate the virulence of APMV while revealing, on the other hand, the battle which occurs between them during the infection of the host. We then examined the virucidal activity of biocides commonly used in clinical practice for the disinfection of hospital equipment. APMV and Marseillevirus show high resistance to chemical biocides, especially to alcohol. Only a temperature of 75°C or glutaraldehyde were able to reduce APMV and Marseillevirus titres to undetectable levels. Whether dried or under ultraviolet, APMV and Marseillevirus demonstrated their lasting stability. Previous pre-treatment of environmental samples by ethanol 70° allowed disappearance of bacterial contaminating bacteria without reducing giant virus load allowing their isolation on amoeba without need the use of antibiotic that may have a deleterious effect on amoebae.

Asymptotic Giant Branch (AGB) stars are stars with low- to intermediate mass in a late stage in their stellar evolution. An important feature of stellar evolution is the ongoing nucleosynthesis, the creation of heavier elements. Unlike main sequence stars, the AGB stars have a thick convective envelope which makes it possible to dredge-up the heavier fused elements from the stellar core to its surface. AGB stars are also pulsating variable stars, meaning the interior expands and contracts, causing the brightness to fluctuate. These pulsations will also play a major role in the mass loss observed in these stars. The mass loss is caused by stellar winds that accelerate gas and dust from the surface of these stars and thereby chemical enrich the interstellar medium. It is important to understand the properties of these pulsations since they play a key role in how stellar winds are produced and then enrich the galaxy with heavier synthesized elements. These pulsation periods can be observed with their corresponding Light-Curves, where the periodic motion of the brightness can be clearly seen. The main goal with this project is to calculate these pulsation periods for different AGB stars and compare these values with the periods listed in the General Catalogue of Variable Stars (GCVS). The comparison between these values gives a better understanding of methods of determining these periods and the uncertainties that follow. / Asymptotiska jättegrenen är en del av slutstadiet för låg- till medelmassiva stjärnor (AGB stjärnor). Ett viktigt kännetecken hos stjärnutvecklingen är den pågående nukleosyntesen, sammanslagningen av tyngre ämnen i stjärnans inre. Till skillnad mot stjärnor på huvudserien har AGB stjärnor ett tjockt konvektivt lager som gör det möjligt att dra upp dessa nybildade ämnen till stjärnans yta. AGB stjärnor är pulserande variabla stjärnor där variationer i stjärnans radie gör att ljusstyrkan varierar. Dessa pulsationer kommer även att spela en viktig roll för den massförlust som observeras hos dessa stjärnor. Massförlusten orsakas av stjärnvindar som accelererar gas och stoft från stjärnans yta och därmed kemiskt berikar det interstellära mediet. Det är viktigt att förstå dessa pulsationer eftersom de är en viktig komponent för hur stjärnvindar uppstår och sedan berikar galaxer med tyngre ämnen. Dessa pulsationsperioder kan studeras genom att observera stjärnornas ljuskurvor, där man tydligt ser det periodiska beteendet hos ljusstyrkan. Det huvudsakliga målet med detta projekt är att beräkna dessa perioder för olika AGB stjärnor och att sedan jämföra dem med värden från General Catalogue of Variable Stars (GCVS). Jämförelsen mellan dessa värden ger en bättre förståelse för metoderna som används för att bestämma dessa perioder och hur osäkra dessa värden är.

O sucesso da gestação depende da adequada comunicação materno-fetal, que em algumas espécies têm um contato mais íntimo devido à capacidade migratória de populações de células trofoblásticas. Nos bovinos esse mecanismo é realizado pelas células trofoblásticas gigantes (CTGs), com invasão limitada até a lâmina basal do epitélio materno. Apesar dessa leve invasão das CTGs, é possível encontrar células fetais circulantes no sangue periférico da vaca gestante, levando ao microquimerismo fetal. Além de toda uma sinalização local e sistêmica e mudanças conformacionais, a migração das CTGs também é dependente da tolerância imunológica do epitélio materno que possui uma baixa expressão de MHC de classe I. Em contrapartida, o trofoblasto expressa MHC de classe Ib para impedir a ativação das células natural killers uterinas (uNK) contra ele mesmo. Neste contexto, o objetivo desse trabalho foi estudar a ocorrência e contribuir para o entendimento dos mecanismos da migração celular na placenta bovina, com marcadores exclusivos do cromossomo Y e de um modelo de clone transgênico expressando a proteína GFP. A hipótese testada foi que o microquimerismo fetal observado mediante a detecção do gene TSPY no sangue periférico da vaca gestante de embrião macho, e de GFP nos tecidos placentários maternos, associado à expressão de MHC classe 1b (Qa2) na interface materno-fetal. Para tanto, 153 embriões produzidos por fertilização in vitro (FIV) foram transferidos, resultando em 34 embriões machos e 31 fêmeas no dia 62 de gestação, quando foi realizada a coleta de sangue periférico da receptora. Dentre estas gestações, foram selecionadas de 25 machos, 4 fêmeas e 5 perdas gestacionais (confirmadas no D39 por ultrassonografia) para detecção de TSPY. Também foram produzidas gestações de clones transgênicos, expressando GFP com 30, 60 e 90 dias que foram utilizadas para a detecção de mRNA e a proteína GFP. Nas gestações de FIV 60% dos embriões machos, 50% das fêmeas e 40% das perdas gestacionais foram positivos para TSPY. A detecção de TSPY nas gestações de fêmeas possivelmente é resultante da persistência do microquimerismo de gestações anteriores. Nas gestações de clones transgênicos, observou-se a presença de mRNA e proteína GFP no endométrio, também indicando migração nesta região ou o transporte da GFP, e outros conteúdos do trofoblasto, para o epitélio materno. Nos placentônios, usando anticorpo anti-GFP pode-se ver a marcação positiva tanto no trofoblasto como no epitélio materno, possivelmente decorrente de liberação das CTGs no estroma endometrial após a fusão. As CTGs, quando em formação sincicial, têm a sua expressão de GFP diminuída, o que também foi observado, utilizando-se anticorpo anti-Qa2 (antígeno murino para MHC classe Ib). O epitélio materno e o trofoblástico também foram marcados para Qa2. Mediante as técnicas utilizadas, observamos que o microquimerismo pôde ser identificado nas gestações analisadas com o uso dos marcadores TSPY no sangue e o GFP nos tecidos placentários maternos. Este estudo mostra que na placenta bovina ocorre uma migração de células fetais além do epitélio materno e abre novas perspectivas para estudos das características da interação materno-fetal ainda pouco explorada nos bovinos. / The pregnancy success depends of adequate materno-fetal communication, that in some species are have a more intimate contact due migratory capacity of trophoblastic cells populations. In bovines, this mechanism is realized by trophoblast giant cells (TGC) with limited invasion until basal lamina of maternal epithelium. Besides this light invasion of TGCs, is possible to encounter circulate fetal cells in peripheral blood of pregnant cow, leading to fetal microchimerism. Beyond local and systemic sinalization and conformational changes, TGC migration is also dependent of immunologic tolerance of maternal epithelium that possess a downregulation of classe I MHC. In complement, the trophoblast express classe Ib MHC to inhibit natural killers cells activation. In this context, the objective of this work was to study the occurrence and contribute for knowledge cellular migration mechanisms in bovine palcenta, with Y-specific markers and a model of transgenic clone expressing GFP. The tested hypothesis was that fetal microchimerism observed by detection od TSPY gene in peripheral blood of cow pregnant of male embryo, and of GFP in maternal placental tissues associated by expression of class Ib MHC (Qa2) in materno-fetal interface. For this, 153 embryos produced by in vitro fertilization (IVF) were trasnfered, resulting in 34 male embryos and 31 female in day 62 of pregnancy, when recipient peripheral blood was collected. Among these pregnancies, 25 males, 4 females and 5 pregnancy losses (confirmed at 39 days of pregnancy by ultrassonography) was selected for TSPY detection. Also were produced pregnancies of transgenic cloned, embryos expressing GFP, with 30, 60 and 90 days of pregnancy that utilized for GFP mRNA and protein detection. In IVF pregnancies, 60% of male embryos, 50% of females and 40% of pregnancy losses were positive for TSPY. The detection of TSPY in female pregnancies possible is resultant of persistence of microchimerism of anterior pregnancies. In transgenic cloned pregnancies, was observed presence of GFP mRNA and protein in endometrium, also indicating migration in this region or GFP transport, and another trophoblast content, to maternal epithelium. In placentomes, using anti-GFP antibody, could be observed positive immunolabeling in trophoblast and maternal epithelium, possible due CTGs liberation in endometrial stroma after fusion. The CTGs, in syncytium formation, have a downregulation of GFP, that also be observed, utilizing anti-Qa2 antibody (murine antigen of classe Ib MHC). The maternal and trophoblastic epithelium also was Qa2 immunolabed. By utilized techniques, microchimerism could be indentified in analyzed pregnancies with use of markers for TSPY in maternal blood and GFP in maternal placental tissues. This study show that in bovine placenta occurs fetal cell migration further maternal epithelium and show new perspectives for studies of materno-fetal interaction characteristics until under explored in bovines.

Resumo: Propósito: É propósito deste trabalho estudar a agressividade da lesão de células gigantes periférica da cavidade bucal (LCGP) através de aspectos clínicos, radiográficos, histopatológicos, histoquímicos e imunohistoquímicos, objetivando melhor conhecer o perfil clínico do paciente e, sobretudo, avaliar o comportamento biológico dessa lesão. Material e Método: Foram estudados retrospectivamente 61 casos de LCGP, procedentes da Faculdade de Odontologia de Araçatuba - UNESP e da Faculdade de Odontologia da Universidade de Passo Fundo - FO-UPF. Primeiramente, realizou-se confirmação diagnóstica histopatológica e avaliação da presença de tecido ósseo imaturo nas lesões. Após, os casos selecionados foram agrupados com base em critérios clínico-radiográficos, em difererentes graus (agressividade nula, leve, moderada, severa e extrema) e seus dados clínico-demográficos e imaginológicos foram avaliados e correlacionados. Em seguida, utilizando-se 15 casos do total estudado (05 casos com agressividade clínico-radiográfica nula, 05 com agressividade moderada e 05 com agressividade severa), investigou-se a proliferação celular das LCGPs por meio da contagem e aferição da área de AgNORs (regiões de organização nucleolar marcadas pela prata) e avaliação da expressão de PCNA (antígeno de proliferação nuclear celular), Ki-67 (Kiel-67) e p53 (proteína p53). Resultados: Dentre os resultados clínico-radiográficos obtidos destacam-se os que dizem respeito à idade dos pacientes (prevalência entre 31 e 40 anos), sexo (prevalência em mulheres), raça (prevalência em brancos) e localização anatômica (prevalência na região mandibular anterior). A maioria das lesões se mostrou como nódulo indolor, de superfície lisa, dois meses de evolução, consistência fibrosa, formato arredondado ou ovóide, coloração púrpura e base séssil... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Purpose: To study the aggressiveness of peripheral giant cell lesion of buccal cavity (PGCL) by means of clinical, radiographic, histopathological, histochemical and immunohistochemical aspects in order to determine the patient's profile and to evaluate the biologic behavior of this lesion. Material and Method: 61 cases of PGCL of patients from Faculdade de Odontologia de Araçatuba - UNESP and Faculdade de Odontologia da Universidade de Passo Fundo - FOUPF were studied retrospectively. A corroborative diagnostic and the evaluation of immature bony tissue in the lesions were accomplished. These cases were divided into different groups according a classification based on grades of clinical-radiographic aggressiveness. The data of each group were evaluated and correlated and 15 cases were selected (5 cases with null clinical-radiographic aggressiveness, 5 with moderate aggressiveness and 5 with severe aggressiveness). An investigation of the cellular proliferation of PGCL was accomplished by count and measurement of the area of AgNORs (nucleolar organization regions marked by silver) and evaluation of PCNA (proliferating cell nuclear antigen), Ki-67 (Kiel-67) and p53 (protein p53). Results: The following conclusions were drawn by the clinical-radiographic results: in respect to the patient's age (prevalence between 31 and 40 years old), sex (prevalence in women), race (prevalence in white people), anatomical location (prevalence in the mandibular anterior region). Clinically the lesions were predominantly observed as painless tissue growths of smooth surface, two months of evolution, "fibrous" consistence, nodular form, purple coloration and sessile basis of implantation. In the greater number of cases the radiographic image of the subjacent bony tissue to the PGCL... (Complete abstract, click electronic address below) / Orientador: Norberto Perri Moraes / Coorientador: Marcelo Macedo Crivelini / Banca: Glauco Issamu Miyahara / Banca: Soluete Oliveira da Silva / Banca: Norberto Perri Moraes / Mestre

Regulation of the actin cytoskeleton is key to cellular function and underlies processes including cell migration, mitosis and endocytosis. Motile cells send out dynamic actin protrusions that enable them to sense and interact with their environment, as well as generating physical forces. Linking of the actin cytoskeleton to the cell membrane is essential for the formation of these protrusions. The proteins that are thought to fulfil such a role have a membrane interacting domain (such as the PH domain in lamellipodin, or I-BAR protein in IRSp53) and a domain which interacts with actin regulatory proteins (such as the SH3 domain of IRSp53, which binds Ena and VASP). I investigated the contribution of the F-BAR protein Toca-1 in linking actin polymerisation to membranes, by characterising a new protein-protein interaction and the interaction of Toca-1 with giant unilamellar vesicles. FBP17, a homologue of Toca-1, can oligomerise to form 2D flat lattices and 3D tubules on membranes. Proteins of the Toca-1 family have previously been implicated in actin polymerisation in cell-free systems and during endocytosis. However, there is emerging evidence that Toca-1 family proteins could also be involved in the formation of outward facing protrusions, lamellipodia and filopodia. In an in vitro system that recapitulates the formation of filopodia-like structures (FLS) on supported lipid bilayers, Toca-1 is recruited early, suggesting a Toca-1 scaffolding mechanism could precede the recruitment of other actin regulators. One prediction of this model is that Toca-1 would bind proteins previously implicated in filopodia formation, such as formins. I found that extracts depleted of Toca-1 binding partners no longer forms filopodia-like structures and subsequently optimised pull-down assays to identify Toca-1 binding partners by mass-spectrometry. I identified four formins, Diaph1, Diaph3, FHOD1 and INF2, and as well as the actin elongation factors and filopodia proteins, Ena and VASP. I further characterised these interactions and found that Toca-1 binds Ena and VASP via its SH3 domain. The interaction is direct and is strongly reduced if the proline-rich region in Ena is deleted. VASP was still able to bind without its proline rich region, suggesting there could be additional binding sites. I discovered that the binding of Ena and VASP was dependent on the clustering state of Toca-1, whilst the binding of the previously identified Toca-1 binding partner N-WASP was not. This further supports the importance of Toca-1 oligomerisation in actin polymerisation. I tested these interactions in the FLS system and found that increasing Toca-1 concentration leads to increased recruitment of N-WASP, as well as the novel binding partner Ena to the structures, whereas an increase in VASP was not observed. SH3-domain mediated interactions are required for Toca-1 recruitment to FLS, suggesting that its membrane and protein binding activities act cooperatively. I showed that unlike N-WASP, which promotes the formation of branched actin, Ena and VASP are not required for actin polymerisation on supported lipid bilayers, suggesting that they are redundant with other factors in the elongation step of FLS formation. Ena and VASP are known to be important for the formation of neuronal filopodia and so I began to further test the role of these interactions in a cellular context using a neuronal cell culture system. As well as recruiting protein binding partners, F-BAR family proteins are implicated in stabilising lipid microdomains and can induce the clustering of phosphoinositides. I investigated the role of Toca-1 in actin polymerisation on PI(4,5)P2-rich giant unilamellar vesicles (GUVs). Actin-rich tails formed on the GUVs only when excess Toca-1 was supplemented into the extracts, and I propose that this is due to lipid organisation by Toca-1. In summary, my work suggests a model in which Toca-1 clusters, stabilises the membrane lipids and recruits regulators of actin polymerisation, such as Ena. This mechanism could be used to link actin polymerisation to the membrane in cellular protrusions, such as filopodia.