Distal-less (Dll) is a master regulator gene responsible for proximal-distal axis formation as well as distal appendage identity. Previous research showed that the expression of Dll is maintained through a feedback loop with Disco, a C2H2 zinc finger transcription factor. In this project I investigate recent suggestions that disco may play additional roles as a cofactor or downstream target of Dll during appendage development. I confirm previous research that the presence of Dll is sufficient to turn on disco. I found that the presence of ectopic Dll in the wing discs activates Dll subordinate genes in cells where they are not normally expressed. I again performed experiments confirming previous reports that ectopic expression of Dll in the wing tissue is sufficient to cause the appearance of ectopic legs. I then showed that when Dll is expressed ectopically in the absence of disco, there ectopic appendages similar to those formed in the presence of disco. Put together, my results suggest that disco is does not function as a cofactor or downstream target required for the development and differentiation of Drosophila ventral appendages. / Thesis / Master of Science (MSc)

Desmopathy of the collateral ligament of the distal interphalangeal joint is a common cause of lameness in the horse and carries a poor prognosis for soundness. Intralesional treatment has been suggested as a way to improve outcome; however, limited reports describe methods for injecting this ligament. This study compares the accuracy of injecting the collateral ligament of the distal interphalangeal joint using magnetic resonance imaging (MRI) versus radiographic guidance. Equine cadaver digit pairs (n=10) were divided by random assignment to injection of the ligament by either technique and assessed using post-injection MRI or gross sections. Images from the proximal, middle, and distal portions of the ligament were blindly evaluated for successful injection. McNemar\'s test was performed to determine statistical difference between injection techniques. Fisher\'s exact test was used to evaluate number of injection attempts and injection of the medial or lateral collateral ligament. MRI-guided injection was successful more frequently than radiographic-guided injection on post-injection MRI (24 of 30 versus 9 of 30; p=0.0006) and gross sections (26 of 30 versus 13 of 30; p=0.0008). At each level of the ligament (proximal, middle, and distal), MRI-guided injection resulted in more successful injections than radiographic guidance. Statistical significance occurred at the proximal aspect of the collateral ligament based on post-injection MRI (p=0.0143) and the middle portion of the ligament based on gross sections (p=0.0253). Based on these results, injection guided by standing, low-field MRI should be considered an option for delivering intralesional regenerative therapy to horses with desmopathy of these collateral ligaments. / Master of Science

Researchers and military organizations have defined military conflicts as being fought in two principal combat environments, namely, close proximity, or the proximal combat environment, and distant proximity, or the distal combat environment. This study used Gal and Jones's psychological model of combat stress as the theoretical framework. The study also used Merriam's generic interpretative qualitative research method, including open-ended interviewing and document review, to obtain data. The study used inductive thematic analysis to analyze the narratives of 10 distal combat veterans who were recruited for the study. The research questions were designed to identify the characteristics of distal combat and the personal narratives of distal combat veterans. The study also was an exploration of the emotional, psychological, behavioral, and physical reactions of distal combat veterans to the onset of combat stress and the type of coping strategies that they used in the face of such challenges. Potential social change implications of the study are that it elucidated current understanding of the psychological consequences of distal combat and identified factors that can help combat soldiers and veterans to develop resiliency to distal combat stress. Given the gap in the combat literature regarding distal combat stress, conducting this study adds to knowledge of the ways in which combat stress affects distal combat veterans during operations.

Η ενδομυελική ήλωση αποτελεί πλέον μια κοινώς αποδεκτή και καταξιωμένη μέθοδο, στην αντιμετώπιση καταγμάτων διάφυσης μακρών οστών (και όχι μόνο). Μετά από μισό και πλέον αιώνα εφαρμογής της, έχει αποκτηθεί σημαντική εμπειρία και για πολλούς Ορθοπαιδικούς χειρουργούς αποτελεί μέθοδο εκλογής στην καθημερινή χειρουργική πρακτική. Πολλές πτυχές της μεθόδου έχουν αποτελέσει αντικείμενο διεξοδικής μελέτης αλλά το ζήτημα της περιφερικής ασφάλισης των ενδομυελικών ήλων, εξακολουθεί να προκαλεί ίσως το μεγαλύτερο προβληματισμό για τον χειρουργό. Η «δια χειρός» (free-hand) αναζήτηση των οπών περιφερικής ασφάλισης του ήλου είναι μια διαδικασία χρονοβόρα, συχνά τραυματική και κυρίως επιβαρυντική με ακτινοβολία Χ λόγω χρήσης ακτινοσκοπικού μηχανήματος διεγχειρητικά, ακόμη και για έμπειρους χειρουργούς. Τα μηχανικά σκόπευτρα (στις διάφορες παραλλαγές που μέχρι τώρα έχουν χρησιμοποιηθεί) μπορούν να βοηθήσουν. Η συχνά αναπόφευκτη όμως παραμόρφωση του ήλου κατά την εισαγωγή του στον αυλό, καθιστά προβληματική τη λειτουργικότητα και μειώνει σημαντικά την αποτελεσματικότητα τους. Αυτό σε συνδυασμό με το συχνά περίπλοκο εξοπλισμό αλλά και την απαιτούμενη εκμάθηση της τεχνικής, αποθαρρύνουν σε πολλές περιπτώσεις το Χειρουργό, που καταφεύγει τελικά εκ νέου στη free-hand τεχνική. Η εκτεταμένη πλέον χρήση των ηλεκτρονικών υπολογιστών και στη χειρουργική, έχει βρει πεδίο εφαρμογής και στο θέμα αυτό. Πολύπλοκα λογισμικά με αντίστοιχο εξοπλισμό και μεθοδολογία, φιλοδοξούν να κατευθύνουν με ακρίβεια το χέρι του Χειρουργού στις οπές ασφάλισης του ήλου. Μέχρι σήμερα τέτοια συστήματα αποτελούν «σενάριο απ’ το μέλλον» για το μέσο Ορθοπαιδικό Χειρουργό (και ειδικότερα για τα δεδομένα του Ελληνικού χώρου). Οι υψηλές απαιτήσεις τους σε τεχνογνωσία και εξοπλισμό σε συνδυασμό με το σημαντικό κόστος τους, είναι λόγοι για τους οποίους δεν μπορούν να θεωρηθούν ρεαλιστική λύση επί του παρόντος. Το σύστημα περιφερικής σκόπευσης του ήλου S2 ανήκει στην κατηγορία των μηχανικών σκοπεύτρων. Χωρίς να διαφέρει σημαντικά στις απαιτήσεις του ως προς την εκμάθηση της συναρμολόγησης και χρήσης του από τον Χειρουργό (και το βοηθητικό προσωπικό του χειρουργείου) σε σχέση με άλλα μηχανικά σκόπευτρα, παρουσιάζει ένα ιδιαίτερο χαρακτηριστικό. Η προσαρμοστικότητα του μηχανισμού σκόπευσης παρέχει τη δυνατότητα απόσβεσης της γεωμετρικής παραμόρφωσης (προσθιοπίσθιας, πλαγιοπλάγιας και στροφικής) του ήλου μετά την εισαγωγή του, εντός φυσικά ορισμένων ορίων με αξιόλογο όμως εύρος. Η παρούσα διατριβή παρουσιάζει τα μετρήσιμα ευρήματα από μια σειρά 190 περιστατικών, στα οποία εφαρμόστηκε κλινικά για πρώτη φόρα ο ήλος S2 σε κατάγματα μηριαίου και κνήμης, καθώς επίσης και συγκριτική μελέτη αποτελεσματικότητας του σκοπεύτρου του ήλου S2 και της «δια χειρός» περιφερικής σκόπευσης για την ασφάλιση του ήλου G-K. Το ενδιαφέρον εστιάστηκε στην ευστοχία του σκοπεύτρου, στην ταχύτητα εφαρμογής της μεθόδου και ιδιαίτερα στην μείωση της χρήσης ακτινοσκοπικών λήψεων. Τα ευρήματα αυτά σε σύγκριση με αντίστοιχα άλλων μεθόδων, όπως προκύπτει από τα έως τώρα βιβλιογραφικά δεδομένα, καθιστούν τον ήλο S2 και το σύστημα σκόπευσης του, μια ιδιαίτερα αποτελεσματική και κυρίως ελάχιστα επιβαρυντική ως προς την ακτινοβολία (για ασθενή, Χειρουργό και βοηθητικό προσωπικό) μέθοδο. / Intramedullary nailing is a well known, successful and widely accepted method in treatment of diaphyseal fractures (and not only) of long bones. After more than a half of century of application of intramedullary nails, important experience has been acquired and for many Orthopaedic surgeons the specific method constitutes the treatment of choice in the daily chirurgical practice. Many aspects of the method have been studied extensively but the part of distal locking of intramedullary nails continues to be troubleshooting for many Surgeons. “Free-hand” technique is excessively used as a targeting method for distal locking despite the fact that it is a time-consuming process, frequently traumatic and mainly aggravating with radiation, as the use of image intensifier is mandatory intra-operatively. Mechanical targeting devices (in many variants) that up to now have been used, can help. However the deformation of nail, usually inevitable during its import in the intramedullary canal, turns out to be the main cause of limited effectiveness. This, in combination with the relatively complex equipment, but also the required learning time of technique, usually discourage surgeon of using these mechanical targeting devices and finally the free-hand technique turns again to be the method of choice in distal locking. The extensive henceforth, use of computers in operating theatre has also found a challenging field of application in this subject. Complicate software with corresponding equipment and methodology aspire to drive surgeon’s hand with precision to distal screw holes of intramedullary nails. Still, such systems seem to be “script of future” for the average Orthopaedic surgeon. High requirements in know-how and equipment, in combination with considerable cost of computer-assisted navigation, are strong reasons for not being considered as a realistic solution on the present. S2 nails with associated distal targeting system, belong in the category of nail mounted, mechanical targeting devices. Without differing considerably in requirements and learning demands from the Surgeon (and the assisting personnel in operating theatre), in comparison with other mechanical devices, it presents one particular characteristic. The adaptability of targeting system provides the ability of damping of nail deformation (during intramedullary application), inside limits with appreciable breadth. This dissertation presents the measurable findings of the first 190 cases of femoral and tibial shaft fractures treated by intramedullary nailing with S2TM nail and S2TM distal targeting device, and also a comparative study between S2 distal targeting device and ‘free-hand’ technique for G-K distal locking. The interest was focused in the effectiveness of the device, the duration of the procedure (distal locking screws placement and total intramedullary nailing time) and particularly in the reduction of image intensifier use (reduction of patient, surgeon and personnel exposition to x-rays). Results were compared to bibliographic data of other well-studied methods of distal targeting in intramedullary nailing and in conclusion, S2TM nail and its distal targeting device is a significantly effective and mainly minimally aggravating with x-ray radiation, method.

Submitted by Adriana Martinez (amartinez@unoeste.br) on 2017-09-05T12:53:44Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Luci.pdf: 503217 bytes, checksum: bcb750cc3835332fed12d8730ecb4254 (MD5) / Made available in DSpace on 2017-09-05T12:53:44Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Luci.pdf: 503217 bytes, checksum: bcb750cc3835332fed12d8730ecb4254 (MD5) Previous issue date: 2017-06-22 / This work aims to study the effects of omega 3 and / or vitamin B12 administration on sperm and histomorphometric parameters of the reproductive organs of Wistar rats and their correlation with the temperature of these regions. Sixteen rats were divided into four groups (n = 4 per group) who received daily injections for 30 days, being: Control Group - saline solution; Group Omega three- fish oil (Equaliv®) 1g / kg; Group B12 - vitamin B12 (Monovin B12 ®) 3μg; And Group Omega + B12 - fish oil 1g / kg and vitamin B12 3μg. Analyzed sperm morphology and the histomorphometries were evaluated by the Motic Images Plus® version 2.0 program. On a daily basis, thermographic images of areas of the body (FLIR E40®, Sweden) were analyzed and analyzed by the Flir Tools 2.1® program. Data were submitted to analysis of variance and Tukey test at 5%. Omega 3 reduced the seminiferous epithelium and B12 minimized this deleterious effect (P ≤ 0.05). The surface temperature of the scrotum was higher in group B12 (P ≤0.05). There was a high positive correlation between scrotum surface temperature and distal cytoplasmic droplet percentage (P = 0.678). / Este trabalho tem como objetivo estudar os efeitos da administração do ômega três e/ou vitamina B12 sobre parâmetros espermáticos e histomorfométricos dos órgãos reprodutivos de ratos Wistar e sua correlação com a temperatura dessas regiões. Utilizou-se 16 ratos, divididos em quatro grupos (n=4 por grupo) que receberam injeções diárias por 30 dias, sendo: Grupo Controle – solução salina; Grupo Ômega três– óleo de peixe (Equaliv®) 1g/kg; Grupo B12 – vitamina B12 (Monovin B12®) 3µg; e Grupo Ômega + B12 – óleo de peixe 1g/kg e vitamina B12 3µg. Analisados morfologia espermática e as histomorfometrias foram avaliados pelo programa Motic Images Plus® versão 2.0. Diariamente, foram obtidas imagens termográficas de áreas do corpo (FLIR E40®, Suécia) e analisadas pelo programa Flir Tools 2.1®. Os dados foram submetidos à análise de variância e teste de Tukey a 5%. O ômega 3 reduziu o epitélio seminífero e a B12 minimizou esse efeito deletério (P ≤ 0,05). A temperatura da superfície do escroto foi superior no grupo B12 (P ≤0,05). Houve alta correlação positiva entre temperatura da superfície do escroto e porcentagem de gota citoplasmática distal (P = 0,678).

Nesta pesquisa, foi realizada uma análise comparativa, por meio do método dos elementos finitos bidimensional linear, das deformações e tensões geradas a partir da aplicação de uma carga de 100N, nas estruturas de suporte (fibromucosa,ligamento periodontal, osso cortical e osso esponjoso) de dois modelos que simulam um hemiarco mandibular associada a uma prótese parcial removível de extremidade livre dista inferiorl, quando da variação da inclinação do rebordo residual no plano sagital. O modelo A (MA) foi desenhado com um rebordo ascendente distal com 15 graus de inclinação e o modelo B (MB) com um rebordo descendente distal com 15 graus negativos, ambos em relação ao plano oclusal. Nos dois modelos aplicou-se uma carga primeiramente na cúspide médio-vestibular do primeiro molar inferior artificial (C1) e depois na cúspide vestibular do segundo pré-molar inferior (C2), por meio do programa I-deas (software de elementos finitos) onde foram feitas as análises de deformações e tensões e processamento das imagens. Fez-se então uma análise comparativa dos carregamentos (C1 e C2) em cada modelo individualmente e entre os dois modelos(MA e MB). Os resultados permitiram concluir que: 1) A inclinação do rebordo residual no plano sagital influenciou na distribuição e na intensidade das tensões das estruturas de suporte; 2) No osso cortical as tensões foram mais localizadas e de maior intensidade que no osso esponjoso; 3) A diferença de deformação entre fibromucosa e ligamento periodontal foi maior quando a carga foi aplicada no primeiro molar, de ambos os modelos, sendo a mais expressiva no rebordo ascendente distal; 4) As tensões de compressão foram maiores em intensidade e melhor distribuídas no osso esponjoso e no osso cortical, quando comparadas as tensões de tração; 5) O rebordo ascendente distal se mostrou mais favorável à distribuição das tensões nas estruturas de suporte quando comparado ao rebordo descendente distal; 6) Nos dois modelos a aplicação da carga no primeiro molar gerou um maior torque sobre o dente suporte para a distal do que quando aplicado no segundo pré-molar, principalmente no rebordo descendente distal; 7) As estruturas de suporte do rebordo residual foram mais solicitadas em C1 que em C2, em ambos os modelos, principalmente no rebordo ascendente distal; 8) No dente suporte as tensões nas estruturas de suporte foram mais axiais quando o carregamento ocorreu no segundo pré-molar inferior, em ambos os modelos. / In this study, a two-dimensional finite element comparative analysis of the stress distribution and of displacement in the supporting structures – oral mucosa,periodontal ligament, compact and cancellous bone – was carried out for a distal extension base removable partial denture (RPD), modeled on two mandibular dental archs with different types of residual ridge when aplaying loads of 100N. The A model (MA) was developed to include a distal ascending alveolar ridge with a 15-degrees positive inclination, while the B model (MB) was prepared with a distal descending alveolar ridge with a 15-degrees negative inclination. Both models had a load applied inicially on the lower first molar median cusp (C1) and then on the lower second premolar buccal cusp (C2). Using the FEA software (I-DEAS, USA) for the stress distribution and displacement analysis of the generated images. A comparative analysis was them performed on the C1 and C2 loads for which and both models (MA and MB).The results showed that: 1 – The alveolar ridge inclination showed some degree of on the stress distribution and intensity generated by the RPD; 2 –The compact bone showed greater stress concentration and intensity than the cancellous bone; 3 – The ratio between oral mucosa and periodontal ligament displacement showed greater values when load was applied on the lower first molar for both models. The ratio was higher for the distal ascending alveolar ridge; 4- The compressive stress showed greater intensity and better range of distribution than the tensile stress for both the compact and cancellous bone; 5 – The distal ascending residual ridge had a more favourable stress distribution than distal descending conterpart ; 6 – The load applied on the lower first molar produced more distal torquing on the supporting tooth than that on the lower second premolar ,specially on the distal descending residual ridge; 7 – the load applied on the lower firt molar generated greater amount of stress on the residual ridge supporting structures for both models, but specially for the distal ascending residual ridge; 8 – The load applied on the lower second premolar produced more vertical forces on the supporting structures of the tooth for both models.

Nesta pesquisa, foi realizada uma análise comparativa, por meio do método dos elementos finitos bidimensional linear, das deformações e tensões geradas a partir da aplicação de uma carga de 100N, nas estruturas de suporte (fibromucosa,ligamento periodontal, osso cortical e osso esponjoso) de dois modelos que simulam um hemiarco mandibular associada a uma prótese parcial removível de extremidade livre dista inferiorl, quando da variação da inclinação do rebordo residual no plano sagital. O modelo A (MA) foi desenhado com um rebordo ascendente distal com 15 graus de inclinação e o modelo B (MB) com um rebordo descendente distal com 15 graus negativos, ambos em relação ao plano oclusal. Nos dois modelos aplicou-se uma carga primeiramente na cúspide médio-vestibular do primeiro molar inferior artificial (C1) e depois na cúspide vestibular do segundo pré-molar inferior (C2), por meio do programa I-deas (software de elementos finitos) onde foram feitas as análises de deformações e tensões e processamento das imagens. Fez-se então uma análise comparativa dos carregamentos (C1 e C2) em cada modelo individualmente e entre os dois modelos(MA e MB). Os resultados permitiram concluir que: 1) A inclinação do rebordo residual no plano sagital influenciou na distribuição e na intensidade das tensões das estruturas de suporte; 2) No osso cortical as tensões foram mais localizadas e de maior intensidade que no osso esponjoso; 3) A diferença de deformação entre fibromucosa e ligamento periodontal foi maior quando a carga foi aplicada no primeiro molar, de ambos os modelos, sendo a mais expressiva no rebordo ascendente distal; 4) As tensões de compressão foram maiores em intensidade e melhor distribuídas no osso esponjoso e no osso cortical, quando comparadas as tensões de tração; 5) O rebordo ascendente distal se mostrou mais favorável à distribuição das tensões nas estruturas de suporte quando comparado ao rebordo descendente distal; 6) Nos dois modelos a aplicação da carga no primeiro molar gerou um maior torque sobre o dente suporte para a distal do que quando aplicado no segundo pré-molar, principalmente no rebordo descendente distal; 7) As estruturas de suporte do rebordo residual foram mais solicitadas em C1 que em C2, em ambos os modelos, principalmente no rebordo ascendente distal; 8) No dente suporte as tensões nas estruturas de suporte foram mais axiais quando o carregamento ocorreu no segundo pré-molar inferior, em ambos os modelos. / In this study, a two-dimensional finite element comparative analysis of the stress distribution and of displacement in the supporting structures – oral mucosa,periodontal ligament, compact and cancellous bone – was carried out for a distal extension base removable partial denture (RPD), modeled on two mandibular dental archs with different types of residual ridge when aplaying loads of 100N. The A model (MA) was developed to include a distal ascending alveolar ridge with a 15-degrees positive inclination, while the B model (MB) was prepared with a distal descending alveolar ridge with a 15-degrees negative inclination. Both models had a load applied inicially on the lower first molar median cusp (C1) and then on the lower second premolar buccal cusp (C2). Using the FEA software (I-DEAS, USA) for the stress distribution and displacement analysis of the generated images. A comparative analysis was them performed on the C1 and C2 loads for which and both models (MA and MB).The results showed that: 1 – The alveolar ridge inclination showed some degree of on the stress distribution and intensity generated by the RPD; 2 –The compact bone showed greater stress concentration and intensity than the cancellous bone; 3 – The ratio between oral mucosa and periodontal ligament displacement showed greater values when load was applied on the lower first molar for both models. The ratio was higher for the distal ascending alveolar ridge; 4- The compressive stress showed greater intensity and better range of distribution than the tensile stress for both the compact and cancellous bone; 5 – The distal ascending residual ridge had a more favourable stress distribution than distal descending conterpart ; 6 – The load applied on the lower first molar produced more distal torquing on the supporting tooth than that on the lower second premolar ,specially on the distal descending residual ridge; 7 – the load applied on the lower firt molar generated greater amount of stress on the residual ridge supporting structures for both models, but specially for the distal ascending residual ridge; 8 – The load applied on the lower second premolar produced more vertical forces on the supporting structures of the tooth for both models.

L’Hypertension Hyperkaliémique Familiale (FHHt) est une forme rare d’hypertension artérielle associée à une hyperkaliémie et une acidose métabolique hyperchlorémique. Ces troubles sont corrigés par les diurétiques thiazidiques qui inhibent le co-transporteur Na+-Cl- NCC exprimé dans le néphron distal. Cette sensibilité aux diurétiques thiazidiques a laissé́ supposer que la FHHt est causée par une activation de NCC. En 2001, des mutations, de type gain-de-fonction, responsables de la FHHt ont été découvertes dans les gènes codant les sérine-thréonine kinases WNK1 et WNK4 [With No (K) lysine]. Des études in vitro ont montré que WNK1 et WNK4 stimulent NCC de façon indirecte, via la phosphorylation et l'activation de la kinase SPAK (Ste20 like Proline-Alanine rich Kinase). In vivo, l’activation de SPAK joue un rôle central dans le développement de la FHHt due aux mutations de WNK4. L'implication de SPAK n'a pas été définie dans le cas des mutations de WNK1. Nous avons donc croisé les souris WNK1+/FHHt, porteuses d'une mutation FHHt de WNK1, avec les souris SPAK243A/243A, porteuses d’une mutation abolissant l’activation de SPAK par les WNK. L’ensemble des phénotypes observés chez les souris WNK1+/FHHt est corrigé chez les souris WNK1+/FHHt:SPAK243A/243A démontrant ainsi le rôle central de SPAK dans la FHHt causée par les mutations WNK1. En 2012, de nouvelles mutations ont été identifiées dans les gènes codant CUL3 et KLHL3, deux composants d’un complexe ubiquitine ligase. Comme les mutations de WNK1 et WNK4, ces mutations entraînent une augmentation de l’expression de WNK1 et de WNK4. De façon inattendue, les patients FHHt porteurs d'une mutation CUL3 présentent un phénotype plus sévère que les autres. Des études suggèrent que ces mutations perturbent la fonction non seulement du néphron distal mais également des artères. Afin de vérifier cette hypothèse, nous avons généré et comparé deux modèles de souris : les souris pgk-Cul3Δ9, exprimant la mutation Cul3 de façon ubiquitaire, comme les patients, et les souris sm22-Cul3Δ9, exprimant la mutation uniquement dans les cellules musculaires vasculaires lisses. Les deux modèles sont hypertendus, mais les souris pgk-Cul3Δ9 le sont significativement plus que les souris sm22-Cul3Δ9, ce qui prouve que l’hypertension causée par les mutations Cul3 résulte du cumul d’une atteinte rénale et vasculaire. Récemment, de nouvelles mutations faux-sens d'un domaine dit acide de WNK1 ont été identifiées dans un petit nombre de patients atteints de FHHt. Ce domaine est nécessaire à la liaison à KLHL3 et donc à l’ubiquitination des WNK. Notre étude montre que, in vitro, ces mutations entraînent une accumulation d'une seule isoforme de WNK1. Chez la souris, la mutation du domaine acide provoque le même phénotype que les patients, ainsi qu’une augmentation de la phosphorylation de SPAK et du co-transporteur NCC. Cette étude a donc permis de démontrer le rôle essentiel de ce domaine dans la régulation de l'abondance de WNK1 dans le néphron distal in vivo. En conclusion, mon travail a permis une meilleure compréhension du rôle joué par SPAK, WNK1 et CUL3 dans le développement de la FHHt et, plus largement, de la physiopathologie de la FHHt. / Familial Hyperkalemic Hypertension (FHHt) is a rare form of hypertension associated with hyperkalemia and hyperchloremic metabolic acidosis. These disorders are all corrected by thiazide diuretics that inhibit the Na+-Cl- NCC cotransporter expressed in the distal nephron. The sensitivity of FHHt patients to thiazide diuretics strongly suggested that FHHt is caused by NCC activation. In 2001, gain-of function-mutations were identified in the genes encoding the serine-threonine kinases WNK1 and WNK4 [With No (K) lysine] in a subst of FHHt patients. In vitro studies demonstrate that WNK1 and WNK4 indirectly stimulate NCC, through the phosphorylation and activation of SPAK (Ste20 like Proline-Alanine rich Kinase). In vivo, SPAK activation plays a central role in the pathogenesis of FHHt caused by WNK4 mutations. However, the implication of SPAK has never been shown for the WNK1 mutations. Thus, we crossed WNK1+/FHHt mice, bearing the WNK1-FHHt mutation, with SPAK243A/243A mice bearing a mutation abolishing SPAK activation by the WNKs. All FHHt phenotypes observed in WNK1+/FHHt were corrected in WNK1+/FHHt:SPAK243A/243A mice demonstrating the central role of SPAK in FHHt caused by WNK1 mutations. In 2012, new mutations have been identified in CUL3 and KLHL3 genes. The products of these genes are both part of a ubiquitin ligase complex. As WNK1 and WNK4 mutations, these mutations lead to an increased expression of L-WNK1 and/or WNK4. Surprisingly, patients with CUL3 mutations display a more severe phenotype. Previous studies have suggested that CUL3 could be involved not only in the regulation of ion transport in the distal nephron but also in the regulation of vascular tone. To verify this hypothesis, we generated and compared two mouse models: pgk-Cul3Δ9 mouse model bearing, as patients, an ubiquitous Cul3 mutation, and sm22-Cul3Δ9 mouse model that express the Cul3 mutation only in vascular smooth muscle cells. Both models are hypertensive, but pgk-Cul3Δ9 mice display a more severe hypertension than sm22-Cul3Δ9 mice. It demonstrates that the hypertension caused by Cul3 mutations results from both renal and vascular disorders. Recently, new missense mutations have been identified in WNK1 acidic motif in a small number of FHHt patients. This acidic motif is necessary for the liaison to KLHL3 and therefore for WNK ubiquitination. Our study shows that, in vitro, these mutations lead to the accumulation of only one isoform of WNK1. In mice, the mutation of WNK1 acidic motif leads to an increased phosphorylation of SPAK and NCC. Therefore, it demonstrates the essential role of the acidic motif in the regulation of WNK1 abundance in vivo in the distal nephron. This work contributes to a better comprehension of the role played by SPAK, WNK1 and CUL3 in FHHt and more generally in the regulation of blood pressure and Na+/K+ homeostasis.

Este projeto, analisou a distribuição dos neurônios nos plexos mioentérico (PM) e submucoso (PS) imunorreativos aos receptores P2X2 (ir) e P2X7 (ir), calbindina (Calb-ir), calretinina (Calr-ir), colina acetil transferase (ChAT-ir) e ao óxido nítrico sintase (NOS-ir) do colo distal de ratos submetidos à desnutrição a renutrição protéica. Utilizaram-se colos distais de ratos nutridos (N42), desnutridos (D42) e renutridos (RN42). Os resultados do plexo PM, demonstraram que 100% dos neurônios Calb-ir, Calr-ir, ChAT-ir e NOS-ir, expressavam os receptores P2X2-ir e P2X7-ir nos três grupos. A densidade neuronal no PM, demonstrou um aumento de 20% a 97% dos neurônios receptores P2X2-7-ir, Calr-ir, ChAT-ir e NOS-ir e no PS foi de 29% a 75%, ambos D42 e recuperação no RN42. O perfil neuronal P2X7-ir, Calb-ir, Calr-ir e ChAT-ir do PM demonstrou diminuição de 28% a 40% e no PS os neurônios P2X2-7-ir, Calb-ir e ChAT-ir de 19% a 47% no D42. Concluí-se que, a desnutrição afeta os neurônios entéricos havendo recuperação na renutrição, podendo influenciar nas funções gastrintestinais. / The aim of the work was to analyze the distal colon myenteric (MN) and submucous (SN) neurons immunoreactive for P2X2-7 receptors, calbindin (Calb-ir), calretinin (Calr-ir), choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) of the animals submitted to undernutrition and refeeding proteic. Distal colon was used from nourished (N42), undernourished (D42) and refeeding (RN42) rats. The results have shown 100% coexpression of the myenteric and submucous Calb-ir, Calr-ir, ChAt-ir e NOS-ir neurons with P2X2-7-ir receptors. The MN density have shown increase of the 20% and 97% of the P2X2-7-ir, Calr-ir, ChAT-ir e NOS-ir neurons of the D42 group, and the SN have been increased 29% a 75% in the D42 group. In the MN neuronal profile have shown decrease P2X7-ir, Calb-ir, Calr-ir and ChAT-ir neurons of the 28% to 40% and in the PS P2X2-7-ir, Calb-ir and ChAT-ir of the 19% a 47% neurons in the D42 group. I concluded that, the undernutrition affects the enteric neurons and there was recuperation in the refeeding, this can influence the gastrintestinal functions.

A alta frequência das fraturas da extremidade distal do rádio estimula o contínuo estudo e desenvolvimento dos métodos de tratamento, buscando melhor qualidade de vida com menos sequelas e limitações. A placa volar bloqueada tem se tornado o método de escolha no tratamento nos últimos anos. Menos morbidade e a reabilitação mais precoce têm aumentado muito a utilização dessas placas. Esse método, contudo, não é isento de complicações. As mais frequentes estão relacionadas aos tendões extensores. Tendinites e rupturas têm sido relatadas. Com o objetivo de se proteger os tendões extensores, estudos têm sugerido a utilização de parafusos unicorticais na parte distal da placa e recomendam que os mesmos tenham pelo menos 75% do comprimento do parafuso bicortical. Esses estudos são restritos a fraturas extra-articulares. Esta pesquisa objetivou estudar e comparar as propriedades mecânicas dos modelos fixados com placas volares utilizando parafusos unicorticais e bicorticais em fraturas intra-articulares da extremidade distal do rádio classificadas como AO 23C3 sob cargas fisiológicas e a possibilidade de recomendar o parafuso unicortical na prática clínica. Objetivou também comparar, pelo método dos elementos finitos, as tensões geradas nos modelos após diferentes carregamentos com validações realizadas com os resultados dos ensaios mecânicos. Foram avaliados 42 modelos divididos em seis grupos de sete modelos, três com parafusos unicorticais e três com parafusos bicorticais. Cada grupo foi submetido a um único tipo de ensaio: compressão axial, flexão dorsal e flexão volar. Foram feitos dois ensaios estáticos intercalados por um carregamento cíclico e por último um ensaio até a falência. Os resultados demonstraram similaridade entre os respectivos grupos e confirmaram nossas hipóteses de que: a) o comportamento mecânico do modelo utilizando parafusos unicorticais é equivalente ao modelo usando parafusos bicorticais; b) o carregamento cíclico afeta o comportamento mecânico dos modelos das fixações das placas volares bloqueadas na extremidade distal do rádio. A análise por elementos finitos mostrou que as fixações unicortical e bicortical não resultaram em qualquer região de concentração de tensão crítica, sendo as duas indicadas para a estabilização de fraturas do rádio. / The high frequency of distal radius fractures stimulates the continuous study and development of treatment methods seeking a better quality of life and fewer sequels and limitations. The volar locking plate has become the chosen method for the treatment in recent years. The lower morbidity and earlier rehabilitation have greatly increased the use of these plates. This method, however, is not free from complications, the most common being related to extensor tendons. Tendinitis and rupture have been reported. In order to protect the extensor tendons, studies have suggested the use of unicortical screws in the distal part of the plate, and recommended that they be at least 75% of the length of the bicortical screw. These studies are restricted to extra articular fractures. This study aimed at studying and comparing the mechanical properties of the models fixed with volar plates using unicortical and bicortical screws in intra-articular fractures of the distal radius classified as AO 23C3 under physiological loads and the possibility of recommending the unicortical screw in clinical practice. The tensions generated in the models after different loads with validations performed with the results of the mechanical tests were also compared. We studied 42 models divided into six groups of seven models, three with unicortical screws and three with bicortical screws. Each group underwent a single type of test: axial compression, dorsiflexion and volar flexion. Two static tests were performed, intercalated by a cyclic loading, and finally a test until bankruptcy. Our results demonstrated a similarity between the respective groups and confirmed both our hypotheses that: a) the mechanical behavior of the model using unicortical screws is equivalent to the model using bicortical screws, and b) that the cyclic loading affects the mechanical behavior of the models of the fixations of the volar locked plates in the distal radius. The finite element analysis showed that the unicortical and bicortical fixations did not result in any regions of critical stress concentration, so they are both indicated for the stabilization of radius fractures.