Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury in both native kidneys and renal allografts. Previous studies in our lab have shown that a subset of Eph family receptor tyrosine kinases, including EphA2, is strongly and persistently upregulated in renal tubular cells in both in vitro and in vivo models of the renal IRI. Src kinases are necessary and sufficient for upregulation of EphA2. We have proposed that IRI-induced EphA2 upregulation may serve as a necessary step in renal tubular remodelling. / In this study, we have further defined the mechanism of Src kinase-induced EphA2 upregulation by identifying the -145/+137 EphA2 promoter region as the minimal region required for basal and Src kinase-induced activation of the promoter. Moreover, we have identified within this region, at position -45, a canonical cAMP response element (CRE) (Nowakowski et al.), which is essential for EphA2 promoter activation. However, we also found that the prototypical CRE-binding transcription factor, CREB, was not necessary for activation of the EphA2 promoter, suggesting that CREB-related or -unrelated transcription factors are responsible for EphA2 upregulation.

The evolution of the blood feeding adaptation has required precise coordination of multiple physiological processes in the insect, such as reproduction, behavior, digestion and diuresis. These processes are under careful synchronous hormonal control. For rapid excretion, multiple diuretic hormones are known. Although originally described based on their ability to stimulate hindgut contractions, the Aedes kinins have been shown to stimulate fluid secretion in female mosquitoes of Aedes aegypti. Aedes kinins are leucokinin-like neuropeptides released from neurosecretory cells in the brain and abdominal ganglia. They act by binding to the Aedes kinin receptor, a G proteincoupled receptor (GPCR). The Aedes kinin receptor has been cloned, sequenced, functionally characterized, and immunolocalized to stellate cells in the Malpighian tubules of Ae. aegypti. In addition to their myotropic and diuretic roles, leucokinin-like peptides and/or their receptors have been also been discovered in the nervous, digestive, and reproductive systems of other arthropod species. Therefore, the Aedes kinins have the potential to function in several simultaneous physiological processes that are stimulated by blood feeding. This thesis aims to understand better their role in the whole mosquito by investigating the Aedes kinin receptor's global expression as well as its in vivo contribution to post-prandial diuresis. Presence of the Aedes kinin receptor was investigated in the head, posterior midgut (stomach), hindgut, ovaries, and Malpighian tubules of both non blood-fed and blood-fed females by western blot using anti-receptor antibodies. The receptor was then immunolocalized in the posterior midgut and rectum. Finally, RNAi was employed to knock down kinin receptor expression, followed by measurement of in vivo urine excretion post blood feeding in a precision humidity chamber. Transcript and protein knockdown were confirmed by qPCR and immunohistochemistry, respectively. Results indicate widespread expression of the Aedes kinin receptor protein in organs novel for hematophagous insects and demonstrate the receptor's fundamental role in rapid diuresis. These findings strongly point to the Aedes kinins as integrative signaling molecules that could coordinate multiple physiological systems. The Aedes kinins could therefore have contributed to the success of the blood feeding adapation in mosquitoes.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Cornell University, August, 2008. / Vita. Includes bibliographical references (leaves 116-140).

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Physiology and Biophysics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (M.S.)--University of Alabama at Birmingham, 2006. / Title from first page of PDF file (viewed on June 30, 2007). Includes bibliographical references (p. 69-73).

The present research had the objective to analyze qualitatively though histobacteriology, the remaining dentine of upper human premolars after removal of clinically decayed tissue, according to presence, location and distribution of microorganisms in dental tubules in different degrees of depth of carious lesion. It was selected representative histological blades of histological cut in series, obtained during the research of Biffi et al (1982), extracted from patients of both genera and age from 20 to 40 years old. The teeth should present proximal caries and/ or occlusal caries and the removal of decayed tissue would be done in vitro. The three faces (dental, mesial and occusal) were clinically classified according to the depth of the caries in degrees 0, 1, 2, 3, 4 or 5 and microscopically assessed. The blades were tinged using the technique of Hematoxylin and Eosine, Trichrome of Masson and histobacteriological of Gram, modified by Brown and Brenn. The interpretation of histological cuts was realized with the use of a trinocular and photomicroscope. In the descriptive analysis it was possible to check the bacterial penetration in a superficial way and/ or in a deep way in the dentinal tubules both in the pulpal floor or in the amelodentinal junction, as well as the presence of bacterial niches in the cavity preparation. The contaminated tubules showed morphologically inaltered, and in pulp exposure during the removal of deep caries it was verified the introduction of a contaminated dentinal fragment in the pulp tissue. In order to have a statistical analysis of the number of microorganisms, it was applied the Pearson s correlation coefficient, respectively among the different degrees of caries and localization in the amelodentinal junction and pulp floor. It was brought to a conclusion that, in 56,7% of the decayed faces the microorganisms were placed in dentine considered clinically healthy, in which they lodged in dentinal tubules morphologically inaltered; the localization and the distribution of microorganisms in the tubules were variable and independent of the depth of the caries. / A presente pesquisa teve como objetivo realizar analise histobacteriológica da dentina remanescente de pré-molares humanos após a remoção do tecido cariado, quanto à presença, localização e distribuição dos microrganismos nos túbulos dentinários em diferentes graus de profundidade da lesão cariosa. Foram selecionadas lâminas histológicas representativas de cortes histológicos seriados de 20 dentes, obtidos durante a pesquisa de Biffi et al. (1982), extraídos de pacientes de ambos os gêneros e faixa etária entre 20 a 40 anos. Estes dentes apresentaram cárie proximal e/ou oclusal e a remoção do tecido cariado foi realizada in vitro. As três faces do dente (mesial, distal e oclusal) foram clinicamente classificadas quanto à profundidade de cárie em graus 0, 1, 2, 3, 4 ou 5 e microscopicamente avaliadas. As lâminas foram coradas pela técnica Hematoxilina e Eosina, Tricrômico de Masson e histobacteriológico de Gram, modificado por Brown e Brenn. A analise dos cortes histológicos foi realizada utilizando o microscópio de luz. Na análise histológica foi observada a presença bacteriana superficial e/ou profunda nos túbulos dentinários tanto no assoalho pulpar quanto na junção amelo-dentinária, bem como a presença de nichos bacterianos no preparo cavitário. Os túbulos contaminados apresentavam-se morfologicamente inalterados e na exposição pulpar, durante a remoção profunda da cárie, constatou-se a introdução de fragmento dentinário contaminado no tecido pulpar. Para análise estatística do número de microrganismos, foi aplicado o coeficiente de correlação de Pearson, respectivamente entre os diferentes graus de cárie e localização na junção amelo-dentinária e assoalho pulpar. Conclui-se que, em 56,7% das faces cariadas os microrganismos encontravam-se situados na dentina considerada clinicamente como sadia, os quais alojavam-se em túbulos dentinários morfologicamente inalterados; a localização e a distribuição dos microrganismos nos túbulos foi variável e independente da profundidade da cárie. / Mestre em Odontologia

Un des objectifs majeurs de la neuroimagerie moderne est l'identification de nouveaux marqueurs qui puissent aider au diagnostic et au suivi des pathologies neurologiques. L'imagerie par résonnance magnétique multimodale (IRMm) est une approche permettant l'évaluation de plusieurs biomarqueurs complémentaires au cours d'un seul examen d'IRM. Cette approche a montré son efficacité dans de nombreuses études récentes et notamment dans la maladie de Parkinson. A l'approche IRMm précédemment utilisée, nous avons introduit un nouveau biomarqueur i.e. les changements de forme des structures sous-corticales à partir d'images pondérées en T1. Ce dernier marqueur vient enrichir l'approche IRMm composée de la quantification de : (i) la volumétrie à partir d'images pondérées en T1 (ii) de l'intégrité et l'orientation microstructurales à partir des images pondérées en diffusion et (iii) des dépôts de métaux à partir de la relaxométrie T2*. Nous avons appliqué l'approche IRM multimodale à une autre maladie neurodégénérative, la maladie d'Alzheimer à un stade précoce. Les résultats de cette étude préliminaire nous ont permis de suggérer la présence de processus physiopathologiques différents à la phase prodromique de la maladie d'Alzheimer. En effet nous avons observé pour l'hippocampe et l'amygdale une atrophie avec modification de l'intégrité microstructurale alors que seule une atrophie a été observée pour le thalamus et le putamen. Ces résultats nous ont aussi permis de confirmer l'importance d'une approche multimodale pour les études portant sur les maladies neurodégénératives. Parmi les marqueurs de l'IRMm, la quantification du fer intracérébral par relaxométrie T2* est une des méthodes qui a été développé ces dernières années à l'unité Inserm U825. Le dérèglement du métabolisme du fer et son accumulation sont en effet impliqués dans la physiopathologie de nombreuses maladies neurodégénératives telles que la maladie de Parkinson. L'expérience acquise à travers les différentes validations cliniques de cette méthode ces dernières années nous a conduit à améliorer cette dernière. Nous avons dirigé nos travaux sur l'amélioration de la méthode de relaxométrie R2* en optimisant l'acquisition d'une part et le traitement des images d'autre part. Nous avons donc comparé différentes résolutions, antennes, facteurs d'accélération, et nombres d'acquisitions par temps d'écho afin de déterminer les paramètres permettant d'obtenir le plus haut rapport signal sur bruit. Pour la partie traitement des images nous avons comparé la méthode utilisée comme référence, la méthode des moindres carrés par algorithme de Levenberg-Marquardt, à une autre méthode, la décomposition en valeur singulière pour estimer avec le plus de justesse le taux relaxation R2*. Nous avons ainsi pu mettre au point une séquence de relaxométrie T2* optimisée que nous avons comparé à celle utilisée lors de la première étude, dans le modèle du vieillissement physiologique. Au final en plus de permettre la discrimination entre sujets âgés et jeunes, les résultats obtenus avec cette nouvelle séquence se sont révélés être beaucoup moins sensibles au bruit. / One of the main goals of modern neuroimaging is the identification of new markers that can help in the diagnosis and monitoring of neurological pathologies. Multimodal magnetic resonance imaging (MRIm), is an approach allowing the evaluation of several complementary biomarkers within one MRI. This approach has already demonstrated its efficiency in several recent studies, and in particular in Parkinson's disease. We added a new biomarker to the MRIm approach previously used i.e. shape changes of subcortical structures based on T1 images. This marker is now a part of our MRIm approach along with: (i) volumetry from T1 images, (ii) microstructural integrity and orientation from diffusion images and (iii) metal deposits from T2* relaxometry. We applied this multimodal MRI approach to an other neurodegenerative disease, the Alzheimer's disease at a prodromal stage. Results of this preliminary study gave us the opportunity to suggest the existence of two different physiopathological processes at the prodromal phase of the Alzheimer's disease. In fact we observed atrophy with modification of the microstructural integrity for the hippocampus and the amygdala, while only atrophy has been observed for the thalamus and the putamen. Those results also confirmed the necessity of studying neurodegenerative diseases in a multimodal way. Among MRIm markers, the T2* relaxometry for the quantification of intracerebral iron is one of the methods which has been developed lately at the Inserm U825. Dysregulation of iron metabolism and its accumulation are involved in the physiopathology of several neurodegenerative diseases like Parkinson's disease. The experience gained through the different clinical validation of this method in recent years has led us to improve it. Our work was to improve T2* relaxometry by optimizing the acquisition of the images on one hand, and the processing of the images on the other hand. We compared several resolutions, acquisition antennas, number of acquisition by echo time, to determine which parameters gave the higher signal to noise ratio. For the part about the process of the images, we compared the method used as a reference, the least square method using a Levenberg-Marquard algorithm, to an other method, the singular value decomposition to obtain the best estimation of the relaxation rate R2*. Then we were able to develop an optimized T2* relaxometry sequence, which we compared to the one used in the first study, but in the physiological ageing model. Finally in addition to allowing discrimination between elderly and young people, the results obtained with this new sequence were found to be much less sensitive to noise.

O objetivo do presente estudo foi avaliar a remoção da smear layer e a penetração intratubular do cimento obturador AH Plus utilizando diferentes protocolos de irrigação. Após aprovação do Comitê de Ética em Pesquisa da Faculdade de Odontologia de Ribeirão Preto - FORP/USP, noventa incisivos centrais superiores foram selecionados e fornecidos pelo banco de dentes da mesma instituição. Estes foram radiografados no sentido mésio - distal e vestíbulo - lingual para comprovar a presença de canais únicos, sem curvaturas abruptas, reabsorções ou tratamentos endodônticos prévios. Após a remoção da porção coronal para a obtenção de raízes com comprimentos padronizados de 15 milímetros, os diâmetros anatômicos foram determinados e as instrumentações realizadas por meio da técnica coroa ápice estabelecendo os diâmetros cirúrgicos utilizando 3 instrumentos acima dos anatômicos. Durante este processo, os espécimes foram divididos em seis grupos de acordo com os protocolos de irrigação: Grupo 1 (controle - n. 15) - 2,5ml de água destilada a cada troca de instrumentos e 2,5ml de água destilada por 3 minutos ao final da instrumentação; Grupo 2 (n. 15) - 2,5ml de hipoclorito de sódio a 2,5% a cada troca de instrumentos e 2,5ml de hipoclorito de sódio a 2,5% por 3 minutos ao final da instrumentação; Grupo 3 (n. 15) - 2,5ml de hipoclorito de sódio a 2,5% a cada troca de instrumentos e 2,5ml de EDTA a 17% por 3 minutos ao final da instrumentação; Grupo 4 (n. 15) - 2,5ml de hipoclorito de sódio a 2,5% e 2,5ml de EDTA a 17% a cada troca de instrumentos + 2,5ml de EDTA a 17% por 3 minutos ao final da instrumentação; Grupo 5 (n. 15) - 2,5ml de hipoclorito de sódio a 2,5% a cada troca de instrumentos e 2,5ml de ácido cítrico a 10% por 3 minutos ao final da instrumentação; Grupo 6 (n. 15) - 2,5ml de hipoclorito de sódio a 2,5% e 2,5ml de ácido cítrico a 10% a cada troca de instrumentos + 2,5ml de ácido cítrico a 10% por 3 minutos ao final da instrumentação. Após esta fase, cinco espécimes de cada grupo foram clivados no sentido mésio - distal para avaliação da remoção da smear layer em microscopia eletrônica de varredura por meio da análise de 3 imagens de cada terço em aumentos de 300, 1000 e 2000 vezes. Esta variável foi determinada por um score qualitativo estabelecido pelo consenso de 3 avaliadores calibrados. Os demais espécimes foram obturados pela técnica de condensação lateral com cones principais correspondentes ao último instrumento associados ao cimento AH Plus corado com Rodamina B, cones acessórios B8 e mantidos em ambiente com 100% de umidade por 7 dias. Em seguida, os espécimes foram seccionados transversalmente para obtenção de slices de aproximadamente 2mm de espessura em cada terço (cervical, médio e apical) e as faces cervicais submetidas a um tratamento metalográfico para análise da penetração intratubular do cimento obturador em microscopia confocal a laser. Os melhores índices de remoção de smear layer e as maiores porcentagens de penetração intratubular do cimento foram obtidos pelos grupos onde as soluções quelantes foram utilizadas (G3, G4, G5 e G6) com diferenças estatisticamente significantes (p<0,05) em relação aos grupos onde utilizou - se somente a água destilada (G1) e o hipoclorito de sódio a 2,5% (G2) durante o processo de instrumentação. Não foram observadas vantagens em relação ao uso associado do EDTA a 17% e do ácido cítrico a 10% ao hipoclorito de sódio pela ausência de diferenças estatísticas significantes entre os grupos 3 e 4 e 5 e 6, respectivamente. / The aim of this study was to evaluate the smear layer removal and tubular dentine sealer penetration of AH Plus after different irrigation protocols. After approval of the study by the Ethics Committee of the Ribeirão Preto Dental School - FORP/USP, ninety maxillary central incisors were selected and supplied by the tooth bank of the same institution. The teeth were radiographed in the mesiodistal and buccolingual directions to verify the presence of single canals, without abrupt curvatures, resorption or previous endodontic treatment. The coronal portion was removed and roots lengths were standardized at 15 mm. The anatomic diameters were then determined and instrumentations were carried out by the crown-down technique, using 3 higher-diameter instruments to establish the surgical diameters. During this process, the specimens were divided into six groups, according to the following irrigation protocols: Group 1 (control - n. 15) - 2.5ml of distilled water at each change of instruments + 2.5ml of distilled water for 3 minutes at the end of the instrumentation; Group 2 (n. 15) - 2.5ml of 2.5% sodium hypochlorite at each change of instruments + 2.5 ml of 2.5% sodium hypochlorite for 3 minutes at the end of the instrumentation; Group 3 (n. 15) - 2.5ml of 2.5% sodium hypochlorite at each change of instruments + 2.5ml of 17% EDTA for 3 minutes at the end of the instrumentation; Group 4 (n. 15) - 2.5ml of 2.5% sodium hypochlorite and 2.5ml of 17% EDTA at each change of instruments + 2.5ml of 17% EDTA for 3 minutes at the end of the instrumentation; Group 5 (n. 15) - 2.5ml of 2.5% sodium hypochlorite at each change of instruments + 2.5ml of 10% citric acid for 3 minutes at the end of the instrumentation; Group 6 (n. 15) - 2.5ml of 2.5% sodium hypochlorite and 2.5 ml of 10% citric acid at each change of instruments + 2.5 ml of 10% citric acid for 3 minutes at the end of the instrumentation. Following this phase, five specimens from each group were cleaved in the mesiodistal direction to evaluate the smear layer removal by scanning electron microscopy. Evaluation was performed by analyzing 3 images of each third at 300, 1000 and 2000X. This variable was determined by a qualitative score established by the consensus of 3 calibrated examiners. The remaining specimens were filled using lateral condensation with AH Plus sealer labeled with Rhodamine B and kept in an environment with 100% humidity for 7 days. Then, the specimens were sectioned to obtain approximately 2 mm thick slices of each third (cervical, middle and apical). The cervical surfaces were subjected to metallographic treatment for analysis of the tubular dentine sealer penetration by confocal laser microscopy. The highest smear layer removal rates and tubular dentine sealer penetration percentages were obtained by the groups that used chelating solutions (G3, G4, G5 and G6). These showed statistically significant differences (p<0.05) compared with the groups that used just distilled water (G1) and 2.5% sodium hypochlorite (G2) during instrumentation. No benefits were observed for the combined use of 17% EDTA or 10% citric acid with 2.5% sodium hypochlorite, considering that there were no statistically significant differences between groups 3 and 4, and between 5 and 6, respectively.

Está bem estabelecido na literatura que o NHE3 é ativado, no intestino, pelo transporte de glicose mediado pelo SGLT1, e que esta ativação não dependente do metabolismo da glicose. Acredita-se que a co-ativação do NHE3 e do SGLT1 ocorra para maximizar a reabsorção de nutrientes no período pós-prandial. Porém, ainda não foi determinado se a captação de glicose através dos SGLTs é capaz de regular a atividade do NHE3 no túbulo proximal renal. Levando-se em conta que este segmento renal também expressa o SGLT2 e que os rins e intestinos apresentam significativas diferenças na disponibilidade de glicose ao longo do dia, o objetivo do presente trabalho foi o de determinar o efeito da glicose e da atividade dos SGLTs renais sobre a atividade do NHE3. Experimentos de microperfusão estacionária demonstraram que a perfusão luminal de glicose 5mM estimula o NHE3 via o metabolismo glicolítico. A perfusão de concentrações suprafisiológicas de glicose inibe o NHE3 por promover aumento de volume celular. A inibição farmacológica dos SGLTs, utilizando-se o inibidor inespecífico Florizina, ocasionou acentuada inibição do NHE3, mesmo na ausência de glicose. Além disso, experimentos de imunofluorescência determinaram que o NHE3 é co-expresso com o SGLT2, mas não com o SGLT1. Conclusão: os resultados deste trabalho demonstram que a glicose apresenta um efeito bimodal sobre o NHE3: em concentrações fisiológicas este açúcar estimula o NHE3, enquanto que em concentrações suprafisiológicas a glicose inibe o trocador. Além disso, a inibição farmacológica do transporte de glicose ocasiona acentuada inibição do NHE3 demonstrando que estes transportadores interagem funcionalmente no túbulo proximal. / It is well established that SGLT1-mediated glucose uptake leads to NHE3 activation in the intestine. This co-activation is thought to be important for postprandial nutrient uptake. However, it remains to be determined whether SGLT-mediated glucose uptake is capable of regulating NHE3-mediated NaHCO3 reabsorption in the renal proximal tubule. Considering that this nephron segment also expresses another SGLT isoform, SGLT2, and that the kidneys and intestine show significant variations in daily glucose availability, the goal of the present work was to determine the effect of SGLT-mediated glucose uptake on NHE3 activity in the renal proximal tubule. Stationary in vivo microperfusion experiments demonstrated that luminal perfusion with 5 mM glucose stimulates NHE3-mediated bicarbonate reabsorption. This stimulatory effect was mediated by glycolytic metabolism but not through ATP production. Conversely, luminal perfusion with 40 mM glucose inhibited NHE3 due to cell swelling. Interestingly, the pharmacological inhibition of SGLT activity by phlorizin produced a marked inhibition of NHE3, even in the absence of glucose. Furthermore, immunofluorescence experiments showed that NHE3 co-localizes with SGLT2, but not with SGLT1, in the rat renal proximal tubule. Collectively, the findings of this work demonstrate that glucose exerts a bimodal effect on NHE3. The physiological metabolism of glucose stimulates NHE3 transport activity, whereas supraphysiological glucose concentrations inhibit this exchanger. Additionally, phlorizin-sensitive SGLT transporters and NHE3 interact functionally in the proximal tubule.