Dissertations / Theses on the topic 'In vivo'

Com objetivo de avaliar os efeitos sistêmicos da contaminação alimentar por micotoxinas, enfatizando aspectos morfológicos e imunohistoquímicos, dois experimentos foram realizados. No primeiro experimento, 20 leitões de cinco semanas de idade foram divididos em quatro grupos experimentais. Os animais receberam durante 28 dias os seguintes tratamentos: dieta controle, dieta contaminada com desoxinivalenol (DON) (1,5 mg/kg), dieta contaminada com DON (2 mg/kg) + nivalenol (NIV) (1,3 mg/kg) + zearalenona (ZEA) (1,5 mg/kg) ou dieta contaminada com DON (3 mg/kg) + NIV (1,3 mg/kg) + ZEA (1,5 mg/kg). A dieta mono-contaminada não alterou o ganho de peso, entretanto os animais expostos à dieta multi-contaminada apresentaram uma diminuição significativa no ganho de peso final. A ingestão crônica das dietas contaminadas induziu alterações histológicas no intestino, como atrofia e fusão de vilosidades, diminuição na altura de vilosidades e na profundidade de criptas, redução no número de células caliciformes e linfócitos. O fígado, os linfonodos mesentéricos e o baço dos animais expostos às dietas contaminadas apresentaram um aumento significativo de lesões. Um aumento significativo na expressão de caspase-3 nos órgãos linfoides foi observado nos animais que receberam as dietas contaminadas. No segundo experimento, 12 leitões de 4 a 5 semanas de idade foram eutanasiados para obtenção de explantes jejunais, os quais foram expostos a cinco tratamentos por 4 horas, sob agitação constante a 37ºC em atmosfera umidificada a 5% de CO2. No tratamento controle foi utilizado meio de cultura William E sem ou com o diluente DMSO a 0,1%. Nos demais tratamentos os explantes foram expostos a DON, NIV (1, 3, 10 µM) e fusarenona X (FX) (0,3, 1 e 3 µM). Após incubação, as amostras de tecidos foram processadas histologicamente e analisadas utilizando-se escore histológico. Não foi observada diferença significativa no escore histológico entre amostras não incubadas e incubadas com meio de cultura na ausência ou presença de DMSO. As principais alterações observadas nos explantes expostos às micotoxinas foram atrofia de vilosidades, células epiteliais cúbicas e pavimentosas, edema na lâmina própria e desnudamento apical com perda de enterócitos. Os tratamentos individuais com DON, NIV e FX induziram uma diminuição do escore histológico a partir das doses de 3µM, 1µM e 0,3 µM, respectivamente. Em conclusão, os dados obtidos fornecem uma melhor compreensão dos efeitos das fusariotoxinas isoladas ou em combinação sobre a morfologia intestinal e de órgãos linfoides, as quais podem predispor os animais a infecções secundárias.
In order to evaluate the systemic effects of food contamination by mycotoxins, emphasizing morphological and immunohistochemical aspects, two experiments were conducted. In the first experiment, 20 5-week-old piglets were randomly assigned to four groups. The animals received for 28 days the following treatments: control diet, a diet contaminated with deoxynivalenol DON (1.5 mg/kg), a diet contaminated with DON (2.0 mg/kg) + nivalenol (NIV) (1.3 mg/kg) + zearalenone (ZEA) (1.5 mg/kg) or a diet contaminated with DON (3.0 mg/kg) + NIV (1.3 mg/kg) + ZEA (1.5 mg/kg). The mono-contaminated diet showed no difference in weight gain, however the animals fed the multi–contaminated diets presented a significant decrease in final weight gain. The chronic ingestion of these contaminated diets induced histological changes in the intestine as show by atrophy and fusion of villi, decreased villi height and crypth depth, and reduced number of goblet cells and lymphocytes. The liver, mesenteric lymph nodes and spleen of animals exposed to contaminanted diets showed a significant increase of lesions. A significant increase in caspase-3 expression in lymph nodes and spleen was observed in animals receiving the contaminated diets. In the second experiment, 12 4 to 5-week-old piglets were euthanized to obtain jejunal explants, which were exposed to 5 treatments for 4 hours, under constant stiring at 37ºC and 5% CO2 humidified atmosphere. In the control treatment were used the William’s medium E without or with the diluent DMSO to 0.1%. In the other treatments the explants were exposed to DON, NIV (1, 3, 10 µM) and fusarenone X (FX) (0.3, 1 and 3 µM). After incubation, the tissue samples were histologically processed and analyzed using histological score. The analyse of samples non-incubated and incubated with culture medium in the absence or presence of DMSO showed no significant different of histopathological score. The main lesions observed in the explants exposed to mycotoxins were villi atrophy, cuboidal and squamous epithelial cells, areas of oedema in the lamina propria and apical denudation with loss of enterocytes. The individual treatment with DON, NIV and FX resulted in a significant decrease of the histopathologic score from doses of 3µM, 1µM and 0.3 µM, respectively. In conclusion, the data obtained provide a better understanding of the possible effects of Fusarium toxins, alone or in combination on the morphology of the intestine and lymphoid organs, which would may predispose animals to secondary infections.

La dopamine est un neuromodulateur impliqué dans une grande diversité de fonctions physiologiques et il est étonnant de constater qu'un faible nombre de neurones dopaminergiques puisse encoder une telle richesse d'informations. Ceci s'explique par l'organisation anatomique complexe des projections dopaminergiques, l'encodage temporel de la libération de dopamine et la diversité des récepteurs. La contribution de l'intégration post-synaptique du signal dopaminergique est mal connue. Les récepteurs D1 activent la cascade AMPc/PKA qui, jusqu'à présent, ne pouvait être étudiée qu'en dehors de son contexte spatio-temporel. Les biosenseurs FRET permettent désormais d'accéder à ces dimensions et nous avons utilisé l'imagerie optique pour suivre cette cascade de signalisation dans des neurones. Dans des tranches de cerveau, nous montrons que, par-rapport aux neurones pyramidaux du cortex, les neurones du striatum présentent une activité AC plus forte, pas d'activité PDE4 et une régulation par la DARPP-32. Ces particularités permettent aux neurones striataux de répondre efficacement à des stimulations dopaminergiques de moins d'une seconde, telles celles associées à la récompense. Ces résultats démontrent que deux types de neurones peuvent intégrer différemment un même signal dopaminergique via la même cascade de signalisation, démontrant l'importance du niveau post-synaptique dans l'intégration de l'information véhiculée par la dopamine. Pour analyser l'intégration du signal dopaminergique in vivo, nous avons utilisé la microscopie par fibre optique avec la détection simultanée de fluorescence dans deux longueurs d'onde. Nous avons mis au point et validé un nouveau biosenseur avec GFP/dTomato. Ce biosenseur et l'imagerie par fibre optique nous ont permis de suivre par imagerie in vivo, pour la première fois, l'activation de la PKA dans des neurones de régions profondes du cerveau

El Carprofè s’utilitza com a agent antiinflamatori, analgèsic i antipirètic. Pertany a la família de medicaments antiinflamatoris no esteroideus. S’utilitza en medicina veterinària en nombroses espècies, tot i així està molt poc estudiat el seu ús en l’espècie porcina. D’altra banda, alguns efectes indesitjables s’associen a la seva administració sistèmica. Les rutes locals alternatives són especialment útils per facilitar la seva administració en animals. La hipòtesis d’aquest treball és trobar noves formes de dosificació del Carprofè per diverses vies en l’espècie porcina. Per això, ens plantegem diversos objectius: hem volgut conèixer la capacitat de permeació del Carprofè a través d’estudis ex vivo vehiculitzat en nanopartícules o en solució en mucoses i teixits oculars per aconseguir conèixer la seva possible eficàcia, tolerància i seguretat per aquestes vies i oferir alternatives terapèutiques. D’altra banda, volem conèixer el comportament farmacocinètic del Carprofè en l’espècie porcina i així, poder avaluar tots el paràmetres per la via intravenosa i intramuscular. Finalment, ens proposem validar diferents mètodes d’anàlisis per donar fiabilitat als resultats obtinguts. En aquesta tesis hem realitzat tres treballs per donar resposta als notres objectius: 1. L’objectiu principal del primer article és validar la idoneïtat dels experiments de permeació ex vivo de Carprofè en membranes mucoses porcines (bucals, sublinguals i vaginals) i teixits oftàlmics (còrnia, esclera i conjuntiva) destinats a ser representatius de condicions in vivo. Els resultats es poden consultar en el següent article publicat: - Gómez-Segura, L.; Parra, A.; Calpena, A.C.; Gimeno, Á.; Boix-Montañes, A. Carprofen Permeation Test through Porcine Ex Vivo Mucous Membranes and Ophthalmic Tissues for Tolerability Assessments: Validation and Histological Study. Veterinary Sciences. 2020, 7, 152. 2. L’objectiu principal del segon treball és la investigació de la permeació ex vivo de Carprofè mitjançant cèl·lues de Franz a través de diferents tipus de membranes mucoses porcines i teixits oftàlmics (prèviament esmentats) per comparar la formulació de nanopartícules de Carprofè i Carprofè en solució. A més, es van realitzar estudis in vivo per verificar que les formulacions no afectaven l’estructura cel·lular i establir la quantitat retinguda als teixits. Els resultats es poden consultar en el següent article publicat: - Gómez-Segura, L.; Parra, A.; Calpena-Campmany, A.C.; Gimeno, Á.; Gómez de Aranda, I.; Boix-Montañes, A. Ex Vivo Permeation of Carprofen Vehiculated by PLGA Nanoparticles through Porcine Mucous Membranes and Ophthalmic Tissues. Nanomaterials. 2020, 10, 355. 3. El tercer treball es base en l’objectiu de proporcionar un estudi per primera vegada de la farmacocinètica del Carprofè en porcs Yorkshire-Landrace. A més, un nou mètode d’anàlisi mitjançant cromatografia de líquids per espectrometria de masses ens ha permès calcular les concentracions plasmàtiques, avaluar els paràmetres farmacocinètics i la biodisponibilitat (també hem validat aquest nou mètode analític). Els resultats es podran consultar pròximament en el següent article pendent de ser publicat: - Gómez-Segura, L.; Parra, A.; Gimeno, Á; Calpena-Campmany, A.C.; Bellido, D.; Soriano-Ruiz J.L.; Boix-Montañes A. Application of liquid chromatography/mass spectrometry for bioanalysis of Carprofen in swine: pharmacokinetics and bioavailability. Pendent d’enviar. Com a conclusions finals s’ha demostrat la idoneïtat d’aquest test per quantificar la distribució de Carprofè amb una bona tolerabilitat histològica en els teixits porcins estudiats. Tanmateix, es va concloure que les nanopartícules de Carprofè poden ser una eina útil per al tractament tòpic de la inflamació local en medicina veterinària i humana. Per finalitzar, s’ha descrit i validat un nou mètode per caracteritzar la farmacocinètica del Carprofè en porcs i proposar un règim de dosificació en aquesta espècie.
El Carprofè utiliza como agente antiinflamatorio, analgésico y antipirético. Pertenece a la familia de medicamentos antiinflamatorios no esteroideos. Se utiliza en medicina veterinaria en numerosas especies, aún así está muy poco estudiado su uso en la especie porcina. Por otra parte, algunos efectos indeseables se asocian a su administración sistémica. Las rutas locales alternativas son especialmente útiles para facilitar su administración en animales. La hipótesis de este trabajo es encontrar nuevas formas de dosificación del Carprofè por diversas vías en la especie porcina. Por ello, nos planteamos varios objetivos: hemos querido conocer la capacidad de permeación del Carprofè a través de estudios ex vivo vehiculizado en nanopartículas o en solución en mucosas y tejidos oculares para lograr conocer su posible eficacia, tolerancia y seguridad por estas vías y ofrecer alternativas terapéuticas. Por otra parte, queremos conocer el comportamiento farmacocinético del Carprofè en la especie porcina y así, poder evaluar todos los parámetros para la vía intravenosa e intramuscular. Finalmente, nos proponemos validar diferentes métodos de análisis para dar fiabilidad a los resultados obtenidos. En esta tesis hemos realizado tres trabajos para dar respuesta a los Notro objetivos: 1. El objetivo principal del primer artículo es validar la idoneidad de los experimentos de permeación ex vivo de Carprofè en membranas mucosas porcinas (bucales, sublinguales y vaginales) y tejidos oftálmicos (córnea, esclera y conjuntiva) destinados a ser representativos de condiciones in vivo . Los resultados se pueden consultar en el siguiente artículo publicado: - Gómez-Segura, L .; Parra, A .; Calpena, A.C .; Gimeno, Á .; Boix-Montañes, A. Carprofen Permeation Test through Porcine Ex Vivo Mucous Membranas and Ophthalmic Tissues for Tolerability Assessments: Validation and Histological Study. Veterinary Sciences. 2020, 7, 152. 2. El objetivo principal del segundo trabajo es la investigación de la permeación ex vivo de Carprofè mediante Cellu de Franz a través de diferentes tipos de membranas mucosas porcinas y tejidos oftálmicos previamente mencionados para comparar la formulación de nanopartículas de Carprofè y Carprofè en solución. Además, se realizaron estudios in vivo para verificar que las formulaciones no afectaban la estructura celular y establecer la cantidad retenida a los tejidos. Los resultados se pueden consultar en el siguiente artículo publicado: - Gómez-Segura, L .; Parra, A .; Calpena-Campmany, A.C .; Gimeno, Á .; Gómez de Aranda, Y .; Boix-Montañes, A. Ex Vivo Permeation of Carprofen Vehiculated by PLGA nanoparticles through Porcine Mucous Membranas and Ophthalmic Tissues. Nanomateriales. 2020, 10, 355. 3. El tercer trabajo se base en el objetivo de proporcionar un estudio por primera vez de la farmacocinética del Carprofè en cerdos Yorkshire-Landrace. Además, un nuevo método de análisis mediante cromatografía de líquidos por espectrometría de masas nos ha permitido calcular las concentraciones plasmáticas, evaluar los parámetros farmacocinéticos y la biodisponibilidad (también hemos validado este nuevo método analítico). Los resultados se podrán consultar próximamente en el siguiente artículo pendiente de ser publicado: - Gómez-Segura, L .; Parra, A .; Gimeno, Á; Calpena-Campmany, A.C .; Bellido, D .; Soriano-Ruiz J.L .; Boix-Montañes A. Application of liquid chromatography / mass spectrometry for Bioanalysis of Carprofen in swine: pharmacokinetics and Bioavailability. Pendiente de enviar. Como conclusiones finales se ha demostrado la idoneidad de este test para cuantificar la distribución de Carprofè con una buena tolerabilidad histológica en los tejidos porcinos estudiados. Sin embargo, se concluyó que las nanopartículas de Carprofè pueden ser una herramienta útil para el tratamiento tópico de la inflamación local en medicina veterinaria y humana. Para finalizar, se ha descrito y validado un nuevo método para caracterizar la farmacocinética del Carprofè en cerdos y proponer un régimen de dosificación en esta especie.
Carprofen is used as an anti-inflammatory, analgesic and antipyretic agent. It belongs to the family of non-steroidal anti-inflammatory drugs. It is used in veterinary medicine in many species, although its use in the porcine species is very little studied. On the other hand, some side effects are associated with its systemic administration. Alternative local routes are especially useful for ease of administration in animals. The hypothesis of this work is to find new ways of dosing Carprofen in various ways in porcine species. Therefore, we set ourselves several objectives: we wanted to know the permeation capacity of Carprofen through ex vivo studies carried in nanoparticles or in solution in mucous membranes and eye tissues to get to know its possible efficacy, tolerance and safety by these routes offer therapeutic alternatives. On the other hand, we want to know the pharmacokinetic behavior of Carprofen in the porcine species and thus be able to evaluate all the parameters intravenously and intramuscularly. Finally, we propose to validate different methods of analysis to give reliability to the results obtained. In this thesis we have carried out three works to respond to our objectives: 1. The main objective of the first article is to validate the suitability of ex vivo permeation experiments of Carprofen in porcine mucous membranes (buccal, sublingual and vaginal) and ophthalmic tissues (cornea, sclera and conjunctiva) intended to be representative of in vivo conditions. . The results can be consulted in the following published article: - Gómez-Segura, L .; Parra, A .; Calpena, A.C .; Gimeno, Á .; Boix-Montañes, A. Carprofen Permeation Test through Porcine Ex Vivo Mucous Membranes and Ophthalmic Tissues for Tolerability Assessments: Validation and Histological Study. Veterinary Sciences. 2020, 7, 152. 2. The main objective of the second work is the investigation of the ex vivo permeation of Carprofen by Franz cells through different types of porcine mucous membranes and ophthalmic tissues previously mentioned to compare the formulation of nanoparticles of Carprofen and Carprofen in solution. In addition, in vivo studies were performed to verify that the formulations did not affect cell structure and to establish the amount retained in tissues. The results can be consulted in the following published article: - Gómez-Segura, L .; Parra, A .; Calpena-Campmany, A.C .; Gimeno, Á .; Gómez de Aranda, I .; Boix-Montañes, A. Ex Vivo Permeation of Carprofen Vehiculated by PLGA Nanoparticles through Porcine Mucous Membranes and Ophthalmic Tissues. Nanomaterials. 2020, 10, 355. 3. The third work is based on the aim of providing a first study of the pharmacokinetics of Carprofen in Yorkshire-Landrace pigs. In addition, a new method of analysis by liquid chromatography by mass spectrometry has allowed us to calculate plasma concentrations, evaluate pharmacokinetic parameters and bioavailability (we have also validated this new analytical method). The results will be available soon in the following article pending publication: - Gómez-Segura, L .; Parra, A .; Gimeno, Á; Calpena-Campmany, A.C .; Bellido, D .; Soriano-Ruiz J.L .; Boix-Montañes A. Application of liquid chromatography / mass spectrometry for bioanalysis of Carprofen in swine: pharmacokinetics and bioavailability. Pending submission. Final conclusions have been shown to be suitable for this test to quantify the distribution of Carprofen with good histological tolerability in swines tissues studied. However, it was concluded that Carprofen nanoparticles may be a useful tool for the topical treatment of local inflammation in veterinary and human medicine. Finally, a new method has been described and validated to characterize the pharmacokinetics of Carprofen in swines and to propose a dosing regimen in this species.
Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina i Sanitat Animals

So far, the large majority of in vivo computation research has been based on the detection and conditional manipulation of protein concentrations inside cells, which is the biological method of gene expression. In contrast, this thesis describes how a computational program, encoded in genetic material inside a bacterium, can be triggered by external stimuli to reassemble itself in a directed manner to create a newly arranged computational program. In order to investigate the potential utility of in vivo self-arranging programs, software was designed to explore a search space of candidate computational programs, encoded in genetic material, which are able to rearrange themselves; to simulate these candidates and to evaluate their behaviour against a set of criteria. Rearrangements were facilitated by biological catalysts which can selectively sever and rejoin genetic material in a cooperative manner. Their ability to perform compound operations was found to allow for a general purpose mechanism. As proof of concept, one of the candidate computational programs, a two-colour switch which can be set irreversibly through its rearrangement, was encoded in genetic material. Measurements of in vivo expression were observed resulting from in vitro rearrangement manipulations, to illustrate its operation.

A proton nuclear magnetic resonance spectrum of the brain in vivo contains peaks from every proton-containing molecule in the brain. Sensitivity limitations mean that only those molecules present at concentrations of at least a few millimolar are detectable in a reasonable period of time; this still leaves many important molecules such as amino acids and other small metabolites. Most of their resonance frequencies fall in the region between 1.0 and 4.5 p.p.m. A typical linewidth in vivo is about 0.05 p.p.m., so the number of distinct peaks observable is restricted. The use of two-dimensional NMR techniques such as COSY can spread peaks out into a second dimension enabling otherwise overlapping peaks to be resolved. This thesis describes the development, testing and application of two such 2D NMR pulse sequences, dubbed ISIS-COSY and ISIS-JRES. They are based on an existing magnetisation localisation sequence and excite detected magnetisation in a manner analogous to the high-resolution sequences COSY and 2D J-resolved spectroscopy. A method for quantifying the metabolites visible in an ISIS-COSY spectrum from their cross-peak intensities is described, and results presented from both control rat brains and those of animals treated with vigabatrin, an inhibitor of GABA-transaminase that has the effect of increasing brain γ-amino butyric acid (GABA) levels. Further applications mentioned are in the study of neutrophil-infiltrated rat brain and adaptation of the ISIS-COSY technique for human use.

Genetic engineering of microbes has developed rapidly along with our ability to synthesize DNA de novo. Yet, even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. While technological advances have resulted in powerful techniques for in vitro and in vivo assembly of DNA, each suffers inherent disadvantages. Here, an ex vivo DNA cloning suite using crude cellular lysates derived from E. coli is demonstrated to amplify and assemble DNA containing small sequence homologies. Further, the advantages of an ex vivo approach are leveraged to rapidly optimize several parameters of the ex vivo DNA assembly methodology testing lysates from different engineered strains of E. coli, with various buffer components and using titrations of purified cloning enzymes. Finally, in order to complete the cloning suite, a vector expressing the Pyrococcus furiosis (Pfu) DNA polymerase was designed, constructed and expressed in E. coli to create a ‘functionalized lysate’ capable of ex vivo PCR. Not only do we demonstrate ex vivo cloning methodology as a complete cloning package capable of replacing the expensive cloning reagents currently required by synthetic biologists, but also establish ex vivo as an overarching approach for conducting molecular biology.

I Draw the Body Because I Live is the name of this research that intends to approach the artistic creation as a course of lines that draw themselves. It concerns thinking of the drawing through an expanded conception as any body made up of lines, that is to say, a drawing which does not necessarily follow the traditional techniques, categories and conceptions. Therefore, this study is centered on two main objects: the series Silhouettes, photographic actions produced by the artist Ana Mendieta; and the book The Passion According to G.H., written by Clarice Lispector. The lines of thought for this dissertation find resonances and convergences with the philosophy of Gilles Deleuze, whose conceptsconstitute fundamental tools to follow the weave of a drawing in its process of composition and dissolution.8
Desenho Corpo Porque Vivo é o nome desta pesquisa que propõe abordar a criação artística como um percurso de linhas que se desenham. Trata-se de pensar o desenho através de uma concepção expandida como qualquer corpo composto por linhas, ou seja,que não esteja necessariamente dentro das categorias, concepções e técnicas tradicionais.Portanto, as investigações são centradas em dois objetos principais: a série Silhuetas, ações fotográficas produzidas pela artista Ana Mendieta, e o livro A Paixão Segundo G.H., escrito por Clarice Lispector. As linhas de pensamento desta dissertação encontram ressonâncias econvergências com a filosofia de Gilles Deleuze, cujos conceitos constituem ferramentas fundamentais para acompanhar as tramas de um desenho em seu processo de composição e dissolução.

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Os localizadores eletrÃnicos foraminais (LEFs) disponÃveis atualmente utilizam diversos mÃtodos de determinaÃÃo eletrÃnica da posiÃÃo foraminal, qual seja a medida entre o forame apical e uma referÃncia incisal/oclusal, definindo consequentemente o comprimento do canal radicular. Cada um desses mÃtodos procura oferecer maior precisÃo e menor sensibilidade a possÃveis interferÃncias no sistema de canais radiculares. Desta forma, procurou-se avaliar a precisÃo de alguns destes LEFs ex vivo e in vivo na realizaÃÃo de odontometrias eletrÃnicas em diferentes posiÃÃes (0,0 mm e -1,0 mm) e em condiÃÃes de uso clÃnico, respectivamente. No estudo ex vivo, 42 prÃ-molares inferiores tiveram seus comprimentos reais comparados a odontometrias eletrÃnicas realizadas com os LEFs Root ZX, Mini Apex Locator, Propex II, iPex e RomiApex A-15. Inicialmente, em funÃÃo dos displays dos aparelhos, determinaram-se mediÃÃes 1,0 mm aquÃm do forame apical (FA), e posteriormente as mediÃÃes no FA. Para o estudo in vivo, dez pacientes que apresentavam prÃ-molares com indicaÃÃo de exodontia como parte de seus planejamentos clÃnicos ortodÃnticos tiveram odontometrias eletrÃnicas realizadas com os LEFs Propex II e Root ZX previamente a exodontia. Os Ãltimos instrumentos utilizados foram fixados aos dentes que foram entÃo extraÃdos e tiveram 4,0 mm apicais de suas raÃzes expostos e analisados quanto à distÃncia entre as pontas dos instrumentos e os FA. No estudo ex vivo, considerando as mediÃÃes realizadas por cada um dos aparelhos a 0,0 mm e a -1,0 mm, a precisÃo dos LEFs foi: 70,6% e 47,1% (Root ZX), 61,8% e 52,9% (Mini Apex Locator), e 67,6% e 38,2% (Propex II), 61,8% e 38,2% (iPex), e 73,5% e 38,2% (RomiApex A-15), respectivamente (Â0,5 mm). DiferenÃas estatÃsticas foram encontradas para o Propex II, iPex e RomiApex A-15, quando comparadas suas leituras nas duas posiÃÃes (0,0mm X -1,0 mm). NÃo foram encontradas diferenÃas entre os LEFs a 0,0 mm, porÃm, a -1,0 mm o iPex foi estatisticamente inferior aos demais. Jà no estudo in vivo, o FA foi localizado em 75% (Root ZX) e 66,7% (Propex II), considerando margem de Â0,5 mm, tendo sido encontrada diferenÃa estatisticamente significante entre os LEFs. Diante do exposto, nas condiÃÃes do estudo, pode-se concluir que os LEFs sÃo ferramentas confiÃveis na determinaÃÃo de comprimentos reais, todavia, nÃo sÃo infalÃveis; que em condiÃÃes ex vivo, quando mantidos aquÃm do FA, todos os LEFs reduziram sua precisÃo, tendo o Propex II, iPex e RomiApex A-15 apresentado diferenÃas significantes; e que em condiÃÃes de uso clÃnico, o Root ZX apresentou maior confiabilidade do que o Propex II.
The electronic foramen locators (EFLs) currently available are based on different methods for determination of the distance between the apical foramen and a coronal reference, consequently presenting the real root canal length. Each of these methods aim to offer greater precision while presenting lower sensitivity to potential interferences found in the root canal system. With this in mind, the goal of this work was to evaluate the precision of some of these EFLs ex vivo and in vivo for electronic measurement of the root canal length at two different positions (0.0 mm and -1.0 mm) and under clinical conditions, respectively. In the ex vivo study, 42 mandibular bicuspids had their actual lengths compared to electronic measurements performed by the following EFLs: Root ZX, Mini Apex Locator, Propex II, iPex, and RomiApex A-15. Initial measurements were performed to positions identified by the devices as 1.0 mm short of the apical foramen (AF), and subsequent measurements were at the AF (0.0 mm). For the in vivo study, ten patients with bicuspids referred for extraction as part of their orthodontic clinical planning had electronic root length measurements using two EFLs, Propex II and Root ZX, prior to extraction. The last files used were fixated to the teeth, which were then extracted. Then, the apical 4 mm of the canals were exposed to allow assessment of the distance between the tip of the file and the AF. The percentages of precision from the ex vivo electronic measurements at 0.0 mm and -1.0 mm considering each device were: 70.6% and 47.1% (Root ZX); 61.8% and 52.9% (Mini Apex Locator); 67.6% and 38.2% (Propex II); 61.8% and 38.2% (iPex); and 73.5% and 38.2% (RomiApex A-15), respectively (Â0.5 mm). Statistical differences were observed for Propex II, iPex, and RomiApex A-15 when measurements at both positions were compared (0.0 mm X -1.0 mm). No significant differences between the EFLs were observed at 0. 0 mm. However, at -1.0 mm, the precision of iPex was statistically lower compared with the other devices. Regarding the in vivo study, the AF was located in 75% (Root ZX) and 66.7% of the teeth (Propex II), under a tolerance margin of Â0.5 mm. Statistically significant differences were observed between the two EFLs. Based on the results obtained and considering the conditions of this work, it was concluded that EFLs are reliable tools for determining the real length of the canal, but are not infallible. It was also observed in the ex vivo experiments that all EFLs had decreased precision in measurements with the instruments short of the AF, with significant differences observed between Propex II, iPex, and RomiApex A-15. Moreover, it was concluded that under clinical conditions, Root ZX was more reliable than Propex II.

VASCONCELOS, Bruno Carvalho de. Confiabilidade das determinações de localizadores eletrônicos foraminais : estudo ex vivo e in vivo. 2011. 75 f. Tese (Doutorado em Odontologia) - Universidade Federal do Ceará. Faculdade de Farmácia, Odontologia e Enfermagem, Fortaleza, 2011.
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The electronic foramen locators (EFLs) currently available are based on different methods for determination of the distance between the apical foramen and a coronal reference, consequently presenting the real root canal length. Each of these methods aim to offer greater precision while presenting lower sensitivity to potential interferences found in the root canal system. With this in mind, the goal of this work was to evaluate the precision of some of these EFLs ex vivo and in vivo for electronic measurement of the root canal length at two different positions (0.0 mm and -1.0 mm) and under clinical conditions, respectively. In the ex vivo study, 42 mandibular bicuspids had their actual lengths compared to electronic measurements performed by the following EFLs: Root ZX, Mini Apex Locator, Propex II, iPex, and RomiApex A-15. Initial measurements were performed to positions identified by the devices as 1.0 mm short of the apical foramen (AF), and subsequent measurements were at the AF (0.0 mm). For the in vivo study, ten patients with bicuspids referred for extraction as part of their orthodontic clinical planning had electronic root length measurements using two EFLs, Propex II and Root ZX, prior to extraction. The last files used were fixated to the teeth, which were then extracted. Then, the apical 4 mm of the canals were exposed to allow assessment of the distance between the tip of the file and the AF. The percentages of precision from the ex vivo electronic measurements at 0.0 mm and -1.0 mm considering each device were: 70.6% and 47.1% (Root ZX); 61.8% and 52.9% (Mini Apex Locator); 67.6% and 38.2% (Propex II); 61.8% and 38.2% (iPex); and 73.5% and 38.2% (RomiApex A-15), respectively (±0.5 mm). Statistical differences were observed for Propex II, iPex, and RomiApex A-15 when measurements at both positions were compared (0.0 mm X -1.0 mm). No significant differences between the EFLs were observed at 0. 0 mm. However, at -1.0 mm, the precision of iPex was statistically lower compared with the other devices. Regarding the in vivo study, the AF was located in 75% (Root ZX) and 66.7% of the teeth (Propex II), under a tolerance margin of ±0.5 mm. Statistically significant differences were observed between the two EFLs. Based on the results obtained and considering the conditions of this work, it was concluded that EFLs are reliable tools for determining the real length of the canal, but are not infallible. It was also observed in the ex vivo experiments that all EFLs had decreased precision in measurements with the instruments short of the AF, with significant differences observed between Propex II, iPex, and RomiApex A-15. Moreover, it was concluded that under clinical conditions, Root ZX was more reliable than Propex II.
Os localizadores eletrônicos foraminais (LEFs) disponíveis atualmente utilizam diversos métodos de determinação eletrônica da posição foraminal, qual seja a medida entre o forame apical e uma referência incisal/oclusal, definindo consequentemente o comprimento do canal radicular. Cada um desses métodos procura oferecer maior precisão e menor sensibilidade a possíveis interferências no sistema de canais radiculares. Desta forma, procurou-se avaliar a precisão de alguns destes LEFs ex vivo e in vivo na realização de odontometrias eletrônicas em diferentes posições (0,0 mm e -1,0 mm) e em condições de uso clínico, respectivamente. No estudo ex vivo, 42 pré-molares inferiores tiveram seus comprimentos reais comparados a odontometrias eletrônicas realizadas com os LEFs Root ZX, Mini Apex Locator, Propex II, iPex e RomiApex A-15. Inicialmente, em função dos displays dos aparelhos, determinaram-se medições 1,0 mm aquém do forame apical (FA), e posteriormente as medições no FA. Para o estudo in vivo, dez pacientes que apresentavam pré-molares com indicação de exodontia como parte de seus planejamentos clínicos ortodônticos tiveram odontometrias eletrônicas realizadas com os LEFs Propex II e Root ZX previamente a exodontia. Os últimos instrumentos utilizados foram fixados aos dentes que foram então extraídos e tiveram 4,0 mm apicais de suas raízes expostos e analisados quanto à distância entre as pontas dos instrumentos e os FA. No estudo ex vivo, considerando as medições realizadas por cada um dos aparelhos a 0,0 mm e a -1,0 mm, a precisão dos LEFs foi: 70,6% e 47,1% (Root ZX), 61,8% e 52,9% (Mini Apex Locator), e 67,6% e 38,2% (Propex II), 61,8% e 38,2% (iPex), e 73,5% e 38,2% (RomiApex A-15), respectivamente (±0,5 mm). Diferenças estatísticas foram encontradas para o Propex II, iPex e RomiApex A-15, quando comparadas suas leituras nas duas posições (0,0mm X -1,0 mm). Não foram encontradas diferenças entre os LEFs a 0,0 mm, porém, a -1,0 mm o iPex foi estatisticamente inferior aos demais. Já no estudo in vivo, o FA foi localizado em 75% (Root ZX) e 66,7% (Propex II), considerando margem de ±0,5 mm, tendo sido encontrada diferença estatisticamente significante entre os LEFs. Diante do exposto, nas condições do estudo, pode-se concluir que os LEFs são ferramentas confiáveis na determinação de comprimentos reais, todavia, não são infalíveis; que em condições ex vivo, quando mantidos aquém do FA, todos os LEFs reduziram sua precisão, tendo o Propex II, iPex e RomiApex A-15 apresentado diferenças significantes; e que em condições de uso clínico, o Root ZX apresentou maior confiabilidade do que o Propex II.

Chez la souris, la néphrogenèse débute par l'apparition du blastème metanéphrogène à 9.5 dpc. Une transition mésenchymo-épithéliale, comportant 5 étapes, débute a 11.5 dpc et aboutit au rein mature, composé de 3 structures : glomérules, tubules, et capillaires glomérulaires. Les étapes initiales du développement rénal peuvent être récapitulées en culture ex vivo; toutefois, l'organogenèse terminale et la maturation rénale sont incomplètes, et les structures rénales obtenues ex vivo ne sont pas fonctionnelles. Une étude du développement vasculaire in vivo au cours du développement rénal montre une angiogenèse (cellules Pecam-1 positives) et une vasculogenèse (cellules VEGFR-1 positives) précoces, dès 10.5 dpc. Une analyse quantitative par qRT-PCR confirme le rôle de Hif1α et VEGF dans la vasculogenèse rénale. En outre, la voie PGC1α, inductrice de VEGF indépendante de HIF, est activée en conditions hypoxiques. Pour améliorer le développement vasculaire rénal ex vivo, nous proposons un modèle de culture avec micro-perfusion rénale. L'étude morphologique par immunofluorescence des reins après culture micro-perfusée montre une survie tissulaire normale (TUNEL), et une intégrité anatomique (Néphrine, Cytokératine, WT1), en particulier vasculaire (Pecam-1). Une perfusion de vivo-morpholinos WT1 aboutit à une perte d'expression de WT1, confirmant le caractère fonctionnel de notre modèle. En conclusion, nous montrons le rôle précoce de l'angiogenèse et de la vasculogenèse au cours du développement rénal ; nous identifions le rôle de PGC1α dans la vasculogenèse rénale en conditions hypoxiques, et nous proposons une nouvelle technique de culture rénale ex vivo
In mice, nephrogenesis starts with the formation of the metanephric mesenchyme, at e9.5 dpc. A mesenchymal epithelial transition, consisting of 5 steps, starts at e11.5 dpc, and leads to a mature kidney, composed of 3 main structures: glomeruli, tubules, and capillaries. The initial steps of renal development can be recapitulated ex vivo; however, terminal organogenesis and maturation are impaired, and the explants are not functional. A study of vascular development in vivo during renal development shows that angiogenesis (Pecam-1 positive cells) and vasculogenesis (VEGF-R1 positive cells) occur early, at e10.5 dpc. A quantitative analysis, by qRT-PCR, shows that Hif1α and VEGF play a major role in renal vasculogenesis. Moreover, the PGC1α signaling pathway, a HIF independent VEGF inductor, is activated under hypoxic conditions. To improve ex vivo vascular development, we propose a novel culture technique, with micro-perfusion of the explant. A morphologic analysis of the kidneys obtained by micro-perfused cultures shows no apoptosis (TUNEL), a conserved parenchymal structure (Nephrin, Cytokeratin, WT1), and a proper vascular development (Pecam-1). A micro-perfusion of WT1 vivo-morpholinos leads to a decrease in WT1 expression, thus validating our model. In conclusion, we showed the early role of angiogenesis and vasculogenesis in renal development, we analyzed PGC1α role in hypoxic kidney cultures, and we proposed a novel kidney culture model

Chez la souris, la néphrogenèse débute par l'apparition du blastème metanéphrogène à 9.5 dpc. Une transition mésenchymo-épithéliale, comportant 5 étapes, débute a 11.5 dpc et aboutit au rein mature, composé de 3 structures : glomérules, tubules, et capillaires glomérulaires. Les étapes initiales du développement rénal peuvent être récapitulées en culture ex vivo; toutefois, l'organogenèse terminale et la maturation rénale sont incomplètes, et les structures rénales obtenues ex vivo ne sont pas fonctionnelles. Une étude du développement vasculaire in vivo au cours du développement rénal montre une angiogenèse (cellules Pecam-1 positives) et une vasculogenèse (cellules VEGFR-1 positives) précoces, dès 10.5 dpc. Une analyse quantitative par qRT-PCR confirme le rôle de Hif1α et VEGF dans la vasculogenèse rénale. En outre, la voie PGC1α, inductrice de VEGF indépendante de HIF, est activée en conditions hypoxiques. Pour améliorer le développement vasculaire rénal ex vivo, nous proposons un modèle de culture avec micro-perfusion rénale. L'étude morphologique par immunofluorescence des reins après culture micro-perfusée montre une survie tissulaire normale (TUNEL), et une intégrité anatomique (Néphrine, Cytokératine, WT1), en particulier vasculaire (Pecam-1). Une perfusion de vivo-morpholinos WT1 aboutit à une perte d'expression de WT1, confirmant le caractère fonctionnel de notre modèle. En conclusion, nous montrons le rôle précoce de l'angiogenèse et de la vasculogenèse au cours du développement rénal ; nous identifions le rôle de PGC1α dans la vasculogenèse rénale en conditions hypoxiques, et nous proposons une nouvelle technique de culture rénale ex vivo
In mice, nephrogenesis starts with the formation of the metanephric mesenchyme, at e9.5 dpc. A mesenchymal epithelial transition, consisting of 5 steps, starts at e11.5 dpc, and leads to a mature kidney, composed of 3 main structures: glomeruli, tubules, and capillaries. The initial steps of renal development can be recapitulated ex vivo; however, terminal organogenesis and maturation are impaired, and the explants are not functional. A study of vascular development in vivo during renal development shows that angiogenesis (Pecam-1 positive cells) and vasculogenesis (VEGF-R1 positive cells) occur early, at e10.5 dpc. A quantitative analysis, by qRT-PCR, shows that Hif1α and VEGF play a major role in renal vasculogenesis. Moreover, the PGC1α signaling pathway, a HIF independent VEGF inductor, is activated under hypoxic conditions. To improve ex vivo vascular development, we propose a novel culture technique, with micro-perfusion of the explant. A morphologic analysis of the kidneys obtained by micro-perfused cultures shows no apoptosis (TUNEL), a conserved parenchymal structure (Nephrin, Cytokeratin, WT1), and a proper vascular development (Pecam-1). A micro-perfusion of WT1 vivo-morpholinos leads to a decrease in WT1 expression, thus validating our model. In conclusion, we showed the early role of angiogenesis and vasculogenesis in renal development, we analyzed PGC1α role in hypoxic kidney cultures, and we proposed a novel kidney culture model

Orientador: Mary Luci de Souza Queiroz
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Neste trabalho avaliamos os efeitos de compostos derivados do nitroestireno, NTS1 e NTS2, sobre a resposta imunohematopoética de camundongos normais e portadores do tumor ascítico de Ehrlich (TAE). O estudo dos mecanismos envolvidos no tratamento de camundongos com NTS1 e NTS2 frente às alterações induzidas pelo TAE mostra efeitos imunológicos distintos dos dois compostos. O composto NTS1 induziu aumentos significativos na atividade de células NK, na proliferação de células mononucleares esplênicas e na produção de citocinas com padrão Th1 (IL-2 e IFN-?) liberadas por células mononucleares do baço. Em contrapartida, a atividade de NTS2 esteve relacionada com ativação macrofágica. Nossos resultados demonstram que o tratamento com NTS2 promove aumento significativo nos níveis de TNF-a e IL-1 no sobrenadante da cultura de macrófagos peritoneais. Outro efeito importante de NTS2 sobre macrófagos peritoneais foi o estímulo na produção de NO2-. Além dos parâmetros imunológicos citados, avaliamos, os efeitos de NTS1 e NTS2 sobre o crescimento e diferenciação de precursores hematopoéticos. Tanto NTS1 como NTS2 foram capazes de reverter a mielossupressão provocada pela evolução do tumor, além de aumentar a atividade estimuladora de colônias (CSA) no soro de camundongos Balb/c. Os resultados obtidos demonstram que os compostos compartilham da habilidade de regular positivamente os desequilíbrios hematopoéticos e imunológicos envolvidos na evolução temporal do TAE. A partir disso, testamos a capacidade dos compostos no processo de diferenciação mielocítica utilizando um sistema de cultura ex- vivo no qual células humanas CD34+ foram tratadas com NTS1 e NTS2. Nossos resultados apresentaram atividade dose-dependente de NTS1 e NTS2 na proliferação e viabilidade de células CD34+. Além de aumentar significativamente o número de progenitores mielóide comum e para granulócitos de macrófagos. Os compostos apresentaram diferentes efeitos durante o processo de diferenciação. Inibiram a formação de neutrófilos maduros, porém, NTS1 aumentou significativamente a produção de metamielócitos e NTS2 de monócitos. Estes efeitos fenotípicos na proliferação e na diferenciação observados após o tratamento com NTS1 e NTS2 podem estar relacionados com a via p38MAPK e o fator transcricional CEBP-a
Abstract: In this work, we have investigated the effects of two nitrostirene derivatives compounds, NTS1 and NTS2, in the immune-hematopoeitic system in normal and Ehrlich ascites tumor (EAT)-bearing mice. The study of the mechanisms involved in the treatment produced by the NTS1 and NTS2 against induced alterations by EAT were different, showing significant improvements in the NK cells activity, proliferation and Th1 (IL-2 and INF-?) by mononuclear spleen cells. On the other hand, NTS2 activity was related to macrophage activation. Our results show that treatment with NTS2 promotes significant increase in the TNF-a and IL-1 levels in supernatants of the cultures of peritoneal macrophages. Another important effect of NTS2 on peritoneal macrophage was the stimulation in the production of NO2-. Beyond immunological parameters, we investigated the NTS1 and NTS2 effects on hematopoietic progenitors. Treatment with NTS1 and NTS2 protected the host of myelosuppression caused by tumor development and increase the colony-stimulating activity (CSA) in the serum of Balb/c mice. The results showed that NTS1 and NTS2 share the ability of regulating positively the hematopoietic and immunological unbalance involved in the TAE development. Concerning their effects on myelopoiesis, we investigated the compounds utilizing an ex-vivo differentiation system in which umbilical cord blood derived CD34+ cells were treated with NTS1 and NTS2. Our results show that NTS1 and NTS2 have concentration dependent effects on proliferation and viability of CD34+ cells. Moreover, NTS1 and NTS2 significantly increase common myeloid and granulocyte/macrophage progenitors. The compounds have differential effects on terminal differentiation, inhibiting mature neutrophil, however, NTS1 significantly increased metamyelocytes and NTS2 monocytes. The phenotypic effects on proliferation and differentiation observed after NTS1 and NTS2 treatment can be explained by changes in p38MAPK cell signaling and with CEBP-a transcription factor
Doutorado
Farmacologia
Doutor em Farmacologia

La @peau est notre organe le plus important de par sa surface et son poids. Véritable barrière aux agressions extérieures, elle représente notre première ligne de défense naturelle. C'est un organe complexe qui présente une forte hétérogénéité, et qui peut être altéré par des désordres cutanés liés à des pathologies. Les techniques de spectroscopie vibrationnelle représentent un outil innovant pour l'analyse de la peau. Elles reposent sur l'interaction non-destructive lumière-matière ; ce qui permet d'extraire une "empreinte moléculaire" caractéristique de l'état physiopathologique de l'échantillon analysé. Nous avons évalué le potentiel de ces techniques pour la caractérisation des lésions cutanées. A partir de coupes de biopsies paraffinées, la micro-imagerie infrarouge, combinée à des méthodes puissantes de statistiques multivariées, a été appliquée avec succès pour le diagnostic différentiel des carcinomes cutanés (carcinomes basocellulaires et spinocellulaires) et pour la caractérisation du mélanome cutané. De plus, la microspectroscopie Raman polarisée a été mise en œuvre pour l'examen des lésions de type carcinome basocellulaire. Nous avons analysé plus précisément les foyers tumoraux et le tissu péritumoral. Les résultats montrent que le potentiel discriminant de la microspectroscopie Raman est renforcé par l'analyse de la polarisation du signal. Enfin, nous avons développé en milieu clinique la spectroscopie Raman in vivo pour l'analyse de pathologies cutanées chez les patients pris en charge au sein du service de Dermatologie du CHU de Reims

Unconjugated and conjugated bilirubin are molecules of physiological importance with increasing evidence demonstrating multipotent cardiovascular protective effects in conditions of benign hyperbilirubinemia. Individuals with Gilbert’s Syndrome, a condition typified by benign hyperbilirubinemia resultant to impaired bilirubin conjugation, report reduced cardiovascular disease risk, increased serum antioxidant capacity, increased anti-inflammatory profiles and favourable lipid-profiles. Unconjugated bilirubin also possesses established antiplatelet characteristics, with reduced activation and aggregation reported at physiologically relevant concentrations. Despite previous works demonstrating that endogenous and synthetic bilirubin compounds possess antioxidant potential, the specific impact of these compounds on platelet function more globally is poorly understood. The main aims of this thesis were to assess whether bilirubin ditaurate, a synthetic water-soluble analogue of bilirubin diglucuronide, induces acute antiplatelet effects and, if so, could inclusion during platelet storage preserve platelet characteristics? This thesis also aimed to investigate if increased in vivo serum total bilirubin concentrations inhibit platelet activation in vivo, in efforts to establish if a widely available natural product could induce an Iatrogenic Gilbert’s Syndrome. In the first study, platelet function and viability were examined following acute ex vivo bilirubin ditaurate exposure. The impact of a synthetic water-soluble form of bilirubin diglucuronide, bilirubin ditaurate, was investigated in isolated platelets over a period of 15 to 240 minutes. Granule exocytosis, activation, aggregation, mitochondrial membrane polarisation, mitochondrial reactive oxygen species production and viability was investigated. Bilirubin ditaurate attenuated both dense (n=3) and α-granule (n=8) exocytosis following agonist stimulation, with resultant reductions in αIIbβ3 upregulation (n=8) observed, despite no significant alterations in platelet aggregation (n=8). Bilirubin ditaurate itself did not perturb basal platelet P-selectin, phosphatidylserine, activated αIIbβ3 expression, general platelet viability (n=5) or mitochondrial membrane potential (n=4). Additionally, both basal mitochondrial superoxide production (n=4) nor general cellular reactive oxygen species production (n=6) were perturbed following exposure. However, following induced mitochondrial oxidative stress, bilirubin ditaurate dose dependently attenuated superoxide production (n=4). These findings suggest that acute bilirubin ditaurate exposure does not modulate resting basal platelet function and is not acutely cytotoxic, but demonstrates antiplatelet activity following agonist stimulation. These data suggest that effects were mediated in pathways that involved reactive oxygen species production. These results supported the rationale to examine the inclusion of bilirubin ditaurate, at physiologically relevant concentrations, in platelet units during storage given that increased reactive oxygen species production are a hallmark of the platelet storage lesion. In the second study various biochemical, activation, metabolic and functional parameters were examined in stored platelet units following inclusion of 35μM bilirubin ditaurate over a total of seven days of storage. This model assessed the impact of extended bilirubin ditaurate exposure given promising acute results observed in the previous study. Extended bilirubin ditaurate exposure was undertaken in full size platelet units donated by the Australian Red Cross Blood Service. Platelet units, pooled from four individual donors that were leukocyte depleted and supplemented with the platelet additive solution SSP+, were prepared as clinically and legislatively required, stored with constant agitation between 20°C and 24°C in the dark until seven days post collection. Within twenty-four hours of bilirubin ditaurate inclusion, platelet mitochondrial membrane potential was first significantly decreased, with complete glucose exhaustion observed after seventy-two hours. These results, in concert with the considerable loss of media bicarbonate, increased lactate accumulation and increased lactate dehydrogenase activity, support perturbed platelet metabolism following extended bilirubin ditaurate exposure during storage. Additionally, bilirubin ditaurate supplemented platelets also exhibited a substantial loss of viability, a reduced ability to aggregate and degranulate following agonist stimulation, increased mitochondrial superoxide production and upregulated basal expression of both phosphatidylserine, P-selectin and integrin αIIbβ3. These results support a conclusion that extended bilirubin ditaurate exposure is deleterious to platelets stored at room temperature in units with SSP+ as an additive solution. The third study, the Effect of Milk thistle extract (Legalon®) On circulating unconJugated bilirubin levels and markers of Oxidative stress and inflammation (the MOJO trial, ACTRN12619001296123) aimed to investigate the impact of elevated in vivo serum total bilirubin concentrations on platelet function. Our lab provided the first evidence for the antiplatelet activity of unconjugated bilirubin following ex vivo exposure, with similar attenuated platelet activity observed in individuals with Gilbert’s Syndrome. Gilbert’s Syndrome presents clinically as a mild unconjugated hyperbilirubinemia that is associated with reductions in all-cause mortality (including cancer), a decreased risk of developing coronary artery disease and a greater serum antioxidant status with less susceptibility to serum lipid oxidation. Interestingly, serum total bilirubin concentrations above 10μM are associated with substantial reductions in risk factors associated with such pathologies. Therefore, increasing circulating bilirubin concentrations is an attractive and potential therapeutic strategy for protection. Increased serum bilirubin concentrations are observed following Milk thistle ingestion in previous clinical trials and is not surprising given the attenuation of key hepatic transporters responsible for bilirubin excretion by the major flavonolignan constituents. Milk thistle (Legalon®) was administered for fourteen days, at the manufacturer’s recommended maximum dosage, to a population of healthy males in a randomised, placebo-controlled cross over trial design. An average increase of 0.76μM serum total bilirubin was observed with Legalon® treatment, albeit it not statistically significant when compared to placebo following unblinding and analysis of seventeen participants’ results. The MOJO trial was adequately powered to detect a ≥5μM increase in serum total bilirubin concentrations in response to Legalon® administration (two sided, α<0.01, 1-β=0.95) requiring a minimum of 15 male participants, accounting for a 50% drop-out rate. Platelet aggregation and activation were also not perturbed in concert with unremarkable impact on participant coagulation or haematological profiles. These results demonstrate that Legalon® (at the dosage recommended by the manufacturer) does not modulate circulating bilirubin or perturb platelet function in a cohort of healthy males. This is contrast to previous works detailing the induction of mild hyperbilirubinemia following Milk thistle ingestion and impaired platelet function following ex vivo exposure of platelets to Milk thistle constituents. This thesis provides the first evidence for the antiplatelet characteristics of bilirubin ditaurate, a conjugated bilirubin analogue, building on previous observations of similar activity of unconjugated bilirubin from our lab. This thesis contributes the first evidence that bilirubin ditaurate possesses significant antioxidant properties in platelets, potently scavenging mitochondrial derived superoxide following induced oxidative stress. This thesis also provides unique future directions highlighting the need for investigations towards assessing the impact of conjugated hyperbilirubinemias on platelet function given the first results of deleterious impact following extended exposure at physiologically relevant concentrations.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
Full Text

Les femmes enceintes sont exposées à de nombreux médicaments et les essais cliniques sont difficilement réalisables dans cette population, c'est pourquoi avoir une méthode qui permet d'estimer l'ampleur des modifications de pharmacocinétique chez la femme enceinte et le passage transplacentaire est essentiel. En effet les modifications physiologiques prennent place durant cette période clé. Nous avons développé des modèles pharmacocinétiques basé sur la physiologie (PBPK) et intégré les modifications physiologiques connues survenant durant la grossesse. Ils décrivent bien la pharmacocinétique de 3 antirétroviraux éliminés par le rein, le ténofovir (TFV), l'emtricitabine (FTC) et la lamivudine (3TC) et d'une molécule métabolisée par le CYP3A4, 2D6 et 2B6, la névirapine (NVP) et ceci pour différentes voies d'administration et pour des populations enceintes et non enceintes. De plus les clairances individuelles disponibles pour le TFV, le FTC et le 3TC tout au long de la grossesse ont permis d'explorer l'évolution de la sécrétion rénale. Celle-ci évoluerait proportionnellement au débit plasmatique rénal. L'intégration dans les modèles PBPK, des paramètres estimés à partir de l'expérience ex-vivo de cotylédon humain perfusé, a permis la prédiction de la cinétique foetale en fin de grossesse du TFV, FTC et NVP. Les prédictions ont été validées en les comparants aux concentrations mesurées au sang de cordon à l'accouchement. De plus, pour la névirapine nous avons exploré le métabolisme foetal et en avons conclu que même si celui-ci existe et est proche voir un peu supérieur à celui du nouveau-né, il n'influence pas la cinétique foetale
Pregnant women are exposed to numerous drugs and for obvious ethical reasons studies in this sensitive population arelimited. Information about the maternal pharmacokinetic (PK) changes and transplacental transfer of drugs prior to theiradministration to pregnant women would be highly useful. Indeed is it known that physiological changes during pregnancycan affect drug disposition. Time-varying pregnancy-related physiological parameters changes were implemented in fullPBPK models. They successfully predicted the disposition of 3 renally excreted drugs tenofovir (TFV), emtricitabine (FTC)and lamivudine (3TC) and one metabolized drug, nevirapine (NVP) for non-pregnant and pregnant populations. We foundthat both renal secretion and filtration changed during pregnancy. Changes in renal clearance secretion were related tochanges in renal plasma flow. Transplacental parameters estimated from ex vivo human placenta perfusion experiments implemented in PBPK models allowed good prediction of foetal TFV, FTC and NVP PK. Predictions were compared to observed cord blood concentrations to validate these models. Moreover, we have explored nevirapine foetal metabolism and concluded that even if the foetal metabolism is the same than the newborn one or a little more important, it is notlikely to impact foetal PK

Thesis (M.S.)--Southern Illinois University Carbondale, 2006.
"Department of Molecular Biology, Microbiology, Biochemisty and Cell Biology." Includes bibliographical references (leaves 76-86). Also available online.

Thesis (M.S.)--Southern Illinois University Carbondale, 2007.
"Department of Molecular Biology, Microbiology, and Biochemisty." Includes bibliographical references (leaves 61-72). Also available online.

Single neurons form the fundamental information processing units of the brain. Yet despite our growing knowledge of the electrophysiological and molecular properties of single neurons and their dendrites, little is known about their contribution to information processing in vivo, which depends strongly on the patterns of synaptic input and the way in which they engage active dendritic mechanisms. Here, I address the input-output function of single neurons in vivo and the role played by active dendrites in pyramidal neurons of mouse primary visual cortex using a dual modelling and experimental approach. First, I developed and tested new experimental techniques to map functional synaptic input in vivo: I have established an ultrastructural method to label active synapses received by the dendrites of an identified neuron during sensory processing in vivo, while measuring its output with somatic whole-cell recording. I show how to use the styryl dye FM1-43FX as an activity-dependent in vivo label which can be read out using focused ion-beam electron microscopy (FIBSEM) and I developed a pipeline for the semi-automated segmentation of the resulting FIBSEM volumes. Second, to study dendritic computation and the role of dendritic spikes in vivo, I have constructed a detailed biophysical model of a layer 2/3 pyramidal neuron, and a model of the synaptic input it receives during visual stimulation in vivo. Both models are tightly constrained by experimental data from the literature, including direct patch-clamp recordings from layer 2/3 dendrites in vivo. My work answers longstanding questions about the synaptic input patterns received by single neurons in vivo and the computations implemented by their integration in active dendrites: I provide a detailed biophysical mechanism for the transformation of synaptic inputs, via dendritic nonlinearities, to somatic output and elucidate the role of active dendrites for the function of the neuron.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2017.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Nutrient requirements for cancer cells are different from those of most normal cells. Understanding these differences, and the factors that are responsible for creating different metabolic dependencies, is critical to understanding the basic requirements of rapid proliferation and identifying potential therapeutic vulnerabilities. In particular, oncogene status, tissue-of-origin, cell-of-origin, and microenvironmental factors all can contribute to defining these requirements, but how to characterize which dependencies exist in various physiological setting are unknown. In this thesis, we address key challenges to study cancer metabolism in the living organism. We present novel experimental and analytical methods to study tumor metabolism in mouse models. This includes: (1) an approach to determine the metabolic fate of any radioactive or stable-isotope tracer in tumors and tissues (2) strategies to create stable-isotope macromolecular (protein) tracers (3) a plasmapheresis strategy to deliver labeled protein in vivo (4) qualitative and quantitative methods to define kinetics of extracellular protein catabolism in live tumors in real-time (5) the characterization of a conditional allele to test the requirements for isoform-specific pyruvate kinase expression. We applied these methods to three mouse models of cancer (models of human lung, pancreas, and prostate cancer) to better characterize metabolism and identify potential metabolic vulnerabilities in these tumors. Our results show that tumors in vivo utilize a metabolic program that is distinct from cells in tissue culture derived from those same tumors. Additionally, we identified that the uptake and subsequent catabolism of extracellular protein provides a substantial proportion of free intracellular amino acids for pancreatic cancer cells in tumors. Finally, we have identified prostate cancer as a tumor type that may benefit from pharmacological activation of pyruvate kinase. Overall, our results demonstrate the importance of studying cancer metabolism in the correct physiological setting. Data generated from patients corroborates our findings in these mouse models and suggests that these approaches can be used to define more effective cancer therapeutic strategies. The methods presented here are adaptable to study the metabolic phenotypes in any tumor or tissue of interest. Further use of these methods enable the examination of cell autonomous and non-cell autonomous metabolic consequences of oncogene status, microenvironment, and in organismal metabolism as a function of cancer initiation and progression.
by Shawn Michael Davidson.
Ph. D.

Long DNA palindromes cannot be propagated in wild-type Escherichia coli. Either a replicon with a long DNA palindrome is so poorly replicated that it is available, or the palindrome itself is unstable so that it undergoes partial or complete deletion. The deleterious effect of long DNA palindromes is alleviated in strains mutant for sbsC and D: each mutation by itself is necessary and sufficient to allow the plating of a λ phage containing a long palindrome. In this thesis, the mechanism by which long DNA palindromes cause inviability is studied, and it is discussed whether inviability is a consequence of an unusual DNA structure. An analysis of the effects of palindromic central asymmetry on the propagation of a λ phage across a broad range of E. coli host strains is described. Palindromes carrying an 8bp asymmetry confer a less severe phenotype than do perfect palindromes, arguing that a centre-dependent pathway for palindrome-mediated inviability exists that is independent of the host strain. For further study, a set of long DNA palindromes with paired changes in the central sequence was constructed in bacteriophage λ. Identical palindrome centres were previously used by others to test the S-type model for cruciform extrusion in vitro. The plaque areas produced by the palindrome-containing λ phage were compared on an E. coli sbsC lawn. Central sequence changes had a greater effect upon the plaque area than peripheral changes, implying that the residual palindrome-mediated inviability in E. coli sbcC is centre-dependent and could be due to the formation of a cruciform structure. The results argue strongly that intrastrand pairing within palindromes is critical in determining their effects in vivo.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós Graduação em Enfermagem, Florianópolis, 1998.
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El presente trabajo de investigación versa sobre la caracterización in vitro, ex vivo e in vivo de dos sistemas nanoestructurados de liberación sostenida (PF-F1NPs y PF- F2NPs) conteniendo pranoprofeno (PF) como principio activo. Estas formulaciones fueron optimizadas en estudios previos desarrollados por Abrego y colaboradores (a) como una estrategia innovadora para el tratamiento del dolor y los procesos inflamatorios asociados a patologías oculares. Inicialmente se prepararon y caracterizaron morfológica y fisicoquímicamente las formulaciones PF-NPs bajo las condiciones descritas por Abrego y colaboradores. Los resultados obtenidos revelaron que las formulaciones PF-NPs presentaban características apropiadas para su administración ocular. Se llevó a cabo entonces un ensayo in vitro de citotoxicidad para las formulaciones PF-NPs, las mismas formulaciones pero sin fármaco (NPs blancas) y para una solución de PF en tampón fosfato (PBS) usando la línea celular Y-79 de retinoblastoma humano. Este análisis evidenció que cuando las células fueron tratadas con concentraciones de 150 µg/mL para la formulación PF-F1NPs y de 200 µg/mL para PF-F2NPs, la viabilidad celular resultó cercana al 20% en comparación con el grupo control transcurridas 48 h de ensayo. Por otra parte, la exposición de las células Y-79 a una concentración superior a 750 µg/mL de las NPs blancas y de la solución de PF en PBS, mostró una viabilidad celular inferior al 50% respecto al grupo control. Los resultados obtenidos podrían atribuirse a que el fármaco en solución es susceptible de degradarse originando metabolitos menos tóxicos para las células, mientras que el fármaco encapsulado en las NPs se encuentra protegido de la degradación y se libera continuamente en su forma estable para interactuar con las células causando mayor toxicidad. Adicionalmente, concentraciones altas de las formulaciones PF-NPs y de las NPs blancas limitan el suministro de oxígeno y nutrientes a las células, lo que interfiere en el metabolismo y la proliferación de las mismas. Asimismo, en esta investigación se dedicaron esfuerzos significativos a validar una metódica bioanalítica para la cuantificación del PF por cromatografía líquida de alta eficacia (HPLC) en muestras que hubieran estado en contacto directo con tejidos oculares. Los resultados demostraron que el método escogido es lineal, exacto y preciso para el intervalo de concentraciones ensayadas (6.25 – 100 µg/mL), además de establecerse como selectivo y robusto. Se llevaron a cabo estudios ex vivo en córnea y esclera de cerdo para evaluar el perfil de permeación del PF a partir de una solución a saturación del fármaco. El análisis reveló que la cantidad de PF permeada a través de la esclerótica es superior a la observada para la córnea, quedando una mayor cantidad del PF retenido en esta última. Estos resultados podrían deberse a las diferencias anatómicas existentes entre las dos membranas oculares. La idoneidad del método extractivo escogido fue evaluada a través de la recuperación y cuantificación del PF retenido en las membranas biológicas tras los ensayos ex vivo de permeación. Los datos obtenidos revelaron que el método validado es adecuado para tal fin. Además, se desarrolló un ensayo ex vivo en córnea de conejo con el propósito de evaluar el perfil de permeación del PF desde las formulaciones PF-NPs en comparación con la formulación comercial de PF (Oftalar®) y con una solución de PF en PBS (1.0 mg/mL). El estudio demostró que tanto la cantidad permeada como la cantidad retenida de PF a partir de las formulaciones PF-NPs es superior respecto a la presentada por el Oftalar® y la solución de PF en PBS. Estos resultados se encuentran en concordancia con los obtenidos en el ensayo in vivo de eficacia antiinflamatoria en el que la formulación PF-F2NPs evidenció la mayor eficacia antiinflamatoria respecto al resto de formulaciones sometidas a ensayo. Finalmente, no se detectó ningún signo de irritación corneal, del iris o de la conjuntiva tras un ensayo in vivo de tolerancia ocular para las formulaciones PF-NPs. a) Abrego G, Alvarado HL, Egea MA, Gonzalez-Mira E, Calpena AC, Garcia ML. Design of Nanosuspensions and Freeze-Dried PLGA Nanoparticles as a Novel Approach for Ophthalmic Delivery of Pranoprofen. Journal of Pharmaceutical Sciences. 2014;103(10):3153-64.

Upconversion nanoparticles (UCNPs) have been utilized for biological applications. Unlike conventional linear excitation molecules, UCNPs are excited by 980nm and emit photon in visible and near infrared region. The unique photophysical property offers superior penetration depth and lower photo-cytotoxicity. With the aid of various vectors such as target-specific peptides and photosensitizers, the UCNPs can precisely interact selectively with designated proteins (Cyclin D1 and Polo-like Kinase 1) and cancer cells so as to achieve theranostic effect. This thesis illustrated the upconversion mechanism and anti-cancer effect by UCNPs conjugated with peptides. Two research studies focus on Cyclin D1 or Polo-like kinase 1 (Plk1) specific peptides coated UCNPs function as key cell cycle inhibitors, in vitro imaging agent and in vivo tumor suppressor. Apart from inorganic nanomaterials, graphitic phase carbon nitride (g-C3N4) nanoparticles coupled with porphyrin moieties act as cancer directional photodynamic therapy agents was also described in the aspects of detailed photophysical measurements and in vitro theranostic studies.

L'anévrisme aortique abdominal s'accompagne d'une dégradation du réseau élastique et d'une augmentation des métalloprotéases. Nous avons essayé de transposer les qualités de réparations du fibroblaste gingival sur ces artères dans des modèles ex-vivo et in vivo. Un modèle de culture d'artère de lapin en gel de collagène est évalué puis utilisé en coculture avec des fibroblastes gingivaux pour évaluer l'effet de ces fibroblastes sur le remodelage artériel. Les fibroblastes gingivaux sont également cultivés avec des artères issues d'anévrismes aortiques humains. Enfin notre hypothèse est testée sur un modèle d'anévrisme chez le lapin où les cellules sont transférées par voie endoluminale. En coculture, les fibroblastes gingivaux inhibent la MMP-9 par une augmentation de son inhibiteur le TIMP-1. La même inhibition est présente dans des cocultures avec des artères anévrismales humaines. La MMP-7 est également inhibée par augmentation du TIMP-1 mais aussi au niveau transcriptionnel par une augmentation du TGF-pl. Ces cocultures permettent la préservation du réseau élastique artériel. Le transfert des fibroblastes dans des anévrismes créés chez le lapin entraîne la diminution de leurs diamètres et de la MMP-9. Ces résultats obtenus sur des modèles ex vivo et in vivo montrent la capacité des fibroblastes gingivaux à préserver le réseau élastique et à moduler l'activité de protéases impliquées dans la pathologie. La transplantation de fibroblastes gingivaux semble être une approche intéressante dans le traitement des anévrismes aortiques. Néanmoins des expériences complémentaires sont nécessaires pour confirmer nos résultats et comprendre comment le fibroblaste gingival influe positivement le remodelage
Aortic abdominal aneurysm is accompanied by a degradation of the elastic network and an increase of the metalloproteinases. We tried to transpose repair qualities of the gingival fibroblast on these arteries in ex-vivo and in vivo models. A culture model of rabbit artery in collagen gel is evaluated then used in coculture with gingival fibroblasts to evaluate the effect of these fibroblasts on arterial remodeling. The gingival fibroblasts are also cultivated with human aneurismal aortas. Finally our hypothesis is tested on an in vivo model of rabbit aneurism where the cells are transplanted into the lumen. In coculture, the gingival fibroblasts inhibit MMP-9 by an increase of its inhibitor: TIMP-1. Same inhibition is present in cocultures with human aneurismal aortas. The MMP-7 is also inhibited by increase in the TIMP-1 but also at a transcriptional level by an increase of TGF-pl. These cocultures allow the preservation of the arterial elastic network. The transfer of the fibroblasts in aneurisms created in rabbit reduced their diameters and the MMP-9. These results obtained on ex vivo and in vivo models show the capacity of the gingival fibroblasts to preserve the elastic network and to modulate the activity of proteases implied in pathology. The transplantation of gingival fibroblasts seems to be an interesting approach in the treatment of aortic aneurisms. Nevertheless complementary experiments are necessary to confirm our results and to understand how the gingival fibroblast influences remodeling

Each year, approximately 1,200,000 cases of colorectal cancer are diagnosed worldwide. Patients with early-stage disease, without lymph node metastatic, evolve with tumor recurrence or relapse in up to a quarter of cases, likely due tounderstaging. The objective is to research sentinel lymph nodes in patients with colon adenocarcinomas, comparing in vivo and ex vivo techniques used in their identification, and to detail anatomical-pathological examination, with multisectionand immunohistochemistry, of these lymph nodes. Thirty-three patientsundergoing curative cancer surgery were studied. The marker used to identify sentinel lymph nodes was patent blue dye. In 18 of these patients, the dye was injected in vivo into the peritumoral subserosa, while with 15 patients, it was injected ex vivo into the peritumoral submucosa. The visual identification of sentinel lymph nodes with the dye was possible in 72.2% of the in vivo cases, and in 33.3% of the ex vivo cases. In routine histopathological analysis, the sensitivity for detection of metastases in sentinel lymph nodes was 66.7% in vivo, and 100% ex vivo; alse- negatives were 33.3% for the in vivo group, and zero ex vivo. Amongpatients without metastases by routine histopathological examination with hematoxylin-eosin, examination with multisection and immunohistochemistry of sentinel lymph nodes diagnosed metastases in one (9%) subject from the in vivo group and in one subject (14.3%) from the ex vivo group, leading to a consideration of restaging. We conclude that in the identification of sentinel lymph nodes, in vivo research yielded better results than the ex vivo approach. Thediagnosis of metastases by routine histopathology was proportionally the same in sentinel and non-sentinel lymph nodes. The antomical- pathological techniques of multisection and immunohistochemistry improved the staging of tumors, compared to routine histopathological examination.
No mundo, a cada ano são diagnosticados cerca de 1.200.000 casos de câncer colorretal. Pacientes com doença em estágio inicial, sem linfonodo metastático, evoluem com recorrência ou recidiva do tumor em até um quarto dos casos, por provável subestadiamento. O objetivo é pesquisar sobre linfonodo-sentinela em pacientes com denocarcinoma de cólon, comparando as técnicas in vivo e ex vivo utilizadas na sua identificação, e detalhar o exame anatomopatológico com multissecção e imunoistoquímica desses linfonodos. Foram estudados 33pacientes submetidos à cirurgia oncológica curativa. O marcador utilizado para identificação de linfonodo-sentinela foi o corante azul patente. Em 18 deles o corante foi injetado in vivo na subserosa peritumoral e em 15 foi injetado ex vivo na submucosa peritumoral. A identificação visual de linfonodo-sentinela com o corante foi possível in vivo em 72,2% e ex vivo em 33,3% dos pacientes. Na análise histopatológica de rotina, a sensibilidade para detecção de metástasesnos linfonodos-sentinela foi de 66,7% in vivo e de 100% ex vivo; e o falsonegativo foi de 33,3% no grupo in vivo e zero no grupo ex vivo. Entre os pacientes sem metástases pelo exame histopatológico de rotina com hematoxilina-eosina, o exame com multissecção e imunoistoquímica dos linfonodos-sentinela diagnosticou metástase em um (9%) indivíduo do grupo in vivo e em um (14,3%) do grupo ex vivo, sendo considerados reestadiamentos. Concluiu-se que na identificação de infonodo- sentinela a pesquisa in vivo obteve melhores resultados que a ex vivo. O diagnóstico de metástases pela histopatologia de rotina foi proporcionalmente o mesmo nos linfonodos-sentinela e nos não sentinela. As técnicas anatomopatológicas de multissecção e imunoistoquímica aprimoraram o estadiamento dos tumores em relação ao exame histopatológico de rotina.