This diploma thesis summarizes results of the detailed morphometric analysis of extensive fossil materials of the genus Myotis from the upper Pliocene deposits of Javoříčské caves (Czech Republic) and Urwista cave (Poland). Patterns of phenotype variation of particular fossil populations were analyzed with aid of uni- and multivariation techniques, compared with type series of fossil taxa described from MN15 site Gundersheim and a comparative series of extant European species. Nine fossil and 10 extant species were identified and further reexamined in terms of their phenotype variation and possible phylogenetic relations. The phenotype structure of fossil assemblages were compared to that of Gundersheim and extant European fauna: samples from Urwista cave show clear resemblances to the former one, those from Javoříčské cave exhibit certain similarities to the extant European fauna despite clear differences in variation patterns of particular species. Technical preparation, documentation by photographic material, measurement and statistical analysis is included in this work. Key words: bats, Pliocene, phenotype dynamics, community structure

Depuis plusieurs décennies, l’étude des conséquences des mutations pathogéniques a permisnon seulement de définir l’origine de nombreuses maladies génétiques humaines, héréditaires ou non,mais également de contribuer à l’interprétation de la variabilité phénotypique inter-individus au seind’une pathologie donnée. Les mutations induisant une perte de fonction de la protéine synthétisée ouune protéine incomplète ont été et sont encore les plus étudiées. Avec l’introduction des technologiesde séquençage à haut-débit, le nombre de variants faux-sens, silencieux ou introniques détectés auniveau des gènes humains augmente de façon continue. Distinguer les variations nucléotidiques quivont modifier significativement un phénotype de celles qui seront neutres est un véritable défi pour larecherche.De nombreuses variations nucléotidiques à signification clinique inconnue ont été identifiéesparmi les près de 2000 mutations décrites au niveau du gène CFTR (Cystic Fibrosis Transmembraneconductance Regulator) responsables de la mucoviscidose. Certaines de ces variations vont avoir unimpact sur les transcrits ARN modifiant leur qualité et leur quantité et par conséquent l’expression dugène CFTR. Elles vont notamment affecter l’épissage de l’ARN pré-messager en altérant des signauxreconnus par la machinerie cellulaire et perturber la fidélité de ce mécanisme. Malgré un recul de plusde 20 ans dans la description de la corrélation génotype-phénotype dans cette maladie, de nombreuxphénotypes inattendus et atypiques sont observés. Ils peuvent être dus à des facteurs autres que desvariations génotypiques, mais sans l’accès direct aux transcrits des individus porteurs de tels variants,il est difficile de mesurer les conséquences de ces variations sur la synthèse et la maturation de l’ARN.L’objectif de ce travail de thèse a été de montrer, par des approches in vitro etbioinformatiques, comment des variations nucléotidiques au sein du gène CFTR peuvent impacter surl’épissage de l’ARN pré-messager. Ce travail a permis dans un premier temps la découverte demécanismes d’épissage inattendus modificateurs de phénotypes sévères attendus pour une mutationnon-sens. En effet, par un épissage alternatif subtil de trois nucléotides au niveau d’un site d’épissageen tandem, le codon stop se retrouve délété et aboutit à des transcrits fonctionnels expliquant lesphénotypes modérés. Dans un second temps, nous avons montré que plusieurs mutations non-sensportées par le même exon 15 impactaient différemment sur l’épissage de l’ARN en modifiantsignificativement le taux d’inclusion de cet exon. La connaissance préalable des ratios de transcritsincluant ou non l’exon peut améliorer l’efficacité des traitements correcteurs de codons stopprématurés en les combinant avec des traitements modulateurs de l’épissage augmentant le taux detranscrits correctibles. Dans un troisième et dernier temps, une combinaison d’analyses in silico et invitro des exons du gène CFTR a permis de détecter des exons porteurs de signaux d’épissage plusfaibles les rendant plus sensibles à des mutations exoniques d’épissage. Des mutations faux-sens auniveau de l’exon 3 ont par exemple été montrées comme favorisant l’exclusion de cet exon, diminuantle taux de transcrits fonctionnels.L’ensemble de ces travaux a contribué à comprendre les conséquences de mutations au niveaude l’épissage du gène CFTR sur les variations du phénotype. La connaissance améliorée des variationspossibles du phénotype rattaché à un génotype donné permettra non seulement de prédire l’évolutionde la maladie, mais également d’ajuster et de proposer des thérapies personnalisées selon la mutationportée par le patient. / Over the past decades, studying the consequences of pathogenic mutations has allowed not only todefine the origin of several genetic diseases, but also to contribute to understand the phenotypicvariability between individuals within a disease. The CFTR gene was extensively analyzed since 1989,but among the over 1,900 mutations identified, the current challenge is to classify them as diseasecausingor neutral. These variants of unknown clinical significance (UVs) can alter multiple processes,from gene transcription to RNA splicing or protein function. The CFTR gene needs to include intactversions of all its 27 exons to be functional, and any mutation affecting its splicing process will reducethe amount of functional full-length transcripts. Previous studies have shown that mutations withinsome CFTR exons increase exon skipping. We hypothesized that a number of UVs occurring in otherCFTR exons could indeed affect splicing. By combining in vitro and bioinformatics approaches, weshowed how nucleotide variations within the CFTR gene could impact on the splicing of its premRNA.This work enabled us to provide the first experimental evidence of a premature terminationcodon removal by alternative splicing at a NAGNAG acceptor splice site. This unexpected phenotypemodifyingmechanism explains the much milder phenotype severity than expected for a nonsensemutation. The correction of premature termination codons (PTCs) by agents that promote readthroughrepresents a promising emerging tool for the treatment of many genetic diseases. Having demonstratedthat nonsense mutation could cause aberrant splicing, we postulated that the efficiency of thereadthrough treatment could be due not only to the stop codon itself but also to the amount ofcorrectible transcripts. We showed that a subset of nonsense mutations within the CFTR exon 15 has adifferent impact on the splicing efficiency by modifying the inclusion rate of this exon anddemonstrated that the total amount of transcripts together with the splicing profile should be assessedto anticipate and improve efficacy of readthrough therapy in CF patients. Finally, in order to anticipatethe occurrence of “splicing-causing” mutations, we used a combination of in silico and in vitroanalyses of all CFTR exons and pinpointed those harboring weak splicing signals, which render themmore sensitive to exonic splicing mutations.All these studies contribute to expand our knowledge on the phenotypic variability due to alternativesplicing of the CFTR gene. These studies will lead not only to predict the evolution of a disease, butalso to adjust therapy according to the mutation of each patient.

La chimiorésistance des cancers est caractérisée par une résistance pléïotropique à de multiples médicaments. Ce mécanisme est en partie causé par la surexpression des transporteurs à «ATP binding cassette» (Pgp, MRP1, BCRP…). Les inhibiteurs connus de ces transporteurs comme la cyclosporine A, le vérapamil et le RU486 sont toxiques à doses élevées. Dans cette étude, de nombreux dérivés stéroïdiens synthétisés au laboratoire à base de progestérone ou d’acides biliaires ont été évalués pour leur capacité à inhiber les transporteurs ABC et plus spécifiquement les fonctions de transport par la Pgp ou la BCRP. Plusieurs de ces dérivés synthétisés se sont avérés capables de restaurer complètement la sensibilité des cellules résistantes d’une manière plus importante que la cyclosporine A in vitro. De plus, le meilleur des nos dérivés testés s’est avéré capable in vivo de diminuer significativement la progression tumorale de xénogreffe sur les souris et d’augmenter la durée de survie des souris. Cette étude a ainsi permis d’ouvrir la voie au développement de nouveaux dérivés stéroïdiens peu toxiques ayant la capacité d’inhiber le phénotype MDR et de restaurer la sensibilité des cellules cancéreuses vis-à-vis des agents chimiothérapeutiques utilisés, avec un perspective d’application clinique / The chemoresistance of cancer is characterized by a pleitropic resistance to multiple drugs. This mechanism is partly caused by the overexpression of “ATP Binding Cassette” transporters (Pgp, MRP1, BCRP…). Known inhibitors of these transporters such as cyclosporin A, verapamil and RU486 rather toxic at high doses. In this study, many steroid derivatives synthesized in the laboratory (from progesterone or bile acid precursor) to bind and inhibit ABC transporters, specifically the transport by Pgp or BCRP. Several of these synthetic derivatives were able to completely restore the sensivity of the resistant cells compared to cysclosporine A in vitro. In addition, the efficient of these derivatives could, in vivo significantly reduce tumor growth of xenografts on mice and increase survival time of mice. This study opens a route for development of new steroid derivatives with low toxicity, having the ability to reverse the MDR phenotype and restore sensitivity of cancer cells to the chemotherapeutic agents use, with a perspective at clinical use

Le système de l’immunité innée cérébrale (SIIC) est principalement composé des cellules microgliales. En réponse à des stimuli immuns, inflammatoires ou un trauma neurologique, la microglie s’active et produit des facteurs pro et anti-inflammatoires qui d’une part coordonnent la réponse de l’immunité innée cérébrale et d’autre part modulent l’activité neuronale et, in fine, le comportement. Plus récemment, les cellules microgliales se sont révélées jouer un rôle clé dans le développement cérébral. Ainsi, par leurs activités de phagocytose, elles participent à la maturation des réseaux neuronaux. Si l’activation du SIIC permet de défendre le tissu cérébral des agressions, l’activation prolongée des cellules microgliales a aussi des effets délétères. Ainsi, dans le cerveau adulte, la production soutenue de cytokines inflammatoires contribue au développement de pathologies neurodégénératives. Au cours du développement les stimuli inflammatoires, en perturbant l’activité des cellules microgliales conduisent à une dysfonction de circuits neuronaux qui pourrait être impliquée dans des pathologies neuropsychiatriques à composante neurodéveloppementale. La compréhension de la régulation des cellules microgliales et de leur réponse est donc capitale. L’activité microgliale repose sur ses propriétés morphologique, dynamique et sa communication avec les neurones qui impliquent des profils de synthèse de facteurs (cytokines, chemokines, facteurs de croissance, etc..) et de récepteurs particuliers, la polarisation vers un phénotype pro ou anti-inflammatoire et la phagocytose. Peu d’études ont caractérisé l’ensemble des propriétés morphofonctionnelles des cellules microgliales in vivo. Par la combinaison d’approches de FACS, immunohistochimie, microscopie confocale et reconstruction en 3D, microscopie bi-photonique et dosage des facteurs de communication, il est aujourd’hui possible de mieux caractériser ces cellules afin de comprendre leur régulation par l’environnement et l’impact (bénéfique ou délétère) sur les fonctions neuronales. L’objectif général de cette thèse a été d’étudier les propriétés morphofonctionnelles des cellules microgliales in vivo dans deux situations physiopathologiques, une inflammation induite par l’administration périphérique de lipopolysaccharide (LPS) et une déficience alimentaire en acides gras polyinsaturés (AGPI) de type n-3, connus pour leurs propriétés immunomodulatrices. La première étude nous a permis de développer des outils nécessaires à l’étude de la plasticité morphofonctionnelle de la microglie et d’apporter de nouveaux éléments de compréhension de l’impact d’une inflammation périphérique sur l’activité de ces cellules in vivo. Dans la deuxième partie de cette thèse, nous avons montré pour la première fois que le statut alimentaire maternel en AGPI n-3 influence les propriétés morphofonctionnelles des cellules microgliales au cours du développement post-natal ainsi que l’activité des réseaux neuronaux. De façon générale, nos résultats apportent des éléments de compréhension des relations entre plasticité morphologique et fonctionnelle des cellules microgliales in vivo. / The brain innate immune system is mainly composed of microglial cells. Microglia are activated in response to an immune or inflammatory stimuli or a trauma, and then produce pro- and anti-inflammatory factors. These factors drive the innate immune response and can modulate neuronal activity and in fine, learning and memory. Recently, microglia have been shown to play a key role during brain development. Via their phagocytic activity, microglial cells can participate to neuronal networks maturation. Although brain innate immune system defends brain tissue from aggression, chronic activation of microglia can also be deleterious. In the adult brain, chronic production of inflammatory cytokines can contribute to the pathogenesis of neurodegenerative diseases. During development, inflammatory stimuli modifying microglia activity and homeostasis could lead to neuropsychiatric diseases with a neurodevelopmental origin. Understanding how microglia are regulated and how they respond to various stimuli is therefore crucial.Microglia activity is characterized by morphological and dynamic properties of microglia,by its communication with neurons by its polarization into a specific phenotype, and by their phagocytic profile. Few studies have characterized all the morphofunctional properties of microglial cells in vivo. Using a combination of approaches including FACS, immunohistochemistry, confocal microscopy, 3D reconstruction, two-photon microscopy and communication factors assays, it is now possible to better characterize these cells in order to understand their regulation by the environment and the resulting impact (beneficial or deleterious) on neuronal functions. The main goal of this thesis was to study the morphofunctional properties of microglial cells in vivo in two pathophysiological states: a peripheral inflammation induced by a peripheral injection of lipopolysaccharide (LPS) and in an n-3 PUFAs nutritional state. In the first study, we developed tools to investigate microglial morphofunctional plasticity and gained a better understanding of the impact of peripheral inflammation on the activity of these cells in vivo. In the second part of this thesis, we showed for the first time that maternal nutritional status in n-3 PUFAs affect the morphofunctional properties of microglial cells and the establishment of neural circuits during the postnatal development of the pups. Overall, our results provide new insights in the relationship between morphological and functional plasticity of microglial cells in vivo.

No description available.

The relationship between leaf and its environment has resulted in a tremendous diversification of leaf shape within and between plants species, which is important to cope with the differing environmental conditions. Arabidopsis lyrata is a prime model plant that shows leaf shape variation within species and between related species. In Cardamine and Capsella, the RCO genes (homeodomain leucine zipper family transcription factors) are involved in shaping leaves, yielding more complex shaped leaves (leaflets). In A. thaliana, over the course of evolution, the RCO-A and RCO-B paralogous genes have been deleted that led to the loss of lobes (leaf simplification). Based on previous quantitative trait locus (QTL) mapping results, these gene family members are thought to control leaf shape variation also in A. lyrata. Since the paralog involved in leaf shape variation is unknown, both copies of the RCO genes (AL6G13350 and AL6G13360) were sequenced. The study aimed to identify whether DNA sequence variation exists in the two paralogous RCO genes, which could explain the phenotype variation both within population and between A. lyrata populations, along with related species A. arenosa. The results showed limited variation in the coding regions in the form of indels, single nucleotide polymorphisms (SNPs) and amino acid substitutions resulting in no significant difference in phenotype between genotype (p>0.139). The most variants were rare and increasing the number of individuals within the populations, to cover the full phenotypic spectrum, may lead to different results. Not being able to obtain the nucleotide sequence of the promotor region, further analysis is required since it is an important region for gene expression, which could explain phenotype variation for leaf shape in Arabidopsis lyrata.

A Síndrome de Marfan (SMF) é uma doença de caráter autossômico dominante que acomete o tecido conjuntivo. As principais manifestações clínicas afetam o sistema cardiovascular, ótico e ósseo. A SMF é causada por mutações no gene FBN1, que codifica a proteína extracelular fibrilina-1, componente principal das microfibrilas, que formam as fibras elásticas. Estudos mostraram que mutações no gene da fibrilina-1 levam a um aumento indiscriminado do TGF-Β ativo na matriz, que resulta nos principais fenótipos da doença. O Sistema Renina Angiotensina (RAS) tem como produto principal a Angiotensina II (Ang-II), envolvida na regulação da massa óssea e da atividade do TGF-Β. Estratégias terapêuticas para a SMF utilizando fármacos que agem no RAS têm sido alvos de estudos em modelos animais. Em camundongos, Ramipril, inibidor da ACE (Angiotensin-converting enzyme inhibitor, ACEi), aumenta a transcrição do gene Fbn1 em 35% e melhora a cifose, característica do fenótipo ósseo no modelo animal mgΔloxPneo. O mecanismo de ação do Ramipril no sistema ósseo ainda não está totalmente elucidado, sendo que pode agir por diminuição na produção de Ang-II e consequente diminuição nos níveis de TGF-Β, ou pela inibição de degradação da bradicinina (BK) pela Ang-II. A bradicinina ativa diretamente seu receptor B2R, que induz ações fisiológicas opostas às da Ang-II. O objetivo deste trabalho foi avaliar e compreender os mecanismos gerais da patogênese óssea do modelo murino mgΔloxPneo para a SMF. Para tanto, foi analisado o fenótipo ósseo dos animais mgΔloxPneo e selvagens controle e tratados com Ramipril. Foi verificado que, além da cifose, os animais mutantes apresentaram pior estrutura óssea. O tratamento melhorou a cifose, porém não alterou a qualidade óssea dos animais mutantes. Portanto, o efeito benéfico do Ramipril na cifose dos animais mgΔloxPneo não se deve a uma melhora da estrutura óssea, e pode estar relacionado à integridade do ligamento que sustenta a coluna vertebral. Com o intuito de testar a hipótese de que a sinalização pelo receptor B2R da BK possa estar envolvida no desenvolvimento do fenótipo ósseo dos animais mgΔloxPneo, foram gerados animais mgΔloxPneo e knockout para o receptor B2R. Os resultados mostram que o receptor B2R não interfere no desenvolvimento da cifose, sendo apenas o genótipo para Fbn1 o fator determinante para a manifestação desse fenótipo. Foi realizada a análise de RNA-seq para verificar genes e vias diferencialmente expressas que possam explicar o mecanismo de desenvolvimento do fenótipo ósseo dos animais mgΔloxPneo. Foram encontradas vias como da adesão focal, interação receptor-meio extracelular (MEC), junção ocludente, reparação por excisão de nucleotídeo e de reparação missmatch, que podem explicar alterações no metabolismo de células ósseas. Além disso, foram encontradas diferenças de expressão de genes relacionados ao metabolismo muscular esquelético, o que está de acordo com a hipótese de regulação parácrina entre o tecido muscular e ósseo, levando a uma pior estrutura óssea / Marfan syndrome (MFS) is an autosomal dominant disease that affects the connective tissue. The main clinical manifestations affect the cardiovascular, optical and bone systems. MFS is caused by mutations in the FBN1 gene, that encodes the extracellular protein fibrillin-1, a major component of microfibrils, which form elastic fibers. Studies have shown that mutations in the fibrillin-1 gene lead to an indiscriminate increase in active TGF-Β in the matrix, which results in the major phenotypes of the disease. The Renin Angiotensin System (RAS) has as its main product Angiotensin II (Ang-II), involved in bone mass regulation and TGF-Β activity. Therapeutic strategies using drugs targeting the RAS have been studied in animal models. Ramipril, an ACE inhibitor (Angiotensin-converting enzyme inhibitor, ACEi), increase Fbn1 gene expression in 35% and improve kyphosis index in the mgΔloxPneo mouse model for SMF. Its mechanism of action in bone tissue is not completely elucidated, and it may act by decreasing Ang-II production and consequent reduction in TGF-Β levels, or by inhibiting degradation of bradykinin (BK) by Ang II. BK directly activates its B2R receptor, which induces opposite physiological actions to Ang-II. This study aims to evaluate and understand the general mechanisms of bone pathogenesis in the mgΔloxPneo mouse model. We analyzed the bone phenotype of mgΔloxPneo and wildtype animals treated, or not, with Ramipril by measuring the kyphosis index (KI), micro computed tomography (μCT) and Real-time PCR (RT-PCR). We found that mutant animals showed a greater degree of kyphosis and an altered bone structure. Ramipril improved kyphosis but did not alter bone quality of mutant animals, while in wild type animals Ramipril decreased bone structure without altering KI. Therefore, the beneficial effect of Ramipril on mgΔloxPneo animals\' kyphosis is not due to an improvement in bone structure. In order to test the hypothesis where signaling through BK B2R receptor may be involved in the development of bone phenotype of mgΔloxPneo animals, a mouse model with the mgΔloxPneo mutation and knockout for B2R receptor was generated. The analysis of these animals show that the B2R receptor does not interfere with the development of kyphosis, with Fbn1 genotype as sole determinant for this phenotype manifestation. RNA-seq analysis was performed to verify differential expression of genes and altered cellular pathways, which could reveal mechanisms of bone phenotype development in mgΔloxPneo animals. Altered pathways found included focal adhesion, receptor- extracellular matrix (ECM) interaction, tight junction, nucleotide excision repair and missmatch repair, which may explain changes in bone cells metabolism. In addition, there were differences in gene expression related to skeletal muscle metabolism, which is in agreement with the paracrine regulation of bone and muscle tissue, leading to worst bone structure

A doença renal crônica (DRC) está associada com inibição de sistemas enzimáticos e de transportadores de fármacos. O nebivolol, um bloqueador de terceira geração, seletivo para receptores 1 adrenérgicos e com atividade vasodiladora, é metabolizado principalmente por hidroxilação aromática dependente do CYP2D6, hidroxilação alicíclica e por glicuronidação. O estudo avalia a influência da DRC estágios 3 e 4 e da hemodiálise na farmacodinâmica e na farmacocinética dos isômeros do nebivolol. Os pacientes investigados divididos nos Grupos controle (n=12), DRC estágios 3 e 4 (n=12) e Hemodiálise (n=11) receberam dose única p.o. de 10 mg de nebivolol racêmico. As amostras seriadas de sangue foram coletadas até 48h após a administração do fármaco. Em cada tempo de colheita de sangue, a frequência cardíaca foi avaliada na situação de exercício isométrico durante 2 min com o handgrip, a 30% da contratilidade voluntária máxima. Todos os pacientes foram fenotipados como metabolizadores rápidos do metoprolol, exceto um paciente do Grupo controle fenotipado como metabolizador lento. Os isômeros do nebivolol foram analisados em plasma como concentração total empregando LC-MS/MS e coluna de fase quiral. O método foi linear no intervalo de concentrações de 25-2500 pg de cada isômero do nebivolol/mL de plasma. Os parâmetros farmacocinéticos foram calculados empregando o programa WinNonlin. O teste de Wilcoxon foi empregado para avaliar as razões isoméricas diferentes da unidade (p<0,05) e o teste de Kruskal-Wallis foi empregado para comparar os parâmetros farmacocinéticos entre os três Grupos investigados. A disposição cinética do nebivolol nos pacientes do Grupo controle é enantiosseletiva com acúmulo plasmático (Cmax 1,32 vs 0,88 ng/mL e AUC0-¥ 8,02 vs 4,25 ng.h/mL), menores valores de clearance oral aparente (623,58 vs 1176,40 L/h) e menores valores de volume aparente de distribuição (5383,30 e 6397,70 L) para o isômero lnebivolol. Os parâmetros farmacocinéticos do l-nebivolol e d-nebivolol para os pacientes do Grupo DRC (n=12) permitem inferir, à semelhança do Grupo controle, maiores valores de Cmax e AUC (Cmax 2,40 vs 1,67 ng/mL e AUC0- 10,20 vs 8,37 ng.h/mL) e menores valores de clearance oral aparente (491,51 vs 604,58 L/h) e de volume aparente de distribuição (3527,00 e 5232,50 L) para o isômero l-nebivolol. Semelhante aos Grupos controle e DRC, o Grupo Hemodiálise também apresenta enantiosseletividade com acúmulo acúmulo plasmático (Cmax 1,35 vs 0,78 ng/mL e AUC0- 6,74 vs 4,50 ng.h/mL), menores valores de clearance oral aparente (742,26 vs 1112,10 L/h) e menores valores de volume aparente de distribuição (5704,70 vs 9477,10 L) para o isômero l-nebivolol. A farmacocinética dos isômeros do nebivolol no paciente investigado do Grupo controle, fenotipado como metabolizador lento, difere dos dados apresentados para os pacientes do Grupo controle (n=11), Grupo DRC (n=12) e Grupo Hemodiálise (n=11), fenotipados como metabolizadores rápidos, com observação de razão isomérica de AUC l/d de 4,77 e redução nos valores de clearance oral aparente de ambos os isômeros do nebivolol (67,24 vs 122,07 L/h, respectivamente para os isômeros l-nebivolol e d-nebivolol). Concluindo, a DRC estágios 3 e 4 e a Hemodiálise não alteram a farmacocinética de ambos os isômeros do nebivolol, o volume aparente de distribuição de ambos os isômeros do nebivolol não mostra correlação com o peso dos pacientes, assim como os valores de clearance aparente de ambos os isômeros não mostram correlação com o peso ou com os valores de clearance da creatinina dos pacientes investigados. No entanto, os valores de clearance aparente de ambos os isômeros do nebivolol mostram correlação significativa com a atividade do CYP2D6 avaliada através da RMplasma ix metoprolol/-hidroximetoprolol. A análise PK-PD foi realizada incluindo os 34 pacientes fenotipados como metabolizadores extensivos. O modelo Emax inibitório descreveu a análise PK-PD empregando a variação da frequência cardíaca como parâmetro farmacodinâmico em função das concentrações plasmáticas do d-nebivolol, resultando em valores de Emax de 15,42 bpm e de EC50 de 2,26 ng/mL, seguindo a administração de dose única oral de 10 mg de nebivolol racêmico. / Chronic kidney disease (CKD) is related to inhibition of enzyme systems and drug transporters. Nebivolol, a third generation -blocker, is a selective 1-adrenoceptor antagonist and has vasodilatory properties. It undergoes aromatic hydroxylation through the CYP2D6, alicyclic hydroxylation and glucuronidation. The study evaluates the influence of CKD stages 3 and 4 and hemodialysis on the pharmacodynamics and pharmacokinetics of nebivolol isomers. The investigated patients were divided into 3 groups: control group (n = 12), CKD stages 3 and 4 (n = 12) and hemodialysis (n = 11). They received a single oral dose of 10 mg of racemic nebivolol and serial blood samples were collected up to 48h. At each time of blood sampling, heart rate was assessed in the situation of isometric exercise during 2 min with handgrip at 30% of maximal voluntary contractility. All patients were phenotyped as extensive metabolizers (EM) of metoprolol, except one patient in the control group phenotyped as poor metabolizer (PM). The isomers of nebivolol were analyzed in plasma samples by LC-MS/MS using a chiral phase column. The method was linear over the concentration range of 25-2500 pg of each isomer of nebivolol/mL of plasma. Pharmacokinetic parameters were calculated using the WinNonlin program. The Wilcoxon test was used to assess isomeric ratios different from unit (p <0.05) and the Kruskal-Wallis test was used to compare the pharmacokinetic parameters among the 3 groups investigated. The kinetic disposition of nebivolol was stereoselective in control group with plasma accumulation (Cmax 1.32 vs 0.88 ng/mL and AUC0 0- 8.02 vs 4.25 ng.h/mL), lower values of apparent clearance (623.58 vs 1176.40 L/h) and apparent volume of distribution (5383.30 and 6397.70 L) for the l-nebivolol isomer. The kinetic disposition of nebivolol was also stereoselective in CKD group (n=12) showing higher Cmax and AUC (Cmax 2.40 vs 1.67 ng/mL and AUC0- 10.20 vs 8.37 ng.h/mL) and lower values of apparent clearance (491.51 vs 604.08 L/h) and apparent volume of distribution (3527.00 vs 5232.50 L) for the l-nebivolol isomer. Similarly to CKD and control groups, the Hemodialysis Group showed stereoselectivity with plasma accumulation (Cmax 1.35 vs 0.78 ng/mL and AUC0- 6.74 vs 4.50 ng.h/mL), lower values of apparent clearance (742.26 vs 1112.10 L/h) and apparent volume of distribution (5704.70 vs 9477.10 L) for the l-nebivolol isomer. The pharmacokinetics of nebivolol isomers in the patient phenotyped as PM differed from the data presented to the patients phenotyped as EM, with observation of isomeric ratios AUC l/d of 4.77 and reduced values of apparent clearance of both nebivolol isomers (67.24 vs 122.07 L/h, respectively for l- and d-nebivolol). Concluding, CKD stages 3 and 4 and Hemodialysis did not alter the pharmacokinetics of both nebivolol isomers (Kruskal-Wallis test, p> 0.05), the apparent volume of distribution of both nebivolol isomers showed no correlation with the weight of the patients, the apparent clearance of both isomers also showed no correlation with the weight or with the creatinine clearance of the investigated patients. However, the values for apparent clearance for both nebivolol isomers showed a significant correlation with the CYP2D6 activity evaluated by the metabolic ratios plasma metoprolol/-hidroximetoprolol. PK-PD analysis was evaluated including all the investigated patients phenotyped as EM (n=34). The inhibitory Emax model described the PK-PD analysis using heart rate variation as a pharmacodynamic parameter plotted against the plasma concentrations of the isomer dxi nebivolol, showing Emax values of 15.42 bpm and EC50 of 2.26 ng/mL, following administration of a single oral dose of 10 mg of racemic nebivolol.

Existe um relevante interesse em pesquisar a ocorrência de polimorfismos no genoma bovino por diferentes motivos, e mais recentemente, com a finalidade de agregar mais informações ao estudo de características quantitativas visando selecionar animais geneticamente superiores com considerável valor comercial. Os polimorfismos de base única (SNPs) neste estudo foram identificados como RFLP/MboII e RFLP/HpaII sendo que o polimorfismo RFLP/MboII está situado no exon 6 do gene IGF2 (insulin-like growth factor 2), localizado no cromossomo 29 em bovinos, e desempenha um papel importante na proliferação e diferenciação celular para o crescimento e desenvolvimento dos mamíferos. O polimorfismo RFLP/HpaII encontra-se no elemento Bov-A2 (considerado um elemento SINE - Short Interspersed Nucleotide Element) presente na região promotora do gene CYP21 (Steroid 21-hydroxylase gene) no cromossomo 23 em bovinos. Para avaliar a ocorrência dos SNPs utilizou-se a técnica de PCR-RFLP em amostras de DNA a partir de sangue/sêmen de cerca de 300 animais bovinos das raças zebuínas Gir, Guzerá e Nelore. As frequências alélicas mostraram maior incidência do alelo T quando comparado ao C enquanto que as frequências genotípicas apresentaram alta ocorrência do heterozigoto TC em comparação aos homozigotos CC e TT para o polimorfismo IGF2 - RFLP/MboII. Com relação ao polimorfismo CYP21 RFLP/HpaII, a frequência alélica revelou alto valor do alelo T. A população encontrou-se em equilíbrio de Hardy-Weinberg para os SNPs estudados. Ferramentas de bioinformática foram utilizadas para investigações in silico revelando que os sítios polimórficos estão em regiões com potencial regulatório. A associação desses polimorfismos com DEPs das características reprodutivas e produtivas foram investigadas, entretanto mostrou-se significativas apenas para DP550 (IGF2 - RFLP/MboII) e DP450 (CYP21 - RFLP/HpaII). Os resultados obtidos sugerem que protocolos de Biologia Molecular in vitro podem ser usados para identificar novos marcadores moleculares, como SNPs funcionais adicionando informações que certamente contribuirão para estratégias de melhoramento dessas raças bovinas de grande importância para a produção de carne e leite em nosso país. Este foi o primeiro estudo sobre a ocorrência desses polimorfismos em raças zebuínas criadas no Brasil. / There is a considerable interest in researching the occurrence of polymorphisms in the bovine genome for different reasons, and more recently, in order to add more information to the study of quantitative traits to select genetically superior animals with considerable commercial value. The single nucleotide polymorphisms (SNPs) in this study were identified as RFLP/MboII and RFLP/HpaII polymorphisms being the RFLP/MboII is situated in exon 6 of the IGF2 gene (insulin-like growth factor 2), located on chromosome 29 in cattle, perform an important role in cell proliferation, differentiation for growth and in the development of mammals. Polymorphism RFLP/HpaII is the element Bov-A2 (considered an element SINE - Short Interspersed Nucleotide Element) present in the promoter region of CYP21 gene (Steroid 21-hydroxylase gene) on chromosome 23 in cattle. To evaluate the occurrence of SNPs, we used the PCR-RFLP method on DNA samples from blood/semen of about 300 cattle breeds from Zebu Gyr, Guzerat and Nellore. The allele frequencies showed a higher incidence of T allele compared to C while the genotype frequencies showed high incidence of heterozygous CT compared to CC and TT homozygous for the IGF2 polymorphism - RFLP/MboII. On the subject of the CYP21 polymorphism - RFLP/HpaII, the allele frequency showed high value T. The population was found in Hardy-Weinberg equilibrium for the SNPs studied. Bioinformatics tools, used for in silico investigations, revealed that the polymorphic sites are in regions with regulatory potential. The association of these polymorphisms with EPDs of reproductive and productive traits were investigated, but proved to be significant only for DP550 (IGF2 - RFLP/MboII) and DP450 (CYP21 - RFLP/HpaII). The results suggest that protocols of molecular biology in vitro can be used to identify new molecular markers, such as functional SNPs adding information that certainly will contribute to the improvement strategies of these breeds of great importance for the production of meat and milk in our country. It has been the first study on the occurrence of these polymorphisms in Zebu breeds raised in Brazil.

INTRODUÇÃO: O grau histológico é um dos principais fatores prognósticos em câncer de mama. Entretanto, os carcinomas pouco diferenciados ainda constituem um grupo bastante heterogêneo de tumores, pois podem corresponder a qualquer um dos subgrupos segundo a classificação genética: basal-símile, HER2, luminal ou mama-normal. OBJETIVOS: Neste estudo nós analisamos a freqüência dos perfis imunoistoquímicos básicos quanto à expressão de receptores de estrógeno e progesterona e HER2 em carcinomas de mama com menos de 10% de formação tubular e suas relações com fatores prognósticos clássicos e com a expressão de p63, c- Kit, EGFR, VEGF-A e citoqueratinas basais e luminais. MATERIAL E MÉTODOS: Este estudo retrospectivo incluiu 134 carcinomas de mama selecionados dos arquivos da Divisão de Patologia Cirúrgica da Faculdade de Medicina da Universidade de São Paulo, de 2000 a 2003. Os tumores foram revistos e classificados quanto à expressão imunoistoquímica de receptor de estrogênio, receptor de progesterona e HER2 nos perfis luminal (receptores hormonais positivos), HER2 (HER2 positivo e receptores hormonais negativos) e triplo-negativo. O subgrupo luminal foi subdividido quanto à expressão ou não de HER2. O exame imunoistoquímico para p63, citoqueratinas basais 5, 14 e 17,VEGF-A, EGFR, c-Kit e citoqueratinas luminais 8/18 foram feitos em bloco construídos pela técnica de arranjo em matriz de amostras teciduais. As variáveis prognósticas clássicas: idade da paciente, tamanho do tumor, estado linfonodal, grau nuclear, necrose tumoral, contagem mitótica e embolização vascular linfática, foram comparadas nos diferentes subgrupos. RESULTADOS: Os carcinomas mamários invasivos pouco diferenciados confirmaram sua heterogeneidade. O subtipo luminal foi o mais freqüente (73 casos), seguido pelo subtipo triplonegativo (39 casos) e do subtipo HER2 (22 casos). O subgrupo triplonegativo apresentou a maior proporção de tumores maiores que 2,0 cm , maior freqüência de necrose e a maior taxa de expressão de citoqueratinas basais. Este subgrupo juntamente com o subgrupo HER2 expressaram maiores índices de proliferação celular. O subgrupo HER2 mostrou maior número de casos associados à presença de componente in situ e maior freqüência de detecções de embolização vascular linfática. O subgrupo luminal apresentou o maior número de casos com expressão de citoqueratinas luminais 8/18 e o menor número de tumores maiores que 2,0 cm. Quanto à expressão de citoqueratinas basais, 81 casos não as expressaram e 53 expressaram pelo menos uma delas, a maioria destes casos pertencentes ao subgrupo triplo-negativo. O fenótipo triplo-negativo esteve intimamente relacionado à expressão de citoqueratinas basais. Dentre todas as variáveis estudadas, o único fator preditivo independente associado ao perfil triplo-negativo foi a expressão de citoqueratinas basais (p<0.0001) e o único fator preditivo independente associado à expressão de citoqueratinas basais foi a presença do fenótipo triplo-negativo (p<0,0001). O subgrupo basal-símile (tumores triplo-negativo com expressão de pelo menos uma citoqueratina basal) mostrou a maior freqüência de tumores > 2,0 cm e conjuntamente com o subgrupo triplo-negativo não basal-símile, a maior freqüência de tumores com presença de necrose e com alta contagem mitótica. A comparação entre os carcinomas HER2(+) quanto à expressão de receptores hormonais mostrou maiores indicadores de atividade proliferativa no grupo de receptores hormonais (-). Por outro lado, tumores positivos para os receptores de estrógeno e/ou progesterona com o sem expressão HER2 não mostraram qualquer diferença. CONCLUSÕES: O subgrupo de tumores com receptores hormonais positivos foi o mais freqüente e com características morfológicas e imunoistoquímicas mais favoráveis que os outros subgrupos, independente da expressão de HER2, sugerindo que carcinomas de mama pouco diferenciados com expressão de pelo menos um dos receptores hormonais correspondem ao subtipo luminal B genético. Os carcinomas pouco diferenciados com imunofenótipo triplonegativo e HER2(+) são similares quanto às características morfológicas associadas à doença com potencial agressivo. No entanto, a expressão das citoqueratinas basais diferenciam os dois subgrupos, e pode sugerir o fenótipo basal-símile e o padrão de doença associado a este fenótipo. Nosso estudo mostrou que entre os carcinomas de mama pouco diferenciados de mama, o painel imunoistoquímico clássico (receptores hormonais e HER2) associado à expressão de pelo menos uma das citoqueratinas basais permitem a identificação dos distintos subtipos, equivalentes aos vistos na classificação genética / INTRODUCTION: Histological grade is one of the main prognostic factors for breast cancer. However, poorly differentiated carcinomas still make up a quite heterogeneous group of tumors since they can belong to any of the subgroups according to genetic classification: basal-like, HER2, luminal or normal breast. OBJECTIVES: In this study we have analyzed the frequency of the basic immunohistochemical profiles as for the expression of estrogen and progesterone receptors and HER2 in breast cancer with less than 10% of tubular formation, and their relation with classic prognostic factors, expression of p63, c-Kit, EGFR, VEGF-A, basal and luminal cytokeratins. MATERIAL AND METHODS: This retrospective study included 134 breast carcinomas dated between 2000 and 2003, selected from the files from the Division of Surgical Pathology, University of São Paulo Medical School. All slides were revised and classified according to immunohistochemical expression of estrogen receptor, progesterone receptors and HER2 in luminal (positive hormonal receptors), HER2 (positive HER2 and negative hormonal receptors) and triple-negative. The luminal subgroup was further divided according to expression or absence of HER2. Immunohistochemical marking for p63, cytokeratins 5, 14 and 17 and 8/18, VEGF-A, EGFR, c-Kit was done through tissue microarray. The classical prognostic variables: age, tumor size, lymph node state, nuclear grade, tumor necrosis, mitotic count and lymphatic vascular embolization were compared among the different subgroups. RESULTS: The poorly differentiated invasive breast carcinomas confirmed their heterogeneity. The luminal subtype was the most frequent (73 cases), followed by the triple-negative (39 cases) and HER2 (22 cases). The triple-negative subgroup presented the highest proportion of tumors bigger than 2.0 cm, necrosis and higher basal cytokeratin rate expression. This subgroup as well as HER2 subgroup expressed higher cellular proliferation scores. The HER2 subgroup presented the highest number of cases associated with in situ component and higher frequency of lymphatic vascular embolization detection. The luminal subgroup presented the highest expression of luminal cytokeratins 8/18 and the least number of tumors larger than 2.0 cm. As for basal cytokeratin expression, 81 cases did not present any cytokeratin at all and 53 at least one, most of them belonging to the triple-negative subgroup. The triple-negative phenotype was intimately related to the expression of basal cytokeratins. Among all the variables studied, the only independent predictive factor associated with triple-negative profile was the presence of basal cytokeratin (p<0.0001), and the only independent predictive factor associated with basal cytokeratin expression was the presence of triple-negative phenotype (p<0.0001). The basal like subgroup (triple-negative tumors with the expression of at least one basal cytokeratin) showed the highest frequency of tumors larger than 2.0 cm, and together with the non-basal like, the highest frequency of tumors with necrosis and high mitotic count. The comparison between HER2 (+) carcinomas as far as hormonal receptors, showed higher proliferative activity in the group of negative hormonal receptors. On the other hand, tumors positive for estrogen and/or progesterone receptors with or without HER2 expression did not present any difference. CONCLUSIONS: The tumor subgroup with positive hormonal receptors was the most frequent and with more favorable morphological and imunohistochemical characteristics than the other subgroups, independent of HER2 expression, suggesting poorly differentiated breast carcinomas with the expression of at least one of the hormonal receptors correspond to luminal B genetic subgroup. The poorly differentiated carcinomas with triple-negative and HER2(+) immunophenotype are similar in relation to morphological characteristics associated to the disease with aggressive potential. However, the expression of basal cytokeratins differs the two subgroups and can suggest the basal-like phenotype and the disease evolution associated with it. Our study showed that among poorly differentiated breast carcinomas, the classic immunohistochemical panel (hormonal receptors and HER2) associated with the expression of at least one of the basal cytokeratins, allow the identification of the specific subtypes, similar to the ones seen in genetic classification