Mother-to-child transmission of HIV is the leading cause of pediatric AIDS; however, mechanisms by which HIV compromises pregnancy are not understood. CD4+CD25+ T-regulatory (Treg) cells play a role in pregnancy maintenance. RNA from early and late gestation placentas and fetuses from FIV-infected and control cats were probed for expression of FIV gag and Treg markers CD25, FOXP3, and CTLA4, using real time reverse-transcriptase (RT)-PCR. High rates of vertical transmission and reproductive failure were detected in early and late pregnancy. In control animals, both FOXP3 and CTLA4 expression decreased with gestational stage, indicating a natural decline in Tregs. Expression of FOXP3 and CTLA4 was decreased at early gestation in FIV-infected queens and a trend toward decreased expression of CD25, FOXP3, and CTLA4 in placentas from FIV-infected queens producing non-viable pregnancies was observed as well. Our results suggest that FIV infection may alter placental Treg function and adversely affect pregnancy outcome.

No description available.

El principal objectiu del treball de recerca d'aquesta tesi fou l'estudi de l'efecte del tractament dels espermatozoides caprins frescs sobre diferents paràmetres seminals i la seva relació amb els resultats de fecundació in vitro (FIV) d'oòcits de cabres prepúbers madurats in vitro.Per tal d'assolir aquest objectiu, es varen fixar els següents objectius concrets: 1) Estudiar l'efecte de diferents sistemes de rentat i selecció dels espermatozoides (swim-up, gradient discontinu de Percoll, rentat per centrifugació i centrifugació mitjançant una solució de Ficoll) sobre els resultats de FIV, 2) Estudiar l'efecte de la inducció in vitro de la capacitació espermàtica mitjançant diferents concentracions (5, 50 i 100 μg/mL) i temps de preincubació (1, 15, 30, 45 i 60 min) amb heparina, 3) Determinar la concentració òptima d'espermatozoides en el medi de fecundació, 4) Valorar la incidència de reacció acrosòmica espontània i la seva relació amb la taxa de penetració dels oòcits de cabra i la seva divisió embrionària i 5) Analitzar l'evolució de la motilitat, viabilitat i integritat de l'acrosoma dels espermatozoides durant las diferents fases de la FIV i la seva relació amb els resultats de fecundació. Els anàlisis seminals realitzats en aquest treball van consistir en l'anàlisi subjectiu de la motilitat individual dels espermatozoides, la viabilitat d'aquests mitjançant la tinció d'eosina-nigrosina, la incidència de reacció acrosòmica verdadera mitjançant la Triple Tinció i la integritat de l'acrosoma mitjançant l'ús de la lectina Pisum Sativum marcada amb fluoresceïna durant tot el procés de la FIV.Els resultats obtinguts permeteren concloure que en les nostres condicions: 1) No és necessari la realització de tècniques de rentat i selecció dels espermatozoides frescs procedents de bocs de provada fertilitat per a l'obtenció d'embrions a partir de oòcits madurats i fecundats in vitro. 2) La utilització d'heparina per a induir la capacitació espermàtica incrementa significativament els resultats de FIV, obtenint els millors resultats de divisió embrionària amb concentracions altes d'heparina (50 i 100 µg/mL) en el medi de pre-incubació dels espermatozoides, no observant diferències significatives en els resultats de FIV antre els diferents temps de pre-incubació. 3) Una concentració de 4x106 de espermatozoides viables/mL a les gotes de FIV proporciona els millors resultats de divisió embrionaria. 4) La incidència de la reacció acrosòmica espontània no està relacionada con la taxa de penetració, encara que es veu afavorida per la presencia dels complexes cumulus-oòcit, observant un increment en el nombre d'espermatozoides vius que pateixen dita reacció entre las 8-12 hores de co-cultiu de les gàmetes i 5) Els paràmetres seminals avaluats (motilitat, viabilitat i integritat de l'acrosoma) durant el procés de preparació dels espermatozoides frescs de boc no influeixen sobre els resultats de FIV de oòcits de cabres prepúbers madurats in vitro. / El principal objetivo del trabajo de investigación de esta tesis fue el estudio del efecto del tratamiento de los espermatozoides caprinos sobre distintos parámetros seminales y su relación con los resultados de fecundación in vitro (FIV) de ovocitos de cabras prepúberes madurados in vitro.Para ello se fijaron los siguientes objetivos concretos: 1) Estudiar el efecto de diferentes sistemas de lavado y selección de los espermatozoides frescos (swim-up, gradiente discontinuo de Percoll, lavado mediante centrifugación y centrifugación mediante una solución de Ficoll) sobre los resultados de FIV, 2) Estudiar el efecto de la inducción in vitro de la capacitación espermática mediante distintas concentraciones (5, 50 y 100 μg/mL) y tiempos de preincubación (1, 15, 30, 45 y 60 min) con heparina, 3) Determinar la concentración óptima de espermatozoides en el medio de fecundación, 4) Valorar la incidencia de reacción acrosómica espontánea y su relación con la tasa de penetración de los ovocitos de cabra y división embrionaria y 5) Analizar la evolución de la motilidad, viabilidad e integridad del acrosoma de los espermatozoides durante las distintas fases de la FIV y su relación con los resultados de fecundación. Los análisis seminales realizados en este trabajo consistieron en el análisis subjetivo de la motilidad individual de los espermatozoides, la viabilidad de éstos mediante la tinción de eosina-nigrosina, la incidencia de reacción acrosómica verdadera mediante la Triple Tinción y la integridad del acrosoma mediante el uso de la lectina Pisum Sativum marcada con fluoresceína a lo largo de todo el proceso de la FIV.Los resultados obtenidos nos permitieron concluir que en nuestras condiciones: 1) No es necesario la realización de técnicas de lavado y selección de los espermatozoides frescos procedentes de machos cabríos de probada fertilidad para la obtención de embriones a partir de ovocitos madurados y fecundados in vitro. 2) La utilización de heparina para inducir la capacitación espermática incrementa significativamente los resultados de FIV, obteniéndose los mejores resultados de división embrionaria con concentraciones altas de heparina (50 y 100 µg/mL) en el medio de pre-incubación de los espermatozoides, sin observarse diferencias significativas en los resultados de FIV al utilizar diferentes tiempos de pre-incubación. 3) Una concentración de 4x106 de espermatozoides viables/mL en las gotas de FIV proporciona los mejores resultados de división embrionaria. 4) La incidencia de la reacción acrosómica espontánea no está relacionada con la tasa de penetración, aunque se ve favorecida por la presencia de los complejos cúmulos-ovocitos, observándose un incremento en el número de espermatozoides vivos que sufren dicha reacción entre las 8-12 horas del co-cultivo de los gametos y 5) Los parámetros seminales evaluados (motilidad, viabilidad e integridad del acrosoma) a lo largo del proceso de preparación de los espermatozoides caprinos frescos no influyen sobre los resultados de FIV de ovocitos de cabras prepúberes madurados in vitro. / The main aim of the research work of this thesis was to study the effect of the treatment of fresh caprine spermatozoa on different sperm parameters and its potential relationship to the in vitro fertilization (IVF) of in vitro matured (IVM) prepubertal goat oocytes. Therefore the following specific aims were fixed: 1) To study the effect of different methods of washing and selection of spermatozoa (swim-up, centrifugation in Percoll gradients, centrifugation and centrifugation in Ficoll medium) on the IVF of IVM oocytes from prepubertal goats. 2) To study the effect of the induction of sperm capacitation after exposure of spermatozoa to different concentrations (5, 50 and 100 μg/mL) and different exposure intervals (1, 15, 30, 45 and 60 min) of heparin. 3) To determine the optimal sperm concentration on the fertilization medium, 4) To assess the spontaneous acrosome reaction and its relationship with IVF results and 5) To analyze the sperm motility, viability and acrosome integrity evolution during the different phases of the IVF and its relation with the results of penetration and embryo cleveage rate.The different sperm parameters analysis were the subjective assessment of the percentage of sperm progressive motility, the viability of the spermatozoa evaluated by eosine-nigrosine stain, the incidence of spontaneous acrosome reaction using the simplified triple stain technique or TST and the evaluation of the acrosome status using Pisum Sativum lectin labelled with FITC along the IVF process.The obtained results allowed to us to conclude that in ours conditions: 1) washing and selection methods of fresh spermatozoa from male of tested fertility are not necessary for obtaining embryos from in vitro matured and fertilized oocytes. 2) Using heparin to induce the in vitro sperm capacitation increase significantly the IVF results, getting the best percentages of cleavage rate with high concentrations of heparin (50 and 100 µg/mL) in the sperm pre-incubation medium, not observing significant differences on IVF percentages among the different exposure intervals of preincubation. 3) A concentration of 4x106 of viable sperm/mL in the IVF microdrop gives the best cleavage rate. 4) The incidence of spontaneous acrosome reaction is not related to the penetration rate, although is facilitated by the presence of cumulus-oocyte complex, observing an increase on the live acrosome reacted sperm between 8-12 hours post-insemination and 5) the evaluated sperm parameters (motility, viability and acrosome integrity) along the sperm treatment don't influence on IVF of goat prepubertal in vitro matured oocytes.

Para avaliar a encefalopatia em gatos infectados experimentalmente pelo vírus da imunodeficiência dos felinos, foram obtidas amostras de encéfalo após necropsia de nove gatos previamente inoculados pelo subtipo B do vírus e monitorados por quatro anos. As amostras foram fixadas em formalina 10% para coloração em Hematoxilina-eosina, e em metacarn para avaliação análise imunohistoquímica. Através da técnica de imunohistoquímica, as lâminas de encéfalo foram incubadas com anticorpos específicos para os antígenos Proteína Glial Fibrilar Ácida (GFAP) e Vimentina para marcação de astrócitos e com anticorpos para a proteína p24 do capsídeo viral do FIV. Nas lâminas marcadas para GFAP foram observados astrócitos em substância branca e cinzenta em quantidade moderada, sugerindo astrocitose reativa. Houve marcação mais evidente da região subpial e em alguns animais das regiões perivasculares. A marcação para vimentina mostrou raras células distribuídas pelo neurópilo e forte marcação de astrócitos na região subependimária nos ventrículos laterais e IV ventrículo, o que pode indicar um aumento da proliferação e migração de células tronco. Também foram observadas alterações como: nódulos gliais, vacuolização da substância branca, satelitose e focos de calcificação de meninge. A satelitose indica a presença de processo degenerativo em desenvolvimento. A marcação para a proteína p24 confirmou a presença de células microgliais infectadas. Raros astrócitos com marcação citoplasmática foram vistos em apenas um animal. Curiosamente, houve marcação dos neurônios da camada granulosa do cerebelo. Esta técnica deve ser mais bem explorada para o uso em diagnóstico post mortem. Esses resultados sugerem que os animais infectados experimentalmente pelo FIV subtipo B apresentam alterações microscópicas mesmo na ausência de sinais clínicos neurológicos. / To evaluate the encephalopathy in cats experimentally infected with feline immunodeficiency vírus, were obtained brain samples from autopsies of nine cats previously inoculated with subtype B virus and monitored for four years. The samples were fixed in 10% formalin for hematoxylin-eosin staining, and in metacarn for immunohistochemical analysis. Throuh the immunohistochemistry technique, the brain slides were incubated with antibodies against the glial fibrillary acidic protein (GFAP) and vimentin to mark astrocytes and against the viral capsid protein p24 of FIV. On the GFAP marked slides, astrocytes were observed in gray and white matter in moderate amounts, suggesting reactive astrocytosis. There was evident reactivity in the subpial area and in some animals, around blood vessels. The markup for vimentin showed rare cells distributed throughout the neuropil, and strong labeling of astrocytes in the subependymal region of the lateral ventricules and fourth ventricule, which may indicate an increased rate of proliferation and migration of stem cells. The histopathological changes observed were: glial nodules, white matter vacuolization, sattelitosis and foci of meningial calcification. The sattelitosis indicates the presence of degenerative process in developing. The use of antibodies against the protein p24 confirmend the presence of infected microglia in the brain of infected animals. Rare astrocytes with cytoplasmic staining were seen in only one animal. Interestling, there was labeling of neurons in the granular layer of the cerebellum. This technique should be further exploited for use in post-mortem diagnosis. These results suggest that animals experimentally infected with FIV subtype B show microscopic changes in the absence of clinical neurologic signs.

A parede celular de levedura (PCL) parece exercer importante papel na modulação da microbiota intestinal. Sua inclusão pode inibir a colonização por bactérias patogênicas e resultar em possíveis benefícios à saúde intestinal. O vírus da imunodeficiência felina (FIV) apresenta relativa disseminação ambiental e, frente ao crescente número de gatos domiciliados no Brasil, reconhecer alterações microbiológicas dos animais acometidos por este vírus é de grande importância. Neste sentido, o presente trabalho objetivou avaliar os efeitos da ingestão de PCL na composição da microbiota fecal de gatos saudáveis e de gatos com FIV pelo emprego da técnica de sequenciamento Illumina. Foram utilizados 19 gatos adultos, distribuídos em dois grupos experimentais: GS (10 animais saudáveis) e GI (9 animais infectados naturalmente pelo FIV); e duas dietas experimentais: D0 (dieta controle, sem adição de PCL) e D4 (dieta teste com adição de 0,4% de PCL), totalizando quatro tratamentos (GSD0, GSD4, GID0 e GID4), em delineamento inteiramente casualizado (DIC). Os resultados apresentaram como mais abundantes os filos Firmicutes e Actinobacteria; as ordens Clostridiales e Coriobacteriales; a família Veillonellaceae; e o gênero Megasphaera spp.. O GS apresentou menor concentração de Fusobacteria (p=0,0037), Clostridiales (p=0,0001) e Fusobacteriales (p=0,0330), maior de Coriobacteriales (p<0,0001), Collinsella spp. (p=0,0135) e Peptococcus spp. (p=0,0265) em comparação ao GI. O consumo da PCL reduziu a porcentagem de Bacteroidetes (p =0,0421) e Catenibacterium spp. (p=0,0252). No GS, o consumo da PCL aumentou a concentração de Actinobacteria (p<0,0001) e Bifidobacterium spp. (p=0,0420) e, diminuiu a de Firmicutes (p=0,0205), Aeromonadales (p=0,0027), Dialister spp. (p=0,0137), Megasphaera spp. (p=0,0005) e Anaerobiospirillum spp. (p=0,0084). No GI, o consumo de PCL aumentou Proteobacteria (p=0,0005), Lactobacillales (p=0,0095), Aeromonadales (p=0,0027), Streptococcus spp (p=0,008) e Anaerobiospirillum spp. (p=0,0084) e reduziu Bifidobacterium spp. (p=0,0420). Com relação aos coeficientes de digestibilidade aparente (CDA), o GI apresentou menor CDA da proteína bruta (p=0,0305) e extrativos não-nitrogenados (p=0,0078) e, maiores do extrato etéreo em hidrólise ácida (p<0,0001), em comparação ao GS. Não houve diferença na concentração de ácidos graxos de cadeia curta (AGCC) ou mesmo de ácido lático entre os tratamentos, grupos e doses. A modulação bacteriana exercida pelo consumo da PCL aparentemente é positiva, porém mais estudos se fazem necessários para fortalecer esta hipótese na espécie alvo e, principalmente, esclarecer as diferenças existentes em sua resposta entre gatos doentes e saudáveis. / The yeast cell wall (YCW) seems to play an important role on the modulation of the intestinal microbiota. Their inclusion can inhibit pathogenic bacteria colonization and possible result in benefits on intestinal health. The feline immunodeficiency virus (FIV) shows relative environmental dissemination and due to the growth of the population of resident cats in Brazil, acknowledge of microbiological changes in the affected FIV + animals are of great importance. Therefore, this study aims to evaluate the effects of YCW intake in the composition of fecal microbiota of healthy cats and cats affected by FIV (FIV+), and to evaluate the fecal bacterial composition of cats FIV+ and compare it with the fecal microbiota of healthy cats by the use of Illumina sequencing technique. For this we used 19 adult cats, distributed in two experimental groups: GH (10 healthy animals) and GI (9 animals naturally infected by FIV); and two experimental diets: D0 (control diet, without addition of YCW) and D4 (test diet with addition of 0.4% of YCW), total of four treatments (GSD0, GSD4, GID0 and GID4), in a randomized design. The results presented as the most abundant phyla Firmicutes and Actinobacteria; the orders Clostridial and Coriobacterial; the family Veillonellaceae; and the genus Megasphaera spp. GH showed lower concentrations of Fusobacteria (p=0.0037), Clostridiales (p=0.0001), and Fusobacteriales (p=0.0330), higher of Coriobacteriales (p<0.0001), Collinsella spp. (p=0.0135) and Peptococcus spp. (p=0.0265) compared to GI. The consumption of PCL reduced the percentage of Bacteroidetes (p=0.0421) and Catenibacterium spp. (p=0.0252). In GH, PCL consumption positively modulated the concentration of Actinobacteria (p<0.0001) and Bifidobacterium spp. (p=0.0420), negatively to Firmicutes (p=0.0205), Aeromonadales (p=0.0027), Dialister spp. (p=0.0137), Megasphaera spp. (p=0.0005) and Anaerobiospirillum spp. (p=0.0084). In the GI, PCL consumption positively modulated Proteobacteria (p=0.0005), Lactobacillales (p=0.0095), Aeromonadales (p=0.0027), Streptococcus spp. (p=0.008) and Anaerobiospirillum spp. (p=0.0084) and negatively Bifidobacterium spp. (p=0.0420). In relation to the apparent digestibility coefficients (ADC), the GI present lower ADC of the crude protein (p=0.0305) and extractive non-nitrogenous (p=0.0078) and higher ADC of the ethereal extract in acid hydrolysis (p<0.0001), compared to GS. There was no statistical difference between the concentration of short chain fatty acids (SCFA) or even lactic acid between the treatments, groups or doses. The bacterial modulation exerted by PCL consumption appears to be positive, but more studies are needed to strengthen this hypothesis in the species and to clarify the differences in its response between sick and healthy cats.

Para avaliar a encefalopatia em gatos infectados experimentalmente pelo vírus da imunodeficiência dos felinos, foram obtidas amostras de encéfalo após necropsia de nove gatos previamente inoculados pelo subtipo B do vírus e monitorados por quatro anos. As amostras foram fixadas em formalina 10% para coloração em Hematoxilina-eosina, e em metacarn para avaliação análise imunohistoquímica. Através da técnica de imunohistoquímica, as lâminas de encéfalo foram incubadas com anticorpos específicos para os antígenos Proteína Glial Fibrilar Ácida (GFAP) e Vimentina para marcação de astrócitos e com anticorpos para a proteína p24 do capsídeo viral do FIV. Nas lâminas marcadas para GFAP foram observados astrócitos em substância branca e cinzenta em quantidade moderada, sugerindo astrocitose reativa. Houve marcação mais evidente da região subpial e em alguns animais das regiões perivasculares. A marcação para vimentina mostrou raras células distribuídas pelo neurópilo e forte marcação de astrócitos na região subependimária nos ventrículos laterais e IV ventrículo, o que pode indicar um aumento da proliferação e migração de células tronco. Também foram observadas alterações como: nódulos gliais, vacuolização da substância branca, satelitose e focos de calcificação de meninge. A satelitose indica a presença de processo degenerativo em desenvolvimento. A marcação para a proteína p24 confirmou a presença de células microgliais infectadas. Raros astrócitos com marcação citoplasmática foram vistos em apenas um animal. Curiosamente, houve marcação dos neurônios da camada granulosa do cerebelo. Esta técnica deve ser mais bem explorada para o uso em diagnóstico post mortem. Esses resultados sugerem que os animais infectados experimentalmente pelo FIV subtipo B apresentam alterações microscópicas mesmo na ausência de sinais clínicos neurológicos. / To evaluate the encephalopathy in cats experimentally infected with feline immunodeficiency vírus, were obtained brain samples from autopsies of nine cats previously inoculated with subtype B virus and monitored for four years. The samples were fixed in 10% formalin for hematoxylin-eosin staining, and in metacarn for immunohistochemical analysis. Throuh the immunohistochemistry technique, the brain slides were incubated with antibodies against the glial fibrillary acidic protein (GFAP) and vimentin to mark astrocytes and against the viral capsid protein p24 of FIV. On the GFAP marked slides, astrocytes were observed in gray and white matter in moderate amounts, suggesting reactive astrocytosis. There was evident reactivity in the subpial area and in some animals, around blood vessels. The markup for vimentin showed rare cells distributed throughout the neuropil, and strong labeling of astrocytes in the subependymal region of the lateral ventricules and fourth ventricule, which may indicate an increased rate of proliferation and migration of stem cells. The histopathological changes observed were: glial nodules, white matter vacuolization, sattelitosis and foci of meningial calcification. The sattelitosis indicates the presence of degenerative process in developing. The use of antibodies against the protein p24 confirmend the presence of infected microglia in the brain of infected animals. Rare astrocytes with cytoplasmic staining were seen in only one animal. Interestling, there was labeling of neurons in the granular layer of the cerebellum. This technique should be further exploited for use in post-mortem diagnosis. These results suggest that animals experimentally infected with FIV subtype B show microscopic changes in the absence of clinical neurologic signs.

Les Vibrations Induites par Frottement (FIV) sont un phénomène complexe qui surgit chaque fois deux surfaces subissent un glissement relatif. Pendant les dernières décennies, une quantité significative de œuvres expérimentales et numériques a traité des Vibrations Induites par Frottement, tandis que la simulation de l'excitation dynamique de contacts frictionnels a été toujours un vrai défi dans beaucoup de domaines de recherche industrielles. Dans ce cadre de recherche, ce travail est adressé à l'analyse des Vibrations Induites par Frottement, en développant des analyses en même temps expérimentales et numériques ; on propose une nouvelle approche numérique pour reproduire l'excitation dynamique locale du contact et son effet sur la réponse vibrationnel du système, sans augmentation significative des coûts de calcul. Le système mécanique, l'objet de l'analyse, est composé par deux poutres en acier en contact frictionnel dans un mouvement relatif ; la dynamique simple du système tient compte de la distinction entre la réponse de dynamique du système et l'excitation à haut débit venant du contact. Une campagne expérimentale paramétrique a été conduite pour analyser les effets de trois paramètres de contact principaux (la vitesse de glissement, la charge normale et la rugosité superficielle) sur la réponse du système vibrationnel, c'est-à-dire sur les vibrations induites. En parallèle, un modèle numérique a été mis en œuvre pour reproduire l'excitation dynamique locale du contact et son effet sur la réponse vibrationnel du système. Une nouvelle loi de friction a été présentée dans le modèle, proposant l'utilisation d'un terme provoquant une perturbation dans le coefficient de frottement pour simuler les effets de l'excitation au contact. Les inclusions de l'excitation dynamique locale, en raison des phénomènes de contact, par le terme de perturbation du coefficient de frottement, permettent de reproduire correctement les Vibrations Induites par Frottement sans présenter une représentation de la topographie superficielle réelle, qui a besoin d'un grand nombre d'éléments, économisant donc le temps de calcul. Des signaux différents pour le terme provoquant la perturbation ont été testés pour simuler correctement les vibrations mesurées. L'évolution du terme provoquant la perturbation récupérée par une méthode inverse a surligné les contributions spectrales différentes de l'excitation locale du contact. La comparaison entre les Vibrations Induites par Frottement mesurées et ceux simulés numériquement a montré une bonne corrélation, validant la loi de frottement proposée. Finalement, l'effet d'un changement de rugosité e de vitesse de glissement a été aussi simulé numériquement et corrélé avec les résultats expérimentaux. / Friction-Induced Vibrations (FIV) are a complex phenomenon which arises each time two surfaces undergo relative sliding. During the last decades, a significant amount of experimental and numerical works dealt with Friction-Induced Vibrations, while the simulation of the dynamic excitation from frictional contacts has always been a real challenge to face in many industrial research areas. In this research framework, this work is addressed to the investigation of the Friction-Induced Vibrations, carrying on at the same time experimental and numerical analyses; a new numerical approach is proposed to reproduce the local dynamic excitation from the contact and its effect on the vibrational response of the system, without significant increase of the computational time costs. The mechanical system, object of the investigation, is composed by two steel beams in frictional contact during relative motion; the simple dynamics of the system allows for distinguishing between the dynamics response of the system and the broadband excitation coming from the contact. A parametrical experimental campaign has been conducted to analyse the effects of three main contact parameters (the relative sliding velocity, the normal load and the surface roughness) on the system vibrational response, i.e. on the induced vibrations. In parallel, a numerical model has been implemented to reproduce the local dynamic excitation from the contact and its effect on the vibrational response of the system. A new friction law has been introduced in the model, proposing the use of a perturbative term in the friction coefficient in order to simulate the effects of the contact excitation. The inclusions of the local dynamic excitation, due to the contact phenomena, by the perturbation term of the friction coefficient allows to correctly reproduce the Friction-Induced Vibrations without introducing a representation of the real surface topography, which usually needs a large number of elements, saving then computational time. Different signals for the perturbative term have been tested to simulate correctly the measured vibrations. The evolution of the perturbative term recovered by an inverse method allowed for highlighting the different spectral contributions of the local excitation coming from the contact. The comparison between the measured Friction-Induced Vibrations and the ones simulated numerically showed good correlation, validating the proposed friction law. Finally, the effect in a change of the sliding velocity and surface roughness have been simulated numerically too and correlated with experimental results.

O Brasil é o país que mais realiza aspiração folicular de oócitos guiada por ultrasonografia (OPU) para fins de produção in vitro de embriões bovinos. Com isso é possível aumentar consideravelmente o número de progênies por fêmea por ano. O objetivo desse trabalho foi identificar polimorfismos em genes envolvidos na foliculogênese e associar alterações gênicas com o número de folículos pré-antrais recrutados em bovinos, estimados pelo número de oócitos recuperados por OPU. Dados de aspiração folicular feitas a campo em fêmeas Nelore (Bos taurus indicus) foram obtidos de duas empresas diferentes, sendo que 30 fêmeas foram utilizadas para a prospecção de polimorfismos de nucleotídeos únicos (SNPs), 218 para estudos populacionais e dados de 193 fêmeas foram utilizados para a análise de efeitos das variações genéticas com o número de oócitos viáveis colhidos por OPU. Vinte fêmeas da raça Holandesa (Bos taurus taurus) foram genotipadas para a identificação de alelos taurinos ou zebuínos específicos. Regiões dos genes Gdf9, Fgf8, Fgf10 e BmprII foram amplificadas por meio da reação em cadeia da polimerase (PCR) e seqüenciadas para prospecção de SNPs. Quatro polimorfismos foram genotipados por meio de restrição enzimática de DNA, (PCR-RFLP; genes: Gdf9, Fgf8, Lhr e DNA mitocondrial-mtDNA) ou PCR em tempo real utilizando sondas específicas para cada alelo (BmprII). A avaliação de efeitos dos marcadores sobre a característica foi realizada por análise de variância utilizando modelos mistos com dados repetidos, em que o número de oócitos viáveis foi considerado variável dependente, as fêmeas foram consideradas variável aleatória, e local de aspiração, ano de aspiração e o SNP foram efeitos fixos. O mesmo modelo foi utilizado para estimar efeito médio de substituição alélica, em que o marcador foi substituído por uma co-variável numérica. A diferença entre médias de quadrados mínimos foram estimadas por meio de contrastes e a significância avaliada por teste t. Os efeitos ou as diferenças entre as médias foram consideradas significativas quando P<0,05. Foram identificados 19 SNPs sendo que 10 polimórficos entre as fêmeas Nelore, 04 causadores de substituição de aminoácidos na proteína. Entre os marcadores nucleares genotipados (Gdf9, A318C; Fgf8, C1027G; BmprII, A40048G; Lhr C62478T), todos apresentaram equilíbrio de segregação alélica dentro do marcador e genotípica entre os marcadores (P>0,05). Os SNPs nos genes Gdf9, BmprII e Lhr genotipados em fêmeas Nelore e Holandesas apresentaram-se fixados na segunda raça. Foram identificados efeitos dos polimorfismos dos genes Gdf9, Fgf8, BmprII e Lhr (P<0,05), entretanto os variantes de mtDNA não foram significativos. As diferenças entre médias confirmaram os resultados da ANOVA. Para os polimorfismos no gene Gdf9, BmprII e Lhr, observou-se a tendência de os indivíduos heterozigotos terem menores médias de oócitos viáveis quando comparados aos homozigotos, já o polimorfismo no gene Fgf8, observou-se a tendência de interação aditiva entre os alelos, em que foi estimado um efeito médio de 1,13±0,01 oócitos viáveis por troca de alelo C por G. Conclui-se que variações genéticas são causas de variabilidade do número de folículos presentes nos ovários, estimados por OPU. / Brazil is the country with the biggest number of ovum pick up (OPU) collection for bovine in vitro embryo production. This strategy is used to increase the number of calves born per cow per year. The objective of this work was to identify polymorphisms in genes related in folliculogenesis and associate some of the resulted gene variants with the number of pre antral follicle recruited in bovines, estimated by the number of oocytes collected by OPU. Records of open filed OPUs performed in Nelore cows (Bos taurus indicus) were obtained from two independent enterprises, and 30 females were used for single nucleotide polymorphism (SNP) prospection, 218 females for genotype and allelic population analysis and 193 females used for identification of genetic variation effects over the number of viable oocytes collected by OPU. Twenty Holstein cows (Bos taurus taurus) were also genotyped for identification of taurine and zebuine specific alleles. Segments of genes Gdf9, Fgf8, Fgf10 and BmprII were amplified by polymerase chain reaction (PCR) and sequenced for SNP prospection. Four polymorphisms were genotyped by means of enzymatic DNA digestion (PCR-RFLP, genes: Gdf9, Fgf8, Lhr and mitochondrial DNA - mtDNA) or real time PCR using allelic specific probes (Gene: BmprII). Genetic marker effect over the analyzed characteristic was realized by analysis of variance using mixed models with repeated data, in which the number of viable oocytes were set as dependent variable, the females were considered random variable, OPU place, OPU year and SNP were fixed effects. The same model was used for average allelic substitution effect estimation, where the genetic marker class was changed by a numeric covariant. The least square means (LSM) differences were estimated by contrasts and the significance evaluated by t test. Fixed effects or LSMs differences were considered significant when P<0.05. Nineteen SNPs were identified, from which ten were polymorphic among Nelore cows, four were expected to cause amino acid changes in protein. Among the nuclear genome markers genotyped (Gdf9, A318C; Fgf8, C1027G; BmprII, A40048G; Lhr C62478T, all resulted in allelic segregation equilibrium within marker and genotypic segregation equilibrium between markers (P>0.05). The SNPs in genes Gdf9, BmprII and Lhr genotyped in Nelore and Holstein cows were fixed in the last breed. SNPs in genes Gdf9, Fgf8, BmprII and Lhr affected the characteristic (P<0.05), however the mtDNA variants did not. The LSMs differences confirmed the ANOVA results. Heterozygote females for SNPs in genes Gdf9, BmprII and Lhr tend to have smaller LSMs than homozygous ones. On the other hand, there is indication of additive allelic interaction between alleles of SNP in gene Fgf8, for which an average allele substitution was estimated in 1.13±0.01 viable oocytes for a C/G change. We concluded that genetic variations were responsible for variable number of follicles present in ovaries, estimated by OPU.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-03-17T11:52:18Z No. of bitstreams: 2 Dissertação - Tárik Kassem Saidah - 2015.pdf: 2186450 bytes, checksum: ff29c97a86ff6cb315b6365052160a3f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-03-17T11:54:08Z (GMT) No. of bitstreams: 2 Dissertação - Tárik Kassem Saidah - 2015.pdf: 2186450 bytes, checksum: ff29c97a86ff6cb315b6365052160a3f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-03-17T11:54:08Z (GMT). No. of bitstreams: 2 Dissertação - Tárik Kassem Saidah - 2015.pdf: 2186450 bytes, checksum: ff29c97a86ff6cb315b6365052160a3f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-09-20 / Aims: To make a review article on ectopic pregnancy (EP) in high complexity fertilization, to define the epidemiological and reproductive profile of women carriers of EP, to evaluate the prevalence of EP after in vitro fertilization (IVF) and to verify if the transfer of frozen embryos reduces the rates of EP. METHODS: All fresh and frozen embryos transfers obtained through IVF via ICSI conducted between January 2007 and December 2014 were reviewed. Clinical pregnancies characterized by the visualization of an intrauterine gestational sac or ectopic pregnancy were included in the evaluation. Ectopic pregnancies were diagnosed by ultrasonography and / or laparoscopy. The prevalence of EP and the comparison of the rates between fresh and frozen embryos transfers were analyzed by logistic regression using the SPPS software version 15.0. Results: Of the 933 embryos obtained through IVF that resulted in clinical pregnancies, 19 cases of EP were found. The fertilization with fresh embryos resulted in 772 clinical pregnancies (group I), and 161 with frozen embryos (group II). The global prevalence of EP was 2.02%. When considered by group, group one had 16 EP that represented a rate of 2.1% and group two had a rate of 1.9%. There was a lower rate of ectopic pregnancy with frozen embryos in comparison to fresh embryos, although this did not demonstrate statistical significance (p=0.86)(OR=0.89, IC=0.258-3.11). CONCLUSIONS: The reproductive profile of patients that exhibit EP was of young female with young partner, good ovarian response and that had more than 3 embryos transferred. The prevalence of EP in this study was 2.02. It was observed that there was no significant difference in the occurrence of EP when both groups, frozen and fresh embryos, were compared. / Objetivos: fazer um artigo de revisão sobre gestação ectópica (GE) em fertilização de alta complexidade, definir o perfil epidemiológico e reprodutivo das mulheres portadores de GE, avaliar a prevalência de GE pós-fertilização de alta complexidade e verificar se a transferência de embriões congelados reduz as taxas de GE. Métodos: foram revisadas todas as transferências de embriões frescos e congelados realizadas através de fertilização in vitro pela técnica de injeção intracitoplasmática de espermatozoides no período compreendido entre janeiro de 2007 a dezembro de 2014. Foram incluídas as gestações clínicas, sendo consideradas as gestações com visualização de saco gestacional intraútero e gestações ectópicas. As gestações ectópicas foram diagnosticadas por ultrassonografia e/ou laparoscopia. A prevalência de ectópica e a comparação das taxas entre os grupos de embriões frescos e congelados foram verificadas por meio da análise de regressão logística utilizando o programa Software SPSS versão 15.0. Resultados: foram analisadas 933 fertilizações in vitro que resultaram em gestações clínicas e verificou-se 19 casos de gestações ectópicas. As fertilizações com embriões frescos totalizaram 772 (grupo I) e com embriões congelados 161 (grupo 2). A prevalência de gestação ectópica foi de 2,02 %. No grupo I ocorreram 16 gestações ectópicas representando uma taxa de 2,1 % das fertilizações, no grupo 2 a taxa foi de 1,9 %. Uma menor taxa de gestação ectópica com embriões congelados foi verificada em comparação com embriões frescos, porém, não apresentou significância estatística (p=0,86) (OR= 0,89, IC 0,258 – 3,11). Conclusões: o perfil reprodutivo das pacientes que apresentaram GE foi de mulheres jovens com parceiros jovens, com boa resposta ovariana e que tiveram mais de três embriões transferidos. A prevalência de GE verificada no presente estudo foi de 2,02 %. Observou-se que não houve diferença significativa quanto à GE quando foram comparadas as transferências de embriões frescos com os congelados.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder of which lung disease is the leading cause of morbidity and mortality. One attractive strategy for the treatment of CF lung disease is to directly deliver CF transmembrane conductance regulator gene to airway epithelia. Although promising results have been reported, barriers present in the lung make successful gene transfer to the respiratory tract difficult. In order to improve gene transfer strategies in the intrapulmonary airways, we need to identify the bottlenecks of transduction for the vector system. A previous study reported that feline immunodeficiency virus (FIV)-mediated gene transfer was more efficient in the nasal airways in mice than the intrapulmonary airways (Sinn, P.L. et al. 2008, J. Viol). Our first goal was to identify barriers to lentiviral gene transfer in the murine airways. We demonstrate that host immune response is not the major barrier preventing efficient FIV-mediated transduction in the intrapulmonary airways. We show that the FIV vector transduces murine primary nasal epithelial cell cultures with greater efficiency than murine primary tracheal epithelial cell cultures. In addition, GP64 pseudotyped vesicular stomatitis virus (VSV) transduces better in nasal epithelia compared to intrapulmonary airways in mice. On the other hand, we observed that VSVG glycoprotein-pseudotyped VSV transduces the intrapulmonary airway as well as nasal epithelia in mice with similar efficiency. Our results suggest that differentially expressed cellular factor(s) specific for GP64 or FIV vector may be the major barrier(s) for FIV vector-mediated gene transfer in the murine intrapulmonary airways. The recent development of CF porcine models prompted us to investigate possible barriers for lentiviral vector-mediated gene transfer in porcine cells. Our preliminary results showed that HIV transduction was restricted in porcine but not human lung-derived cell lines. Porcine TRIM5 has sequences similar to restrictive bovine TRIM5 orthologs. Therefore, our second goal was to investigate the possible restriction of lentiviral vectors by porcine TRIM5. We demonstrate that transient overexpression or knockdown of endogenously expressed porcine TRIM5 does not affect HIV or FIV transduction. Lastly, we characterized a mucin domain-deleted EBOV (EBOVΔO) glycoprotein mutant with increased transduction. This EBOVΔO 5-mer mutant was generated based on mutants with an increased transduction as identified by alanine scanning mutagenesis (Brindlay, M.A. et al. 2007. J. Viol). We show that VSV pseudotyped with the 5-mer mutant increased transduction both in vitro and in mice when compared to the wild-type EBOVΔO. Structural analysis demonstrated that 5 mutations were located proximal to the GP1-GP2 interface. Enhanced transduction likely results from a lower energy metastable state of the glycoprotein. FIV pseudotyped with 5-mer also shows increased transduction in multiple cell lines. Identification of barriers in intrapulmonary airways and improvements of vector systems will help the advancement of gene therapy for CF.