Les infections rickettsiales et la fièvre aiguë indifférenciée (FAI) sont des problèmes de santé publique importants sous les tropiques. Ce projet a été réalisé dans le but d'explorer les agents de rickettsies et autres étiologies de la FAI au Vietnam.Dans la première cohorte de 67 participants se concentrant sur les patients cliniquement suspectés de typhus des broussailles, le diagnostic de typhus des broussailles a été confirmé pour 32 patients (47,8%) par qPCR ou IFA. Parmi les 20 patients pour lesquels des escarres et du sang total ont été obtenus, 17 (85%) des échantillons d'escarre et 5 (25%) des échantillons de sang total étaient positifs pour O. tsutsugamushi par qPCR. L'analyse génétique des séquences du gène TSA 56-kDa a démontré que les 14 séquences étaient apparentées à 4 génotypes: Karp, Kawasaki, Gilliam (JG-v et TG-v) et TA716. Ensuite, une analyse prospective des étiologies de la FAI au Vietnam a été réalisée avec 378 patients adultes fébriles (FAIs) et 384 patients adultes fébriles (Contrôles). L'étiologie la plus courante était le virus de la grippe (20,9% dans les FAIs versus 0% dans les Contrôles), suivi des rickettsies (11,11% vs 0,26%), virus de la dengue (7,67% vs 0,52%), Leptospira (4,76% vs 0,78%), adénovirus (4,76% vs 1,04%) et entérovirus (2,12% vs 0%) (p<0,05).Par conséquent, de telles données sur les rickettsies et autres étiologies obtenues au Vietnam pourraient être très utiles pour l’établissement d’une méthode d’approche pour les patients atteints de FAI dans les aspects de diagnostic, de gestion et de prévention au Vietnam et dans les régions tropicales dans l'avenir. / Rickettsial infections and acute undifferentiated fever (AUF) are the important public health issues in the tropics. This project was performed with the aim to explore the rickettsial agents and other etiologies of AUF in Vietnam. In the first 67-participant cohort of patients who were clinically suspected of having scrub typhus, 32 (47.8%) patients were confirmed as scrub typhus by either qPCR or IFA. Among the 20 patients for whom both eschar and whole blood were obtained, 17 (85%) of the eschar specimens and 5 (25%) of the whole blood specimens tested positive for O. tsutsugamushi by qPCR. Genetic analysis of the 56-kDa TSA gene sequences demonstrated that the 14 sequences were related to 4 groups: Karp, Kawasaki, Gilliam (JG-v and TG-v) and TA716.Subsequently, a prospective case-control analysis of the etiologies of AUF in Vietnam was performed. A total of 378 febrile adult patients (AUFs) and 384 afebrile adult patients (Controls) were enrolled. The most common etiology was influenza virus (20.9% in AUFs versus 0% in Controls), followed by rickettsial agents (11.11% in AUFs versus 0.26% in Controls), dengue virus (7.67% vs 0.52%), Leptospira (4.76% vs 0.78%), adenovirus (4.76% vs 1.04%), and enterovirus (2.12% vs 0%) (p<0.05). Consequently, such data of rickettsial agents and other etiologies in Vietnam obtained from this project are extremely useful in guiding the diagnosis, management and preventive-health methods for patients with AUF in the future in Vietnam as well as in the tropics worldwide.

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Specialized skin scales (generation glands) and undifferentiated skin scales were compared in three species of cordylid lizard, viz. Cordy/us cordy/us, Pseudocordy/us micro/epidotus and P. capensis. These skin derivatives were histologically examined and compared, using data existing in literature (Maderson, 1966; 1967; 1968a) with similar structures in gekkonids. Histological descriptions of cordylid skin structure were the same as those shown for gekkonids. Three types of generation glands were identified in cordylids (Van Wyk & Mouton, 1992), and these proved to be different to those existing in gekkonid species. These three types of generation glands were further compared regarding their ecdysis histology, which proved to be identical, thus, these glands differ only on gross morphological structure. Skin of all three species was also compared with regard to ecdysis, and while the histology of all three was identical, shedding activity was shown to differ in all three species. P. micro/epidotus displayed dormancy in the germinative layer for much longer periods of the year than either of the other two species, while P. capensis displayed very little dormancy. C. cordy/us displayed an intermediate pattern of ecdysis activity and dormancy. Autoradiography was performed on all species in both ecdysis activity and dormancy periods, to compare mitotic activity patterns in these periods. Generation gland activity appeared to correlate well with known testosterone peaks in the testicular cycle in all three species. Asynchrony between generation glands and skin scales was shown in all three species. / AFRIKAANSE OPSOMMING: GENERASIEKLIERE EN VERVELLINGSIKLUSSE IN GORDELAKKEDISSE (SAURIA: CORDYLIDAE) Drie spesies gordelakkedisse, Gordy/us cordy/us, Pseudocordy/us micro/epidofus en P. capensis, se gespesialiseerde velskubbe (generasiekliere) en gewone velskubbe word vergelyk. Hierdie velskubbe word deur histologiese metodes geëksamineer en vergelyk, met gebruik van data wat reeds in die literatuur bestaan (Maderson, 1966; 1967; 1968a), met soortgelyke strukture in Gekkonidae. Histologiese beskrywing van Cordylidae veistruktuur was identies met dié van Gekkonidae. Drie tipes generasiekliere was geïdentifiseer in gordelakkedisse (van Wyk & Mouton, 1992), en hulle verskil gedeeltlik van naverwante strukture in Gekkonidae. Hierdie drie tipes generasiekliere was verder vergelyk met betrekking tot hulle vervellingsiklus histologie, wat identies was. Dus verskil hulle net met betrekking tot hulle vorm. Vel is ook vergelyk met dieselfde metodes. Alhoewel daar geen verskille was met betrekking tot hulle histology nie, was die tye van aktiwiteit van kiem-sellae beduidend verskillend in alle spesies. In P. micro/epidofus het die kiem-sellaag russtadium baie langer geduur as in die ander twee spesies, terwyl P. cepensis amper geen russtadium getoon het nie. G. cordy/us het 'n intermediêre gedragspatroon vertoon tussen aktiewe en rustende fase met betrekking tot sy vervellingsiklus. Outoradiografie is gebruik op elke spesie, in beide aktiewe en rustende vervellingsfases, om verskille in mitotiese aktiwiteit te wys. Generasieklier aktiwiteit blyk te korreleer met pieke van testosteroonvlakke van die testikulêre siklus in al drie spesies. Asinkronie is aangetoon tussen generasiekliere en velskubbe in al drie spesies.

Ao se observar ao microscópio de luz e eletrônico de transmissão as espermatogônias indiferenciadas de lambari (Astyanax altiparanae), espécie de grande valor econômico e comercial, as mesmas se subdividem em Aund* e Aund. A do tipo Aund* apresentou núcleo irregular, excêntrico, cromatina descondensada e nuages próximas ao envoltório nuclear e/ou ainda, associadas às mitocôndrias e, a do tipo Aund, apresentou núcleo esférico, central, com cromatina levemente condensada e nuages. Análise por meio de morfometria demonstrou que o nicho espermatogonial das espermatogônias indiferenciadas do tipo Aund* é próximo ao interstício, enquanto as do tipo Aund, próximas às regiões entre dois túbulos (intertúbulo). Adicionalmente, de acordo com a técnica de incorporação e retenção de BrdU foi possível analisar a existência de possíveis candidatas às espermatogônias-tronco, chamadas de labeling-retaining cells, visto que estas células retiveram BrdU ao longo de todo período avaliado, demonstrando um longo ciclo celular, característica esta de célula-tronco. / Observing by light and transmission electron microscope, undifferentiated spermatogonia of lambari (Astyanax altiparanae), species of great economic and commercial value, they are divided into Aund * and Aund. The type Aund* presents irregular nucleus, eccentric and decondensed chromatin, and nuages near the nuclear envelope and/or associated to mitochondria. The type Aund presents spherical central nucleus, with slightly condensed chromatin and nuages. Morphometric analysis showed that the niche of the type Aund* spermatogonia is near the interstitium, while the type Aund, next to the regions between two tubules (intertubule). Additionally, by the BrdU incorporation and retention technique, we found the possible candidates to spermatogonial stem cells, called \"labeling-retaining cells\", since these cells have retained BrdU over the entire study period (21 days), showing a long cell cycle, a stem cells characteristic.

Orientador: Rafael Henrique Nóbrega / Resumo: A decisão se a gônada indiferenciada irá se desenvolver em um testículo ou um ovário é uma etapa crítica na diferenciação sexual dos vertebrados. Este processo consiste em uma complexa cascata gênica que culmina na diferenciação das células somáticas presente na gônada indiferenciada. Neste sentido, o hormônio anti-Mülleriano é um importante efetor nesta decisão. Estudos com o peixe medaka mutante hotei, no qual o receptor do Amh tipo 2 (amhrII) é mutado, mostram elevado índice gonadossomático, e reversão sexual de macho para fêmea. Com base nestas informações, acredita-se que o Amh possa regular o número de células germinativas e diretamente ou indiretamente este fato pode influenciar na decisão da gônada bipotencial em se tornar um testículo ou ovário. Sendo assim, o objetivo do presente trabalho foi de se avaliar a sinalização do Amh durante a diferenciação sexual no medaka. Para tanto, estudos de perda de função gênica foram empregados, no qual o gene amh foi deletado através do sistema CRISPR/Cas9. Também foi avaliada a expressão dos genes que atuam na janela de diferenciação sexual por RT-qPCR. Inicialmente, identificamos dois tipos de mutação nos animais heterozigotos (amh (-/+) da F1. A mutação mais interessante foi a deleção de 759 pares de base entre os exons 6 e 7 que compreende parte dos domínios Amh_N e TGF-β. Na geração F2, os mutantes homozigotos amh (-/-) machos XY apresentaram 100% de reversão sexual para fêmeas fenotípicas. Além disso os animais amh (-/-) ap... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The decision whether the undifferentiated gonad will develop in a testicle or an ovary is a critical step in the sexual differentiation of vertebrates. This process consists of a complex genetic cascade that culminates in the differentiation of the somatic cells present in the undifferentiated gonad. In this context, the anti-Müllerian hormone is an important effector in this decision. Studies with the medaka hotei fish, in which the Amh type 2 receptor (amhrII) is mutated, show high gonadosomatic index, and sexual reversion from male to female. Based on this information, it is postulated that the Amh regulates the number of germ cells, and directly or indirectly this fact may influence the decision of the bipotential gonad to become a testicle or ovary. Therefore, the objective of the present study was to evaluate the signaling of Amh during sexual differentiation in medaka. For that, studies of loss of gene function were employed, in which the amh gene was deleted through the CRISPR / Cas9 system. We also evaluated the expression of genes that act in the window of sexual differentiation by RT-qPCR. We initially identified two types of mutation in F1 heterozygous (amh -/+) animals. The most interesting mutation was the deletion of 759 base pairs between exons 6 and 7 which comprises part of the Amh_N and TGF-β domains. In the F2 generation, the homozygous amh (-/-) mutants XY males presented 100% of sexual reversion. In addition, the amh (-/-) animals presented gonadal hyper... (Complete abstract click electronic access below) / Doutor

The cognitive and affective characteristics and responses to stimulant medication of children who were diagnosed as having attention deficit-hyperactivity disorder (AD-HD) or undifferentiated-attention deficit disorder (UADD) were investigated using a pretest-posttest experimental design. Nineteen AD-HD and 17 UADD children were compared using unpaired t-tests, prior to initiation of stimulant medication, on measures of intellectual functioning, impulsivity, problem behavior, and self-reported depression and self-esteem. Children from both the AD-HD (n = 12) and UADD (n = 12) groups were then compared before and after a 3-month trial of stimulant medication on measures of impulsivity, problem behavior, and self-reported depression and self-esteem using repeated measures analyses of variance. No significant differences were found between groups in cognitive ability, impulsivity, depression, self-esteem, anxiety, peer relationships, or social withdrawal. AD-HD children were found to exhibit more hyperactive, aggressive, and delinquent problem behavior. Significant improvement was found in both groups in self-reported depression and self-esteem following a trial of stimulant medication. A trial of stimulant medication was found to reduce hyperactive problem behavior in AD-HD children. Stimulant medication had a beneficial effect on peer relationships and aggressive problem behavior in UADD children but did not produce similar positive effects in AD-HD children. These results are discussed as in relationship to the issue of whether AD-HD and UADD are separate syndromes and to the previous literature regarding attention deficit disorder with hyperactivity (ADD/H) and attention deficit disorder without hyperactivity (ADD/WO). Ramifications regarding appropriate treatment are also discussed.

No description available.

Ao se observar ao microscópio de luz e eletrônico de transmissão as espermatogônias indiferenciadas de lambari (Astyanax altiparanae), espécie de grande valor econômico e comercial, as mesmas se subdividem em Aund* e Aund. A do tipo Aund* apresentou núcleo irregular, excêntrico, cromatina descondensada e nuages próximas ao envoltório nuclear e/ou ainda, associadas às mitocôndrias e, a do tipo Aund, apresentou núcleo esférico, central, com cromatina levemente condensada e nuages. Análise por meio de morfometria demonstrou que o nicho espermatogonial das espermatogônias indiferenciadas do tipo Aund* é próximo ao interstício, enquanto as do tipo Aund, próximas às regiões entre dois túbulos (intertúbulo). Adicionalmente, de acordo com a técnica de incorporação e retenção de BrdU foi possível analisar a existência de possíveis candidatas às espermatogônias-tronco, chamadas de labeling-retaining cells, visto que estas células retiveram BrdU ao longo de todo período avaliado, demonstrando um longo ciclo celular, característica esta de célula-tronco. / Observing by light and transmission electron microscope, undifferentiated spermatogonia of lambari (Astyanax altiparanae), species of great economic and commercial value, they are divided into Aund * and Aund. The type Aund* presents irregular nucleus, eccentric and decondensed chromatin, and nuages near the nuclear envelope and/or associated to mitochondria. The type Aund presents spherical central nucleus, with slightly condensed chromatin and nuages. Morphometric analysis showed that the niche of the type Aund* spermatogonia is near the interstitium, while the type Aund, next to the regions between two tubules (intertubule). Additionally, by the BrdU incorporation and retention technique, we found the possible candidates to spermatogonial stem cells, called \"labeling-retaining cells\", since these cells have retained BrdU over the entire study period (21 days), showing a long cell cycle, a stem cells characteristic.

BACKGROUND: Soft tissue sarcomas (STSs) are a diverse group of cancers that originate from mesenchymal tissue and are estimated to result in 13,130 new cases and 5,350 deaths this year. These neoplasms are hard to detect, which results in physicians struggling to treat late-stage STSs with a limited number of ineffective treatments. Currently, surgical excision is the primary treatment with radiation therapy administered when possible. However, even with optimal margins, the rate of recurrence is high, and the overall survival is low. There is a desperate need for new, more effective treatments. Immune checkpoint blockade (ICB) has recently had widespread success in treating melanoma, and in recent trials, SARC028 and Alliance A091401, have shown demonstrated activity of ICBs in STS in the neoadjuvant setting. Two histological subtypes of STS showed more promise than others: dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS). Another issue plaguing the field of STS is that there is no universal indicator of response. The percentage of hyalinization found within the tumor was recently identified as a better marker of response than radiographic imaging or percent viable tumor. METHODS: This study was an investigator-initiated, single-center, randomized, open-label, phase II study (NCT03307616), in which 23 patients with either DDLPS of the retroperitoneum (RP) or UPS of the extremity/trunk (ET) were separated by disease into Cohort 1 and 2, respectively. Subjects in each cohort were randomized into two neoadjuvant treatment arms per cohort. Arm A (n=7) of Cohort 1 received nivolumab (anti-PD-1) monotherapy, while Arm B (n=7) of Cohort 1 received nivolumab and ipilimumab (anti-CTLA-4) combination therapy. Arm C (n=5) of Cohort 2 received nivolumab monotherapy and radiation therapy, whereas Arm D (n=4) of Cohort 2 received nivolumab/ipilimumab combination therapy and radiation therapy. A tumor biopsy was obtained before treatment, and another sample was taken during the primary treatment of surgical excision. These samples were processed and analyzed by a pathologist who determined the percentage of viable tumor, hyalinization, and necrosis in each sample. Radiographic imaging was also taken throughout to make RECIST 1.1 response determinations. RESULTS: The average treatment response (1 - % viable tumor) for Cohort 1 was 25 ± 23 and there was no difference between Arm A and Arm B, p=0.48. The average treatment response in Cohort 2 was higher at 85 ± 27, but there was also no significant difference between the arms, p = 0.46. The mean percent hyalinization for Cohort 1 was 13 ± 13%, and for Cohort 2 was 69 ± 35%. Again, there was no significant difference between the arms in the Cohort 1 or 2, p = 0.45 and p = 0.54, respectively. Lastly, the mean % necrosis in Cohort 1 was 13 ± 13 %, and in Cohort 2 was 17 ± 24%, and neither had significantly different results in the arms, p = 0.60 and p = 0.92. The RECIST 1.1 results were independent of the arms of the study, and the radiographic response (percent image change) did not correlate to any metric of histologic response. Those who received Ipilimumab had higher rates of adverse events. CONCLUSION: There is significant evidence that ICBs elicited a response in RP DDLPS and ET UPS, and the response of ET UPS was profound. However, there was no apparent benefit from the combination therapy compared to the monotherapy in either cohort. The higher response in ET UPS may be due to the additional radiation therapy or to the nature of UPS itself. Finally, radiographic imaging does not show the response which is apparent at the histological level, so treatment regimens and future experiments should no longer rely on radiographic imaging as a marker for response. / 2021-06-07T00:00:00Z

Indiana University-Purdue University Indianapolis (IUPUI) / Chromosomal mechanisms of sex determination vary greatly in phylogenetically closely related species, indicative of rapid evolutionary rates. Sex chromosome karyotypes are generally conserved within families; however, many species have derived sex chromosome configurations. Insects display a plethora of sex chromosome systems due to rapid diversification caused by changes in evolutionary processes within and between species. A good example of such a system are insects in the blow fly family Calliphoridae. While cytogenetic studies observe that the karyotype in blow flies is highly conserved (five pairs of autosomal chromosomes and one pair sex chromosome), there is variation in sex determining mechanisms and sex chromosome structure within closely related species in blow flies. The evolutionary history of sex chromosomes in blow fly species have not been fully explored. Therefore, the objective of this research was to characterize the sex chromosome structures in four species of blow flies and investigate the selective forces which have played a role in shaping the diverse sex chromosome system observed in blow flies. The blow fly species used in this study are Phormia regina, Lucilia cuprina, Chrysomya rufifacies and Chrysomya albiceps. Phormia regina,and Lucilia cuprina have a heteromorphic sex chromosome system and are amphogenic (females produce both male and female offspring in equal ratio). In contrast, Chrysomya rufifacies and Chrysomya albiceps, have a homomorphic sex chromosome system, are monogenic (females produce unisexual progeny), have two types of females (arrhenogenic females – male producers and thelygenic females – female producers), and sex of the offspring is determined by the maternal genotype. To accomplish these tasks, a total of nine male and female individual draft genomes for each of the four species (including three individual draft genomes of Chrysomya rufifacies – male, and the two females) were sequenced and assembled providing genomic data to explore sex chromosome evolution in blow flies. Whole genome analysis was utilized to characterize and identify putative sex chromosomal sequences of the four blow fly species. Genomic evidence confirmed the presence of genetically differentiated sex chromosomes in P. regina and L. cuprina; and genetically undifferentiated sex chromosomes in C. rufifacies and C. albiceps. Furthermore, comparative analysis of the ancestral Dipteran sex chromosome (Muller element F in Drosophila) was determined to be X-linked in P. regina and L. cuprina contributing to sex chromosome differentiation but not sex-linked in C. rufifacies and C. albiceps. Evolutionary pressures are often quantified by the ratio of substitution rates at non-synonymous (dN) and synonymous (dS) sites. Substitution rate ratio analysis (dN/dS) of homologous genes indicated a weaker purifying selection may have contributed to the loss of sex-linked genes in Muller element F genes of the undifferentiated sex chromosome as compared to the differentiated sex chromosome system. Overall, the results presented herein greatly expands our knowledge in sex chromosome evolution within blow flies and will reinforce the study of sex chromosome evolution in other species with diverse sex chromosome systems.

Em mamíferos, a espermatogênese é mantida pela autorrenovação e diferenciação das células-tronco espermatogoniais (SSC). Apesar da grande importância do SSC para a fertilidade masculina, em Bos taurus pouco se sabe sobre a sua identificação e biologia celular. Para roedores, mais de 30 marcadores para células germinativas indiferenciadas já foram descritos. No entanto, ainda não é conhecido um marcador específico apenas para SSC. Quase todos são também expressos por gonócitos, espermatogônias mais diferenciadas ou mesmo células somáticas. Yin Yang 2 (YY2) é um factor de transcrição expresso nas células com a morfologia de gonócitos e SSC, sendo um candidato a marcador de SSC. Assim, a identificação de novos marcadores para SSC e factores que afectam a sua expressão, tais como a idade, são fundamentais para o desenvolvimento da biotecnologia como transgenia e tratamento de infertilidade, nos quais as SSC poderiam ser ferramentas biológicos importantes. Assim, nesta tese temos duas hipóteses principais: 1) a idade do dador afeta a expressão de marcadores moleculares específicos de SSC bovinas assim como potencial de células-tronco dessas células e que as sequências de DNA em que se associa YY2 regulam a expressão génica de SSC em camundongos. Os objetivos específicos, organizados em 4 artigos científicos, foram: identificar a melhor plaqueamento diferencial para enriquecer SSC bovina (artigo 1), verificar se a expressão de marcadores moleculares de SSC bovina difere entre adultos pré-púberes (artigo 2 e 3), identificar novos marcadores específicos para SSC em Bos taurus (artigo 3), verificar que a idade afeta o potencial de célula-tronco de SSC bovinas (artigo 3), descrever YY2 como um marcador específico para SSC em camundongos e verificar se as sequências DNA associadas YY2 são loci de importância para SSC. Assim, definimos o melhor plaqueamento diferencial para o enriquecimento de SSC bovinas, que idade afeta a expressão marcadores já estabelecidos assim como genes específicos do transcriptoma de SSC bovinas e que idade também afeta o seu potencial de células-tronco (ensaio de repopulação). Concluímos também que YY2 é um marcador para SSC de camundongo em cultivo, em animais adultos e que as sequências do genoma que se associam YY2 possivelmente tem capacidade de regulação génica em SSC murina. / Mammalian spermatogenesis is sustained by self-renewal and differentiation of spermatogonial stem cells (SSC). Despite the importance of the SSC for male fertility, in Bos taurus líttle is known about their identification identity and cell biology. For rodents, more than thirty markers for undifferentiated germ cells have already been described. However, none of these represents a marker specific for SSC, as most are also expressed in gonocytes, differentiated spermatogonia or even somatic cells. Yin Yang 2 (YY2) is a transcription factor specifically expressed in cells with the morphology of gonocytes and SSC in the mouse, being a candidate marker for SSC. For the use of SSC as an important biological tool in the development of biotechnology such as transgenesis and the treatment of infertility, it is important to identify new markers for SSC and factors that affect their expression, such as the age of donors. Therefore, the experimental work described in this thesis was based on two main hypothesis: (1) the donor age affects the expression of specific molecular markers in SSC as well their potential as stem cells and 2) YY2 exerts important functions in SSC and genomic targets correspond to loci relevant for gene regulation or genome management in SSC. The specific goals have been organized in 4 manuscripts as follows: optimization of differential plating to enrich for bovine SSC (Article 1), check if the expression of molecular markers of bovine SSCs differs between prepubertal and adult donors (Article 2 and 3), identify new markers specific for SSC in Bos taurus (Article 3), continuation of the initial description of YY2 as a specific marker for SSC in mice and check if sequences bound by YY2 in vivo harbor the capacity to influence gene expression in SSC. As conclusions, we present an optimized differential plating protocol for the enrichment of bovine SSC, we conclude that age effects the expression of SSC markers, the expression of specific genes of bovine SSC and that age also affects their potential as stem cells (measured in repopulation assays). We also contribute to the description of the restricted expression of YY2 in prepuberal and adult SSC in mice and we show that YY2 binding sites represent genomic sequences relevant for control of gene expression.