No description available.

La cellule est l’unité à la base de la vie. Elle est généralement délimitée par sa membrane et contient un noyau et du cytoplasme en plus d’autres composantes. Les cellules se divisent afin de maintenir et de perpétuer la vie par duplication de leur matériel génétique et par leur séparation en deux cellules physiquement distinctes durant la cytocinèse. Cependant, la division cellulaire est parfois modifiée et aboutit à la formation d’un tissu contenant plusieurs noyaux bordés d’une membrane unique appelé syncytium. Les syncytia sont fréquemment retrouvés chez les organismes vivants, bien que leurs fonctions et mode de formation restent peu compris. L’organisation en syncytium est conservée chez tous les animaux étudiés à ce jour au niveau de la lignée germinale dans laquelle les cellules partagent un cytoplasme commun par l’intermédiaire d’un pont intercellulaire stable. Dans la majorité des lignées germinales étudiées, les cellules sont directement connectées l’une à l’autre par un pont intercellulaire stable qui émerge de cytocinèses incomplètes. Cependant, certaines lignées germinales sont organisées autour d’une cavité commune à laquelle chaque cellule germinale est connectée. Dans ces lignées germinales, les mécanismes qui mènent à l’expansion du syncytium sont peu compris. Ma thèse décrit l’utilisation de la lignée germinale primordiale de C. elegans à son premier stade larvaire pour mieux comprendre l’organisation, l’expansion et la fonction des lignées germinales syncytiales. En utilisant la microscopie électronique et confocale, j’ai découvert que l’organisation du syncytium est fixée au premier stade larvaire. En effet, les deux cellules germinales primordiales (CGP) sont chacune individuellement connectée à une cavité cytoplasmique centrale par le biais de ponts intercellulaires stables. Nous avons nommé cette cavité le proto-rachis car l’organisation des CGP est identique à l’organisation de la gonade adulte. Chez l’adulte, les ponts intercellulaires qui connectent les cellules germinales au rachis sont stabilisés par des régulateurs d’actomyosine. Nous avons vérifié si cela était également le cas dans la gonade au premier stade larvaire. Tous les régulateurs présents dans la gonade adulte, sont aussi présent dans les ponts intercellulaires des CGP, mais la lignée germinale primordiale est réfractaire à la perturbation de la fonction de ces régulateurs. Ce résultat suggère que les régulateurs d’actomyosine sont organisés de manière très stable au premier stade larvaire. Afin de mieux comprendre comment le syncytium se développe dans la lignée germinale de C. elegans, j’ai ensuite suivi la première division des CGP par microscopie à temps réel. J’ai mis en évidence que l’anneau de cytocinèse se stabilise, puis se déplace vers le proto-rachis jusqu’à qu’il s’y intègre. Ces résultats indiquent que le syncytium se développe par cytocynèse incomplète. De plus, mes résultats montrent que la connexion au proto-rachis est maintenue durant la division des CGP. C’est pourquoi nous proposons un modèle pour l’expansion du syncytium dans lequel l’anneau de cytocinèse stabilise pour connecter une des cellules filles au proto-rachis, tandis que l’autre cellule fille est connecté par l’anneau stable qu’elle aura hérité de la cellule mère. Enfin, pour s’assurer que les mécanismes d’expansion du syncytium observés durant la division des CGP sont conservés au cours du développement de la gonade, j’ai conceptualisé et créé un dispositif de micro-fluidique qui en théorie permettrait de suivre plusieurs séries de division des CGP. En somme, mon travail de doctorat a fourni une caractérisation détaillée de la structure du syncytium dans la lignée germinale de C. elegans au premier stade larvaire, ainsi qu’un modèle pour l’expansion du syncytium. Mes découvertes indiquent que malgré des différences dans l’organisation des syncytia, la cytocinèse incomplète est un mécanisme conservé dans toutes les lignées germinales animales. Des travaux futurs seront nécessaires pour découvrir quelles voies de signalisation moléculaires sont sous-jacentes aux mécanismes de formation des syncytia, et ainsi de mieux comprendre quelle est la fonction de ces structures fascinantes. / The cell constitutes the basic unit of life. It is generally delimited by its membrane and contains a nucleus and cytoplasm amongst other components. To maintain and perpetuate life, cells divide by duplicating their genetic material, and by physically separating into two distinct cells during the process called cytokinesis. However, cell division is sometimes modified and leads to the formation of a tissue in which several nuclei are delimited by a single membrane, called a syncytium. Syncytial tissues are common amongst living organisms, but why and how they form remains unclear. The syncytial architecture is conserved in all studied animal germlines where germ cells share a common cytoplasm through stable intercellular bridges. In most animal germlines, the germ cells are directly connected with one another, and the stable intercellular bridges that connect the cells are known to arise from regulated incomplete cytokinesis. However, some germlines are organized around a central common cavity to which each germ cell is connected. In such germlines, the mechanisms of syncytium expansions remain unknown. My thesis describes the use of the C. elegans germline primordium at the first larval stage to better understand the organization, the expansion, and the function of germline syncytia. Using electron and confocal microscopy I found that the organization of the syncytium is established at the first larval stage. The two germ cells called the primordial germ cells (PGCs) each connect to a central cytoplasmic cavity through stable intercellular bridges. Because this organization is identical to the adult germline where each germ cell is connected to the central rachis, we termed the cavity between the PGCs proto-rachis. In the adult gonad, the intercellular bridges that connect the germ cells to the rachis are stabilized by actomyosin regulators, so I verified if this was also the case in the first larval stage gonad. All the regulators that localize to adult intercellular bridges were also present between the PGCs, but the primordial germ line is refractory to perturbation of these regulators. This suggests that the actomyosin regulators are organized in a very stable manner in the first larval stage germline. I next tracked the first division of the PGCs with live imaging to better understand how the syncytium expands in the C. elegans germline. I found that the cytokinetic ring stabilizes, then displaces towards the proto-rachis until it integrates into the syncytial structures. This finding suggests the syncytium expands by incomplete cytokinesis. In addition, my results indicate that the connection to the proto-rachis was maintained during PGCs division. We therefore propose a model in which the cytokinetic ring stabilizes and connects one of the daughter cells to the proto- rachis while the other cell is connected through the inherited stable ring from the mother cell. Finally, I designed and a created a microfluidic device that in theory would allow us to live image several rounds of PGCs division. This would confirm if the mechanisms of syncytium expansion that we observed during the first division of the PGCs are conserved in further development. My work has provided a detailed characterization of the syncytial structure in the C. elegans germline primordium as well as a model for syncytium expansion. My findings indicate that despite differences in the organization of the syncytium, incomplete cytokinesis is conserved as the mechanism for syncytium expansion in all animal germlines. Further research will be necessary to bring to light the molecular pathways underlying syncytium formation to have a better understanding of the function of these fascinating structures.

Mounting concerns about drug-resistant pathogenic bacteria have rekindled the interest in bacteriophages (bacterial viruses). As bacteria’s natural predators, bacteriophages offer a critical advantage over antibiotics, namely that they can be highly specific. This means that phage therapeutics can be designed to destroy only the infectious agent(s), without causing any harm to our microbiota. However, the potential secondary effects on the balance of microbiota through bacteriophage-induced genome evolution remains as one of the critical apprehensions regarding phage therapy. There exists a significant gap in knowledge regarding the direct and indirect effect of phage therapeutics on the microbiota. The aim of this thesis was to: (1) establish an in vivo model for investigation of the evolutionary dynamics and co-evolution of therapeutic phage and its corresponding host bacterium in the gut; (2) determine if phage therapy can affect the composition of the gut microbiota, (3) observe the differences of phage-resistant bacteria mutants evolved in vivo in comparison to those evolved in vitro. We used germ-free mice colonized with a consortium of eight known bacteria, known as the altered Schaedler flora (ASF). The colonizing strain of choice (mock infection) was a non-pathogenic strain E. coli K-12 (JM83) known to co-colonize the ASF model, which was challenged in vivo with T7 phage (strictly lytic). We compared the composition of the gut microbiota with that of mice not subject to phage therapy. Furthermore, the resistant mutants evolved in vivo and in vitro were characterized in terms of growth fitness and motility. / Thesis / Master of Applied Science (MASc) / Bacteriophages are viruses that infect bacteria. After their discovery in 1917, bacteriophages were a primary cure against infectious disease for 25 years, before being completely overshadowed by antibiotics. With the rise of antibiotic resistance, bacteriophages are being explored again for their antibacterial activity. One of the critical apprehensions regarding bacteriophage therapy is the possible perturbations to our microbiota. We set out to explore this concern using a simplified microbiome model, namely germ-free mice inoculated with only 8 bacteria plus a mock infection challenged with bacteriophage. We monitored this model for 9 weeks and isolated a collection of phage-resistant bacterial mutants from the mouse gut that developed post phage challenge, maintaining the community of mock infection inside the gut. A single dose of lytic phage challenge effectively decreased the mock infection without causing any extreme long-term perturbations to the gut microbiota.

Die Arbeit enthält Videoaufnahmen. / Die vorliegende Arbeit befasst sich mit Aspekten der frühen Entwicklung der Phoronida („Hufeisenwürmer“). An drei Arten wird der Furchungsprozess untersucht (Phoronis pallida, Phoronis muelleri, Phoronis vancouverensis). Dies erfolgt sowohl mithilfe der 4D-Mikroskopie als auch anhand von immunocytochemischen Markierungen der Mitosespindeln und konfokaler Laser-Scanning-Mikroskopie. Verschiedene morphologische Merkmale des Furchungsprozesses werden quantitativ erfasst und innerhalb sowie zwischen den Arten verglichen. Die Ergebnisse zeigen eine weitgehend übereinstimmende Furchung bei P. pallida und P. muelleri Embryonen: Ab dem dritten Zellzyklus teilen sich die Blastomeren meist schräg – und alternierend dextral und sinistral – zur animal-vegetativ Achse. Dieses Muster zeigt überraschende Übereinstimmungen mit dem Muster der Spiralfurchung. Dies kann als morphologische Unterstützung molekular-phylogenetischer Befunde einer Stellung der Phoronida innerhalb der Spiralia/Lophotrochozoa interpretiert werden. Die Furchung bei P. vancouverensis unterscheidet sich von der Furchung der anderen beiden Arten; sie weist jedoch auch Unterschiede zu einer Radiärfurchung auf. Generell zeigt die Furchung aller drei Arten einen gewissen Grad an Variabilität. Anhand von in-vivo Einzelzellmarkierungen untersucht die Studie darüber hinaus das Schicksal der Blastomeren früher P. pallida Embryonen bis zu späten Gastrulationsstadien. Diese Analysen zeigen, dass die ersten beiden Furchungsteilungen durch die spätere Achse Blastoporus-Apikalplatte, jedoch in keinem konstanten Orientierungsverhältnis zur Ebene der Bilateralsymmetrie der Gastrula verlaufen. Dies unterscheidet sich von der Situation, wie sie von spiralfurchenden Tieren bekannt ist. Die Unterschiede und die beobachtete Variabilität des Furchungsprozesses werden im Licht unterschiedlicher Mechanismen der Spezifizierung von Zellschicksalen und Körperachsen bei verschiedenen Taxa der Spiralia und den Phoronida diskutiert. / This study addresses aspects of the early development of Phoronida (“horseshoe worms”). The cleavage process is analyzed for three species (Phoronis pallida, Phoronis muelleri, Phoronis vancouverensis). These investigations are performed using 4D-microscopy as well as immunocytochemical stainings of the mitotic spindle apparatuses in combination with confocal laser-scanning microscopy. Different morphological features of the cleavage process are quantified and compared within as well as between the species. The results reveal a highly consistent cleavage of P. pallida and P. muelleri embryos: from the third cell cycle onward, the blastomeres divide mostly obliquely – and alternatingly dextral and sinistral – with respect to the animal-vegetal axis. This cleavage pattern shows surprising correspondences to the pattern of spiral cleavage. The finding can be interpreted as morphological support for recent molecule-based phylogenies, which indicate a position of Phoronida within the Spiralia/Lophotrochozoa clade. The cleavage of P. vancouverensis differs from the cleavage in the other two species; however, it also shows differences to a radial cleavage pattern. In all three species, the cleavage process also involves some degree of variability. Furthermore, the study traces the cell fates of early P. pallida embryos up to the state of late gastrulation, by the use of fluorescent in-vivo single cell markings. These analyses reveal that the first two cleavage divisions both pass through the later axis blastopore-apical plate of the gastrula, yet they do not pass in a constant relationship with respect to the later plane of bilateral symmetry. This differs from the situation known from spiral cleaving animals. The differences and the encountered variability of the cleavage process are discussed with respect to different mechanisms of the specification of cell fates and body axes in different taxa of the Spiralia and the Phoronida.

This project is an exploration of 20th century dystopian literature through the lens of germ theory. This scientific principle, which emerged in the late 19th century, asserts that microorganisms pervade the world; these invisible and omnipresent germs cause specific diseases which are often life threatening. Additionally, germ theory states that vaccines and antiseptics can prevent some of these afflictions and that antibiotics can treat others. This concept of a pervasive, invisible, infection-causing other is not just a biological principle, though; in this paper, I argue that one can interpret it as an ideological framework for understanding human existence as a whole. Particularly, I believe that authors of prominent 20th century dystopian novels applied the tenets of germ theory in order to explore the potential “pathogens” that furtively exist within the human mind. These pseudo-germs are various human tendencies that, when left “untreated” by governments, create nonnormative members of society. In the eyes of dystopian regimes, it is precisely this nonnormativity that poses a lethal threat, in that it challenges the continued existence of society with the current ruling body at the helm. In this paper, I trace love (both sexual and familial) and individuation (as a function of social hierarchy, recreational activities, and the use of language) as social disease-causing pathogens in George Orwell’s 1984 and in Aldous Huxley’s Brave New World.

No description available.

Thesis summary The process of B cell lymphogenesis in swine remains uncertain. Some reports indicate that pigs belong to a group of animal that use ileal Peyers's patches (IPP) for the generation of B cells while others point to the possibility that the bone marrow is functional throughout life. The functional subpopulations of B cells in swine are also unknown. Together with other ruminants, and also birds, γδ T cells in swine may account for >70% of all T cells which is in apparent contrast with humans and mice. The purpose of this thesis was to address these discrepancies and unresolved issues. The results disprove the existing paradigm that the IPP is primary lymphoid tissue and that B cells develop in IPP in an antigen-independent manner. On the other hand, it shows that bone marrow is fully capable of B cell lymphogenesis and remains active at least for the same period of time as it had been speculated for the IPP. This thesis also identified functionally different subsets of porcine peripheral B cells, and shows that CD21 molecules can be expressed in differential forms. Finally, this thesis identifies two lineages of γδ T cells that differ in many functional and phenotype features. This finding may explain why γδ T cells constitute of minority of lymphocytes in circulation of humans and mice.

Introdução: A incidência de tumores adrenocorticais em crianças é particularmente elevada nas regiões sudeste e sul do Brasil, correlacionandose com a ocorrência da mutação germinativa p.R337H do supressor tumoral p53, entretanto, o carcinoma adrenocortical é uma neoplasia endócrina maligna rara em todo o mundo com uma incidência aproximada de 0,5 - 2 casos por milhão por ano. Esta condição é uma doença heterogênea, apresentando frequentemente comportamento clínico agressivo e letal. A cascata de sinalização Wnt é uma via importante de transdução de sinal em cânceres humanos e tem sido implicada na tumorigênese adrenocortical. A atividade desta via de sinalização é dependente da quantidade de beta-catenina citoplasmática e nuclear. Mutações ativadoras no gene da beta-catenina (CTNNB1) foram relatadas em diversas neoplasias humanas. Estudos demonstraram que mutações no gene CTNNB1 são os defeitos genéticos mais frequentemente encontrados em adenomas e em carcinomas adrenocorticais. O estudo destas mutações demonstrou que as alterações no gene CTNNB1 localizam-se principalmente exon 3, que codifica a porção amino terminal da beta- catenina. Objetivos: determinar a ocorrência e a frequência das mutações somáticas no exon 3 do gene CTNNB1. Adicionalmente, determinar a imunorreatividade de beta-catenina e de p53 em tumores adrenocorticais benignos e malignos de crianças e adultos. Correlacionar os resultados da análise de mutações gênicas e os dados de imunorreatividade com as características hormonais, a mutação p.R337H do p53, o diagnóstico histológico e a evolução dos tumores adrenocorticais de crianças e adultos. Métodos: Neste estudo, a análise de imunohistoquímica para beta-catenina e p53 foi realizada em 103 tumores adrenocorticais benignos e malignos (40 crianças e 63 adultos), estando as amostras histológicas alocadas em micromatriz tecidual (TMA). A pesquisa de mutações no exon 3 do gene CTNNB1 foi determinada por seqüenciamento automático em 64 tumores adrenocorticais. Resultados: a imunorreatividade para beta-catenina em citoplasma e/ou núcleo foi evidenciada de maneira similar nos tumores adrenocorticais benignos e malignos de crianças e de adultos (15% e 23,8%, respectivamente). O percentual das células neoplásicas imunorreativas para beta-catenina em citoplasma e/ou núcleo não foi significativamente diferente entre os tumores clinicamente benignos e malignos pediátricos (15,6% vs. 12,5%, respectivamente; p=0,93) e entre adenomas e carcinomas adrenocorticais de adultos (28,5% vs. 17,8%, respectivamente; p=0,38). A síndrome endócrina causada pelo perfil de secreção hormonal foi similar entre os tumores adrenocorticais com presença ou ausência de acúmulo citoplasmático e/ou nuclear de beta-catenina em crianças e adultos. A associação entre acúmulo anormal de beta-catenina e diminuição de sobrevida foi avaliada nos pacientes adultos portadores de carcinomas adrenocorticais isoladamente (n=25), sendo observada na curva de Kaplan- Meier uma tendência de significância (log-rank p=0,07). A análise do gene CTNNB1 revelou mutações somáticas em heterozigose em 10 tumores adrenocorticais (4 crianças e 6 adultos). As mutações encontradas no gene CTNNB1 foram, sobretudo do tipo missense (p.Ser45Pro, p.Ser45Phe, p.Asp32Asn, p.Pro44Ala_Ser45Pro; p.His36Gln_Ser37Lys). Outras mutações encontradas compreenderam: a inserção de um único nucleotídeo (p.E9GfsX14), dando origem a uma desvio de leitura do exon 3; além da deleção dos três nucleotídeos do códon 45 (p.Ser45del). Todos os tumores com mutações somáticas no gene CTNNB1 mostraram acúmulo anormal para ?-catenina, com exceção de um caso. A presença de alterações no gene CTNNB1 não se associou ao tamanho do tumor (Teste de Mann-Whitney: p=0,75), desfecho desfavorável tanto no grupo pediátrico (log-rank p=0,29) como no grupo de pacientes adultos (log-rank p=0,77). Todos os pacientes portadores da mutação germinativa do gene TP53 apresentaram imunorreatividade nuclear de p53 nas células tumorais. Não foi encontrada correlação entre a presença de acúmulo anormal de beta-catenina e imunorreatividade nuclear de p53, considerando os grupos de crianças e de adultos portadores de tumores adrenocorticais. Adicionalmente, não foi observada correlação entre mutações no gene CTNNB1, bem como acúmulo anormal de beta-catenina, com a imunorreatividade nuclear de p53 no grupo de tumores adrenocorticais de pacientes adultos, porém, interessantemente, avaliando isoladamente o grupo de tumores adrenocorticais pediátricos, foi observada relação entre a presença de mutações no gene CTNNB1 e a presença de acúmulo nuclear de p53 (X2: p=0,009). Conclusões: Estes dados confirmam a participação da via Wnt na tumorigênese adrenocortical de crianças e de adultos, que apresenta uma prevalência de ativação semelhante entre crianças e adultos. O acúmulo citoplasmático e/ou nuclear de beta-catenina provavelmente é um marcador biológico de mau prognóstico do carcinoma de adrenocortical de adultos. Adicionalmente, observamos evidências de uma correlação positiva entre mutações no gene CTNNB1 e acúmulo nuclear de p53 em tumores adrenocorticais pediátricos, confirmando uma possível relação destas duas vias na tumorigênese do córtex da glândula suprarrenal / Introduction: The incidence of adrenocortical tumors in children is particularly high in the southeastern and southern regions of Brazil, correlating with the occurrence of p.R337H p53 tumor suppressor germline mutation. However, adrenocortical carcinoma is a worldwide rare endocrine malignancy with an approximate incidence of 0.5 to 2 cases per million per year. This condition is a heterogeneous disease and is often lethal. The Wnt signaling pathway is an important signal transduction pathway in human cancers and has been implicated in adrenocortical tumorigenesis. The activity of this signaling pathway is dependent on the amount of nuclear and cytoplasmic beta-catenin. Activating mutations of ?-catenin (CTNNB1) gene have been reported in several human malignancies. Studies have shown that CTNNB1 mutations are the most common genetic defect found in adrenocortical adenomas and carcinomas. The study of these mutations demonstrated that the changes in CTNNB1 gene are mainly located in exon 3, which encodes the amino terminal portion of the beta- catenin. Objectives: to determine the occurrence and frequency of CTNNB1 somatic mutations and the abnormal beta-catenin and p53 accumulation in benign and malignant adrenocortical tumors in both children and adults. We also evaluated the correlation of the gene mutations analysis and immunohistochemistry data with the hormonal characteristics, the p.R337H germline mutation, the histological diagnosis and the prognosis of adrenocortical tumors in children and adults. Methods: In this study, immunohistochemistry for beta-catenin and p53 was performed in 103 benign and malignant (40 children and 63 adults) adrenocortical tumors. The histological samples were allocated in a tissue microarray (TMA). The study of the CTNNB1 gene was performed by direct sequencing of 64 adrenocortical tumors. Results: The beta-catenin abnormal accumulation was similar in benign and malignant adrenocortical tumors of children and adults (15 % and 23.8 %, respectively). The percentage of cells with beta-catenin abnormal accumulation was not significantly different between benign and malignant pediatric adrenocortical tumors (15.6% vs. 12.5 %, respectively; P=0.93) and between adrenocortical adenomas and carcinomas in adults (28.5% vs 17.8 %, respectively; p=0.38). The endocrine syndrome caused by hormonal tumor secretion was similar in patients with and without beta-catenin abnormal accumulation both in pediatric and adult patients. The association between beta-catenin abnormal accumulation and decreased survival was evaluated in adult patients with adrenocortical carcinomas (n=25) and a trend toward significance was observed (log-rank p=0,07). The analysis of the CTNNB1 gene revealed heterozygous somatic mutations in 10 adrenocortical tumors (6 adults and 4 children). The mutations found in CTNNB1 gene were mainly missense (p.Ser45Pro, p.Ser45Phe, p.Asp32Asn, p.Pro44Ala_Ser45Pro; p.His36Gln_Ser37Lys). Other mutations found included: a single nucleotide insertion (p.E9GfsX14) and a deletion within codon 45 of exon 3 of CTNNB1 gene, (p.Ser45del). All tumors with somatic mutations in the CTNNB1 gene showed abnormal beta -catenin accumulation, except for one case. The mutations in CTNNB1 gene was not associated with tumor size (Mann - Whitney: p=0.75), unfavorable outcome in both pediatric (log -rank p=0.29) and adult group of patients (log-rank p=0.77). All patients with TP53 germline mutation showed p53 nuclear accumulation in the tumor cells. No correlation was found between the presence of beta-catenin abnormal accumulation and p53 nuclear accumulation in adrenocortical tumor cells of children and adults. In addition, no correlation was observed between CTNNB1 mutations, as well as beta-catenin abnormal accumulation, with p53 nuclear accumulation in adults adrenocortical tumors. Interestingly, the evaluation of pediatric adrenocortical tumors revealed a relationship between the occurrence of CTNNB1 mutations and the presence of p53 nuclear accumulation (X2: p=0.009). Conclusions: These data confirm the involvement of the Wnt pathway in adrenocortical tumorigenesis of children and adults, which has a prevalence similar activation between children and adults. We observed that abnormal beta-catenin accumulation in adults adrenocortical carcinoma is probably associated with a dismal prognosis. Additionally, we found evidence of a positive relationship between CTNNB1 mutations and p53 nuclear accumulation in pediatric adrenocortical tumors, confirming a possible connection of these two pathways in the pediatric adrenocortical tumorigenesis

Introdução: O termo distúrbios do desenvolvimento gonadal (DDG) inclui condições congênitas nas quais o desenvolvimento gonadal é atípico. Estudos feitos em camundongos observaram que alguns genes como o Cbx2 e o Tcf21 interferem na fase inicial do desenvolvimento gonadal, afetando tanto gônadas XX quanto XY. O gene Dhh, por sua vez, codifica o fator de transcrição Dhh, produzido pelas células de Sertoli, que é fundamental para a diferenciação das células de Leydig em gônadas XY. Nos ovários, o gene FOXL2 atua na foliculogênese, sendo fundamental para a formação dos ovários. Objetivos: Analisar clinicamente e pesquisar anormalidades nos genes CBX2, TCF21, DHH e FOXL2 em pacientes portadores de distúrbios do desenvolvimento gonadal 46, XY e 46, XX. Material e Métodos: Foram estudados 60 pacientes (41 com DDG 46, XY e 19 com DDG 46, XX). A análise molecular foi realizada a partir da amplificação gênica por PCR e sequenciamento direto. Resultados: Várias alterações alélicas foram encontradas nos quatro genes, algumas ainda não descritas na literatura. Uma alteração intrônica no gene DHH foi encontrada em um paciente com DDG 46, XY e não foi encontrada em nenhum dos 360 alelos normais estudados (g.IVS2 +29G>A). Estudamos essa variante através da extração do RNA do testículo do paciente afetado, mas não encontramos alteração no RNA; portanto ela parece não ser uma mutação. No gene TCF21, a variante encontrada foi identificada em controles normais. No gene CBX2, das treze alterações encontradas, uma não foi identificada em 206 alelos normais, e há troca de aminoácidos (p.C132R / g.394 T>C). Trata-se de uma variante que pode ter relação com o fenótipo do paciente, portador de DDG 46, XY. No gene FOXL2, das três alterações encontradas, uma não foi identificada em 206 alelos normais; contudo, não há troca de aminoácidos (p.A181A / g.543 C>T). Conclusão: Esse estudo sugere que mutações nos genes CBX2, TCF21, FOXL2 e DHH são causas raras de distúrbios do desenvolvimento gonadal. / Introduction: Congenital disorders of gonadal development (DGD) include conditions whose gonadal development is atypical. Studies in mice found that some genes such as Cbx2 and Tcf21 interfere in the initial phase of gonadal development, affecting both XX and XY gonads. Dhh gene, in turn, encodes the transcription factor Dhh, produced by Sertoli cells, which is essential for the differentiation of Leydig cells in XY gonads. In the ovaries, genes as FOXL2 act in folliculogenesis, fundamental to the development of the ovaries. Objectives: To analyze patients with disorders of gonadal development (DGD) 46, XY and 46, XX and research mutations in CBX2, TCF21, DHH and FOXL2 genes. Methods: We analyzed 60 patients (41 DGD 46, XY patients and 19 DGD 46, XX patients). The whole coding region of CBX2, TCF21, DHH and FOXL2 genes were amplified by PCR and direct sequenced. Results: Several allelic variations have been found in the four genes, some not even described by literature. One intronic variation in DHH was described in one patient with 46, XY DGD and it wasnt found in any of the 360 normal control alleles studied (g.IVS2 +29G>A). We studied this variant through RNA extraction from the affected patients testes, but we didnt find any alteration in the RNA, so it doesnt seem to be a mutation. In TCF21 gene, the single variant that was found was identified in normal controls. In CBX2 gene, among the 13 alterations described, one wasnt identified in 206 normal control alleles, and there is aminoacid change (p.C132R / g.394 T>C). This is a variant that may be a mutation, causing the patients phenotype that had 46, XY DGD. In FOXL2, among the 3 variations described, one wasnt indentified in 206 normal control alleles, but there wasnt amino acid change (p.A181A / g.543 C>T).Conclusion: This study suggests that mutations in CBX2, TCF21, FOXL2 and DHH genes are rarely causes of disorders of gonadal development.

<p><p><em>Transforming the Brute</em> addresses the ethical acceptability of creating painless animals for usage in biomedical experimentation.  In recent decades the possibility of creating genetically decerebrate animals or AMLs for human ends has been discussed in scientific, academic, and corporate communities.  While the ability to create animals that cannot feel, experience, and are more plant than animal remains science fiction, biomedicine may now be able to eliminate or significantly reduce the capacity to feel pain and nociception through genetic engineering.  With this new technology comes the opportunity to vastly increase the welfare of animals used in biomedical experimentation, yet this possibility has largely been ignored by the scientific and academic community.  This work seeks to reveal the moral necessity of creating painless animals for usage in biomedical experimentation for animal welfare ends.  Intrinsic objections relating to animal integrity, rights, companionship, the alteration of telos, humility and virtue are considered.  The benefit of eliminating nociceptive pain in experimental animals is addressed, and differences are examined between biomedical experimentation and other usage of animals for human ends which makes the proposed creation of painless animals ethically unique.  Finally, an argument is presented for the moral necessity of replacing normal animals with painless animals in biomedical experimentation with consideration given to genetically decerebrate animals.</p></p>