Dynamic molecular interactions between the microbiota and the intestinal mucosa play an important role in the establishment and maintenance of mucosal homeostasis. Aberrant host- microbiota interaction could lead to many diseases such as inflammatory bowel disease. The aim of our study was to evaluate the commensal and probiotic bacteria activities and their ability to induce pathological or exert beneficial effects. The most important trigger for immune system development is an exposure to microbial components. Here, we show that there is a time window at about three weeks of age, which enables the artificial colonization of germ free mice by a single oral dose of cecal content. The delayed colonization by either inoculation or co-housing causes permanent changes in immune system reactivity, which may downgrade the results of experiments performed on first generation of colonized animals. In this thesis we report that even non-living commensal bacteria such as Parabacteroides distasonis (mPd) or well known probiotics such as L. casei DN-114 001 (Lc) possess anti-inflammatory effects in experimental model of colitis. The mechanisms that this effect is achieved by the lysate of L. casei DN-114 001 comprise: a) improvement in the gut barrier function, b) correction of the dysbiosis, and c) modulation of the...

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Diversos genes expressos durante a diferenciação das células germinativas atuam no desenvolvimento ovariano. A diferenciação das células somáticas ovarianas depende do número de células germinativas pré-meióticas que migram para a fenda gonadal. A expressão espaço-temporal de genes envolvidos na diferenciação dessas células e a posterior sobrevivência dos oócitos meióticos são de interesse no estudo dos distúrbios do desenvolvimento sexual (DDS) 46,XX. Entre os genes envolvidos nesses processos estão o NANOS3, BMP15 e STRA8. O NANOS3, uma molécula de ligação ao RNA que bloqueia a via apoptótica, assegura a sobrevivência das células germinativas durante sua migração para o interior da gônada. O STRA8 atua no início da meiose das células germinativas na gônada de embriões XX, sendo o primeiro sinal de dimorfismo gonadal. Por outro lado a subseqüente sobrevivência dos oócitos é controlada por fatores de transformação e crescimento como o BMP15, que promove a diferenciação das células da granulosa que por sua vez participam indiretamente da diferenciação dos oócitos e das células da teca. Neste trabalho pesquisamos a presença de mutações inativadoras nos genes NANOS3 e BMP15 em 45 pacientes com disgenesia gonadal (DG) 46,XX (10 casos familiais) e 40 pacientes com amenorréia secundária sem mutação nos genes FSHR e SF1. Também pesquisamos mutações nas regiões promotora proximal e codificadora do gene STRA8 de 45 pacientes com DG 46,XX, 16 pacientes com DDS ovotesticular 46,XX e 5 pacientes com DDS testicular 46,XX todos SRY negativo nos quais foram afastados defeitos moleculares nos genes DAX1, WNT4 e SOX9. No NANOS3 identificamos a mutação p.E120K em homozigose, a primeira associada ao fenótipo de DG 46,XX. Esta mutação missense foi identificada em duas irmãs com DG 46, XX e está localizada no domínio de ligação do tipo dedo de zinco da proteína. A nova mutação não foi identificada em 200 alelos controles pesquisados. No BMP15, uma nova mutação nonsense p.Q115X foi identificada em homozigose em duas irmãs com DG 46XX e em heterozigose em uma paciente com amenorréia secundária não familial. O códon de parada prematuro está localizado na região do pré-peptídeo da proteína. A nova mutação não foi identificada em 200 alelos controles pesquisados. No gene STRA8, um único polimorfismo previamente descrito na literatura (rs7805859) foi identificado na região codificadora e nenhuma alteração na região promotora proximal foi identificada. Em conclusão, identificamos pela primeira vez uma mutação no gene NANOS3 associado á DG 46,XX e confirmamos a participação do BMP15 neste fenótipo. Distúrbios do desenvolvimento gonadal 46, XX podem ser causados por mutações em genes envolvidos tanto na diferenciação quanto manutenção das células germinativas ovarianas. / Several genes expressing during the germ cell differentiation act in ovary development. The differentiation of somatic ovary cells depends of a pool of pre meiotic germ cells migration into the gonad. The space and temporal expression pattern of some genes involved with germ cell differentiation and the subsequently oocyte survival should be investigated in the disorders of sexual development (DSD) 46,XX. Some key genes involved with these processes are: NANOS3, BMP15 and STRA8. The NANOS3, a RNA binding molecule that blocks the apoptotic pathway, ensures the survival during migration into genital ridge. The STRA8 acts in the bigining of germ cells meioses in XX embryos and mark the first sexual gonadal dimorphism. In other hand the subsequently oocyte survival is controlled through transforming growth factor member BMP15, that guarantees granulose cells differentiation that acts indirectly in meiotic oocyte and theca cells differentiation. In this work we searched for the presence of inactivating mutations in NANOS3 and BMP15 in 45 patients with 46XX gonadal dysgenesis (10 familial cases) and 40 patients with secondary amenorrhea without FSHR and SF1 mutation. We also searched for inactivating mutations in coding and proximal promoter region of STRA8 in 45 patients with 46XX gonadal dysgenesis, 16 ovotesticular disorder of sex development (DSD) patients and five 46XX testicular DSD patients all SRY negative and molecular defects in DAX1, WNT4 and SOX9 gene. In NANOS3 we identified the mutation p.E120K in homozygous state, the first associated with DG 46,XX phenotype. This missense mutation was identified in two sisters with 46XX GD and affects a zinc finger domain of the protein. The new variant was absent in 200 control alleles. In BMP15, a new nonsense mutation p.Q115X was identified two sisters in homozygous state and in one sporadic case of secondary amenorrhea in heterozygous state. The premature codon STOP affects the pro-peptide domain of the protein. The new variant was absent in 200 control alleles. In STRA8, only a previously described polymorphism (rs7805859) was identified without any other variation in coding or proximal promoter region. In conclusion, we identified for the fist time mutation in NANOS3 associated with DG 46XX and corroborate the role of BMP15 in this phenotype. Disorders of gonadal development 46,XX may be involved with differentiation and maintenance of ovarian germ cells.

INTRODUÇÃO: O carcinoma basocelular (CBC) é o tipo de câncer cutâneo mais comum no ser humano. O aparecimento de CBC na maioria das vezes se dá de forma esporádica em indivíduos que se expõem cronicamente ao sol. Eventualmente pode estar associado a síndromes, como: Bazex-Dupré- Christol, Rombo e Gorlin-Goltz. Diferente do que ocorre nas síndromes, os casos de CBCs múltiplos familiares não sindrômicos(CBCMFNS) são poucos estudados, tendo na literatura somente cinco relatos de famílias com a doença. O fenótipo é de múltiplos CBCs superficiais sem presença de outras anormalidades. Devido os CBCs esporádicos e os CBCs presentes na Síndrome de Gorlin-Goltz apresentarem mutações no gene PTCH1, possivelmente os CBCs múltiplos também estejam associados a alterações neste gene. Este gene esta localizado na região 9q22.3 possuindo 23 éxons, tem um papel importante na formação embrionária e de supressão tumoral. OBJETIVO: Análise genética dos éxons 9,11, 16, 17 e 23 do PTCH1 de oito componentes da mesma família, pertencentes a três diferentes gerações, sendo três portadores de CBCs múltiplos, e dentre estes dois suspeitos de CBCMFNS. MÉTODOS: Extração de DNA dos leucócitos do sangue periférico; PCR; clonagem dos produtos de amplificação (pGEM T Easy Vector) e seqüenciamento (Big Dye Terminator Kit). As mutações e polimorfismos encontrados foram comparados com a literatura e banco de dados de mutação do gene PTCH1 (www.cybergene.se/PATCH). RESULTADOS: Duas novas mutações foram encontradas nos pacientes suspeitos de CBCMFNS: uma frameshift nt4130(del C) e uma missense nt4261(A->G). Nos familiares foram encontradas cinco novas mutações: Em um primeiro indivíduo uma missense nt1420(G->T); em um segundo a mesma missense nt1420(G->T) e mais uma missense nt2873(C->T); em um terceiro duas frameshift nt1443 (ins T) e nt1468 (ins T), em dois outros indivíduos, irmãos, uma outra mutação missense nt4130(C->T). Foram encontradas ainda dezoito mutações, não descritas anteriormente, nos íntrons 10,15,16 e 17, algumas se repetindo em todos os indivíduos analisados. CONCLUSÃO: Pela primeira vez estão sendo descritas mutações em éxons e íntrons do gene PTCH1 em indivíduos portadores de CBCMFNS e em alguns de seus familiares. / INTRODUCTION: Basal cell carcinomas (BCC) are the most usual skin cancer that affects human beings. Sporadic BCCs are prevalent, often arising in people chronically exposed to UV radiation from the sun. Eventually BCCs may be associated to different syndroms like Bazex-Dupré-Christol, Rambo and Gorlin. Contrarily to syndromic BCCs, the cases of multiple familiar nonsydromic BCCs(MFNSBCC) have only few studies found in the literature. Only five families have been described to date with the disease. Since sporadic and Gorlin BCCs are associated to many mutations in the PTCH1 gene, we hypothesized that the multiple BCCs phenotype is also associated with mutations in this same gene. The PTCH1 tumor suppressor gene is located in the 9q22.3 chromosomal region, contains 23 exons, and has an important role in embryogenesis. OBJETIVE: To perform genetic analysis of PTCH1 exons 9, 11, 16, 17 e 23. METHODS: Eight individuals belonging to different generations from the same family were studied. Three of them bore multiple BCCs, and two of those were suspect to have MFNSBCC. DNA was extracted from blood leukocytes, submitted to PCR, and the PCR products were cloned (pGEM T Easy Vector, Promega) and sequenced (Big Dye Terminator Kit; ABI Prism 3100 sequencer; Applied Biosystems). The polymorphisms and mutations found were analyzed and compared to literature and PTCH1database (www.cybergene.se/PTCH/). RESULTS: In the patients suspect of MFNSBCC were found two new mutation: one frameshift nt4130(del C) and one missense nt4261(A->G). In the relatives were found five new mutation: Three missense nt1420(G->T); nt2873(C->T); nt4130(C->T); and two frameshift nt1443 (ins T) and nt1468 (ins T). In the introns 10,15,16 and 17 were found eighteen new mutations that were not previously reported. CONCLUSION: For the first time mutation in exons and introns of PTCH1 gene have been described in patients bore MFNSBCC and some of their relatives.

Welche Ursachen führen bei Keimzelltumoren zu Spätrezidiven? Sind es ungünstige Tumorkonstellationen, Behandlungsfehler oder individuelle Faktoren, die zu Spätrezidiven von Keimzelltumoren (KZT) führen? Ziel der Untersuchung war es, diese Fragen zu beantworten, um Patienten zu identifizieren, für die sich daraus Konsequenzen in der Therapie und Verlaufskontrolle ergeben. Indem wir Spätrezidive nach 4 Jahren auswerteten, wollten wir die besonderen Merkmale dieser Patientengruppe herausarbeiten. Unter 759 erfassten Patienten sahen wir 165 Frührezidive (< 2 Jahre), 92 Rezidive nach 2 Jahren und 73 Spätrezidive mehr als 4 Jahre nach der Initialdiagnose. Unsere statistische Auswertung bezieht sich auf die Spätrezidive mit einer mittleren Beobachtungszeit von 143 Monaten. Die initiale Konstellation der Tumormarker spielt eine bedeutende Rolle. 66% der Patienten hatten sowohl AFP als auch ß-HCG erhöht (gegenüber 47% bei den Frührezidiven und 33% im Gesamtkollektiv). Das gonadal reine Embryonalzellkarzinom (EZK) führt häufiger zu Spätrezidiven als andere Keimzelltumoren. Risikobehaftet sind höhere Stadien, von denen sich unter den Spätrezidiven 85% fanden; initial aber nur 57%. Demgegenüber ist poor-prognosis nach IGCCCG kein Risikofaktor. Mehr als die Hälfte der spätrezidivierten Patienten hatte bereits vorher mindestens ein Rezidiv gehabt. Eine initial von der heutigen Leitlinie abweichende Behandlung war bei 40% der Spätrezidive zu finden. Gründe hierfür waren eine falsche Histologie, die ungenaue Stadienzuordnung oder eine fehlende bzw. inkomplette Residualtumorresektion. Eine günstige Prognose haben die Patienten, bei denen eine Metastasenresektion vorgenommen werden kann. Die Heilungsrate (NED) war bei den operierten Spätrezidiven (70%) deutlich hoher als denjenigen, die zusätzlich chemotherapiert (46%) oder lediglich mit Chemotherapie behandelt worden waren (36%). Als Grund hierfür, ist die Früherkennung und noch vorhandene Operabilität des Befundes anzunehmen. Alle Seminom-Spätrezidive mit initialer Radiatio haben von der Chemotherapie profitiert. Bis auf einen Todesfall in der Chemotherapie wurden alle geheilt. Spätrezidive von KZT sollten als besondere Entität definiert werden. Krankheitsverlauf, Behandlung und Nachsorge weichen von dem üblichen Muster ab. Nur Seminome oder chemonaive Patienten sind zytostatisch zu behandeln. Bei den Nichtseminomen, die bereits initial oder im Frührezidiv eine Chemotherapie erhalten haben, sollte wenn möglich, eine operative Behandlung erwogen werden. Patienten mit initial erhöhten Tumormarkern (AFP + ß-HCG), reinem gonadalem EZK, höheren Krankheitsstadien oder einem Rezidiv innerhalb der ersten 4 Jahre haben eine ungünstige Prognose und erfordern eine jährliche lebenslange spezielle Nachsorge. Die Nachsorge sollte neben der klinischen Untersuchung und AFP-Bestimmung ein Schnittbildverfahren des Thorax und Retroperitoneums beinhalten. / What are the reasons for late relapse of testicular germ cell tumor? Do unfavourable tumor constellations, mismanaged treatment or individual factors; lead to the late relapse of testicular germ cell tumor? The aim of this investigation was to find an answer to this question and to identify the patients, who would benefit from modified treatments and follow-up modalities. By researching the late relapses diagnosed at least 4 years later, we wanted to identify the special characteristics of this group of patients. Among the 759 patients with testis cancer we found 165 early relapses (

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.

Diversos genes expressos durante a diferenciação das células germinativas atuam no desenvolvimento ovariano. A diferenciação das células somáticas ovarianas depende do número de células germinativas pré-meióticas que migram para a fenda gonadal. A expressão espaço-temporal de genes envolvidos na diferenciação dessas células e a posterior sobrevivência dos oócitos meióticos são de interesse no estudo dos distúrbios do desenvolvimento sexual (DDS) 46,XX. Entre os genes envolvidos nesses processos estão o NANOS3, BMP15 e STRA8. O NANOS3, uma molécula de ligação ao RNA que bloqueia a via apoptótica, assegura a sobrevivência das células germinativas durante sua migração para o interior da gônada. O STRA8 atua no início da meiose das células germinativas na gônada de embriões XX, sendo o primeiro sinal de dimorfismo gonadal. Por outro lado a subseqüente sobrevivência dos oócitos é controlada por fatores de transformação e crescimento como o BMP15, que promove a diferenciação das células da granulosa que por sua vez participam indiretamente da diferenciação dos oócitos e das células da teca. Neste trabalho pesquisamos a presença de mutações inativadoras nos genes NANOS3 e BMP15 em 45 pacientes com disgenesia gonadal (DG) 46,XX (10 casos familiais) e 40 pacientes com amenorréia secundária sem mutação nos genes FSHR e SF1. Também pesquisamos mutações nas regiões promotora proximal e codificadora do gene STRA8 de 45 pacientes com DG 46,XX, 16 pacientes com DDS ovotesticular 46,XX e 5 pacientes com DDS testicular 46,XX todos SRY negativo nos quais foram afastados defeitos moleculares nos genes DAX1, WNT4 e SOX9. No NANOS3 identificamos a mutação p.E120K em homozigose, a primeira associada ao fenótipo de DG 46,XX. Esta mutação missense foi identificada em duas irmãs com DG 46, XX e está localizada no domínio de ligação do tipo dedo de zinco da proteína. A nova mutação não foi identificada em 200 alelos controles pesquisados. No BMP15, uma nova mutação nonsense p.Q115X foi identificada em homozigose em duas irmãs com DG 46XX e em heterozigose em uma paciente com amenorréia secundária não familial. O códon de parada prematuro está localizado na região do pré-peptídeo da proteína. A nova mutação não foi identificada em 200 alelos controles pesquisados. No gene STRA8, um único polimorfismo previamente descrito na literatura (rs7805859) foi identificado na região codificadora e nenhuma alteração na região promotora proximal foi identificada. Em conclusão, identificamos pela primeira vez uma mutação no gene NANOS3 associado á DG 46,XX e confirmamos a participação do BMP15 neste fenótipo. Distúrbios do desenvolvimento gonadal 46, XX podem ser causados por mutações em genes envolvidos tanto na diferenciação quanto manutenção das células germinativas ovarianas. / Several genes expressing during the germ cell differentiation act in ovary development. The differentiation of somatic ovary cells depends of a pool of pre meiotic germ cells migration into the gonad. The space and temporal expression pattern of some genes involved with germ cell differentiation and the subsequently oocyte survival should be investigated in the disorders of sexual development (DSD) 46,XX. Some key genes involved with these processes are: NANOS3, BMP15 and STRA8. The NANOS3, a RNA binding molecule that blocks the apoptotic pathway, ensures the survival during migration into genital ridge. The STRA8 acts in the bigining of germ cells meioses in XX embryos and mark the first sexual gonadal dimorphism. In other hand the subsequently oocyte survival is controlled through transforming growth factor member BMP15, that guarantees granulose cells differentiation that acts indirectly in meiotic oocyte and theca cells differentiation. In this work we searched for the presence of inactivating mutations in NANOS3 and BMP15 in 45 patients with 46XX gonadal dysgenesis (10 familial cases) and 40 patients with secondary amenorrhea without FSHR and SF1 mutation. We also searched for inactivating mutations in coding and proximal promoter region of STRA8 in 45 patients with 46XX gonadal dysgenesis, 16 ovotesticular disorder of sex development (DSD) patients and five 46XX testicular DSD patients all SRY negative and molecular defects in DAX1, WNT4 and SOX9 gene. In NANOS3 we identified the mutation p.E120K in homozygous state, the first associated with DG 46,XX phenotype. This missense mutation was identified in two sisters with 46XX GD and affects a zinc finger domain of the protein. The new variant was absent in 200 control alleles. In BMP15, a new nonsense mutation p.Q115X was identified two sisters in homozygous state and in one sporadic case of secondary amenorrhea in heterozygous state. The premature codon STOP affects the pro-peptide domain of the protein. The new variant was absent in 200 control alleles. In STRA8, only a previously described polymorphism (rs7805859) was identified without any other variation in coding or proximal promoter region. In conclusion, we identified for the fist time mutation in NANOS3 associated with DG 46XX and corroborate the role of BMP15 in this phenotype. Disorders of gonadal development 46,XX may be involved with differentiation and maintenance of ovarian germ cells.

Reproductive containment in farmed fish is highly desired for sustainable aquaculture to prevent genetic introgression with wild conspecifics and enhance productivity by suppressing sexual maturation. A number of strategies have already been implemented or have been tested in commercially important fish (e.g. triploidy, monosexing, hormonal therapies); however, they either do not result in 100% containment, or they cannot be applied to all species. One promising new approach consists in disrupting primordial germ cells (PGCs), at the origin of germline cells, to induce sterility. The work carried out in this doctoral thesis aimed to investigate the genes involved in the survival of germ cells and subsequently conduct a functional analysis of candidate genes using CRISPR/Cas9 gene editing system to ultimately provide the basis for the development of a novel sterilisation technique. Nile tilapia was chosen as the experimental animal as it is a major aquaculture species worldwide and the control of reproduction plays a critical role in the farming productivity in this species. In addition, the species has clear advantages as its whole genome sequence is accessible, the generation time is relatively short and zygotes can be available all year round. Initially, a panel of 11 candidate genes with reported roles in survival of PGCs was investigated during the ontogenic development which led to the selection of piwi-like (piwil) gene as a target for genome editing. Then, high temperature was tested as a means to induce germ cell loss to better understand the mechanism underlying germ cell survival and apoptosis, and this study confirmed the functional importance of piwil genes in relation to germ cell loss and proliferation. In addition, the study suggested potential subfunctionalisation within the Bcl-2 gene family which requires further investigation. The next step aimed to optimise the CRISPR/Cas9 gene editing method by improving the microinjection system and testing different concentrations of sgRNAs. Over 95% of injected embryos showed on-target mutation in piwil2 via zygote injection of CRISPR/Cas9 reagents and complete KO larvae were shown in half of the mutants, producing putative sterile fish. However, there was no clear association between the phenotypes in PGCs and the mutation rate. Further comparative studies of mutant screening methods including T7E1, RGEN, HRMA, fragment analysis and NGS revealed that the genotypes of F0 are highly mosaic, suggesting that deep sequencing is recommended for accurate and high throughput F0 screening and further improvement for predictable genome editing is required for a reliable gene functional analysis in F0. In summary, the current thesis provided new scientific knowledge and supporting evidence for the use of the CRISPR/Cas9 gene editing platform to study gene function associated with sterility, with the ultimate goal to develop an alternative sterilisation method in fish.

Neste trabalho, nos concentramos no estudo da topologia da fibração de Milnor associada a um germe de aplicação polinomial f : (Rn , 0) &rarr; (Rp , 0) com uma singularidade isolada na origem. O primeiro resultado é uma extensão da caracterização de germes de aplicações triviais nos pares de dimensões (n; p) quando n - p = 3: Uma caracterização inicial foi apresentada por Church e Lamotke em 1975. O segundo resultado é a caracterização de NS-pares (S5 , K2), usando a topologia de espaços de configuração. Como uma consequência desta caracterização, mostramos a existência de germe de aplicação polinomial real nos pares de dimensões (6; 3) com uma singularidade isolada na origem tal que sua fibra de Milnor não é difeomorfa a um disco. A existência desses exemplos coloca um fim ao problema da não-trivialidade proposto por Milnor em 1968 e além disso, nos permite apresentar um novo resultado sobre a topologia da fibra de Milnor real nos pares de dimensões (2n; n) e (2n + 1; n); n &ge; 3: Tal resultado garante a existência de germes de aplicações polinomiais (Rn , 0) &rarr; (Rp, 0); n &ge; p &ge; 2; com uma singularidade isolada na origem tais que suas fibras de Milnor têm o tipo de homotopia de um buquê de um número positivo de esferas. / In this work, we focus on the study of the topology of the Milnor fibration associated with a polynomial map germ f : (Rn , 0) &rarr; (Rp , 0) with an isolated singularity at the origin. The first result is an extension of the characterization of trivial map germs in the pairs of dimensions (n; p) when n - p = 3: An initial characterization was presented by Church and Lamotke in 1975. The second result is a characterization of NS-pairs (S5 , K2), using the topology of configuration spaces. As a consequence of this characterization, we show the existence of real polynomial map germs in the pairs of dimensions (6; 3) with an isolated singularity at the origin such that its Milnor fibers are not diffeomorphic to a disc. The existence of such examples ends a non-triviality problem posed by Milnor in 1968 and furthermore, it allows us to show a new result about the topology of the real Milnor fibers in the pairs of dimensions (2n; n) and (2n + 1; n); n &ge; 3. This result ensure the existence of polynomial map germs (Rn , 0) &rarr; (Rp, 0); n &ge; p &ge; 2; with an isolated singularity at the origin such that its Milnor fibers has the homotopy type of a bouquet of a positive number of spheres.

In this study, Naegleria fowleri, a pathogenic amoeba and the causative agent of Primary Amebic Meningoencephalitis (PAM), was utilized to determine the presence or absence of classically conserved Golgi molecules featured in the expression of a Golgi apparatus. Previous studies concluded no Golgi expression via light microscopy and transmission electron microscopy, but a recent report on Naegleria gruberi indicated the presence of dispersed Golgi tubules. Non-pathogenic species of the Naegleria genus such as Naegleria gruberi 30540 and Naegleria lovaniensis 30569 were utilized in Western immunoblot analysis compared to reduced whole-cell lysate proteins of two strains of N. fowleri and Vero CCL-81, Chlorocebus sp. kidney epithelial cells, which were utilized as a positive control for Golgi expression. N. fowleri and N. lovaniensis whole-cell lysates had indications of a 110 kDa reduced protein, associated with the predicted molecular weights of the beta-COPI subunit of the COPI cis-Golgi vesicular transport complex with further Western immunoblot indication of a weak band around 25 kDa corresponding to rabbit polyclonal antibodies specific for ARF1. Serial Dilutions of Wheat Germ Agglutinin Alexa Fluor 488TM were performed on Vero cells, Naegleria fowleri 30894, and N. gruberi 30540 with 1:100 dilution of recommended stock dilution of WGA 488 determined for utilization in sequential immunofluorescence. Sequential immunofluorescence with Wheat Germ Agglutinin Alexa Fluor 488TM and then blocked with 3% BSA:PBS [wt/vol] dilution with subsequent incubation in rabbit anti-beta-COPI primary 1:250, and 1:1000 of Alexa Fluor 594 goat anti-rabbit secondary antibody exposure showed strong indications of organized cis- and trans-punctate Golgi body markers in close association in individual and dividing cells of Naegleria fowleri and conserved Golgi expression in the positive control Vero cells, but further experiments are necessary to verify this finding with N. fowleri.