The transcription factor, Pit-1, is involved in the transcriptional regulation of the mammalian prolactin (Prl), growth hormone (GH) and thyroid stimulating hormone b-subunit genes (TSHb) as well as its own gene. The role of Pit-1 in avian species is unknown. Three turkey (t) Pit-1 isoforms have been identified that arise from alternative transcription initiation and alternative splicing. Splicing of exon 1 to an alternative acceptor splice site in exon 2 results in a 28 amino acid insertion in tPit-1β* relative to tPit-1*. Both isoforms initiate transcription at exon 1. A tPit-1 transcript unique to the turkey has been identified and arises following transcription initiation upstream of the alternative acceptor splice site in exon 2. Western blot analysis of pituitary extracts has revealed two isoforms of 37 and 40 kDa. The ability of Pit-1 to transactivate the Prl, GH, and Pit-1 promoters was determined with cotransfection assays. The tPrl, tGH, tPit-1 and rat (r) Prl promoters were cloned upstream of the luciferase gene in a reporter construct. Turkey Pit-1 isoforms and rPit-1 were expressed under the control of the Avian Sarcoma Virus Long Terminal Repeat (ASVLTR) promoter. Cotransfection analyses in mouse L cells indicate that tPit-1* activates the tPrl, tGH, tPit-1 and rPrl promoters 4.6-, 3.8-, 1.7-, and 29.0-fold, respectively. Similar results were observed when cotransfection assays were performed in a turkey pituitary-derived cell line and in primary turkey pituitary cells. These results indicate that tPit-1 is not a strong activator of the tPrl, tGH, or tPit-1 genes, whereas Pit-1 does activate these genes in mammals. A point mutation at amino acid position 176 (ser ⟹ leu) in the POU-homeodomain results in a mutant tPit-1 that shows decreased activity on all promoters tested. Turkey Pit-1* (ser-176) activates the rPrl promoter 14-fold lower than the wild type tPit-1* (leu-176). / Master of Science

A ce jour, un certain nombre d'adénomes hypophysaires somatotropes échappent encore au contrôle de la maladie. Il reste nécessaire d'étudier plus finement les différentes voies de contrôle de la sécrétion hormonale et de la prolifération tumorale afin d'identifier des traitements alternatifs susceptibles de contrôler ces symptômes tumoraux. De même, un certain nombre de patients porteurs de prolactinomes (environ 10%), à l'origine de dysfonctionnement gonadique et sexuels, échappent au contrôle de l'hypersécrétion de prolactine par les traitements actuels. Pour ces deux types de tumeurs, s'ajoutent des complications neurologiques consécutives à l'accroissement des volumes tumoraux. Dans une perspective de contrôle de l'hypersécrétion tumorale nous nous sommes intéressés à deux stratégies faisant intervenir un transfert de gènes dans les cellules. POU1F1 est un facteur de transcription crucial pour le développement et le maintien des lignages somatotrope, lactotrope et thyréotrope et son expression est retrouvée dans les adénomes hypophysaires. De plus, des mutations de ce gène, notamment le mutant dominant négatif R271W, ont été identifiés chez des patients présentant des déficits hypophysaires combinés en GH, en PRL et en TSH. Une première partie de ma thèse a donc porté sur l'étude du transfert du mutant dominant négatif de POU1F1 par un vecteur lentiviral dans les tumeurs hypophysaires somatotropes et lactotropes afin d'évaluer son impact sur la sécrétion hormonale et sur la viabilité cellulaire. Nous avons montré que l'inactivation de ce facteur de transcription entrainait une diminution de la viabilité cellulaire et de la sécrétion de toutes les tumeurs testées. / To this date, some somatotroph adenomas do not respond to the current treatment of pituitary tumors. It still necessary to study the different ways to control the hormonal secretion and the tumor progression to identify alternate therapy capable to manage these symptoms. Likewise, some patient with prolactinomas (about 10%) responsible of gonad and sexual dysfunction do not respond to the control of prolactin hypersecretion. In addition, some neurological complications are finding due to the neighboring structures compression for both of these tumors. To better control the hormonal secretion we investigate two different strategies involving a gene transfer in the cells. POU1F1 is a major transcription factor involved in the pituitary development and the management of somatotroph, thyreotroph and lactotroph cell lines. Its expression is found in pituitary adenomas belonging of these 3 cell lines. Moreover some mutations like the R271W mutant are identified in patients with combined pituitary hormonal deficiency (CPHD). In the first part of my thesis we transfer this dominant negative mutant via a lentiviral transfer in the somatotroph and lactotroph adenomas to evaluate its impact on hormonal secretion and cell viability. We show an inhibitory effect of the POU1F1 inactivation on the secretion and the cell viability of all our tumors. Somatostatin analogs are the major medical therapy for somatotroph adenomas and control GH secretion in 60% of these patients. Octreotide resistance is highly correlated to a low expression of the sst2 receptor.

Growth hormone (GH) is a vital pituitary hormone controlling somatic cell growth and development. GH has a multitude of effects on the body: deficiency can lead to dwarfism while excess can cause conditions such as gigantism. Human patients with mutations in the transcription factor Pit-1 show decreased GH and prolactin levels. However, Pit-1 is known to control multiple pituitary hormones, so what other factors lead to the specificity of transcriptional regulation of the GH gene via its promoter? In order to study this, our lab has been analyzing rat pituitary cell lines to understand the role of ZNF292 (formerly called Zn-16), a selective GH transcription regulator with 16 zinc fingers that bind to the GH promoter DNA. This work has used rat MtT/S cells that are unique in that they exclusively express GH. MtT/S cells were procured from Riken Cell Bank in Japan, cultured, and examined for GH hormone and RNA expression. Results confirmed that the MtT/S cells are terminally differentiated as somatotrophs. To understand the role of ZNF292 in transcription of the GH gene, recent rat genomic data was analyzed to determine the positions of 7 exons upstream from the large exon 8 that contains the important zinc finger-encoding portions of the protein. First, MtT/S RNA was reverse transcribed into DNA, then PCR amplification was performed using primer pairs encompassing various sections of the exon 1 – 7 region. Specific PCR products were found with distinct products ranging in size from 130 to 960, all of which agreed with the predicted sizes of these exons. It had previously been theorized that ZNF292 contained a single large exon; however, these results show that splicing of the primary transcript does occur in this upstream region. Characterizing this exonic region was performed because it has been shown that ZNF292 produces circular RNA (circRNA) of unknown function in human endothelial cells and in certain types of cancer. CircRNAs are thought to be created by the “back-splicing” of exons, so that rather than a linear transcript, the ends are circularized for a portion of the transcript. Having determined the sequence and organization of these upstream exons, we are now testing primer sets that will demonstrate productive amplification only from circRNAs. Further, we are removing linear RNAs using RNAse R treatment to selectively enhance circRNA presence in the reactions. Finally, data from RNA-Seq analysis of the MtT/S cells will be scrutinized to determine if additional exon/intron boundaries or alternative splice sites exist in this upstream 7 exon region. The study of circular RNAs could be very important in understanding its role in acting as a microRNA sponge or RNA-binding protein sponge during their regulation of downstream gene transcription. Also, analysis of this mechanism shows potential as a clinical tool in cancer treatment because ZNF292 functions as a tumor suppressor in colorectal and chronic lymphatic leukemia.

No description available.

Poor reproductive performance by turkey hens compared with chickens is partially due to the early cessation of egg production associated with the onset of incubation behavior. Prolactin (Prl) is involved in the induction and maintenance of incubation behavior in birds, and understanding the regulatory mechanism(s) of Prl gene expression will provide fundamental information to manipulate Prl production for better reproductive performance in turkey hens. / Ph. D.

Os craniofaringiomas são os tumores mais frequentes da região hipotálamohipofisária na faixa etária pediátrica. Apesar de serem histologicamente benignos, sua tendência infiltrativa e seu comportamento agressivo resultam em significante morbimortalidade. Histologicamente podem ser divididos em dois subtipos: adamantinomatosos e papilíferos. A patogênese dos craniofaringiomas é pouco compreendida. Mutações no gene CTNNB1, que codifica a proteína beta-catenina, são a única alteração molecular conhecida até o momento implicada na tumorigênese dos craniofaringiomas adamantinomatosos. Tais mutações afetam o sítio de degradação da beta-catenina, que passa a se acumular no citoplasma e no núcleo, ativando excessivamente a via de sinalização WNT, através da ligação aos fatores de transcrição da família LEF/TCF, levando a tumorigênese. Recentemente foi descoberto um novo mecanismo de determinação da linhagem celular hipofisária regulado pela beta-catenina, através do qual ela interage diretamente com o PROP1 para determinar a diferenciação celular hipofisária. De acordo com esse modelo, o complexo protéico PROP1/beta- catenina atua simultaneamente como repressor do HESX1 e ativador do PIT1, dependendo dos co-fatores associados. Pacientes com mutações germinativas inativadoras no PROP1 desenvolvem hipopituitarismo e podem apresentar aumento hipofisário com imagens de ressonância nuclear magnética (RNM) da região selar muitas vezes semelhantes àquelas dos craniofaringiomas, com hiperssinal em T1. Por outro lado, camundongos com expressão persistente do Prop1 exibem defeitos na regulação da proliferação celular hipofisária, incluindo cistos da bolsa de Rathke, hiperplasia adenomatosa e tumores, sugerindo que mutações com ganho de função no PROP1 também poderiam contribuir para a patogênese de tumores hipofisários em seres humanos. A semelhança entre as imagens de RNM dos pacientes com craniofaringiomas e daqueles com aumento hipofisário devido a mutações inativadoras no PROP1, e o fato de que camundongos transgênicos com expressão persistente do Prop1 apresentam aumento da susceptibilidade a tumores hipofisários, deram base a nossa hipótese de que uma desregulação na expressão do PROP1 em humanos poderia estar envolvida na patogênese dos craniofaringiomas adamantinomatosos. Esse trabalho teve como objetivo avaliar a presença de mutação somática no exon 3 do CTNNB1 e avaliar a expressão desse gene e do gene PROP1 em craniofaringiomas adamantinomatosos. Foram obtidas 14 amostras desse tipo de tumor por meio da ressecção terapêutica. As amostras foram submetidas à extração do RNA e posterior transcrição reversa para obtenção de cDNA. A partir do cDNA foi realizada PCR e sequenciamento do exon 3 do CTNNB1 em todas as amostras. Porém, a avaliação por PCR em tempo real foi realizada apenas em 12 amostras, devido à qualidade inadequada de 2 amostras para submissão a essa metodologia. Foram encontradas mutações missense, em heterozigose em 9 das 14 amostras, sendo 5 previamente descritas e 2 ainda não descritas em craniofaringiomas adamantinomatosos. Hiperexpressão do CTNNB1 foi encontrada em 7 amostras, sendo 5 com mutação e 2 sem mutação no CTNNB1.A hiperexpressão variou de 2,5 a 6,2 vezes maior que o pool de hipófise normal. Contudo, a expressão do PROP1 foi indetectável em todas as amostras. Concluímos que o aumento da expressão do CTNNB1 presente em 58% das amostras sugere o envolvimento também da hiperexpressão desse gene na etiopatogenia do craniofaringioma adamantinomatoso, enquanto a ausência de expressão do PROP1 afasta a participação desse gene na etiopatogenia do craniofaringioma adamantinomatoso / Craniopharyngiomas are the the commonest tumors to involve the hypothalamo-pituitary regions in childhood population. Histologically they are benign, and can be divided in two primary subtypes: the adamantinomatous and the papillary. Although histologically benign, their infiltrative tendency and aggressive behavior can result in great morbidity. The pathogenesis of craniopharyngiomas is poorly understood. To date, beta-catenin gene (CTNNB1) mutations have been identified only in the adamantinomatous subtype. These mutations affect the degradation target box of beta-catenin that accumulates in the cytoplasm and the nucleus increasing the transcriptional activity of WNT pathway through interaction with the transcription factors of LEF/TCF family, leading to tumorigenesis. Recently, an interaction between beta-catenin and PROP1 was described as a new mecanism for beta-catenindependent regulation of pituitary cell-lineage determination. According to this novel model, the PROP1/beta-catenin proteic complex would act as a binary switch to simultaneously repress the transcription factor HESX1 and to activate expression of transcription factor PIT1, depending on the associated cofactors. Patients with loss-of-function mutations in PROP1 present combined pituitary hormonal deficiency generally associated with pituitary enlargement and the magnetic resonance imaging (MRI) of the sellar region in these patients sometimes resembles that of the craniopharyngiomas, with T1 hyperintense signal. On the other hand, transgenic mice with persistent Prop1 expression exhibit defects consistent with misregulation of pituitary cell proliferation, including adenomatous hyperplasia with formation of Rathke\'s cleft cysts and tumors suggesting that misregulation of PROP1 expression in human could contribute to pathogenesis of pituitary tumors. The similarity between the MRI images of craniopharyngiomas patients and that of patients with loss-of-function mutations in PROP1, associated with the fact that transgenic mice with persistent Prop1 expression exhibit increased susceptibility to pituitary tumors gave rise to our hypothesis that a misregulation of PROP1 expression could be involved in the pathogenesis of adamantinomatous craniopharyngiomas. The aim of this study was to analyze the presence of somatic mutations in exon 3 of CTNNB1 and the expression pattern of this gene and the PROP1 gene in adamantinomatous craniopharyngiomas. Fourteen samples were obtained from therapeutic surgery and submitted to RNA extraction and reverse transcription in order to produce the cDNA. The cDNA was used as a template to CTNNB1 exon 3 PCR reaction followed by direct sequencing of all samples. However, the real-time RT-PCR analysis was realized only in 12 samples, since 2 of them had an insufficient quality for this method. Missence, heterozygous mutations were found in 9 out of 14 samples; five were previously described and 2 not yet described in adamantinomatous craniopharyngiomas. Overexpression of CTNNB1 was found in 7 samples, which them 5 with CTNNB1 mutation 2 whitout. The overexpression ranged from 2.5 to 6.2 fold more than pituitary normal pool. However, the PROP1 expression was undetectable in all the samples. We could conclude that the amount of 58% CTNNB1 overexpressed samples suggest also a role of this overexpression in the pathogenesis of adamantinomatous craniopharingiomas, while the undetectable levels of PROP1 exclude a role of this gene in the pathogenesis of adamantinomatous craniopharingiomas

O fósforo é um dos minerais mais abundantes no corpo além de ser essencial para muitos processos biológicos. A homeostase do fósforo depende da absorção no intestino, da excreção renal, da remodelação óssea e de hormônios como o paratormônio, calcitriol e FGF-23. Nos pacientes com doença renal crônica, a excreção urinária de fósforo está comprometida levando à hiperfosfatemia, que contribui para o aumento da morbidade e mortalidade desses pacientes. A absorção intestinal de fósforo no intestino delgado, particularmente via cotransportadores NaP-IIb e PiT-1, é pouco estudada. O objetivo desse estudo foi avaliar os efeitos de dietas com diferentes concentrações de fósforo na expressão proteica e gênica dos cotransportadores NaP-IIb e PiT-1, bem como na apoptose dos enterócitos dos diferentes segmentos do intestino delgado de animais controles e urêmicos. Estudamos setenta e seis ratos Wistar machos, inicialmente divididos em dois grupos: controles (C) e urêmicos (Nx). Cada grupo foi subdividido em outros três, de acordo com a concentração de fósforo (P) na dieta: dieta Baixa (0,2%), dieta Padrão (0,54%) e dieta Alta (0,9%). Analisamos parâmetros bioquímicos (creatinina, P, Cai, PTH e FGF-23), a expressão proteica dos cotransportadores, através de Western Blotting, ELISA e imunofluorescência, a expressão gênica por PCR em tempo real e a apoptose dos enterócitos pela técnica TUNEL. Os resultados mostraram que os níveis séricos de creatinina, P, PTH e FGF-23 foram significativamente mais elevados nos animais Nx. Nos animais C com dieta baixa em P observamos aumento da expressão proteica do cotransportador NaP-IIb em todos os segmentos do intestino enquanto, no Nx Baixo, a expressão desse cotransportador foi menor somente no jejuno. Quanto ao PiT-1, sua expressão foi menor no íleo do grupo Nx Alto comparado ao seu respectivo controle. A expressão gênica do cotransportador NaP-IIb foi menor no jejuno do Nx Alto e maior no íleo desse mesmo grupo em relação aos seus respectivos controles. A expressão gênica do PiT-1 foi maior em todos os segmentos do grupo Nx Baixo em relação aos seus controles. Detectamos uma correlação direta entre a expressão gênica do NaP-IIb no jejuno com o fósforo sérico. A expressão gênica do PiT-1 no jejuno correlacionou-se com o fósforo sérico e no duodeno e jejuno, com os níveis séricos de FGF-23. No duodeno e no jejuno, a porcentagem de enterócitos apoptóticos foi maior nos animais Nx Alto comparados aos controles, particularmente no duodeno a porcentagem dessas células também foi maior no grupo Nx Baixo e controle padrão. Já no jejuno, tanto o grupo Nx baixo quanto no Nx Padrão observamos menos células apoptóticas que nos seus respectivos controles. Quando analisamos conjuntamente todos os segmentos intestinais o grupo Nx alto apresentou mais células apoptóticas que o controle e o oposto foi observado nos grupos Nx Baixo e Nx Padrão. Em conclusão, dietas com distintas concentrações de fósforo promovem modificações na expressão proteica e gênica dos cotransportadores NaP-IIb e PiT-1 no intestino delgado que não seguem um padrão uniforme. A sobrecarga de fósforo aumenta a porcentagem de enterócitos apoptóticos nos animais urêmicos / Phosphorus is one of the most abundant minerals in the body besides being essential for many biological processes. Phosphorus homeostasis depends on absorption in the small intestine, renal excretion, bone remodeling and hormones such as parathyroid hormone, calcitriol and FGF-23. In patients with chronic kidney disease phosphorus urinary excretion is compromised leading to hyperphosphatemia, which contributes to increased morbidity and mortality of these patients. Intestinal absorption of phosphorus in the small intestine, particularly of NaP-IIb and PiT-1 cotransporters is poorly studied. The aim of this study was to evaluate the effects of diets with different concentrations of phosphorus in protein expression and gene of NaP-IIb and PiT-1 cotransporters as well as in apoptosis of enterocytes of different segments of intestine in control and uremic animals. We studied seventy-six male Wistar rats initially divided into two groups: controls (C) and uremic (Nx). Each group was subdivided into three others, according to the phosphorus concentration in the diet: Low diet (0.2% P), Standard diet (0.54% P) and High diet (0.9% P). We analyzed biochemical parameters (creatinine, P, iCa, PTH and FGF-23), protein expression of cotransporters, using Western Blot, ELISA, immunofluorescence, real-time PCR and apoptosis of enterocytes using the TUNEL technique. Results showed that serum creatinine, P, PTH and FGF-23 were significantly higher in Nx animals. In C animals with a diet low in P we observed increase in protein expression of NaP-IIb cotransporter in all segments of the intestine while in low Nx expression of this cotransporter was lower only in jejunum. As for PiT-1 expression was lower in the ileum of the high Nx group compared to their respective control. Gene expression of NaP-IIb cotransporter was lower in the jejunum of high Nx and higher in the ileum of the same group as compared to their respective controls. PiT-1 gene expression was higher in all segments of the low Nx group compared to their controls. We detected a direct correlation between the gene expression of NaP-IIb in jejunum and serum phosphorus. Gene expression of PiT-1 correlated with serum phosphorus in the jejunum, and correlated with the serum levels of FGF-23 in the jejunum and duodenum. In the duodenum and jejunum the percentage of apoptotic enterocytes was higher in high Nx animals compared to controls; particularly in the duodenum the percentage of these cells was also higher in low Nx group and standard control. In the jejunum in both low Nx group and standard Nx we observed fewer apoptotic cells than in their respective controls. When we analyze all intestinal segments together the high Nx group had more apoptotic cells than the control, and the opposite was observed in the low Nx group and standard Nx group. In conclusion, diets with different phosphorus concentrations promote changes in protein expression and gene of NaP-IIb and PiT-1 cotransporters in the small intestine that do not follow a uniform pattern. Phosphorus overhead increases the percentage of apoptotic enterocytes in uremic animals

L’embranchement Hemichordata regroupe les classes Enteropneusta et Pterobranchia. Hemichordata constitue, avec l’embranchement Echinodermata, le groupe-frère des chordés. Les entéropneustes sont des organismes vermiformes solitaires qui vivent sous ou à la surface du substrat et s’alimentent généralement par déposivorie, alors que les ptérobranches sont des organismes coloniaux filtreurs habitant dans un réseau de tubes appelé coenecium. Ce mémoire présente trois études dont le point commun est l’utilisation des hémichordés actuels pour répondre à des questions concernant l’évolution des hémichordés, des chordés, et du super-embranchement qui les regroupe, Deuterostomia. Notre première étude démontre que les fentes pharyngiennes, l’organe pré-oral cilié (POCO) et le pharynx de l’entéropneuste Protoglossus graveolens sont utilisés pour l’alimentation par filtration. Le système de filtration de P. graveolens permet la capture de particules jusqu’à 1.3 um, à un débit de 4.05 mm.s-1, pour une demande énergétique de 0.009 uW. Les similarités structurales et fonctionnelles avec le système de filtration des céphalochordés suggèrent que la filtration pharyngienne est ancestrale aux deutérostomes. Lors de notre deuxième étude, nous avons exploré l’hypothèse selon laquelle le POCO des entéropneustes, une structure ciliée pré-buccale au rôle possiblement chémorécepteur, serait homologue au « wheel organ » des céphalochordés et à l’adénohypophyse des vertébrés. Pour cela, nous avons déterminé par immunohistochimie l’expression de Pit-1, un facteur de transcription spécifique à ces deux structures, chez l’entéropneuste Saccoglossus pusillus. Pit-1 est exprimé dans des cellules sensorielles du POCO, mais aussi dans des cellules épithéliales distribuées dans le proboscis, collet et tronc. Ce patron d’expression ne permet pas de confirmer ou rejeter l’homologie du POCO et de l’adénohypophyse des vertébrés. Lors de notre troisième étude, nous avons caractérisé l’ultrastructure du coenecium des ptérobranches Cephalodiscus hodgsoni, Cephalodiscus nigrescens et Cephalodiscus densus par microscopie électronique à transmisison et à balayage. Cephalodiscus est le groupe frère de Graptolithina, un groupe qui inclut les graptolithes éteints ainsi que les ptérobranches du genre Rhabdopleura. Nous avons décrit les types de fibrilles de collagène présents, leur taille et leur organisation, ainsi que l’organisation globale du coenecium. Nous avons ainsi démontré la présence chez Cephalodiscus d’une organisation similaire au paracortex, pseudocortex et eucortex des graptolithes. La présence chez Cephalodiscus de ce type d’organisation suggère que le cortex est ancestral à la classe Pterobranchia. Ces trois études illustrent plusieurs axes importants de la recherche sur les hémichordés, qui en intégrant des données morphologiques, fonctionnelles et moléculaires permet de reconstruire certains évènements clés de l’évolution des deutérostomes. / The phylum Hemichordata comprises the classes Enteropneusta and Pterobranchia. Together with echinoderms, hemichordates are the sister-group to chordates. Enteropneusts are worm-shaped solitary deposit feeders. Pterobranchs are colonial filter feeders that live in a secreted collagenous domicile called a coenecium. In this thesis, three studies are presented. These studies are based on observations of extant hemichordates, and adress a variety of issues relating to the evolution of hemichordates, chordates, and the super-phylum to which they belong: Deuterostomia. Our first study demonstrates that the gill slits, pre-oral ciliary organ (POCO), and lining of the pharynx of the enteropneust Protoglossus graveolens are used in filter feeding. The filter-feeding system of P. graveolens enables particle capture down to 1.3 um, at a rate up to 4.05 mm.s-1, with a power consumption of 0.009 uW. Structural and functional similarities with the cephalochordate filter-feeding system suggest that pharyngeal filter-feeding is ancestral to the deuterostomes. In our second study, we address the hypothesis that the enteropneust POCO, a putative chemosensory structure located anterior to the mouth, is homologous to the cephalochordate wheel organ and vertebrate adenohypophysis. We characterized the expression pattern of the adenohypophysis-specific transcription factor Pit-1 in the adult enteropneust Saccoglossus pusillus with immunohistochemistry. Pit-1 is expressed in sensory cells of the POCO and in scattered epithelial cells of the proboscis, collar and trunk. This expression pattern does not allow to confirm or reject the homology of the POCO with the vertebrate adenohypophysis. In our third study, we characterized the ultrastructure of the coenecium of the pterobranchs Cephalodiscus hodgsoni, Cephalodiscus nigrescens and Cephalodiscus densus using transmission and scanning electron microscopy. Cephalodiscus is the sister-group to the Graptolithina, which includes the extinct graptolites and the extant pterobranch genus Rhabdopleura. We described the fibril types, size and organization, as well as the general organization of the coenecium. We demonstrated that the coenecium of Cephalodiscus shows similarities with the graptolite eucortex, paracortex and pseudocortex. The cortical-like organization of the coenecium of Cephalodiscus suggests that the cortex is ancestral to the Pterobranchia. Together, these three studies illustrate different axes of hemichordate research, and show how integrating morphological, functional and molecular data allows us toinfer key events in the evolution of deuterostomes.

Os craniofaringiomas são os tumores mais frequentes da região hipotálamohipofisária na faixa etária pediátrica. Apesar de serem histologicamente benignos, sua tendência infiltrativa e seu comportamento agressivo resultam em significante morbimortalidade. Histologicamente podem ser divididos em dois subtipos: adamantinomatosos e papilíferos. A patogênese dos craniofaringiomas é pouco compreendida. Mutações no gene CTNNB1, que codifica a proteína beta-catenina, são a única alteração molecular conhecida até o momento implicada na tumorigênese dos craniofaringiomas adamantinomatosos. Tais mutações afetam o sítio de degradação da beta-catenina, que passa a se acumular no citoplasma e no núcleo, ativando excessivamente a via de sinalização WNT, através da ligação aos fatores de transcrição da família LEF/TCF, levando a tumorigênese. Recentemente foi descoberto um novo mecanismo de determinação da linhagem celular hipofisária regulado pela beta-catenina, através do qual ela interage diretamente com o PROP1 para determinar a diferenciação celular hipofisária. De acordo com esse modelo, o complexo protéico PROP1/beta- catenina atua simultaneamente como repressor do HESX1 e ativador do PIT1, dependendo dos co-fatores associados. Pacientes com mutações germinativas inativadoras no PROP1 desenvolvem hipopituitarismo e podem apresentar aumento hipofisário com imagens de ressonância nuclear magnética (RNM) da região selar muitas vezes semelhantes àquelas dos craniofaringiomas, com hiperssinal em T1. Por outro lado, camundongos com expressão persistente do Prop1 exibem defeitos na regulação da proliferação celular hipofisária, incluindo cistos da bolsa de Rathke, hiperplasia adenomatosa e tumores, sugerindo que mutações com ganho de função no PROP1 também poderiam contribuir para a patogênese de tumores hipofisários em seres humanos. A semelhança entre as imagens de RNM dos pacientes com craniofaringiomas e daqueles com aumento hipofisário devido a mutações inativadoras no PROP1, e o fato de que camundongos transgênicos com expressão persistente do Prop1 apresentam aumento da susceptibilidade a tumores hipofisários, deram base a nossa hipótese de que uma desregulação na expressão do PROP1 em humanos poderia estar envolvida na patogênese dos craniofaringiomas adamantinomatosos. Esse trabalho teve como objetivo avaliar a presença de mutação somática no exon 3 do CTNNB1 e avaliar a expressão desse gene e do gene PROP1 em craniofaringiomas adamantinomatosos. Foram obtidas 14 amostras desse tipo de tumor por meio da ressecção terapêutica. As amostras foram submetidas à extração do RNA e posterior transcrição reversa para obtenção de cDNA. A partir do cDNA foi realizada PCR e sequenciamento do exon 3 do CTNNB1 em todas as amostras. Porém, a avaliação por PCR em tempo real foi realizada apenas em 12 amostras, devido à qualidade inadequada de 2 amostras para submissão a essa metodologia. Foram encontradas mutações missense, em heterozigose em 9 das 14 amostras, sendo 5 previamente descritas e 2 ainda não descritas em craniofaringiomas adamantinomatosos. Hiperexpressão do CTNNB1 foi encontrada em 7 amostras, sendo 5 com mutação e 2 sem mutação no CTNNB1.A hiperexpressão variou de 2,5 a 6,2 vezes maior que o pool de hipófise normal. Contudo, a expressão do PROP1 foi indetectável em todas as amostras. Concluímos que o aumento da expressão do CTNNB1 presente em 58% das amostras sugere o envolvimento também da hiperexpressão desse gene na etiopatogenia do craniofaringioma adamantinomatoso, enquanto a ausência de expressão do PROP1 afasta a participação desse gene na etiopatogenia do craniofaringioma adamantinomatoso / Craniopharyngiomas are the the commonest tumors to involve the hypothalamo-pituitary regions in childhood population. Histologically they are benign, and can be divided in two primary subtypes: the adamantinomatous and the papillary. Although histologically benign, their infiltrative tendency and aggressive behavior can result in great morbidity. The pathogenesis of craniopharyngiomas is poorly understood. To date, beta-catenin gene (CTNNB1) mutations have been identified only in the adamantinomatous subtype. These mutations affect the degradation target box of beta-catenin that accumulates in the cytoplasm and the nucleus increasing the transcriptional activity of WNT pathway through interaction with the transcription factors of LEF/TCF family, leading to tumorigenesis. Recently, an interaction between beta-catenin and PROP1 was described as a new mecanism for beta-catenindependent regulation of pituitary cell-lineage determination. According to this novel model, the PROP1/beta-catenin proteic complex would act as a binary switch to simultaneously repress the transcription factor HESX1 and to activate expression of transcription factor PIT1, depending on the associated cofactors. Patients with loss-of-function mutations in PROP1 present combined pituitary hormonal deficiency generally associated with pituitary enlargement and the magnetic resonance imaging (MRI) of the sellar region in these patients sometimes resembles that of the craniopharyngiomas, with T1 hyperintense signal. On the other hand, transgenic mice with persistent Prop1 expression exhibit defects consistent with misregulation of pituitary cell proliferation, including adenomatous hyperplasia with formation of Rathke\'s cleft cysts and tumors suggesting that misregulation of PROP1 expression in human could contribute to pathogenesis of pituitary tumors. The similarity between the MRI images of craniopharyngiomas patients and that of patients with loss-of-function mutations in PROP1, associated with the fact that transgenic mice with persistent Prop1 expression exhibit increased susceptibility to pituitary tumors gave rise to our hypothesis that a misregulation of PROP1 expression could be involved in the pathogenesis of adamantinomatous craniopharyngiomas. The aim of this study was to analyze the presence of somatic mutations in exon 3 of CTNNB1 and the expression pattern of this gene and the PROP1 gene in adamantinomatous craniopharyngiomas. Fourteen samples were obtained from therapeutic surgery and submitted to RNA extraction and reverse transcription in order to produce the cDNA. The cDNA was used as a template to CTNNB1 exon 3 PCR reaction followed by direct sequencing of all samples. However, the real-time RT-PCR analysis was realized only in 12 samples, since 2 of them had an insufficient quality for this method. Missence, heterozygous mutations were found in 9 out of 14 samples; five were previously described and 2 not yet described in adamantinomatous craniopharyngiomas. Overexpression of CTNNB1 was found in 7 samples, which them 5 with CTNNB1 mutation 2 whitout. The overexpression ranged from 2.5 to 6.2 fold more than pituitary normal pool. However, the PROP1 expression was undetectable in all the samples. We could conclude that the amount of 58% CTNNB1 overexpressed samples suggest also a role of this overexpression in the pathogenesis of adamantinomatous craniopharingiomas, while the undetectable levels of PROP1 exclude a role of this gene in the pathogenesis of adamantinomatous craniopharingiomas

O fósforo é um dos minerais mais abundantes no corpo além de ser essencial para muitos processos biológicos. A homeostase do fósforo depende da absorção no intestino, da excreção renal, da remodelação óssea e de hormônios como o paratormônio, calcitriol e FGF-23. Nos pacientes com doença renal crônica, a excreção urinária de fósforo está comprometida levando à hiperfosfatemia, que contribui para o aumento da morbidade e mortalidade desses pacientes. A absorção intestinal de fósforo no intestino delgado, particularmente via cotransportadores NaP-IIb e PiT-1, é pouco estudada. O objetivo desse estudo foi avaliar os efeitos de dietas com diferentes concentrações de fósforo na expressão proteica e gênica dos cotransportadores NaP-IIb e PiT-1, bem como na apoptose dos enterócitos dos diferentes segmentos do intestino delgado de animais controles e urêmicos. Estudamos setenta e seis ratos Wistar machos, inicialmente divididos em dois grupos: controles (C) e urêmicos (Nx). Cada grupo foi subdividido em outros três, de acordo com a concentração de fósforo (P) na dieta: dieta Baixa (0,2%), dieta Padrão (0,54%) e dieta Alta (0,9%). Analisamos parâmetros bioquímicos (creatinina, P, Cai, PTH e FGF-23), a expressão proteica dos cotransportadores, através de Western Blotting, ELISA e imunofluorescência, a expressão gênica por PCR em tempo real e a apoptose dos enterócitos pela técnica TUNEL. Os resultados mostraram que os níveis séricos de creatinina, P, PTH e FGF-23 foram significativamente mais elevados nos animais Nx. Nos animais C com dieta baixa em P observamos aumento da expressão proteica do cotransportador NaP-IIb em todos os segmentos do intestino enquanto, no Nx Baixo, a expressão desse cotransportador foi menor somente no jejuno. Quanto ao PiT-1, sua expressão foi menor no íleo do grupo Nx Alto comparado ao seu respectivo controle. A expressão gênica do cotransportador NaP-IIb foi menor no jejuno do Nx Alto e maior no íleo desse mesmo grupo em relação aos seus respectivos controles. A expressão gênica do PiT-1 foi maior em todos os segmentos do grupo Nx Baixo em relação aos seus controles. Detectamos uma correlação direta entre a expressão gênica do NaP-IIb no jejuno com o fósforo sérico. A expressão gênica do PiT-1 no jejuno correlacionou-se com o fósforo sérico e no duodeno e jejuno, com os níveis séricos de FGF-23. No duodeno e no jejuno, a porcentagem de enterócitos apoptóticos foi maior nos animais Nx Alto comparados aos controles, particularmente no duodeno a porcentagem dessas células também foi maior no grupo Nx Baixo e controle padrão. Já no jejuno, tanto o grupo Nx baixo quanto no Nx Padrão observamos menos células apoptóticas que nos seus respectivos controles. Quando analisamos conjuntamente todos os segmentos intestinais o grupo Nx alto apresentou mais células apoptóticas que o controle e o oposto foi observado nos grupos Nx Baixo e Nx Padrão. Em conclusão, dietas com distintas concentrações de fósforo promovem modificações na expressão proteica e gênica dos cotransportadores NaP-IIb e PiT-1 no intestino delgado que não seguem um padrão uniforme. A sobrecarga de fósforo aumenta a porcentagem de enterócitos apoptóticos nos animais urêmicos / Phosphorus is one of the most abundant minerals in the body besides being essential for many biological processes. Phosphorus homeostasis depends on absorption in the small intestine, renal excretion, bone remodeling and hormones such as parathyroid hormone, calcitriol and FGF-23. In patients with chronic kidney disease phosphorus urinary excretion is compromised leading to hyperphosphatemia, which contributes to increased morbidity and mortality of these patients. Intestinal absorption of phosphorus in the small intestine, particularly of NaP-IIb and PiT-1 cotransporters is poorly studied. The aim of this study was to evaluate the effects of diets with different concentrations of phosphorus in protein expression and gene of NaP-IIb and PiT-1 cotransporters as well as in apoptosis of enterocytes of different segments of intestine in control and uremic animals. We studied seventy-six male Wistar rats initially divided into two groups: controls (C) and uremic (Nx). Each group was subdivided into three others, according to the phosphorus concentration in the diet: Low diet (0.2% P), Standard diet (0.54% P) and High diet (0.9% P). We analyzed biochemical parameters (creatinine, P, iCa, PTH and FGF-23), protein expression of cotransporters, using Western Blot, ELISA, immunofluorescence, real-time PCR and apoptosis of enterocytes using the TUNEL technique. Results showed that serum creatinine, P, PTH and FGF-23 were significantly higher in Nx animals. In C animals with a diet low in P we observed increase in protein expression of NaP-IIb cotransporter in all segments of the intestine while in low Nx expression of this cotransporter was lower only in jejunum. As for PiT-1 expression was lower in the ileum of the high Nx group compared to their respective control. Gene expression of NaP-IIb cotransporter was lower in the jejunum of high Nx and higher in the ileum of the same group as compared to their respective controls. PiT-1 gene expression was higher in all segments of the low Nx group compared to their controls. We detected a direct correlation between the gene expression of NaP-IIb in jejunum and serum phosphorus. Gene expression of PiT-1 correlated with serum phosphorus in the jejunum, and correlated with the serum levels of FGF-23 in the jejunum and duodenum. In the duodenum and jejunum the percentage of apoptotic enterocytes was higher in high Nx animals compared to controls; particularly in the duodenum the percentage of these cells was also higher in low Nx group and standard control. In the jejunum in both low Nx group and standard Nx we observed fewer apoptotic cells than in their respective controls. When we analyze all intestinal segments together the high Nx group had more apoptotic cells than the control, and the opposite was observed in the low Nx group and standard Nx group. In conclusion, diets with different phosphorus concentrations promote changes in protein expression and gene of NaP-IIb and PiT-1 cotransporters in the small intestine that do not follow a uniform pattern. Phosphorus overhead increases the percentage of apoptotic enterocytes in uremic animals