Differentiation is the process of regulated gene expression that gives rise to different phenotypes from a common genotype. Skeletal muscle differentiation, myogenesis, is a good example of this process. Skeletal muscle is susceptible to injury due to direct or indirect causes. If left unrepaired, these injuries may lead to a loss of muscle mass, locomotive deficiency, and even lethality. Thus, understanding the molecular mechanisms behind this process is an important first step in the design of treatment for muscle-related diseases. Once myogenesis is induced, the expression of MRF proteins, such as MyoD and myogenin, is maintained at high levels in myofibers without the need to increase their rates of transcription, suggesting a role for post-transcriptional regulatory mechanisms. HuR is a ubiquitously expressed member of the embryonic lethal, abnormal vision (ELAV) family of RNA binding proteins that is known to post-transcriptionally regulate its target messages. Here, I demonstrate that, in the C2C12 muscle cell line, HuR is a required factor for both the initiation and maintenance of the myogenic process. First in vitro RNA Electro-Mobility Shift Assays (REMSA) and immunoprecipitation experiments demonstrated that HuR specifically binds to the AU-rich elements (AREs) that are present in the 3' untranslated regions (3'UTRs) of the MyoD and myogenin mRNAs. In the absence of HuR at the time of differentiation induction, accomplished using the siRNA technology, the expression of the MyoD and myogenin messages is significantly reduced, leading to inhibition of myogenesis. At this early stage in the differentiation process, HuR, a shuttling protein, is predominantly nuclear; localization that is mediated by the import receptor Transportin2 (Trn2). Nuclear HuR was determined to be required for the negative regulation of nucleophosmin (NPM) translation. Forced overexpression of NPM, resulting in differentiation inhibition, shows that its downregulation is a requirement for induction of the differentiation process. Late in myogenesis, however, NPM RNA is no longer expressed, and HuR is seen to accumulate in the cytoplasm of myotubes. This cytoplasmic accumulation results from dissociation of HuR from Trn2 due to caspase-dependent cleavage within its HNS region. Specifically blocking HuR import through the use of cell-permeable peptides, as well as RNAi-mediated depletion of Trn2, leads to an increase in cytoplasmic HuR, as well as increased cytoplasmic localization and stabilization of the MyoD and myogenin messages, and a corresponding enhancement of differentiation. Overall, we conclude that HuR is required for myogenesis due to its ability to post-transcriptionally regulate genes required for the process, and that HuR itself is regulated at the level of its subcellular localization, mediated by the import receptor TRN2.

No description available.

No description available.

Wolbachia pipientis é uma alfa-protobactéria intracelular obrigatória, endossimbionte de artrópodes e nematódeos que é herdada por via materna ao longo das gerações. A presença desta bactéria nos tecidos germinativos pode provocar nos hospedeiros alterações fenotípicas reprodutivas, como partenogênese, feminização genética de machos, morte de machos, incompatibilidade citoplasmática (IC) e alterações de fitness. Alguns mecanismos moleculares dessas alterações baseiam-se na modulação da expressão gênica do hospedeiro, ou seja, a bactéria pode suprimir ou estimular genes de forma a produzir ambiência favorável à manutenção da endossimbiose. Devido a esse potencial manipulador, Wolbachia tem sido testada como \"ferramenta\" para controle populacional de insetos vetores de patógenos. O mosquito Culex quinquefasciatus, naturalmente infectado por Wolbachia na região Neotropical, é um importante vetor de diversos patógenos que atingem humanos e animais. A presença da bactéria causa IC e altera o fitness no mosquito, mediante alterações temporais na ovogênese, fecundidade e fertilidade reprodutivas. Sabe-se que a presença da Wolbachia altera a expressão do gene grauzone e há fortes indícios de que esta expressão diferencial induza à IC em Cx. quinquefasciatus. Sabe-se também que este gene possui duas cópias parálogas em Cx. quinquefasciatus, porém estudos observaram a relevância de apenas um parálogo como importante regulador dos ciclos celulares da ovogênese e espermatogênese. No entanto, as bases genéticas dos fenótipos IC e \"fitness alterado\" do modelo Wolbachia-Cx. quinquefasciatus Neotropical ainda permanecem desconhecidas. Objetivamos inicialmente neste modelo quantificar e silenciar a expressão do gene grauzone (ambos parálogos) para suportar a hipótese pré-existente de que a superexpressão deste gene em mosquitos infectados por Wolbachia causa as alterações fenotípicas reprodutivas. Durante o desenvolvimento do projeto houve intercorrências que alteraram o rumo do trabalho: a necessidade de substituição da colônia experimental de mosquitos devido a baixas demográficas e à alta variabilidade intraespecífica do gene grauzone. Frente às intercorrências, formulamos como neo-objetivo a comparação filogenética entre as variantes do gene grauzone. Detectamos também variabilidade em um dos parálogos e concluímos que as cópias parálogas do gene encerram proteínas estruturalmente distintas e talvez funcionalmente distintas no tocante à alteração reprodutiva (IC) causada pela presença da Wolbachia. Foi possível observar que fêmeas infectadas apresentam amplificações de grauzone mais intensas quando comparadas com fêmeas não-infectadas no 4° dia de emergência, corroborando dados da literatura. Em conjunto, esses achados indicam que é promissora a continuidade do estudo do papel de grauzone na IC, mas demonstra também que este gene é mais complexo do que se imaginava, o que demandará maior esforço investigativo. A expectativa de uso futuro de Wolbachia como controlador biológico de mosquitos-vetores poderá se beneficiar de estudos como aqui proposto. / Wolbachia pipientis is an obligate intracellular alpha-protobacterium, endosymbiont of arthropods and nematodes, which is inherited through maternal route over generations. The presence of this bacterium in germinative tissues can cause reproductive phenotypic alterations in the hosts, such as parthenogenesis, genetic feminization of males, death of males, cytoplasmic incompatibility (CI) and fitness changes. Some molecular mechanisms of these alterations are based on the modulation of gene expression of the host, that is, the bacterium can suppress or stimulate genes in order to produce favorable environment for the maintenance of the endosymbiosis. Due to this potential manipulator, Wolbachia has been tested as a \"tool\" for population control of pathogen vector insects. The mosquito Culex quinquefasciatus, naturally infected by Wolbachia in the Neotropical region, is an important vector of several pathogens that affect humans and animals. The presence of the bacteria causes CI and changes the fitness in the mosquito, through temporal changes in ovogenesis, reproductive fertility and fertility. The presence of Wolbachia is known to alter the expression of the grauzone gene and there are strong indications that this differential expression induces the CI in Cx. quinquefasciatus. It is known that this gene occurs with two paralogs copies in Cx. quinquefasciatus, but studies have observed the relevance of only one paralog as important regulator of oogenesis and spermatogenesis cell cycles. However, the genetic basis of the phenotypes \"changed fitness\" and CI of Wolbachia-Cx quinquefasciatus Neotropical model remain unknown. We initially aimed at quantifying and knockdown of grauzone gene (both paralogs) to support the preexisting hypothesis that overexpression of this gene in Wolbachia-infected mosquitoes causes reproductive phenotypic changes. During the development of the project there were intercurrences that altered the course of work: the need to replace mosquito populations due to demographic lows and the high intraspecific variability of grauzone gene. In view of the intercurrences we formulated the neo-objective phylogenetic comparison between the variants of the grauzone gene. We also detected variability in one of the paralogs and concluded that the paralogs copies of the gene contain structurally distinct and perhaps functionally distinct proteins with respect to the reproductive alteration (CI) caused by the presence of Wolbachia. It was possible to observe that infected females show more intense grauzone amplifications when compared to uninfected females on the 4th day of emergence, corroborating data from the literature. Taken together, these findings indicate that the continuity of the study of the role of grauzone in CI is promising, but also demonstrates that this gene is more complex than previously thought, which will require more investigative effort. The expectation of future use of Wolbachia as a biological control of mosquito-vectors may benefit from studies as proposed here.

Siu-wai Michael Sung. / Thesis submitted in: December 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 97-111). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations --- p.v / Table of Contents --- p.vii / Chapter Chapter 1 --- General Introduction / Chapter l. 1 --- Drosophila as a model for studying development --- p.1 / Chapter l .2 --- The formation of the body axis in Drosophila --- p.2 / Chapter l .3 --- Maternal genes are essential for development --- p.9 / Chapter 1.4 --- Maternal gene bicoid is essential for formation of the anterior structures in the embryo --- p.13 / Chapter 1.5 --- Establishment of an anterior to posterior bicoid protein gradient --- p.13 / Chapter 1.6 --- The bicoid protein gradient controls the downstream zygotic target genes in a concentration-dependent manner --- p.17 / Chapter 1.7 --- Bicoid protein acts as transcriptional regulators \9 --- p.19 / Chapter 1.8 --- Bicoid protein acts as transcriptional regulators --- p.21 / Chapter 1.9 --- The anterior localization of bcd mRNA --- p.21 / Chapter 1.10 --- Components required for bcd mRNA localization at anterior pole of oocyte / Chapter 1.10.1 --- Cis-acting elements --- p.22 / Chapter 1.10.1.1 --- BLE1 at 3' UTR directs localization of bcd mRNA --- p.23 / Chapter 1.10.2 --- Trans-acting elements / Chapter 1.10.2.1 --- "Exuperantia, swallow, and staufen are necessary for localization for bcd mKNA" --- p.27 / Chapter 1.10.2.2 --- exu protein is an absolute requirement for localization for bcd mRNA --- p.30 / Chapter 1.10.2.3 --- Microtubules dependence of localization --- p.31 / Chapter 1.11 --- Functions of exu in localization of bcd mRNA --- p.32 / Chapter 1.12 --- Characteristics of Bicoid protein and Bic-D gene --- p.33 / Chapter 1.13 --- Aim of Project --- p.36 / Chapter CHAPTER 2 --- Materials and Methods / Chapter 2.1 --- Fly Food --- p.37 / Chapter 2.2 --- Conditions in maintaining the fly stocks and working stocks --- p.37 / Chapter 2.3 --- Localization of exu protein and other intracellular elements by indirect immunofluorescence detection / Chapter 2.3.1 --- Immunohistrochemical distribution of exu and Bic-D protein --- p.38 / Chapter 2.3.2 --- Immunohistrochemical distribution of β-tubulin --- p.39 / Chapter 2.4 --- Preparation of total protein from the female and male flies --- p.41 / Chapter 2.5 --- Analysis of interactions between exu and trans-acting elements / Chapter 2.5.1 --- 35S-methionine metabolic labelling and immunoprecipitation by RIPA buffer --- p.41 / Chapter 2.5.2 --- 35S-methionine metabolic labelling and immunoprecipitation by Mach and Lehmann buffer system --- p.43 / Chapter 2.6 --- Co-immunoprecipitation of exu and Bic-D protein / Chapter 2.6.1 --- Co-immunoprecipitation of exu and Bic-D protein synthesized by in vitro coupled transcription and translation system with modified Mach and Lechmann buffer system --- p.44 / Chapter 2.7 --- in vivo ovary extract co-immunoprecipitation / Chapter 2.7.1 --- in vivo ovary extraction co-immunoprecipitation of exu and Bic-D protein with modified Mach and Lehmann buffer system supplemented with recombinant exu protein --- p.45 / Chapter CHAPTER 3 --- Results / Chapter 3.1 --- Analysis of co-localization of exu and Bic-D protein by double immuno-fluorescence staining on w1118 flies --- p.47 / Chapter 3.2 --- Analysis of co-localization of exu protein and β-tubulin protein by double immuno-fluorescence staining on w1118 flies --- p.51 / Chapter 3.3 --- Analysis of co-localization of exu and Bic-D protein by double immuno-fluorescence staining on Bic-D mutants --- p.55 / Chapter 3.4 --- Co-immunoprecipitation of exu and Bic-D protein synthesized by in vitro coupled transcription and translation system --- p.61 / Chapter 3.5 --- 35S-Methionine metabolic labelling and co-immunoprecipitation of exu and Bic-D protein with RIP A buffer system --- p.65 / Chapter 3.6 --- 35S-Methionine metabolic labelling and co-immunoprecipitation of exu and Bic-D protein with Mach and Lehmann buffer system --- p.68 / Chapter 3.7 --- in vivo ovary extract co-immunoprecipitation of exu and Bic-D protein with modified Mach and Lehmann buffer system supplemented with recombinant exu protein --- p.71 / Chapter CHAPTER 4 --- Discussion / Chapter 4.1 --- Analysis of co-localization of exu protein and other intracellular elements by indirect double immunofluorescence staining detection --- p.74 / Chapter 4.2 --- Analysis of co-localization of exu and BicD protein by double immuno- fluorescence staining on Bic-D mutants --- p.78 / Chapter 4.3 --- Co-immunoprecipitation of exu and BicD protein synthesized by in vitro coupled transcription and translation system --- p.79 / Chapter 4.4 --- Analysis of interactions between exu and trans-acting elements by 35S- Methionine metabolic labelling and immunoprecipitation --- p.82 / Chapter 4.5 --- "in vivo ovary extract coimmunoprecipitation of exu and Bic-D protein with modified Mech and Lehmann buffer system, supplemented with recombinant exu protein" --- p.84 / Chapter 4.6 --- Recent developments on the concept of ribonucleoprotein --- p.86 / Appendix A Supplementary protocols --- p.91 / Appendix B Reagents --- p.95 / Reference --- p.97

Wolbachia pipientis é uma alfa-protobactéria intracelular obrigatória, endossimbionte de artrópodes e nematódeos que é herdada por via materna ao longo das gerações. A presença desta bactéria nos tecidos germinativos pode provocar nos hospedeiros alterações fenotípicas reprodutivas, como partenogênese, feminização genética de machos, morte de machos, incompatibilidade citoplasmática (IC) e alterações de fitness. Alguns mecanismos moleculares dessas alterações baseiam-se na modulação da expressão gênica do hospedeiro, ou seja, a bactéria pode suprimir ou estimular genes de forma a produzir ambiência favorável à manutenção da endossimbiose. Devido a esse potencial manipulador, Wolbachia tem sido testada como \"ferramenta\" para controle populacional de insetos vetores de patógenos. O mosquito Culex quinquefasciatus, naturalmente infectado por Wolbachia na região Neotropical, é um importante vetor de diversos patógenos que atingem humanos e animais. A presença da bactéria causa IC e altera o fitness no mosquito, mediante alterações temporais na ovogênese, fecundidade e fertilidade reprodutivas. Sabe-se que a presença da Wolbachia altera a expressão do gene grauzone e há fortes indícios de que esta expressão diferencial induza à IC em Cx. quinquefasciatus. Sabe-se também que este gene possui duas cópias parálogas em Cx. quinquefasciatus, porém estudos observaram a relevância de apenas um parálogo como importante regulador dos ciclos celulares da ovogênese e espermatogênese. No entanto, as bases genéticas dos fenótipos IC e \"fitness alterado\" do modelo Wolbachia-Cx. quinquefasciatus Neotropical ainda permanecem desconhecidas. Objetivamos inicialmente neste modelo quantificar e silenciar a expressão do gene grauzone (ambos parálogos) para suportar a hipótese pré-existente de que a superexpressão deste gene em mosquitos infectados por Wolbachia causa as alterações fenotípicas reprodutivas. Durante o desenvolvimento do projeto houve intercorrências que alteraram o rumo do trabalho: a necessidade de substituição da colônia experimental de mosquitos devido a baixas demográficas e à alta variabilidade intraespecífica do gene grauzone. Frente às intercorrências, formulamos como neo-objetivo a comparação filogenética entre as variantes do gene grauzone. Detectamos também variabilidade em um dos parálogos e concluímos que as cópias parálogas do gene encerram proteínas estruturalmente distintas e talvez funcionalmente distintas no tocante à alteração reprodutiva (IC) causada pela presença da Wolbachia. Foi possível observar que fêmeas infectadas apresentam amplificações de grauzone mais intensas quando comparadas com fêmeas não-infectadas no 4° dia de emergência, corroborando dados da literatura. Em conjunto, esses achados indicam que é promissora a continuidade do estudo do papel de grauzone na IC, mas demonstra também que este gene é mais complexo do que se imaginava, o que demandará maior esforço investigativo. A expectativa de uso futuro de Wolbachia como controlador biológico de mosquitos-vetores poderá se beneficiar de estudos como aqui proposto. / Wolbachia pipientis is an obligate intracellular alpha-protobacterium, endosymbiont of arthropods and nematodes, which is inherited through maternal route over generations. The presence of this bacterium in germinative tissues can cause reproductive phenotypic alterations in the hosts, such as parthenogenesis, genetic feminization of males, death of males, cytoplasmic incompatibility (CI) and fitness changes. Some molecular mechanisms of these alterations are based on the modulation of gene expression of the host, that is, the bacterium can suppress or stimulate genes in order to produce favorable environment for the maintenance of the endosymbiosis. Due to this potential manipulator, Wolbachia has been tested as a \"tool\" for population control of pathogen vector insects. The mosquito Culex quinquefasciatus, naturally infected by Wolbachia in the Neotropical region, is an important vector of several pathogens that affect humans and animals. The presence of the bacteria causes CI and changes the fitness in the mosquito, through temporal changes in ovogenesis, reproductive fertility and fertility. The presence of Wolbachia is known to alter the expression of the grauzone gene and there are strong indications that this differential expression induces the CI in Cx. quinquefasciatus. It is known that this gene occurs with two paralogs copies in Cx. quinquefasciatus, but studies have observed the relevance of only one paralog as important regulator of oogenesis and spermatogenesis cell cycles. However, the genetic basis of the phenotypes \"changed fitness\" and CI of Wolbachia-Cx quinquefasciatus Neotropical model remain unknown. We initially aimed at quantifying and knockdown of grauzone gene (both paralogs) to support the preexisting hypothesis that overexpression of this gene in Wolbachia-infected mosquitoes causes reproductive phenotypic changes. During the development of the project there were intercurrences that altered the course of work: the need to replace mosquito populations due to demographic lows and the high intraspecific variability of grauzone gene. In view of the intercurrences we formulated the neo-objective phylogenetic comparison between the variants of the grauzone gene. We also detected variability in one of the paralogs and concluded that the paralogs copies of the gene contain structurally distinct and perhaps functionally distinct proteins with respect to the reproductive alteration (CI) caused by the presence of Wolbachia. It was possible to observe that infected females show more intense grauzone amplifications when compared to uninfected females on the 4th day of emergence, corroborating data from the literature. Taken together, these findings indicate that the continuity of the study of the role of grauzone in CI is promising, but also demonstrates that this gene is more complex than previously thought, which will require more investigative effort. The expectation of future use of Wolbachia as a biological control of mosquito-vectors may benefit from studies as proposed here.

Many organs and structures formed during the embryonic morphogenesis of animals derive from epithelia. Epithelia are made up of apicobasally polarized cells which adhere to and communicate with each other, allowing for epithelial integrity and plasticity. During embryonic morphogenesis, epithelia change their shape and migrate in a coordinated manner. How these epithelial processes are regulated is still not fully understood. In a forward genetic screen using the embryo of the fruit fly Drosophila melanogaster, candidate genes influencing the morphogenesis of epithelial structures were identified. Three genes, CG17364, CG17362 and CG9040 were identified as possible regulators of lumen stability in the salivary glands, tubular organs deriving from the embryonic epithelium. Furthermore, the gene Cdep was found to play a crucial role in epithelial sheet migration during dorsal closure of the embryo. Embryos carrying genomic insertions that could affect the expression of CG17364, CG17362 and CG9040 show a luminal penotype of the embryonic salivary glands characterized by alternating bloated and seemingly closed sections. Therefore, one of these genes or a combination of them likely plays a role in stabilizing the salivary gland lumen. However, neither CG17364 nor CG17362 or CG9040 contain any known protein domains, hence their molecular roles remain unknown. Cdep (Chondrocyte-derived ezrin-like protein) is a member of the FERM-FA subclass of proteins. Proteins of the FERM family have been shown to interact with the plasma membrane and membrane-bound proteins as well as cytoskeleton components. Accordingly, they have been implicated in stabilizing the cell cortex, and some of them are involved in signal transduction mechanisms. In addition to a FERM domain, Cdep also contains a RhoGEF domain, although is still not clear whether it actually exerts GEF activity. Genomic insertions in the Cdep locus cause defects in embryonic dorsal closure and atypical migratory behaviour in epithelial tubes. In order to study the molecular role of Cdep, the CRISPR/Cas9 system was employed to establish loss-of-function mutants of Cdep. The mutants show aberrations in germ band retraction, dorsal closure and head involution. Moreover, I found that two mutants carrying a premature STOP codon in the Cdep ORF, CdepE16X and CdepG17X, rescue the defects observed in embryonic cuticles mutant for two other FERM-FA members yurt (yrt) and coracle (cora). A deletion of the full Cdep ORF did not rescue those defects. I hypothesize that CdepE16X and CdepG17X encode Cdep variants with increased activity, which compensates for the loss of yrt or cora function, respectively. In conclusion, this leads to a model in which Cdep acts in parallel to Yrt and Cora during Drosophila embryonic morphogenesis. Many of the defects described in this study are reminiscent of phenotypes found in embryos mutant for components and downstream effectors of the Jun-N-terminal Kinase (JNK) pathway. Hence, my work supports an earlier hypothesis according to which a mouse homologue of Cdep, Farp2, acts as an upstream activator of the JNK pathway during epithelial cell migration in vitro (Miyamoto et al., 2003) The data provided here shows that Cdep plays a role in the morphogenesis of a great number of epithelia-derived organs and structures in vivo. My study therefore elucidates a missing link between cell migration cues and JNK pathway activation.:1 Introduction 1 1.1 Epithelial cell polarity 1 1.1.1 Cellularization and formation of the primary epithelium 1 1.1.1.1 Establishment of epithelial polarity and adhesion 2 1.1.2 The epithelial polarity network 3 1.1.3 Cell-cell adhesion 5 1.1.3.1 Adherens junctions 5 1.1.3.2 Septate junctions 6 1.2 Epithelial movements in Drosophila embryonic morphogenesis 6 1.2.1 Epithelial tube formation during Drosophila embryogenesis 7 1.2.2 Coordinated migration of epithelial sheets during Dros. embryogenesis 7 1.2.2.1 FERM domain proteins in epithelial migration 9 1.2.2.2 Cdep 10 1.3 Mutagenesis with the CRISPR/Cas9 system 12 2 Aim of My PhD Thesis Work 15 3 Preliminary Work 17 4 Results 19 4.1 A screen for novel regulators in Drosophila embryonic morphogenesis 19 4.1.1 Deficiencies on the left arm of chromosome 3 cause defects in SG lumen morphology 19 4.1.2 A locus in the overlap of two deficiencies on the right arm of chromosome 3 causes defects in Drosophila embryonic dorsal closure 20 4.2 Two uncharacterized genes regulate SG lumen diameter in Drosophila embryos 22 4.2.1 Mutations in two uncharacterized genes on chromosome 3 cause intermittent tube closure in the Drosophila SG 22 4.2.2 CG17362/ CG9040/ CG17364 play a role in the maintenance of SG lumen width after lumen expansion 24 4.2.3 CG17362 is exclusively expressed in the embryonic SG 25 4.2.4 CG17362 and CG9040 do not contain known protein domains and are only conserved in Drosophilidae 28 4.3 Loss of Cdep causes different defects in Drosophila embryonic morphogenesis 30 4.3.1 Insertions in the ORF of Cdep cause defects in DC and HI 30 4.3.2 Embryos transheterozygous for PBac{5HPw+}CdepB122 and Mi{MIC} CdepMI00496 show defects in the LE during DC 34 4.3.3 Insertions in the ORF of Cdep cause defects in tracheal and Malpighian tubule morphogenesis 34 4.3.4 The CRISPR/Cas9 system was used to generate loss-of-function mutants of Cdep 35 4.3.5 LOF mutants of Cdep show a variety of phenotypes 37 4.3.6 LOF mutants of Cdep cause denticle belt defects and ventral holes in Drosophila larval cuticles 38 4.3.7 Phenotypes in Cdep−/− mutant embryos are likely not caused by maternal defects 44 4.3.8 LOF mutants of Cdep cause segments to fuse 44 4.3.9 Defects in Cdep−/− mutants are not due to actin mislocalization 51 4.3.10 Cdep genetically interacts with yurt 51 4.3.11 Cdep genetically interacts with cora 55 5 Discussion 57 5.1 The role of CG17362 and CG9040 in SG lumen stability 57 5.1.1 Impairments in the expression of CG17362 and CG9040 could be the cause for the intermittent tube closure in the embryonic SGs 57 5.1.2 CG17362 and CG9040 could be necessary for SG lumen dilation and stability of lumen diameter 57 5.2 The role of Cdep in Drosophila embryonic morphogenesis 59 5.2.1 Cdep is a regulator of the JNK pathway 60 5.2.1.1 Mutations in TGF-_ pathway components cause LE bunching and gaps in the tracheal dorsal trunk 60 5.2.1.2 The JNK pathway, like Cdep, is instrumental in GBR, DC and HI 61 5.2.1.3 Mouse Farp2 acts upstream of the JNK pathway 61 5.2.2 Does Cdep act as a GEF? 62 5.2.3 Cdep might regulate epithelial migration in parallel to Yrt and Cora 63 5.3 Conclusion and Outlook 64 6 Materials and Methods 67 6.1 Cell strains, plasmids and DNA constructs 67 6.2 Culture media 68 6.2.1 Lysogeny Broth (Bertani, 1951) 68 6.2.2 Super Optimal Catabolite repression (SOC) medium (Hanahan, 1983) 68 6.3 Molecular biology methods 69 6.3.1 Amplifying DNA by standard PCR technology 69 6.3.2 Molecular cloning 70 6.3.2.1 Cloning with restriction endonucleases (Old and Primrose, 1980) 70 6.3.2.2 Gibson Assembly (Gibson et al., 2009) 70 6.3.3 Detecting DNA via agarose gel electrophoresis 71 6.3.4 Purifying DNA from E.coli 71 6.3.5 Extracting DNA from Drosophila adults 71 6.3.5.1 Extracting mRNA from Drosophila embryos and reverse transcription into cDNA 72 6.3.6 Making mRNA probes for in situ hybridization 72 6.3.7 Measuring DNA concentrations using the NanoDrop 72 6.3.8 DNA sequencing 72 6.3.9 Transformation 73 6.3.10 Mutagenesis of Cdep with the CRISPR-Cas9 system 73 6.3.10.1 CRISPR/Cas9 tools for mutagenesis in D.melanogaster 73 6.3.11 Strategies for CRISPR/Cas9 mutagensis 74 6.3.11.1 vectors and oligomers used for mutagenesis of Cdep via CRISPR-Cas9 system 75 pCFD3-dU6:3-CdepEx2gRNA 75 CdepSTOP-ssODN 76 pCFD4-CdepExc_2sgRNAs 77 pDsRed-5’HA-3’HA 78 6.3.11.2 Screening for successful mutagenesis events in flies modified with the CRISPR/Cas9 system 79 Insertion of an in-frame stop codon into the Cdep ORF 79 Replacing the entire Cdep locus with dsRed 80 6.3.12 Extracting protein from Drosophila embryos 83 6.3.13 Detecting and separating proteins by mass via SDS-PAGE 83 6.3.14 Detection of specific polypeptides by Western blot analysis (Towbin et al., 1979) 84 6.3.14.1 Transferring polypeptides to nitrocellulose membrane 84 6.3.14.2 Detecting specific polypeptides via immonublotting 85 6.4 Online tools for prediction of protein domains, interactions, sequence alignments, etc. 86 6.5 Cell culture growth and harvest 86 6.5.1 Growing E.coli cells for vector amplification 86 6.5.2 Cell harvest 86 6.6 Work with Drosophila melanogaster 87 6.6.1 Fly stocks used: deficiencies, alleles, duplications and balancers 88 6.6.2 Recombining alleles located on the same chromosome 93 6.6.3 Collecting Drosophila melanogaster embryos 93 6.6.4 Histochemistry 94 6.6.4.1 Embryo dechorionation 94 6.6.4.2 Embryo fixation for light microscopy of whole specimens 94 Heat fixation of Drosophila embryos 94 Formaldehyde fixation of Drosophila embryos 94 6.6.4.3 Embryo fixation for light microscopy of semi-thin sections (Tepass and Hartenstein, 1994) 95 6.6.4.4 Antibody staining of embryos for Immunofluorescence 95 6.6.4.5 Phalloidin and DAPI staining of embryos 96 6.6.4.6 Embryo mounting for analysis via fluorescence microscopy 96 6.6.4.7 Embryo mounting for live imaging 97 6.6.4.8 Embedding of embryos for semi-thin sectioning 97 6.6.4.9 Cuticle preparation from hatched and unhatched Drosophila larvae 97 6.6.4.10 Preparation and staining of ovarian follicles from Drosophila females 98 6.6.4.11 mRNA in situ hybridization of whole-mount Drosophila embryos 99

Made available in DSpace on 2015-07-22T20:50:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-03-25 / O panorama atual do tratamento de pacientes com Anomalias da Diferenciação Sexual (ADS) apresenta o desafio de colocarmos frente a frente o saber do médico e o do paciente e/ou seus familiares — discursos diferentes — de tal forma que estes saberes possam ser escutados. O intuito geral deste trabalho foi o de identificar as angústias, dúvidas e ansiedades dos pacientes e seus pais, bem como da equipe médica que os assiste. O objetivo específico do trabalho foi fazer uma avaliação do que se passava nas entrelinhas do atendimento, para compreender o que o paciente e o familiar entendem ou não entendem sobre atendimento médico e vice-versa. Para isso, foram ouvidos sete médicos especialistas de cinco instituições do Sistema Único de Saúde (SUS), nove familiares e seis pacientes portadores de hiperplasia adrenal congênita (HAC) por deficiência de 21-hidroxilase. / The current scenario regarding treatment of patients with Anomalies of Sex Differentiation (ASD) brings the challenge to put face-to-face the medical knowledge and their patients and/or relatives acquaintance — distinct speeches — so that both these knowledges need to be heard. The general purpose of this work was to identify the anguishes, doubts, distress, and anxieties from patients and their parents, as well as from the medical team that attend them. The specific aim was to evaluate the scenario beyond the medical service in order to appreciate what patients and parents understand or do not understand within the medical attendance and vice-versa. Therefore, interviews were conducted to hear seven specialist physicians from five institutions among the “Sistema Único de Saúde” (SUS), nine parents and six patients bearing the 21-hydroxylase deficiency form of congenital adrenal hyperplasia (CAH). / TEDE / BV UNIFESP: Teses e dissertações

O sistema hormônio de crescimento (GH) / fator de crescimento insulina- símile tipo 1 (IGF-1) é o principal determinante e regulador do crescimento linear pósnatal. O GH é codificado pelo gene Growth Hormone 1 (GH1). Mutações no GH1 com efeito dominante negativo e herança autossômica dominante são as principais causas monogênicas de deficiência isolada de hormônio de crescimento (DIGH), enquanto deleções ou mutações de ponto no GH1 causam formas raras autossômicas recessivas de DIGH. No grupo de pacientes com DIGH do ambulatório de Endocrinologia do Desenvolvimento do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, foram identificadas apenas deleções em homozigose no GH1 mesmo após estudo criterioso deste gene. Esta diferença em relação aos dados descritos na literatura poderia ser justificada pelo critério diagnóstico para a DIGH adotado pelo nosso grupo, sendo utilizado pico de GH em teste de estímulo inferior a 3,3 ug/L, em contraste com os valores de corte descritos na literatura que variam de 7 a 10 ug/L. Devido a esse fator, pacientes com mutações no GH1 com herança autossômica dominante poderiam estar sendo erroneamente diagnosticados como portadores de baixa estatura familiar ou idiopática (BEI) em nosso serviço. Adicionalmente, mutações que originam moléculas de GH biologicamente inativas também poderiam estar presentes nestes pacientes. Pelos fatores acima apresentados, expandimos o estudo do GH1 para um grupo de crianças classificadas como BEI. Foram selecionadas 98 de 487 crianças avaliadas em nosso serviço com baixa estatura utilizando os seguintes critérios: peso e comprimento normais para idade gestacional ao nascimento, escore-Z da altura < -2, escore-Z do IGF-1 < -1 e pico de resposta de GH >= 3,3 ug/L no teste de estímulo. DNA foi extraído de leucócitos periféricos desses pacientes para rastreamento de mutações no gene GH1. Realizamos estudo molecular por reação em cadeia da polimerase e sequenciamento automático de toda a região codificadora do GH1. Segregação familiar foi realizada para as variantes alélicas identificadas. Em nossa casuística, foram identificadas 10 variantes alélicas nos éxons 4 e 5 e no íntron 4 do GH1, sendo três variantes ainda não descritas na literatura (c.407G > A/p.Val122Ile, c.507C > T/p.Tyr169Tyr e c.456+19G > T). A análise in silico de todas as variantes identificadas indicou ausência de predição de efeito deletério sobre a proteína do GH. Estudo complementar realizado pelo nosso grupo identificou em crianças diagnosticadas com DIGH grave apenas uma paciente com mutação no GH1 responsável pela forma dominante desta doença. Em conclusão, mutações no GH1 causadoras da forma autossômica dominante de DIGH ou Tipo II não foram encontradas em nossa casuística, o que sugere que estas mutações sejam infrequentes em nossa população / The growth hormone (GH) / insulin-like growth factor-1 (IGF-1) axis is the most important hormonal regulator of post-natal linear growth. GH is encoded by the Growth Hormone 1 gene (GH1). Mutations in GH1 with dominant inheritance, which exerts a dominant negative effect on the bioactive GH isoforms, are the main causes of monogenic isolated deficiency of growth hormone (IGHD), while deletions or point mutations in GH1 are responsible for a rare autosomal recessive form of IGHD. However, only homozygous deletions were identified in patients with IGHD from Unidade de Endocrinologia do Desenvolvimento do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, even after detailed investigation of GH1. This difference regarding to literature can be caused by different criteria used to diagnose IGHD in our group, which adopted the cutoff value of peak GH < 3.3ug/L in response to stimulation test, in contrast to literature that describes other groups that use the cutoff peak value of the 7 - 10ug/L. Consequently, patients with autosomal dominant inheritance mutations in GH1 could be being erroneously diagnosed, as having idiopathic short stature (ISS) in our group. Additionally, mutations that cause biologically inactive GH can also be responsible for short stature in these patients. Due to the factors described above, we decided to screen mutations in GH1 in a group of children classified as ISS. We selected 98 of 487 children followed in our department with short stature according to the following criteria: normal birth weight and length for gestational age, height SDS <= -2, IGF-1 SDS < -1 and peak GH in stimulation test >= 3.3 ug/L. Genomic DNA was extracted from peripheral blood leucocytes of the patients to screen for mutations in GH1. We performed molecular analysis by polymerase chain reaction and automated sequencing of the entire coding region of the GH1. Segregation analysis was performed in the presence of allelic variations. In our casuistic, we identified 10 allelic variants in exon 4, exon 5 and intron 4 of GH1, three of which have not been described (c.407G > A/p.Val122Ile, c.507C > T/p.Tyr169Tyr and c.456+19G >T). In silico analysis predicted that none of the mutant alleles would result in deleterious effect on the GH protein. An additional study in children diagnosed with severe IGHD, identified just one patient with the pathogenic GH1 mutation responsible for the dominant form of this disease. In summary, defects in GH1 responsible for the autosomal dominant form of IGHD or Type II were not found in our cohort of Brazilian patients, suggesting that these mutations are infrequent in our population

O sistema hormônio de crescimento (GH) / fator de crescimento insulina- símile tipo 1 (IGF-1) é o principal determinante e regulador do crescimento linear pósnatal. O GH é codificado pelo gene Growth Hormone 1 (GH1). Mutações no GH1 com efeito dominante negativo e herança autossômica dominante são as principais causas monogênicas de deficiência isolada de hormônio de crescimento (DIGH), enquanto deleções ou mutações de ponto no GH1 causam formas raras autossômicas recessivas de DIGH. No grupo de pacientes com DIGH do ambulatório de Endocrinologia do Desenvolvimento do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, foram identificadas apenas deleções em homozigose no GH1 mesmo após estudo criterioso deste gene. Esta diferença em relação aos dados descritos na literatura poderia ser justificada pelo critério diagnóstico para a DIGH adotado pelo nosso grupo, sendo utilizado pico de GH em teste de estímulo inferior a 3,3 ug/L, em contraste com os valores de corte descritos na literatura que variam de 7 a 10 ug/L. Devido a esse fator, pacientes com mutações no GH1 com herança autossômica dominante poderiam estar sendo erroneamente diagnosticados como portadores de baixa estatura familiar ou idiopática (BEI) em nosso serviço. Adicionalmente, mutações que originam moléculas de GH biologicamente inativas também poderiam estar presentes nestes pacientes. Pelos fatores acima apresentados, expandimos o estudo do GH1 para um grupo de crianças classificadas como BEI. Foram selecionadas 98 de 487 crianças avaliadas em nosso serviço com baixa estatura utilizando os seguintes critérios: peso e comprimento normais para idade gestacional ao nascimento, escore-Z da altura < -2, escore-Z do IGF-1 < -1 e pico de resposta de GH >= 3,3 ug/L no teste de estímulo. DNA foi extraído de leucócitos periféricos desses pacientes para rastreamento de mutações no gene GH1. Realizamos estudo molecular por reação em cadeia da polimerase e sequenciamento automático de toda a região codificadora do GH1. Segregação familiar foi realizada para as variantes alélicas identificadas. Em nossa casuística, foram identificadas 10 variantes alélicas nos éxons 4 e 5 e no íntron 4 do GH1, sendo três variantes ainda não descritas na literatura (c.407G > A/p.Val122Ile, c.507C > T/p.Tyr169Tyr e c.456+19G > T). A análise in silico de todas as variantes identificadas indicou ausência de predição de efeito deletério sobre a proteína do GH. Estudo complementar realizado pelo nosso grupo identificou em crianças diagnosticadas com DIGH grave apenas uma paciente com mutação no GH1 responsável pela forma dominante desta doença. Em conclusão, mutações no GH1 causadoras da forma autossômica dominante de DIGH ou Tipo II não foram encontradas em nossa casuística, o que sugere que estas mutações sejam infrequentes em nossa população / The growth hormone (GH) / insulin-like growth factor-1 (IGF-1) axis is the most important hormonal regulator of post-natal linear growth. GH is encoded by the Growth Hormone 1 gene (GH1). Mutations in GH1 with dominant inheritance, which exerts a dominant negative effect on the bioactive GH isoforms, are the main causes of monogenic isolated deficiency of growth hormone (IGHD), while deletions or point mutations in GH1 are responsible for a rare autosomal recessive form of IGHD. However, only homozygous deletions were identified in patients with IGHD from Unidade de Endocrinologia do Desenvolvimento do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, even after detailed investigation of GH1. This difference regarding to literature can be caused by different criteria used to diagnose IGHD in our group, which adopted the cutoff value of peak GH < 3.3ug/L in response to stimulation test, in contrast to literature that describes other groups that use the cutoff peak value of the 7 - 10ug/L. Consequently, patients with autosomal dominant inheritance mutations in GH1 could be being erroneously diagnosed, as having idiopathic short stature (ISS) in our group. Additionally, mutations that cause biologically inactive GH can also be responsible for short stature in these patients. Due to the factors described above, we decided to screen mutations in GH1 in a group of children classified as ISS. We selected 98 of 487 children followed in our department with short stature according to the following criteria: normal birth weight and length for gestational age, height SDS <= -2, IGF-1 SDS < -1 and peak GH in stimulation test >= 3.3 ug/L. Genomic DNA was extracted from peripheral blood leucocytes of the patients to screen for mutations in GH1. We performed molecular analysis by polymerase chain reaction and automated sequencing of the entire coding region of the GH1. Segregation analysis was performed in the presence of allelic variations. In our casuistic, we identified 10 allelic variants in exon 4, exon 5 and intron 4 of GH1, three of which have not been described (c.407G > A/p.Val122Ile, c.507C > T/p.Tyr169Tyr and c.456+19G >T). In silico analysis predicted that none of the mutant alleles would result in deleterious effect on the GH protein. An additional study in children diagnosed with severe IGHD, identified just one patient with the pathogenic GH1 mutation responsible for the dominant form of this disease. In summary, defects in GH1 responsible for the autosomal dominant form of IGHD or Type II were not found in our cohort of Brazilian patients, suggesting that these mutations are infrequent in our population