Tesi sul tema "SRD5A1"

Une exposition excessive aux glucocorticoïdes (GC), comme observée chez les patients recevant une corticothérapie, peut entraîner un dysfonctionnement des cellules β et un diabète chez jusqu'à 40% des patients. Dans l'obésité, une surexposition locale au cortisol secondaire à une altération du métabolisme du cortisol contribue à l'apparition du diabète. Des doses élevées de GC comme la dexaméthasone (DEX) inhibent la sécrétion d'insuline stimulée par le glucose (SISG), mais les effets de doses plus faibles et des autres GC, tels que l'hydrocortisone (HC) et la prednisone (PRED), restent peu étudiés. L'enzyme 5α-réductase de type 1 (SRD5A1) est une enzyme cruciale pour la dégradation des GC, modulant ainsi leur biodisponibilité. L'inhibition de SRD5A1 est associée à une altération de la sensibilité à l'insuline et à un risque accru de diabète. La première partie de ma thèse étudie l'impact de doses "thérapeutiques faibles" de PRED (équivalentes à 5 à 10 mg par voie orale) et d'autres GC sur la SISG étudiée par périfusion dans des îlots isolés de pancréas humains. Tous les GCs diminuent significativement la SISG, la DEX ayant un impact plus important que la PRED et l'HC. L'IMC, l'âge ou le sexe n'influencent pas significativement l'impact de la PRED sur la sécrétion d'insuline. La deuxième partie du travail caractérise le métabolisme des GC dans les îlots humains. SRD5A1 est la seule réductase A-ring dans les îlots, et son expression, ainsi que celle de HSD11B1, est localisée dans les cellules β des îlots. Nous avons démontré l'existence d'un métabolisme intracrine du cortisol dans des cultures primaires d’îlots humains. L’expression de HSD11B1 et SRD5A1 est significativement diminuée dans les îlots des donneurs atteints de DT2 par rapport aux donneurs normoglycémiques. La dernière partie visait à prouver que la diminution de la biodisponibilité du cortisol via la surexpression de SRD5A1 dans les îlots humains atténue l'effet inhibiteur des GC sur la SISG. La surexpression de SR5DA1 a permis d’atténuer l'impact de l'HC sur la première phase de la SISG, mais pas de la PRED. En conclusion, même à faibles doses, les GC altèrent la SISG. La diminution de l'expression de SRD5A1 dans les îlots peut contribuer au développement du diabète dans un contexte métabolique. La surexpression de SRD5A1 protège contre l'impact délétère du cortisol sur la SISG. Ces résultats supportent le rôle de SRD5A1 dans la surexposition locale au cortisol et le développement du diabète. Cependant, l'augmentation de l'activité de SRD5A1 ne semble pas efficace pour protéger contre les complications métaboliques induites par la corticothérapie. D'autres aspects de la fonction des cellules β, en particulier la viabilité cellulaire, vont être étudiés. Par ailleurs, le bénéfice potentiel de SRD5A1 dans la modulation de la résistance à l'insuline et de la stéatose hépatique doivent être étudiés. Ces études complémentaires permettront de mieux comprendre le potentiel du gène SRD5A1 dans la modulation de la résistance à l'insuline et de la maladie du foie gras
Excessive glucocorticoid (GC) exposure, as seen in patients receiving GC therapy, can lead to β-cell dysfunction and diabetes in up to 40% of the cases. In obesity, increased local cortisol exposure due to altered metabolism contributes to diabetes onset. High doses of GCs like dexamethasone (DEX) are known to inhibit glucose-stimulated insulin secretion (GSIS), but the effects of lower doses and other GCs, such as hydrocortisone (HC) and prednisone (PRED), remain underexplored. The enzyme 5α-reductase type 1 (SRD5A1) is a crucial enzyme for GC degradation, modulating their bioavailability. Inhibition or knockout of SRD5A1 is associated with impaired insulin sensitivity and increased diabetes risk. This first part of my thesis investigates the impact of “low therapeutic” doses of PRED (equivalent to 5 to 10 mg administrated orally) and other GCs on glucose stimulated insulin secretion (GSIS). We showed that PRED significantly decreases GSIS, with DEX having a worse effect compared to PRED and HC. BMI, age, or sex do not significantly influence the direct impact of PRED on insulin secretion. The second part of the work aimed to characterize GC metabolism in human islets. SRD5A1 is the only A-ring reductase expressed in islets, and its expression, along with HSD11B1, is localized within the β-cells of human islets. We demonstrated evidence of intracrine metabolism of cortisol in intact primary human islets cultured under dynamic experimental settings. Expression data reveals significantly diminished expression of both HSD11B1 and SRD5A1 in T2D donors compared to normoglycemic donors. The last part aimed to provide proof of concept that decreased cortisol bioavailability via the overexpression of SRD5A1 in human islets mitigates the inhibitory effect of GCs on GSIS. SR5DA1 overexpression attenuated the impact of HC on the first phase of insulin secretion, but not the PRED impact. To conclude, even at low doses, GCs impair GSIS. The decrease in SRD5A1 expression in islets may contribute to the development of diabetes in metabolic context. SRD5A1 overexpression protects against the deleterious impact of cortisol on GSIS, providing additional evidence to support the enzyme's role in local cortisol overexposure and the development of diabetes. However, increasing SRD5A1 activity may not be an effective approach to protect against metabolic complications induced by GC therapy. Other aspects of β-cell function, especially cell viability, need to be studied. Moreover, the potential benefits of SRD5A1 in modulating insulin resistance and fatty liver disease should be investigated. These further studies will provide more insight into the potential of SRD5A1 as a therapeutic target

Orientador: Maricilda Palandi de Mello
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Para um correto desenvolvimento sexual masculino em humanos, é necessária a presença, entre outros, de dois hormônios esteróides: a testosterona (T) e a diidrotestosterona (DHT). A T é o hormônio responsável pelo desenvolvimento da genitália interna masculina, já a DHT é o hormônio chave da virilização da genitália externa masculina e responsável pelo estabelecimento dos caracteres sexuais secundários durante a puberdade. Duas enzimas são responsáveis pela produção destes hormônios: a enzima 17?-hidroxiesteróide desidrogenase tipo 3 (gene HSD17B3), a qual é responsável pela conversão do hormônio androstenediona em T, reação realizada na última etapa da biossíntese da T e a enzima 5?-redutase tipo 2 (gene SRD5A2), que é responsável por catalisar a conversão da T em DHT. Mutações nos genes HSD17B3 ou SRD5A2 causam alterações que levam à não produção ou à síntese defeituosa das enzimas 17?-hidroxiesteróide desidrogenase tipo 3 e 5?-redutase tipo 2, promovendo deficiência na virilização de indivíduos 46,XY. Indivíduos estes cujas gônadas são representadas por testículos, apresentam pseudo-hermafroditismo masculino (PHM), agora denominado distúrbio da diferenciação do sexo em indivíduos 46,XY (DDS-XY). Estes podem apresentar genitália ambígua, sendo o sexo de criação predominantemente feminino, com virilização na puberdade. O diagnóstico pode ser confirmado com a identificação de mutações nos genes específicos HSD17B3 e SRD5A2. Assim, o objetivo desse estudo foi investigar alterações moleculares nos genes HSD17B3 e SRD5A2, em pacientes com ambiguidade genital com cariótipo 46,XY, e contribuir para a confirmação do diagnóstico clínico e laboratorial de DDS em indivíduos 46,XY por deficiências nas enzimas 17?-hidroxiesteróide desidrogenase tipo 3 e 5?-redutase tipo 2. A metodologia empregada neste estudo teve por base a amplificação dos 11 exons do gene HSD17B3 e dos 5 exons do gene SRD5A2 pela reação em cadeia da polimerase (PCR), seguida por rastreamento das mutações através do sequenciamento direto dos produtos de amplificação. Das 2 famílias estudadas para diagnóstico de alterações no gene HSD17B, foi encontrada uma alteração, p.R80Q, em homozigose em um paciente. E para o para o gene SRD5A2, foram estudadas 45 famílias, e foi verificada a presença de três mutações: a c.418delT em homozigose em um paciente, a c.278delG em heterozigose em um paciente e a p.Q126R em homozigose em duas irmãs. Vários polimorfismos freqüentes foram observados e, também, algumas riações nucleotídicas novas ou raras foram identificadas. Fez parte do estudo a avaliação in vitro da mutação g.49529G>A, já descrita previamente como uma mutação missense (p.G183S), quanto ao efeito deletério no processo de splicing, uma vez que esta alteração encontra-se no último nucleotídeo do exon 3 do gene SRD5A2. Foi verificado que esta alteração promove a excisão do exon 3, mostrando que o efeito primário desta mutação não é a troca de aminoácidos e sim uma alteração no processo de splicing deste gene.
Abstract: The male sexual development requires the production of normal amounts of two steroid hormones: testosterone (T) and dihydrotestosterone (DHT). The T is responsible for development of male internal genitalia, whereas DHT is the key for the virilization of the male external genitalia and responsible for the establishment of secondary sexual characteristics during puberty. Two enzymes are responsible for the production of these hormones: 17?-hydroxysteroid dehydrogenase type 3 enzyme (HSD17B3 gene), which is responsible for converting androstenedione to T, the last step of the biosynthesis of T; and 5?-reductase type 2 enzyme (SRD5A2 gene), which is responsible for catalyzing the conversion of T into DHT. Individuals with 46,XY karyotype and mutations in either HSD17B3 gene or SRD5A2 gene, present a deficiency of enzyme activity that leads to phenotypes ranging from male with ambiguous genitalia to normal female. Such individuals may be assigned at birth and raised as females. The gonads of those individuals are represented by testes. This condition defines the male-pseudohermaphroditism (MPH), recently renamed as disorder of sexual development in 46,XY karyotype (DSD-XY). The diagnosis of these deficiencies can be confirmed with the identification of mutations in either HSD17B3 or SRD5A2 genes. The aim of this investigation was to identify nucleotide alterations in the HSD17B3 gene or in the SRD5A2 gene in patients with clinical and hormonal characteristics suggestive of 17?-hydroxysteroid dehydrogenase type 3 deficiency, or 5?-redutase type 2 deficiency. Molecular analysis was performed by amplification of the eleven exons of HSD17B3 gene, and the five exons of SRD5A2 gene followed by sequencing. In two families studied for the HSD17B3 gene, one alteration was detect, p.R80Q, in a homozygous patient. Forty-five families were studied for SRD5A2 gene and the presence of three mutations was verified: the c.418delT in one homozygous patient, the heterozygosis for c.278delG in one patient and the homozygosis for p.Q126R in two sisters. Several frequent polymorphisms in the SRD5A2 gene were observed and some novel or rare variations were identified as well. The study in vitro with the g.49529G>A mutation was also performed due to the possibility of deleterious effect on the splicing process, although his mutation had been previously described as a missense mutation (p.G183S). It was verified exon 3 skipping in the mRNA as a result of the mutation, showing that the primary effect is not the change of amino acids but the anomalous splicing process of the SRD5A2 gene.
Mestrado
Genetica Animal e Evolução
Mestre em Genética e Biologia Molecular

Les anomalies cérébelleuses englobent un groupe de maladies rares affectant le fonctionnement du cervelet. La prévalence de ces défauts est estimée à 26 sur 10000 enfants en Europe. Ces maladies entraînent des troubles du mouvement (ataxie) et sont fréquemment associées à des déficiences intellectuelles, des défauts qui empêchent les patients touchés d'avoir une vie normale et qui peux entrainer une mortalité précoce. Pour la plupart de ces conditions, seul un traitement symptomatique est disponible. Outre le diagnostic génétique, utile pour faire face à de nouvelles grossesses, une compréhension profonde de la physiopathologie sous-jacente à l'anomalie est nécessaire pour le développement potentiel des thérapies. Mon travail de thèse avait pour but l'amélioration du diagnostic génétique des défauts cérébelleux et la compréhension de la physiopathologie de l'une des causes les plus fréquentes d'anomalies cérébelleuses : la perturbation de la N-glycosylation des protéines. La perturbation de la N-glycosylation des protéines est à la base des syndromes CDG (troubles congénitaux de la glycosylation ou «congenital disorders of glycosylation»), maladies multisystémiques avec des troubles neurologiques sévères. Une atrophie et une hypoplasie cérébelleuses précoces sont fréquemment observées, en particulier dans les cas des CDG avec des mutations au gène SRD5A3. Pour mieux comprendre comment un défaut général de N-glycosylation affecte le développement cérébelleux, nous avons généré une souris Srd5a3 KO conditionnelle au niveau du cervelet. Ce modèle récapitule le défaut humain avec une N-glycosylation anormale, une hypoplasie cérébelleuse et une altération de la coordination motrice. Une évaluation histologique précise du cervelet a montré que certaines cellules granulaires étaient incapables d'initier leur migration finale lors du développement du cervelet. En combinant une approche protéomique et une approche glycoprotéomique, nous avons montré qu'un défaut de N-glycosylation a un impact variable en fonction du nombre de sites de N-glycosylation sur chaque protéine. Plus le nombre de sites de N-glycosylation d'une protéine est élevé, plus elle est sensible à l'hypoglycosylation et/ou à la dégradation dans un contexte de CDG. Nos données montrent que les molécules d'adhésion cellulaire fortement N-glycosylées avec des domaines d'immunoglobuline (IgSF-CAMs) sont hautement sensibles au défaut de N-glycosylation. En utilisant de l'imagerie en temps réel de cellules granulaires en culture, nous avons identifié un défaut d'extension des neurites liée aux IgSF-CAMs. Ce défaut est lié à une altération de la glycosylation et fonctionnement de L1CAM et NrCAM. Nous avons ensuite évalué si le défaut était conservé dans les neurones humains. Pour étudier cette possibilité, nous avons généré des cellules humaines pluripotentes (hiPCS) SRD5A3-/- qui ont été différenciés vers des neurones corticaux. Ces neurones récapitulent le défaut biochimique trouvé chez la souris (hypoglycosylation de NrCAM et L1CAM). Cette découverte étend nos conclusions à l'ensemble du cerveau humain. Enfin, en utilisant la microscopie électronique, nous avons pu identifier une organisation des fibres parallèles cérébelleuses perturbée chez le mutant, en accord avec le rôle établi de nombreuses IgSF-CAM dans le guidage axonal. Nos résultats fournissent des preuves sur le mécanisme impliqué dans la sensibilité spécifique du cervelet à une altération de la N-glycosylation. De plus, nous montrons comment les défauts de la N-glycosylation affectent principalement l'adhésion cellulaire. Notre travail fournit également de nouvelles preuves sur l'importance critique de la N-glycosylation multiple des IgSF-CAM pour leur stabilité et leur fonctionnalité au cours du développement du cerveau des mammifères
Cerebellar defects encompass a group of rare diseases affecting cerebellar functioning. The prevalence of these defects is estimated to be 26 in 10000 children in Europe. These diseases lead to movement disorders (ataxia) and are frequently associated with intellectual disability, life-threatening conditions that help affected patients from coping with a normal daily life. For most of these conditions, only supportive treatment is available. Besides genetic diagnose, helpful when facing new pregnancies, an in-deep understanding of the physiopathology underlying the disorder is necessary for future therapeutics. My thesis project had as objective to improve the genetic diagnose of cerebellar defects and understanding the physiopathology behind one of the more prevalent cause of cerebellar defects: disruption of protein N-glycosylation. Disruption of protein N-glycosylation causes Congenital Disorders of Glycosylation (CDG), multisystemic disorders with severe neurological disorders. Early-onset cerebellar atrophy and hypoplasia are frequently observed, especially in CDG cases with SRD5A3 mutations. To understand how a general N-glycosylation defect affects cerebellar development, we generated a cerebellum-specific Srd5a3 conditional KO mouse. This model recapitulates the human defect with abnormal N-glycosylation, cerebellar hypoplasia and motor coordination impairment. Careful histological evaluation of the cerebellum proved some granule cells to be unable to initiate their final migration during cerebellar development. By combining a proteomic and a glycoproteomic approach, we showed that a defect in N-glycosylation has a variable impact depending on the number of N-glycosylation sites on each protein. The more N-glycosylation sites that a protein has, the more sensitive it is to hypoglycosylation and/or degradation in a CDG context. Our data suggest the heavily N-glycosylated cell adhesion molecules with immunoglobulin domains (IgSF-CAMs) to be highly sensitive to the glycosylation defect. Using in vitro live granule cells imaging, we identified an IgSF-CAM-dependant neurite extension defect. This defect is linked to impaired glycosylation and functioning of L1CAM and NrCAM. We next evaluated if the defect was conserved in human neurons. To investigate that possibility we generated SRD5A3-/- hiPSCs that were further differentiated towards cortical neurons. Human neuron-like cells recapitulate the biochemical defect in mouse (e.g. L1CAM and NrCAM hypoglycosylation). This finding expands our conclusions to the whole human brain. Finally, using electron microscopy we could identify disrupted cerebellar parallel fiber organization in the mouse mutant, consistent with the established role of numerous IgSF-CAMs in axon guidance. Our results provide important evidence into the molecular mechanism underlying cerebellar sensitivity to an N-glycosylation impairment. Moreover, we show how defects in N-glycosylation will primarily affect cell adhesion. Our work also provides new evidence for the critical importance of the multiple N-glycosylation of IgSF-CAMs for their stability and functionality during mammalian brain development

Orientador: Christine Hackel
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Mestrado

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
CHAPTER I - Prostate cancer is a common disease in the world and in some countries it is one of the main causes of male population mortality. Some molecular markers have been associated with prostate carcinogenesis. To observe changes in transcript levels of the AR, SRD5A2, KLK2, PSMA and PCA3 genes, the mRNA was analyzed in tissues with prostatic adenocarcinoma (PCa, N= 48) and benign prostatic hyperplasia (BPH, N= 25), performed through a differential multiplex RT-PCR assay. Significant differences were observed in the relative expression of these genes between cancerous and non-cancerous tissues. The optical density ratio of the cDNA amplicons between PCa and BPH for the AR gene was 1.6-fold higher for the prostatic adenocarcinoma. On the other hand, the SRD5A2 mRNA levels were associated with BPH and were 1.4-fold higher than that of PCa. For KLK2, PSMA and PCA3, the transcriptional levels were respectively, 1.9-, 1.9- and 5-fold higher in PCa than those in BPH tissues. Of the diagnostics tests carried through individually, the PCA3 gene was that presented higher sensitivity and accuracy, and the inclusion of the serum PSA improved the sensitivity (of 76 to 92%), positive preditive value (of 85 to 94%), negative preditive value (of 60 to 62%) and accuracy (of 74 to 78%). The results suggest that the higher AR, KLK2, PSMA, and PCA3 and/or reduced SRD5A2 genes expression in prostatic tissues may indicate the occurrence of prostate adenocarcinoma; however the PCA3 and serum PSA analysis together are highly promising as auxiliary method in the diagnosis of this cancer. CHAPTER II - Purpose: Prostate cancer (PCa) is the most commonly diagnosed malignancy in men, and it consists of multifactorial and multifocal events. Due to the complexity of the disease process, which includes genome alterations, local invasion, micrometastatic cell extravasations to circulation, invasion of secondary organ tissues, and resistance to hormonal blockage, many markers must be used to represent the multiple and variable events that lead to cancer development. The low specificity of the unique serum marker for prostate cancer diagnostics, PSA, has leaded us to investigate four potential markers in the peripheral blood of patients by detecting their mRNA levels and associating them to clinical parameters. Methods: The expression levels of the KLK2, KLK3, PCA3 and PSMA transcripts were determined by Nested RT-PCR. Patients with PCa (99) and with benign prostatic hyperplasia (BPH, 36), and healthy volunteers (104) were investigated. Results: Significant differences for the RNA relative levels have been found for the KLK2, PCA3 and PSMA transcripts between PCa and BPH patients or healthy volunteers. The KLK2 and PSMA levels also presented a positive association (P<0.05) with extra-prostatic disease (pT3). The combined positive RT-PCR Nested for the KLK2, PCA3, PSMA genes with serum PSA higher than 4ng/mL presented a 10-fold higher chance of cancer occurrence than healthy controls, with sensitivity, specificity and positive predictive value of 57%, 89% and 93%, respectively. Conclusions: The combined analysis of KLK2, PCA3 and PSMA transcripts may become a useful tool for the discrimination of PCa patients from those with benign disease or healthy individuals. Additionally, the KLK2 and PSMA transcripts may also be used as prognostic markers for the presence of extra-capsular disease and assisting in the prediction of the post-operative outcome. CHAPTER III - Purpose: Transcripts of PCA3/DD3 gene are at the moment the most specific molecule found in prostate cancer specimens. This mRNA can be detected in important sample targets for clinical analyses, such as prostatic tissues, urine after prostatic massage, and the peripheral blood. Methods: The present study evaluated the PCA3 gene expression in prostatic tissues and in peripheral blood of BPH and PCa patients, by RT-PCR assays, and based on its detection together with other clinical parameters, we proposed a model for molecular monitoring in order to improve diagnosis as an auxiliary technology. Results: The concomitant use of PCA3 transcript detection in the peripheral blood and in prostate tissues has improved diagnosis, with sensitivity and an accuracy of 77%. For the molecular staging, patients have been classified as: localized disease (PBL-; negative PCA3) and circulating tumors cells disease (PBL+; positive PCA3). The higher frequencies of PBL- had been observed in T1-T2 stages (75%); on the other hand, the higher PCA3 positivity was observed for the T3-T4 staging (43%), while the T1-T2 stages presented 25% positivity. A correlation was found between the molecular staging and serum PSA < 10ng/mL before surgery, and approximately 60% of patients with T3-T4 stages that presented biochemical failure after radical prostatectomy presented a positive PCA3 result (P= 0.05), with an odds ratio of 16-fold higher for the possibility of disease recurrence in relation to the T1-T2 patients, and an accuracy of 82%. Conclusion: These data demonstrated the importance of the PCA3 gene as an auxiliary method in prostate cancer diagnosis, by distinguishing PCa from BPH patients, and also demonstrated its prognostic value in recurrent disease for post-operative patients. CHAPTER IV - Approximately 98% of all the products transcribed in the human genome correspond to non coding RNAs (ncRNA). Many ncRNA functions are attributed to this structural particularity given mainly for the secondary structures formed from its linear sequence of bases. Among the ncRNA types are tRNA, rRNA, small nuclear RNA, small nucleolar RNA, small interference RNA (siRNA), microRNA (miRNA) and catalytic RNAs (ribozymes). The bioinformatics has supplied useful tools in the prediction of optimal or suboptimal secondary structures allowing the design of interference RNA as miRNAs or siRNAs. In human, miRNAs have been associated with the development of diverse complex diseases as cancer. The PCA3 (DD3) gene was molecularly characterized as cancer and prostate specific, and its transcripts are non-coding, once no peptide products have been found. Due to its structural characteristics, the PCA3 gene belongs thus to the increasing family of ncRNA. In the present work, four new variant molecules of the PCA3 gene have been sequence characterized and their frequencies demonstrated in prostate cancer and in benign prostatic hyperplasia patients, as well as in healthy individuals. We have also investigated and predicted the putative secondary structures formed in order to elucidate its role in prostate cancer biology. No association has been found between the frequency of these molecules and prostate pathologies (PCa or BPH). On the other hand, PCA3 variants were found in 10% (12/115) of cases in the general population. Similar analyses of the possible polypeptides of these molecules demonstrated that it remains as a non-coding RNA, and introns presents in the first, second and fourth variants suggesting a possible role as a miRNA with intracellular activity to these molecules to the PCA3 gene. In prostatic tissues, 100% of the prostate cancer cases presented the RNA molecule with an exon 2 splicing. However, further investigation must be carried out to demonstrate the true role of these splicing variants in prostate tumors and in other pathologies, once these molecules have been preferentially found in the peripheral blood.
CAPÍTULO I - O câncer de próstata é uma doença comum no mundo e já assumiu em alguns países uma das principais causas de mortalidade da população masculina. Vários marcadores moleculares têm sido associados à gênese do câncer de próstata. A fim de demonstrar a expressão diferencial dos níveis transcricionais dos genes AR, SRD5A2, KLK2, PSMA e PCA3 em doenças prostáticas, o RNAm foi analisado em tecidos com adenocarcinoma prostático (CaP, N= 48) e hiperplasia prostática benigna (HPB, N= 25) por meio da técnica RT-PCR multiplex semi-quantitativa. Foram observadas diferenças significativas na expressão relativa desses genes entre os tecidos cancerosos e nãocancerosos. A taxa de densidade ótica entre os amplicons para cDNA provenientes do gene AR foi 1.6 vezes maior no adenocarcinoma prostático. Por outro lado, os níveis de RNAm do gene SRD5A2 foi associado com a HPB e foi 1.4 vezes maior do que no CaP. Para os genes KLK2, PSMA e PCA3, os níveis transcricionais foram respectivamente, 1.9, 1.9 e 5 vezes maior no câncer comparado a tecidos benignos. Dos testes diagnósticos realizados, o gene PCA3 individualmente foi o que apresentou as melhores sensibilidade e acurácia, sendo que a inclusão das medidas de PSA sérico melhorou a sensibilidade (de 76 para 92%), o valor preditivo positivo (de 85 para 94%), o valor preditivo negativo (de 60 para 62%) e a acurácia (de 74 para 78%). Os dados sugerem que a maior expressão dos genes AR, KLK2, PSMA e PCA3 ou expressão reduzida do gene SRD5A2 em tecidos prostáticos podem indicar a ocorrência do adenocarcinoma da próstata, sendo que as análises do gene PCA3 juntamente aos do PSA sérico são altamente promissores como método auxiliar no diagnóstico desse tipo de câncer. CAPÍTULO II - O câncer de próstata (CaP) e o mais comumente diagnosticado na população masculina e consiste de eventos multifatoriais e multifocais. Devido a complexidade da doença, a qual inclui alterações genômicas, invasão local, liberação de células micrometastáticas para a circulação, invasão secundaria de tecidos de outros órgãos, e resistência ao bloqueio hormonal, muitos marcadores podem ser usados para representar os eventos múltiplos e variáveis que levam ao desenvolvimento do câncer. A baixa especificidade do único marcador para diagnostico do câncer de próstata, PSA, tem nos levado a investigar quatro potenciais marcadores no sangue periférico de pacientes pela detecção de seus níveis de RNAm e associá-los a parâmetros clínicos. Os níveis de expressão dos transcritos do KLK2, KLK3, PCA3 e PSMA foram avaliados pela RT-PCR Nested, em pacientes com CaP (99), com hiperplasia prostática benigna (HPB, 36) e voluntários saudáveis (104). Diferenças significativas foram encontradas para a expressão dos genes KLK2, PSMA e PCA3 entre os pacientes com CaP e os pacientes com HPB ou voluntários saudáveis. Os níveis do KLK2 e PSMA também apresentaram associação positiva (P<0.05) com doença extra-prostática (pT3). A RT-PCR Nested positiva combinada para os genes KLK2, PCA3 e PSMA com PSA sérico maior que 4ng/mL apresentou uma chance 10 vezes maior de ocorrência do câncer comparado aos controles saudáveis, com sensibilidade, especificidade e acurácia de 57%, 89% e 93%, respectivamente. A análise combinada dos genes KLK2, PCA3 e PSMA pode ser uma ferramenta útil na distinção de pacientes com CaP daqueles com doença benigna ou de indivíduos saudáveis. Ainda, a analise dos transcritos KLK2 e PSMA podem ser usados como marcadores prognósticos para a presença de doença extra-capsular e auxiliando na predição de recidiva da doença no pós-operatório. CAPÍTULO III - Os transcritos do gene PCA3/DD3 são até o momento as moléculas mais específicas encontradas em espécimes de câncer de próstata. Esses RNAm podem ser detectados em importantes alvos para a análise clínica como tecidos prostáticos, na urina após massagem prostática e em sangue periférico. O presente estudo avaliou a expressão do gene PCA3 em tecidos prostáticos e em sangue periférico de pacientes com HPB e CaP, por técnicas de RT-PCR, e baseado na sua detecção juntamente com os parâmetros clínicos, foi proposto um modelo de estadiamento molecular como técnica assessória para melhor o diagnóstico da doença. O uso concomitante da detecção dos transcritos do gene PCA3 no sangue periférico e no tecido prostático melhorou o diagnóstico, com sensibilidade e acurácia de 77%. Para o estadiamento molecular, os pacientes foram classificados como contendo a doença localizada (PBL-) e em doença com células tumorais circulantes (PBL +). Maiores freqüências de tumor localizado pelo estadiamento molecular foram observadas nos estadios T1-T2 (75%), enquanto que 25 e 43% dos cânceres T1-T2 e T3-T4, respectivamente, apresentaram PCA3 positivo (células circulantes). Uma correlação foi encontrada para o estadiamento molecular para doença localizada e PSA sérico pré-cirúrgico < 10ng/mL, e aproximadamente 60% dos pacientes TNM T3-T4 que apresentaram falha bioquímica após a cirurgia radical apresentaram RTPCR positiva do PCA3 (P= 0.05), com um Odds Ratio 16 vezes maior para a possibilidade de recorrência da doença em relação aos pacientes T1-T2 e uma acurácia de 82%. Esses dados demonstram a importância da detecção do gene PCA3 como método no diagnóstico do câncer de próstata, por distinguir pacientes com CaP daqueles com HPB, e também demonstrando seu valor prognóstico na doença recorrente no pósoperatório dos pacientes. CAPÍTULO IV - Aproximadamente 98% de todos os produtos transcritos do genoma humano correspondem a RNAs não codificantes (RNAnc). Muitas funções dos RNAnc são atribuídas a suas particularidades estruturais dadas principalmente pelas estruturas secundárias formadas a partir da sua sequência linear de bases. Dentre os tipos de RNAnc estão os RNAt, RNAr, small nuclear RNA, small nucleolar RNA, small interference RNA (siRNA), microRNA (miRNA) e RNAs catalíticos (ribozimas). A bioinformática tem fornecido ferramentas úteis na predição de estruturas secundárias ótimas ou subótimas permitindo o design de RNAs de interferência como os miRNAs ou siRNAs. Em humanos, os miRNAs tem sido associados ao desenvolvimento de diversas doenças complexas como o câncer. O gene PCA3 (DD3) foi molecularmente caracterizado como câncer- e próstata- específico e os seus RNAs são os responsáveis por essa característica, isso porque nenhum produto protéico tem sido encontrado para esse gene. Devido às suas características estruturais, o gene PCA3, pertence assim à crescente família de RNAnc. No presente trabalho foi analisado as freqüências de quatro moléculas variantes do gene PCA3, além das anteriormente reportadas, como também foram preditas as suas estruturas secundárias na tentativa de elucidar o seu papel na biologia do câncer de próstata. Nenhuma associação foi encontrada entre a freqüência dessas moléculas e as patologias da próstata como hiperplasia benigna ou câncer, sendo que na população geral analisada essas variantes foram encontradas em apenas 10% (12/115) dos casos. As análises de homologia de possíveis polipeptídeos para essas moléculas demonstram que permanece o papel de RNA não-codificante para o gene PCA3. Ainda, a presença de introns nas variantes 1, 2 e 4 podem sugerir um papel intracelular de miRNA para essas moléculas do gene PCA3. Nos tecidos prostáticos, 100% dos casos de câncer foi representando pela molécula com splicing do exon 2. Contudo, para as variantes de splicing, novas pesquisas deverão ser realizadas incluindo outras patologias além das doenças prostáticas e outros tipos tumorais para verificar o real impacto dessas moléculas, uma vez que foram encontradas preferencialmente no sangue periférico.
Doutor em Genética e Bioquímica

Introdução: A pesquisa por marcadores genéticos de susceptibilidade a doenças geralmente focam um gene baseado nas propriedades e vias metabólicas de seus produtos protéicos, assim, genes envolvidos na biossíntese de andrógenos podem identificar possíveis alterações na carcinogênese prostática. Diferenças étnicas em polimorfismos apresentam um papel no metabolismo de hormônios e podem acometer diferentes raças no risco de desenvolver câncer de próstata. A 5 alfaredutase (SRD5A2) é a enzima responsável por catalisar irreversivelmente a conversão da testosterona no principal androgênio prostático, dihidrotestosterona (DHT). Vários polimorfismos associados ao gene SRD5A2 tem sido estudados e relatados quanto à possível associação com o aumento do risco/susceptibilidade do CaP, entre eles, o polimorfismo V89L. Objetivos: Avaliação da associação do polimorfismo V89L do gene SRD5A2 com o risco de câncer de próstata em uma amostra da população do Rio Grande do Sul; avaliação da frequência deste polimorfismo em uma amostra de indivíduos com câncer de próstata e em um grupo controle e correlação da frequência do polimorfismo com os níveis séricos de testosterona total e livre, bem como o PSA. Métodos: Foram coletadas amostras de sangue de 169 casos com câncer de próstata e 177 controles. Os genótipos do polimorfismo V/V, V/L e L/L foram comparados entre casos e controles. Resultados: O genótipo V/V foi significativamente mais frequente nos controles em comparação com os casos, entretanto não encontramos associação entre os genótipos do polimorfismo V89L e o risco de desenvolver o câncer de próstata, porém ao analisarmos somente os indivíduos com idade inferior a 65 anos, verificamos que o genótipo V/V quando comparado com o genótipo V/L e quando comprado com o genótipo (L/L+V/L) apresenta um fator protetor (OR: 0,338; IC: 0,134-0,858; p=0,022 e OR:0,046; IC: 0,166-0,990; p=0,047, respectivamente). Verificamos também que o genótipo V/L quando comparado com o genótipo (V/V+L/L) proporciona um risco para o desenvolvimento de câncer de próstata (OR:1,651; IC: 1,043-2,613; p=0,032). Conclusões: Nosso estudo sugere que entre indivíduos com menos de 65 anos de idade, o genótipo V/L do polimorfismo V89L pode desempenhar um significativo risco de desenvolver CaP e que o genótipo V/V está associado com um fator protetor para o câncer de próstata.
Background: The search for genetic markers of susceptibility to diseases often focus on a gene based on the properties of metabolic pathways and their protein products, thus, genes involved in biosynthesis of androgens can identify possible changes in prostate carcinogenesis. Ethnic differences in polymorphisms have a role in hormone metabolism and may affect different races in the risk of developing prostate cancer. The 5 alpha-reductase (SRD5A2) is the enzyme responsible for irreversibly catalyzing the conversion of testosterone into the main prostatic androgen, DHT. Several polymorphisms associated with the SRD5A2 gene has been studied and reported on the possible association with increased risk or susceptibility of PCa among them, the V89L polymorphism. Objectives: Assessment of association of the V89L polymorphism of SRD5A2 gene with prostate cancer risk in a population sample from Rio Grande do Sul, evaluate the frequency of this polymorphism in a sample of individuals with prostate cancer and a control group and the frequency correlation polymorphism on serum levels of total and free testosterone and PSA. Methods: Blood samples were collected for 169 prostate cancer cases and 177 controls. The genotypes (V/V, V/L and L/L) were compared among cases and controls. Results: The genotype V/V was significantly more frequent in controls compared with cases, however we found no association between the V89L polymorphism genotypes and the risk of developing prostate cancer, but when we analyzed only those individuals under the age of 65 years, we found that the genotype V/V compared with V/L and when purchased with genotype (L/L+V/L) has a protective factor (OR: 0.338 CI:0.134-0.858, p = 0.022 and OR: 0.046; CI: 0.166-0.990,p=0,047, respectively). We also found that genotype V/L compared with genotype (V/V+L/L) gives a risk for developing prostate cancer (OR:1.651; CI:1.043-2.613, p=0.032). Conclusion: Our study suggests that among people under 65 years of age, the genotype V/L may play a significant risk of developing CaP and genotype V/V is associated with a protective factor for prostate cancer.

Prostate cancer constitutes the most common malignancy and the most common cause of cancer‐related death in Swedish men. A large body of evidence suggests that inherited genetic variants contribute to both development and progression of prostate cancer. The aim of this thesis is to identify genetic variants that alter prostate cancer risk and progression. All papers included in this thesis are based on a Swedish population‐based case‐control study (CAPS) comprising 2,965 incident prostate cancer cases and 1,823 controls. In paper I, we investigated if genetic variants in the E‐cadherin gene altered prostate cancer risk. Seven haplotype tagging SNPs(tagSNPs) were selected and genotyped in CAPS and families with hereditary prostate cancer. We confirmed association of a promoter SNP rs16260 previously reported to increase risk of hereditary prostate cancer (OR: 2.6; 95% CI: 1.6‐4.3) for homozygous ‘A’ carriers. In paper II, we assessed 46 polymorphisms earlier reported to be associated with prostate cancer risk. Six polymorphisms in five different genes were replicated. Interestingly, three of these genes were involved in the androgen biosynthesis. In paper III, we followed up on the results from paper II by genotyping 23 tagSNPs located in the hormone regulating genes AR, CYP17 and SRD5A2. Multiple SNPs and haplotypes were associated with prostate cancer risk, especially in the AR gene. Combining risk alleles from all genes revealed a substantial risk increase for each additional allele carried (OR: 1.12; 95% CI: 1.1‐1.2, P=0.00009). In paper IV, we collected information about cause of death for all case patients in CAPS. At time of follow‐up 300 study subjects were deceased from prostate cancer. We assessed AR, CYP17 and SRD5A2 variants for association with lethal prostate cancer and found overall no association. However, one AR promoter SNP was associated with an increased risk of dying from prostate cancer amongst men who received palliative hormonal therapy as primary treatment. In paper V, we assessed common genetic variation at the ERG locus for association between prostate cancer risk and survival. ERG is recognized as a protooncogene frequently overexpressed in prostate cancer. A total of 21 tagSNPs in the 5’ region of ERG were genotyped. There was no correlation between ERG SNPs and prostate cancer risk but common genetic variation located approximately 100,000 basepairs upstream of ERG was significantly associated with prostate cancer specific survival. In summary, our results suggest that common genetic variation in Ecadherin alters prostate cancer risk in Swedish men with a positive family history of prostate cancer. Moreover, common genetic variation in the androgen‐related genes AR, CYP17 and SRD5A2 affects the risk of developing prostate cancer but is unlikely to alter prostate cancer progression. However, genetic variants in AR may affect hormonal therapy response. Finally, ERG polymorphisms are associated with prostate cancer‐specific death but are not likely to play a role in prostate cancer development.

Motion databases are becoming larger with the development of hardware and software, applications based on these data have been widely used in many different areas, such as movies, animations, videos games, sports as well as computer vision and robotics. All those applications had made motion analysis and classification essential for better motion composition. However, in order to achieve a good connectivity between every motion and emotion behind it, it is totally important to understand human behavior, even if human movements are complex and hard to describe completely. In this thesis, we make investigations on connections between various emotional states and different movements with regards to the arousal and valence of the Russell's circumplex model. A variety of different features were applied to describe stylistic characteristics of motion based on Laban Movement Analysis(LMA). Motion capture data from dancers with various background were used for training and classification purpose. In our experiments, we have utilized four methods to finish multi-class classification: Random Forests(RF), Extremely Randomized Trees(ET), Support Vector Machines(SVM) and Spectral Regression Discriminant Analysis(SRDA). The experimental results show that extracted features based on LMA can provide a good description on emotion labels behind different motions. Furthermore, SRDA performed better than the other three classification methods.

The ability to resolve multiple fluorescent emissions from different biological targets in video rate applications, such as endoscopy and intraoperative imaging, has traditionally been limited by the use of filter-based imaging systems. Hyper and multispectral imaging facilitate the detection of both spatial and spectral information in a single data acquisition, however, instrumentation for spatiospectral data acquisition is typically complex, bulky and expensive. This thesis seeks to overcome these limitations by using recently commercialised compact and robust hyper/multispectral cameras based on spectrally resolved detector arrays. Following sensor calibrations, which devoted particular attention to the angular sensitivity of the sensors, we integrated spectrally resolved detector arrays into a wide-field and an endoscopic imaging platform. This allowed multiplexed reflectance and fluorescence imaging with spectrally resolved detector array technology in vitro, in tissue mimicking phantoms, in an ex vivo oesophageal model and in vivo in a mouse model. A hyperspectral linescan sensor was first integrated in a wide-field near-infrared reflectance based imaging set-up to assess the suitability of spectrally resolved detector arrays for in vivo imaging of exogenous fluorescent contrast agents. Using this fluorescence hyperspectral imaging system, we could accurately resolve the presence and concentration of seven fluorescent dyes in solution. We also demonstrated high spectral unmixing precision, signal linearity with dye concentration, at depth in tissue mimicking phantoms, and delineation of four fluorescent dyes in vivo. After the successful demonstration of multiplexed fluorescence imaging in a wide-field set-up, we proceeded to combine near-infrared multiplexed fluorescence imaging with visible light spectral reflectance imaging in an endoscopic set-up. A multispectral endoscopic imaging system, capable of simultaneous reflectance and fluorescence imaging, was developed around two snapshot spectrally resolved detector arrays. In the process of system integration and characterisation, methods to characterise and predict the imaging performance of spectral endoscopes were developed. With the endoscope we demonstrated simultaneous imaging and spectral unmixing of chemically oxy/deoxygenated blood and three fluorescent dyes in a tissue mimicking phantom, and of two fluorescent dyes in an ex vivo oesophageal porcine model. With further developments, this technology has the potential to become applicable in medical imaging for detection of diseases such as gastrointestinal cancers.

La Universidad Peruana de Ciencias Aplicadas (UPC), a través de su carrera de Comunicación Audiovisual y Medios Interactivos y el V Festival de Cine AL ESTE DE LIMA 2014, recibió la visita de la cineasta Dunja Kusturica -hija del laureado cineasta Emir Kusturica- y de Srdan Golubovic, reconocido cineasta serbio, el pasado 9 de mayo para dictar un Master Class sobre sus guiones cinematográficos. Durante la presentación ambos cineastas brindaron a los asistentes pautas, anécdotas y recomendaciones al empezar a escribir un guión. En el transcurso del Master Class hubo variedad de preguntas sobre el quehacer profesional de cada uno, convirtiendo la conversación en un encuentro emotivo e inspirador. Srdan Golubovic centró sus ideas en la importancia que implica el proceso de construcción de la historia al momento de contarla, con un método visual y flexible. Dunja Kusturica, por otro lado, comentó que cada historia se conecta inevitablemente con su autor y que debe existir una sensibilidad entre el autor y la historia que cuenta.

碩士
國立臺灣大學
流行病學研究所
88
Abstract Objective：The specific aims of this study were:1) to assess the risk of hepatocellular carcinoma (HCC) associated with plasma testosterone level and the V89L polymorphism in steroid 5α-reductase type 2 (SRD5A2) gene among HBsAg-positive men in a nested case-control study;and 2) to investigate the association between a microsatellite( (CAG)n) within exon 1 of the androgen receptor(AR) gene and HCC in women by use of case-control study. Methods：A total of 110 incident cases of HCC and 110 matched controls identified from a cohort of 4841 male HBsAg carriers were tested for plasma testosterone level and SRD5A2 V89L genetic polymorphism. AR-CAG-repeat length was determined for 103 women with HCC and 183 healthy women .Genotyping was performed using polymerase chain reaction based methods on peripheral white blood cells or buccal cells. Results：Compared with male HBsAg carriers having LL genotype of the V89L polymorphism at SRD5A2, the multivariate-adjusted odds ratio (OR) of HCC for those who had VL and VV genotype was 2.0 (95% confident interval [CI]：1.0-4.0) and 3.5 (95% CI:1.4-8.6), respectively. The multivariate-adjusted ORs of HCC for male HBsAg carriers in increasing tertiles of plasma testosterone were 1.0, 1.4 (95%CI:0.6-3.3) and 3.1 (95%CI:1.4-6.8). Based on additive model, there was a synergistic interaction between the number of carrying the V allele of the V89L polymorphism and elevated testosterone levels in HCC risk. Compared with male HBsAg carriers in the lowest tertile of plasma testosterone who carried the LL genotype, the highest multivariate-adjusted OR was observed among those with both VV genotype and a testosterone level in the highest tertile (OR:7.6;95%CI:1.5-37.9).The association of HCC risk with plasma testosterone level and SRD5A2 was more striking among male HBsAg carriers who were relatively thin (body mass index≦22.9)、were younger(＜50 years)、had no smoking /drinking habit or had no history of hepatobiliary diseases .Long CAG-repeat sequence (＞22 repeats) in both AR alleles was significantly associated with increased risk of HCC in women (multivariate-adjusted OR: 2.6;95%CI: 1.0-6.5). Significantly increased risk associated with the AR genotype was observed only among female HBsAg carriers. There was no significant interaction of AR genotype with age at menarche/menopause、parity or a history of abortions in HCC risk. Conclusion：These results suggested that male HBsAg carriers who had increased plasma testosterone levels or at least one V allele of the V89L polymorphism in SRD5A2 were at increased risk of HCC. Longer AR-CAG repeats may play a role in the development of HCC among women.

碩士
慈濟大學
原住民健康研究所
94
In Taiwan, the implement of universal hepatitis B (HB) vaccination program (UHBVP) had successfully reduced the rates of HBsAg-seropositivity and hepatocellular carcinoma incidence in children. However, approximately 5-10% of vaccinated-children were low- or non-response to hepatitis B vaccine. Several researches had shown that there were gender and ethnic variations in the response to hepatitis B vaccine. The non-response rate was higher in male children than in female children and the efficacy of HB vaccination was lower in indigenous children than in Hans children. Accordingly, the specific aim of the present study was to investigate the effects of sex hormones and genetic polymorphisms of several important genes that are involved in development and metabolism of sex hormone in male indigenous and Hans adolescents. The inclusion criteria included male students who entered senior high schools during Sep 2003 to Sep 2005, born after the start of HBUVP, had both HBsAg and anti-HBs seronegativities, and received a booster dose of 20 ug of HB vaccine. Based on their responses and ethnicity, eligible subjects were divided into four groups, which were low-response indigenes, non-response indigenes, low-response Hans, and non-response Hans. There were 32 non-response indigenes and 32 non-response Hans. Two folds of the number of non-responders were randomly selected from low-response indigenes and Hans separately. We used polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to determine the IGF2 G820A, IGFBP3 A-202C and SRD5A2 V89L genetic polymorphisms. Concentrations of testosterone, sex hormone-binding globulin (SHBG) and cholesterol in plasma were also detected for each subject. We found that in those subjects carrying SRD5A2 L allele, higher testosterone concentration and higher testosterone-to-cholesterol (TC) ratio were both significantly associated with higher risk of non-response to HB vaccine. Higher SHBG-to-testosterone (ST) ratio was consistently associated with lower risk of non-response to HB vaccine in different ethnic and genetic strata. In conclusion, our results indicate that sex hormones maybe influence the immune response to booster HB vaccination, especially in subjects who carry the SRD5A2 L allele. Our results also suggest that the ST ratio is a good predictor of the responsiveness to HB vaccine.

Role of Nkx2.5 on development and electrophysiology of the mouse heart Prague 2015 Bc. Peter Hámor ABSTRACT The objective of this thesis is to investigate the role of Nkx2.5 gene dosage on electrophysiology of the mouse heart in prenatal stage of its development, in which the physiological functions of the heart fail to function properly. The main goal of this work is to search for differences in conduction of electric impulses through the embryonic mouse heart according to their genotype. Special method of capturing the conduction of electric impulse through myocardium was used for this purpose, called optical mapping. Thanks to this method I was able to construct images and videos capturing transition of the impulse with marked beginning of the activation and its direction in the heart. These outputs, or optical maps, help to define anomalies and defects compared with a normal functioning heart. The thesis focuses on the expression of the transcription factor Nkx2.5 and regulatory components related with the correct formation and physiology of the heart until 9.5 days post coitum. Individuals in this developmental stage were optically mapped and compared according to their genotypes - homozygous non-mutant, heterozygote and homozygous mutant mouse embryos exhibited some degree of similarity, while other...

Role of Nkx2.5 in development and electrophysiology of the mouse heart Prague 2016 Peter Hámor, B.S. ABSTRACT The objective of this thesis is to investigate the role of Nkx2.5 gene dosage on electrophysiology of the mouse heart in prenatal stage of its development. The main goal of this work is to search for differences in conduction of electric impulses through the embryonic mouse hearts of different genotype. Special method of capturing the conduction of electric impulse through myocardium, called optical mapping, was used to visualize the electrical activity. Thanks to this method I was able to construct images and videos capturing the spread of the impulse with identification of the beginning of the activation and its direction in the heart. These outputs, or optical maps, help to define anomalies and defects in mutants compared with a normal functioning heart. The thesis focuses on the expression of the transcription factor Nkx2.5 and regulatory components related with the correct formation and physiology of the heart until 9.5 days post coitum. Embryos at this developmental stage were optically mapped and analysed according to their genotype. While the wild type and heterozygote mouse embryos exhibited high degree of similarity, the homozygous mutants were dramatically different. Considering this work...