Academic literature on the topic 'SRD5A1'

Steroid 5-alpha reductases (SRD5As) are responsible for the conversion of testosterone to dihydrotestosterone, a potent androgen, which is the aetiologic factor of androgenetic alopecia. This study aimed to compare the SRD5A gene expression suppression activity exerted by Thai rice bran extracts and their components and investigate the interactional mechanism between bioactive compounds and SRD5A2 using molecular dynamics (MD) simulation. Bran of Oryza sativa cv. Tubtim Chumphae (TRB), Yamuechaebia Morchor (YRB), Riceberry (RRB), and Malinil Surin (MRB), all rice milling by-products, was solvent-extracted. The ethanolic extract of TRB had the highest sum of overall bioactive compounds (γ-oryzanol; α-, β-, and γ-tocopherol; phenolics; and flavonoids). Among all extracts, TRB greatly downregulated the expression of SRD5A1, SRD5A2, and SRD5A3; there were no significant differences between TRB and finasteride regarding SRD5A suppression. The linear relationship and principal component analysis supported that the α-tocopherol content was correlated with the SRD5A suppression exerted by TRB. Furthermore, MD simulation demonstrated that α-tocopherol had the highest binding affinity towards SRD5A2 by interacting with residues Phe118 and Trp201. Our findings indicate that α-tocopherol effectively downregulates the expression of SRD5A genes and inhibits SRD5A2 activity, actions that are comparable to standard finasteride. TRB, a source of α-tocopherol, could be developed as an anti-hair loss product.

The biological role of steroid 5α-reductase isozymes (encoded by the SRD5A1 and SRD5A2 genes) and angiogenic factors that play important roles in the pathogenesis and vascularization of prostate cancer (PC) is poorly understood. The sub-cellular expression of these isozymes and vascular endothelial growth factor (VEGF) in PC tissue microarrays (n=62) was examined using immunohistochemistry. The effect of SRD5A inhibition on the angiogenesis pathway genes in PC was also examined in prostate cell lines, LNCaP, PC3, and RWPE-1, by treating them with the SRD5A inhibitors finasteride and dutasteride, followed by western blot, quantitative PCR, and ELISA chip array techniques. In PC tissues, nuclear SRD5A1 expression was strongly associated with higher cancer Gleason scores (P=0.02), higher cancer stage (P=0.01), and higher serum prostate specific antigen (PSA) levels (P=0.01), whereas nuclear SRD5A2 expression was correlated with VEGF expression (P=0.01). Prostate tumor cell viability was significantly reduced in dutasteride-treated PC3 and RWPE-1 cells compared with finasteride-treated groups. Expression of the angiogenesis pathway genes transforming growth factor β 1 (TGFB1), endothelin (EDN1), TGFα (TGFA), and VEGFR1 was upregulated in LNCaP cells, and at least 7 out of 21 genes were upregulated in PC3 cells treated with finasteride (25 μM). Our findings suggest that SRD5A1 expression predominates in advanced PC, and that inhibition of SRD5A1 and SRD5A2 together was more effective in reducing cell numbers than inhibition of SRD5A2 alone. However, these inhibitors did not show any significant difference in prostate cell angiogenic response. Interestingly, some angiogenic genes remained activated after treatment, possibly due to the duration of treatment and tumor resistance to inhibitors.

Abstract Context: Despite the importance of dihydrotestosterone in androgen action, polymorphisms in the genes for the two isoforms of 5α-reductase (SRD5A1 and SRD5A2) have not been evaluated as risk factors for polycystic ovary syndrome (PCOS). Objective: The objective of the study was to test the hypothesis that haplotypes in the SRD5A1 and SRD5A2 genes are risk factors for PCOS and the severity of hirsutism in affected women. Design: PCOS and control subjects were genotyped for seven single-nucleotide polymorphisms in SRD5A1 and eight single-nucleotide polymorphisms in SRD5A2. Haplotypes were determined and tested for association with PCOS diagnosis and component phenotypes. Setting: Subjects were recruited from the reproductive endocrinology clinic at the University of Alabama at Birmingham; control subjects were recruited from the general surrounding community. Genotyping took place at Cedars-Sinai Medical Center in Los Angeles. Participants: A total of 287 White women with PCOS and 187 controls participated. Main Measurements: SRD5A1 and SRD5A2 genotype, quantitative hirsutism score, and hormonal and metabolic phenotypes were assessed. Results: Haplotypes within both genes were associated with PCOS risk. The Leu allele of the Val89Leu variant in SRD5A2 was associated with protection against PCOS; this allele is known to modestly reduce 5α-reductase activity. Haplotypes in SRD5A1 but not SRD5A2 were also associated with the degree of hirsutism in affected women. Conclusions: This study presents genetic evidence suggesting an important role of both isoforms of 5α-reductase in the pathogenesis of PCOS. That only SRD5A1 haplotypes were associated with hirsutism suggests that only this isoform is important in the hair follicle.

Oily skin from overactive sebaceous glands affects self-confidence and personality. There is report of an association between steroid 5-alpha reductase gene (SRD5A) expression and facial sebum production. There is no study of the effect of Asparagus racemosus Willd. root extract on the regulation of SRD5A mRNA expression and anti-sebum efficacy. This study extracted A. racemosus using the supercritical carbon dioxide fluid technique with ethanol and investigated its biological compounds and activities. The A. racemosus root extract had a high content of polyphenolic compounds, including quercetin, naringenin, and p-coumaric acid, and DPPH scavenging activity comparable to that of the standard L-ascorbic acid. A. racemosus root extract showed not only a significant reduction in SRD5A1 and SRD5A2 mRNA expression by about 45.45% and 90.86%, respectively, but also a reduction in the in vivo anti-sebum efficacy in male volunteers, with significantly superior percentage changes in facial sebum production and a reduction in the percentages of pore area after 15 and 30 days of treatment. It can be concluded that A. racemosus root extract with a high content of polyphenol compounds, great antioxidant effects, promising downregulation of SRD5A1 and SRD5A2, and predominant facial sebum reduction and pore-minimizing efficacy could be a candidate for an anti-sebum and pore-minimizing active ingredient to serve in functional cosmetic applications.

Background. Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and more prevalent among males than females. However, the biological role of enzyme 5α-reductase (SRD5A3), which plays a critical role in the androgen receptor signaling pathway during HCC development, remains poorly understood. Methods. ONCOMINE, GEPIA, UALCAN, and Kaplan–Meier Plotter were used to analyze the expression and prognostic value of SRD5A3 in HCC. STRING and Metascape were applied to analyze potential target and molecular pathways underlying SRD5A3 in HCC. A real-time quantitative reverse transcription-polymerase chain reaction was used to validate the downstream target expression of SRD5A3. Results. The expression of SRD5A3 was significantly overexpressed in HCC tissues compared with normal tissues, while the expression of SRD5A1 and SRD5A2 were downregulated in multiple public datasets. It may be that the low methylation of the SRD5A3 promoter leads to its overexpression. The level of SRD5A3 tended to be higher expressed in clinical samples with advanced stage and positive node metastasis. Furthermore, the patients with higher SRD5A3 were remarkably associated with poorer overall survival and disease-free survival in the TCGA data. In addition, the increased mRNA expression of SRD5A3 could predict poorer overall survival in Kaplan–Meier Plotter database including different patient cohorts. Moreover, HCC patients with higher level of SRD5A3 had significantly shorter recurrence-free survival, progression-free survival, and disease-specific survival. Furthermore, enrichment analysis demonstrated that multiple processes, such as steroid hormone biosynthesis, lipid biosynthetic process, and androgen metabolic process, were affected by SRD5A1-3 alterations. In vitro experiments showed that the expression of SRD5A3 was increased in HCC tissues than that in adjacent tissues. SRD5A3 silencing promoted the expression of DOLK in two HCC cell lines. Conclusions. This study identified SRD5A3/DOLK as a novel axis to regulate HCC development.

Context: Some men who use finasteride for hair loss report persistent sexual and other symptoms after discontinuing finasteride therapy. Objective: To determine whether these persistent symptoms after discontinuation of finasteride use are due to androgen deficiency, decreased peripheral androgen action, or persistent inhibition of steroid 5α-reductase (SRD5A) enzymes. Participants: Finasteride users, who reported persistent sexual symptoms after discontinuing finasteride (group 1); age-matched finasteride users who did not report sexual symptoms (group 2); and healthy men who had never used finasteride (group 3). Outcomes: Sexual function, mood, affect, cognition, hormone levels, body composition, functional magnetic resonance imaging (fMRI) response to sexually and affectively valenced stimuli, nucleotide sequences of androgen receptor (AR), SRD5A1, and SRD5A2; expression levels of androgen-dependent genes in skin. Setting: Academic medical center. Results: Symptomatic finasteride users were similar in body composition, strength, and nucleotide sequences of AR, SRD5A1, and SRD5A2 genes to asymptomatic finasteride users and nonusers. Symptomatic finasteride users had impaired sexual function, higher depression scores, a more negative affectivity balance, and more cognitive complaints than men in groups 2 and 3 but had normal objectively assessed cognitive function. Testosterone, dihydrotestosterone, 5α-androstane-3α,17β-diol-glucuronide, testosterone to dihydrotestosterone and androsterone glucuronide to etiocholanolone glucuronide ratios, and markers of peripheral androgen action and expression levels of AR-dependent genes in skin did not differ among groups. fMRI blood oxygen level-dependent responses to erotic and nonerotic stimuli revealed abnormal function in brain circuitry linked to sexual arousal and major depression. Conclusions: We found no evidence of androgen deficiency, decreased peripheral androgen action, or persistent peripheral inhibition of SRD5A in men with persistent sexual symptoms after finasteride use. Symptomatic finasteride users revealed depressed mood and fMRI findings consistent with those observed in depression.

Context Post-finasteride syndrome (PFS) occurs in patients with androgenic alopecia after suspension of the finasteride treatment, leading to a large variety of persistent side effects. Despite the severity of the clinical picture, the mechanism underlying the PFS symptoms onset and persistence is still unclear. Objective To study whether epigenetic modifications occur in PFS patients. Methods Retrospective analysis of a multicentric, prospective, longitudinal, case–control clinical trial, enrolling 16 PFS patients, compared to 20 age-matched healthy men. Main outcomes were methylation pattern of SRD5A1 and SRD5A2 promoters and concentration of 11 neuroactive steroids, measured by liquid chromatography-tandem mass spectrometry, in blood and cerebrospinal fluid (CSF) samples. Results SRD5A1 and SRD5A2 methylation analysis was performed in all blood samples (n = 16 PFS patients and n = 20 controls), in 16 CSF samples from PFS patients and in 13 CSF samples from controls. The SRD5A2 promoter was more frequently methylated in CSF of PFS patients compared to controls (56.3 vs 7.7%). No promoter methylation was detected in blood samples in both groups. No methylation occurred in the SRD5A1 promoter of both groups. Unmethylated controls compared to unmethylated SRD5A2 patients showed higher pregnenolone, dihydrotestosterone and dihydroprogesterone, together with lower testosterone CSF levels. Andrological and neurological assessments did not differ between methylated and unmethylated subjects. Conclusions For the first time, we demonstrate a tissue-specific methylation pattern of SRD5A2 promoter in PFS patients. Although we cannot conclude whether this pattern is prenatally established or induced by finasteride treatment, it could represent an important mechanism of neuroactive steroid levels and behavioural disturbances previously described in PFS.

Leaves of guava (Psidium guajava L.) have been used in Thai folk medicine without any supporting evidence as a traditional herbal remedy for hair loss. Androgenetic alopecia (AGA) is chronic hair loss caused by effects of androgens in those with a genetic predisposition, resulting in hair follicle miniaturization. Our objectives were to provide the mechanistic assessment of guava leaf extract on gene expressions related to the androgen pathway in well-known in vitro models, hair follicle dermal papilla cells (HFDPC), and human prostate cancer cells (DU-145), and to determine its bioactive constituents and antioxidant activities. LC-MS analysis demonstrated that the main components of the ethanolic extract of guava leaves are phenolic substances, specifically catechin, gallic acid, and quercetin, which contribute to its scavenging and metal chelating abilities. The guava leaf extract substantially downregulated SRD5A1, SRD5A2, and SRD5A3 genes in the DU-145 model, suggesting that the extract could minimize hair loss by inhibiting the synthesis of a potent androgen (dihydrotestosterone). SRD5A suppression by gallic acid and quercetin was verified. Our study reveals new perspectives on guava leaf extract’s anti-androgen properties. This extract could be developed as alternative products or therapeutic adjuvants for the treatment of AGA and other androgen-related disorders.

Este trabajo revisa los principales artículos relacionados con la actividad de la enzima 5α-reductasa (SRD5A) y las patologías andrógeno-dependientes como la hiperplasia prostática benigna, el cáncer de próstata, acné y la calvicie androgénica entre otras. Con este propósito, se revisaron diferentes plataformas como Medline, Scopus y Science Direct, utilizando como indicadores las palabras 5α-reductasa, isoenzimas y patologías andrógeno- dependientes. Con esta clasificación se encontraron artículos clásicos de décadas anteriores y nuevos artículos en donde aparece ya descrita, la estructura de la 5α-reductasa, que en décadas anteriores no había podido ser cristalizada e identificada por rayos X. Además de las dos isoenzimas principales de la SRD5A, aparece en la literatura una tercera isoenzima de la SRD5A, la de tipo 3, que se expresa en células cancerosas. Se ha demostrado que SRD5A3 está sobreexpresada en varios cánceres con mal pronóstico. En este artículo se revisaron también algunas de las moléculas esteroidales y no esteroidales reconocidas como bloqueadores eficaces de la actividad de esta enzima, logrando mejorar las terapias para la hipertrofia prostática benigna, la calvicie androgénica y el cáncer de la próstata. En conclusión, se ha reconocido que la SRD5A tiene un papel crucial en la reducción de los dobles enlaces carbono-carbono (Δ4) del anillo A del esqueleto esteroidal en los seres vivientes y que la secuencia de aminoácidos de las isoformas SRD5A1 y 2 en humanos, posee una homología con otras especies. Sin embargo, las propiedades bioquímicas y fisiológicas de la isoforma SRD5A3 no han sido tan ampliamente estudiadas. Es importante considerar la dificultad para cristalizar estas reductasas, debido a su ubicación membranal.

Patients suffering from recurrent or metastatic (R/M) salivary duct carcinoma (SDC) are often treated with combined androgen blockade (CAB). However, CAB frequently fails, resulting in a worse prognosis. Therefore, biomarkers that can predict treatment failure are urgently needed. mRNA from 76 R/M androgen receptor (AR)-positive SDC patients treated with leuprorelin acetate combined with bicalutamide was extracted from pre-treatment tumor specimens. AR, Notch, MAPK, TGFβ, estrogen receptor (ER), Hedgehog (HH), and PI3K signaling pathway activity scores (PAS) were determined based on the expression levels of target genes. Additionally, 5-alpha reductase type 1 (SRD5A1) expression was determined. These markers were related to clinical benefit (complete/partial response or stable disease ≥6 months) and progression-free and overall survival (PFS/OS). SRD5A1 expression had the highest general predictive value for clinical benefit and positive predictive value (PPV: 85.7%). AR PAS had the highest negative predictive value (NPV: 93.3%). The fitting of a multivariable model led to the identification of SRD5A1, TGFβ, and Notch PAS as the most predictive combination. High AR, high Notch, high ER, low HH PAS, and high SRD5A1 expression were also of prognostic importance regarding PFS and SRD5A1 expression levels for OS. AR, Notch PAS, and SRD5A1 expression have the potential to predict the clinical benefit of CAB treatment in SDC patients. SRD5A1 expression can identify patients that will and AR PAS patients that will not experience clinical benefit (85.7% and 93.3% for PPV and NPV, respectively). The predictive potential of SRD5A1 expression forms a rational basis for including SRD5A1-inhibitors in SDC patients’ treatment.

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.

This chapter aims in presenting the methods for the accurate estimation of highly non linear phase inductance profile of a switched reluctance motor (SRM). The magnetization characteristics of a test SRM is derived from the SRDaS (Switched Reluctance Design and Simulation) simulation software. Statistical interpolation based regression analysis and Artificial Intelligence (AI) techniques are used for developing the computationally efficient inductance model. Multi Variate Non linear Regression (MVNLR) from the class of regression analysis and Adaptive Neuro Fuzzy Inference System (ANFIS) under the class of AI are implemented and tested on the simulated data. Non linear Inductance profile L(I,θ) of SRM is successfully estimated for the complete working range of phase currents (Iph). At each Iph, L(I,θ) values are estimated and presented for one cycle of rotor position (θ). Estimated inductance profile based on the two proposed methods is observed to be in excellent correlation with the true value of data.

With the rapid development of global economy and great improvement on life quality of consumers, the consumer shopping behaviors have been changed significantly. Modern retailers have put intensive effort on merchandise arrangement in order to satisfy the consumer demands on merchandise shopping. However, most retailers do not provide satisfactory shopping services to customers. For instance, without a customized shopping recommendation for each individual customer, consumers have to spend a lot of time for merchandise selection. Furthermore, most large-spaced retailers merely utilize signs in front the aisles of specific merchandise areas to direct consumers, which cannot provide an accurate guidance for merchandise search. Therefore, regarding the shopping services of a modern retailer, this research develops a customized merchandise recommendation algorithm (CMRA) and a shopping route determination and guidance algorithm (SRDGA). Based on the proposed algorithms, a Shopping Service System (3S-System) is established by integrating the RFID technology. Considering the consumer demands, consumer shopping preferences and market promotion plans, this research proposes an integrated, heuristic methodology to provide a customized shopping list, route recommendation and real-time direction guidance for consumer shopping. Moreover, based on the proposed methodology, a Shopping Service System (3S-System) is established, and a simulated market is created in order to verify the feasibility of the proposed model. The verification results show that the system can offer customers appropriate shopping route recommendation in a short time and could achieve real-time guidance. As a whole, this research provides a methodology and system to provide effective shopping services for consumers and as a result the shopping service quality of modern retailers can be enhanced and the sales volume of merchandises can be increased.

Introdução: O vírus SARS-CoV-2 invade a célula hospedeira através da ligação entre a proteína viral SPIKE e o receptor ECA-2. A resposta antiviral ativa vias inflamatórias do sistema imune. Entretanto, uma resposta exacerbada com hiper-citocinemia pró-inflamatória, na chamada Tempestade de Citocinas (TC), pode evoluir com Síndrome do Desconforto Respiratório Agudo (SRDA) e falência múltipla dos órgãos. Objetivos: Analisar a relação entre a tempestade de citocinas na COVID-19 e a evolução da doença. Material e métodos: Realizou-se revisão narrativa com busca de artigos publicados entre 2020 e 2021, na base de dados PubMed, partindo dos descritores boleanos “Cytokine Storm and COVID-19”. Contabilizou-se 2359 artigos e foram filtradas revisões e revisões sistemáticas cujo título apresentasse os descritores, totalizando 70 trabalhos científicos. Após avaliação criteriosa 11 artigos foram selecionados por convergirem com a temática proposta. Resultados: A TC inicia-se com a expressão de fatores de transcrição indutores de citocinas pró-inflamatórias, como NF-kB. Com isso, padrões sinalizadores resultam na secreção das citocinas de fase aguda (TNF-α, IL1-β e IL-6), responsáveis pela hiperinflamação encontrada na COVID-19. Deficiências na lise de células apresentadoras de antígenos infectadas prolongam as interações entre células do sistema imune, provocando secreção exacerbada de citocinas pró-inflamatórias, como TNF, interferon-γ, interleucinas 1 e 6. TNF e IL-1 induzem o fator de tecido pró-coagulante (TF), ocasionando trombose imunomediada. Já a IL-6 promove uma síndrome de ativação macrofágica, desencadeando hiper-citocinemia pró-inflamatória e danos ao epitélio pulmonar.Conclusão: A rápida replicação viral nos primeiros estágios da infecção resulta em resposta pró-inflamatória exacerbada, com aumento da liberação de IL-6, IL-1 e TNFα e outras citocinas, gerando um quadro grave de hiperinflamação secundária à infecção por COVID-19, frequentemente associado a um pior prognóstico. Surge, assim, o dilema das terapias imunomoduladoras, pois optar pela repressão da atividade do sistema imunológico poderia prejudicar a eliminação viral e produção de anticorpos, além de favorecer o surgimento de infecções oportunistas.