A Síndrome de von Hippel-Lindau é uma doença hereditária multissistêmica, causada por mutações germinativas no gene VHL que predispõe o portador a manifestações benignas e malignas em diversos órgãos. Entre esses eventos, o carcinoma de células claras renais (CRC) é o de pior prognóstico, com uma penetração média de 25% e sendo a principal causa de morte nestes pacientes. Os CRCs são tumores agressivos, pouco responsivos à quimioterapia e imunoterapia, e muitas vezes são diagnosticados em estágios avançados. Podem estar associados a síndromes hereditárias como o VHL ou apresentar a forma esporádica. Caracteristicamente, o CRC é provocado pela inativação dos dois alelos do gene VHL. Nos casos associados ao VHL, um alelo do gene VHL sofre uma mutação germinativa e um segundo evento mutacional somático nas células do tumor. Por outro lado, na forma esporádica, o CRC é resultado de dois eventos somáticos adquiridos, que incluem uma combinação de metilação do promotor, mutações pontuais que afetam a sequência de leitura aberta (ORF) e rearranjos cromossômicos, principalmente perda de heterozigosidade (LOH). Embora os eventos somáticos nos CRCs esporádicos já tenham sido explorados em outros estudos, os mecanismos de inativação somáticos do gene VHL nos CRCs associados à síndrome ainda não foram bem descritos. Este estudo avaliou os eventos somáticos no gene VHL em CRCs retirados em procedimentos cirúrgicos de pacientes portadores da síndrome. Os eventos somáticos em vários tumores de um mesmo paciente foram comparados a fim de verificarmos se essas mutações são independentes e não clonais. Oito pacientes com amostras CRCs previamente armazenadas no BNT tiveram sua mutação germinativa no gene VHL caracterizada por sequenciamento ou MLPA. Todas as amostras foram submetidas a uma revisão da patologia e macrodissecadas sempre que necessário. Para a análise das manifestações somáticas do gene VHL, o DNA foi extraído de 30 CRCs conservados em RNA latter ou formaldeído (parafina). As amostras foram analisadas quanto à metilação da região promotora do gene pelo método MS-PCR e para mutações pontuais por sequenciamento. Fomos capazes de detectar a mutação somática em 25 dos 30 tumores, incluindo uma mutação pontual e dois tumores diferentes de um mesmo paciente, nenhuma microdeleção e 23 grandes deleções. Em contraste com a literatura, nenhum dos tumores apresentou metilação no promotor do VHL. Devido ao grande número de achados LOH e da resolução limitada da técnica de MLPA para avaliar a extensão dos rearranjos cromossômicos em 3p, não foi possível concluir a análise de clonalidade dos tumores. Um estudo exploratório para caracterizar ganhos e perdas genômicas utilizando a técnica CNV array está em andamento em nosso laboratório. / The von Hippel-Lindau syndrome (VHL) is a multissystemic hereditary disease, caused by germline mutations in the VHL gene that predisposes the carrier to benign and malignant manifestations in different organs. Among these events, the clear cell renal carcinoma (RCC) is the most fearful, with an average penetration of 25% being the leading cause of death in these patients. RCCs are aggressive tumors, poorly responsive to chemo- and immunotherapy that are often diagnosed in advanced stages. They can be associated with hereditary syndromes such as VHL or present in a sporadic form. Characteristically, RCCs carrier the inactivation of the two alleles of VHL gene. In cases associated with VHL, one allele of the VHL gene is mutated in the germline, and the second mutational event occurs in the somatic cells of the tumor. On the other hand, in the sporadic form, RCCs results of two acquired somatic events, which includes a combination of methylation of the promoter, point mutations affecting the ORF, and rearrangements mainly loss of heterozigosity (LOH). Although somatic events in sporadic RCC have been explored before by others, the mechanisms of somatic VHL gene inactivation in VHL-associated RCCs have been poorly characterized. This study evaluated the somatic mutational events in the VHL gene of RCCs removed from VHL patients in therapeutic surgical procedures. The somatic events in multiple tumors from the same patient were compared in order to analyze whether these mutations are independent and not clonal. Eight patients with RCCs samples previously stored at BNT had their germline VHL gene mutation characterized by sequencing or MLPA. All samples were submitted to a pathology review and macrodissected whenever necessary. For the analysis of somatic events of VHL gene, DNA from 30 RCCs were extracted from either RNA later or archival formalin-fixed, paraffin-embedded tissue sections. Samples were analyzed for VHL gene promoter methylation by MS-PCR, and for point mutation in the coding DNA by sequencing. We were able to detect the somatic mutation in 25 of the 30 tumors, including one point mutations in two different tumors of the same patient, no micro-deletions, and 23 large deletions. In contrast to the literature, none of the tumors have shown methylation on the VHL promoter. Because of the large number of LOH findings, and the limited resolution of MLPA to evaluate the extension of 3p chromosomal rearrangements, we could not conclude the analysis of tumor clonality. An exploratory study to characterize genomic gains and losses using CNV-array technique are ongoing in our laboratory.

A Síndrome de von Hippel-Lindau é uma doença hereditária multissistêmica, causada por mutações germinativas no gene VHL que predispõe o portador a manifestações benignas e malignas em diversos órgãos. Entre esses eventos, o carcinoma de células claras renais (CRC) é o de pior prognóstico, com uma penetração média de 25% e sendo a principal causa de morte nestes pacientes. Os CRCs são tumores agressivos, pouco responsivos à quimioterapia e imunoterapia, e muitas vezes são diagnosticados em estágios avançados. Podem estar associados a síndromes hereditárias como o VHL ou apresentar a forma esporádica. Caracteristicamente, o CRC é provocado pela inativação dos dois alelos do gene VHL. Nos casos associados ao VHL, um alelo do gene VHL sofre uma mutação germinativa e um segundo evento mutacional somático nas células do tumor. Por outro lado, na forma esporádica, o CRC é resultado de dois eventos somáticos adquiridos, que incluem uma combinação de metilação do promotor, mutações pontuais que afetam a sequência de leitura aberta (ORF) e rearranjos cromossômicos, principalmente perda de heterozigosidade (LOH). Embora os eventos somáticos nos CRCs esporádicos já tenham sido explorados em outros estudos, os mecanismos de inativação somáticos do gene VHL nos CRCs associados à síndrome ainda não foram bem descritos. Este estudo avaliou os eventos somáticos no gene VHL em CRCs retirados em procedimentos cirúrgicos de pacientes portadores da síndrome. Os eventos somáticos em vários tumores de um mesmo paciente foram comparados a fim de verificarmos se essas mutações são independentes e não clonais. Oito pacientes com amostras CRCs previamente armazenadas no BNT tiveram sua mutação germinativa no gene VHL caracterizada por sequenciamento ou MLPA. Todas as amostras foram submetidas a uma revisão da patologia e macrodissecadas sempre que necessário. Para a análise das manifestações somáticas do gene VHL, o DNA foi extraído de 30 CRCs conservados em RNA latter ou formaldeído (parafina). As amostras foram analisadas quanto à metilação da região promotora do gene pelo método MS-PCR e para mutações pontuais por sequenciamento. Fomos capazes de detectar a mutação somática em 25 dos 30 tumores, incluindo uma mutação pontual e dois tumores diferentes de um mesmo paciente, nenhuma microdeleção e 23 grandes deleções. Em contraste com a literatura, nenhum dos tumores apresentou metilação no promotor do VHL. Devido ao grande número de achados LOH e da resolução limitada da técnica de MLPA para avaliar a extensão dos rearranjos cromossômicos em 3p, não foi possível concluir a análise de clonalidade dos tumores. Um estudo exploratório para caracterizar ganhos e perdas genômicas utilizando a técnica CNV array está em andamento em nosso laboratório. / The von Hippel-Lindau syndrome (VHL) is a multissystemic hereditary disease, caused by germline mutations in the VHL gene that predisposes the carrier to benign and malignant manifestations in different organs. Among these events, the clear cell renal carcinoma (RCC) is the most fearful, with an average penetration of 25% being the leading cause of death in these patients. RCCs are aggressive tumors, poorly responsive to chemo- and immunotherapy that are often diagnosed in advanced stages. They can be associated with hereditary syndromes such as VHL or present in a sporadic form. Characteristically, RCCs carrier the inactivation of the two alleles of VHL gene. In cases associated with VHL, one allele of the VHL gene is mutated in the germline, and the second mutational event occurs in the somatic cells of the tumor. On the other hand, in the sporadic form, RCCs results of two acquired somatic events, which includes a combination of methylation of the promoter, point mutations affecting the ORF, and rearrangements mainly loss of heterozigosity (LOH). Although somatic events in sporadic RCC have been explored before by others, the mechanisms of somatic VHL gene inactivation in VHL-associated RCCs have been poorly characterized. This study evaluated the somatic mutational events in the VHL gene of RCCs removed from VHL patients in therapeutic surgical procedures. The somatic events in multiple tumors from the same patient were compared in order to analyze whether these mutations are independent and not clonal. Eight patients with RCCs samples previously stored at BNT had their germline VHL gene mutation characterized by sequencing or MLPA. All samples were submitted to a pathology review and macrodissected whenever necessary. For the analysis of somatic events of VHL gene, DNA from 30 RCCs were extracted from either RNA later or archival formalin-fixed, paraffin-embedded tissue sections. Samples were analyzed for VHL gene promoter methylation by MS-PCR, and for point mutation in the coding DNA by sequencing. We were able to detect the somatic mutation in 25 of the 30 tumors, including one point mutations in two different tumors of the same patient, no micro-deletions, and 23 large deletions. In contrast to the literature, none of the tumors have shown methylation on the VHL promoter. Because of the large number of LOH findings, and the limited resolution of MLPA to evaluate the extension of 3p chromosomal rearrangements, we could not conclude the analysis of tumor clonality. An exploratory study to characterize genomic gains and losses using CNV-array technique are ongoing in our laboratory.

Oxygen homeostasis is essential to the life of aerobes, which is regulated in humans by Hypoxia Inducible Factor-1α (HIF-1α). Under hypoxic conditions, HIF-1α transactivates over a hundred genes related angiogenesis, erythropoiesis, etc. HIF-1α level and function is regulated by four HIF hydroxylase enzymes: three isoforms of prolyl hydroxylase domain (PHD1, PHD2 and PHD3) and factor inhibiting HIF-1α (FIH). PHD2 is the focus of this research. PHD2 is a non-heme Fe(II) 2-oxoglutarate dependent dioxygenase, which controls HIF-1α levels by hydroxylating two proline residues within the ODD domain of HIF-1α, then the hydroxylated prolines are recognized by pVHL, which targets HIF-1α for proteasomal degradation. Under hypoxic conditions PHD2 cannot hydroxylate HIF-1α and its level rises in cells. The aims of this research include understanding how PHD2 chooses its substrate, how the O2 activation occurs, and how certain transition metals inhibit PHD2. Our results revealed that electrostatics play a role in substrate selectivity of PHD2 by provoking a change in the opening and closing rate of β2β3 loop for NODD and CODD substrates. Mutational studies of second coordination sphere residues combined with kinetic studies indicated that decarboxylation of 2OG is the slow step in the chemical mechanism. The removal of a hydrogen-bond by the Thr387aAla mutation revealed a rate 15 times faster than WT-PHD2 by making O2 a better nucleophile. Our results indicate that this hydrogen bonding is essential for proper O2 activation. Previous reports show that certain metals increase HIF-1α levels by inhibiting PHD2. However there are conflicts about how this inhibition occurs, either through metal replacement from the active site or metals binding to a different site causing inhibition. Our competitive and non-competitive kinetic assays showed different inhibition profiles. Under competitive conditions Zn2+, Co2+, Mn2+, and Cu2+ can bind to the enzyme active site and lead to inhibition but under non-competitive conditions Zn2+, Co2+, and Mn2+ partially inhibit PHD2 suggesting that these metals cannot displace the Fe2+ from the active site. XAS experiments with Zn2+ and Fe3+ indicate that Zn2+ binds to the surface of PHD2 in a six-coordinate manner composed of two Cys201, 208, His205, Tyr197 and two water ligands.

La dépigmentation volontaire est une pratique bien connue en Afrique sub-saharienne. Elle se définit comme une pratique par laquelle une personne, de sa propre initiative, tente de diminuer la pigmentation mélanique physiologique de sa propre peau. Les utilisateurs appliquent sur le corps, généralement sans surveillance médicale, de manière soutenue et prolongée, des produits ou des mélanges chimiques composés d’actifs dépigmentants souvent d’une grande nocivité.<p>Cette pratique est documentée dans plusieurs pays d’Afrique sub-saharienne (Sénégal, Mali, Burkina Faso, Togo, Nigéria, ….), et sur d’autres continents. Face à l’absence de données chiffrées pour le Rwanda, nous avons réalisé une étude des pratiques de la dépigmentation volontaire dans la capitale du pays, Kigali. <p>Au Rwanda, certaines plantes étaient utilisées lors des grandes cérémonies comme le mariage, spécialement par les femmes et les jeunes filles, pour éclaircir la peau. Une peau claire semble en fait un critère de beauté dans certaines traditions africaines. Nous avons donc réalisé une enquête ethnobotanique auprès de 61 tradipraticiens rwandais, afin de connaître les plantes qui, avant l’arrivée de la cosmétique moderne, étaient utilisées pour « embellir » (éclaircir) la peau, afin de vérifier si ces plantes pourraient interférer avec la production de la mélanine. <p>Notre enquête nous a permis de documenter 28 espèces, dont cinq [Brillantaisia cicatricosa LINDAU; Chenopodium ugandae (Aellen) Aellen ;Dolichopentas longiflora Oliv. Protea madiensis Oliv. subsp. Madiensis et Sesamum angolense Welw.] se sont distinguées par leur pourcentage de citation par les tradipraticiens. Ces dernières ont fait objet de notre étude de laboratoire. <p>Des extraits de polarité croissante, préparés à partir de ces cinq plantes, ont été testés pour leur modulation de la mélanogénèse et de la tyrosinase (enzyme clé de la mélanogenèse) sur une série de modèles: (i) sur la tyrosinase humaine dans les extraits totaux de mélanocytes normaux; (ii) sur des mélanocytes malins en culture (pour évaluer l’effet global des extraits de plante sur la mélanogenèse); (iii) sur la tyrosinase de champignon en solution et sur chromatoplaque de silice; et enfin (iv) sur l’activité tyrosine hydroxylase de l'enzyme. <p>Deux extraits à l’acétate d’éthyle de Protea madiensis Oliv. et de Sesamum angolense Welw. ont été sélectionnés pour leur activité, respectivement inhibitrice et activatrice de la tyrosinase de champignon. Ces deux extraits ont été soumis à une série de fractionnements dans le but d’isoler et d’identifier des composés actifs. Trois composés ont été isolés de Protea madiensis (2-tridécanone, acide oléique et β-sitostérol). La 2-tridécanone et l’acide oléique ont montré une inhibition de la tyrosinase de champignon sur chromatoplaque et de la tyrosinase humaine dans les extraits cellulaires. De plus, la 2-tridécanone a montré une inhibition de l’activité tyrosine hydroxylase. Le β-sitostérol n’a pas montré d’effet sur nos modèles mais il a déjà été isolé dans d’autres études en tant qu'inhibiteur de la tyrosinase. De l’extrait à l’acétate d’éthyle de Sesamum angolense Welw. nous avons isolé l’acide ursolique qui a montré une augmentation de l’activité de la tyrosinase de champignon sur chromatoplaque.<p>L’enquête ethnobotanique nous a permis de constater que la flore rwandaise regorge de plantes aux vertus cosmétiques intéressantes; celles-ci pourraient représenter une alternative aux actifs dépigmentants connus pour leurs nombreux effets secondaires mais néanmoins largement disponibles sur le marché rwandais. <p>L’enquête réalisée dans la ville de Kigali, nous a permis de constater que 27 % de notre population d’étude sont des utilisateurs conscients de produits dépigmentants. Ce pourcentage nous semble fort élevé et des mesures devraient être prises pour la sensibilisation et la conscientisation de la population quant aux risques encourus et à l’existence de médecines traditionnelles à visée dépigmentante. Ces mesures devraient être combinées avec la recherche de composés naturels dans l'espoir d'identifier des molécules actives et faiblement toxiques, voire atoxiques. <p>L’étude de la modulation de la pigmentation par les extraits des cinq plantes sélectionnées, nous a permis de confirmer l’information reçue des tradipraticiens. Cette étude nous a également montré que ces extraits de plantes renferment des activateurs de la mélanogenèse, qui pourraient être exploités pour le bronzage recherché par les sujets de peau claire.<p>L’isolement et identification de molécules à partir des extraits de deux plantes, nous a permis de constater que notre méthode de bioguidage fonctionne correctement; des mesures de déréplications devraient cependant être prises pour éviter autant que possible de retomber sur des molécules déjà connues./<p><p><p>Voluntary depigmentation, well-known in sub-Saharan Africa, is defined as a practice by which a person, by his/her own initiative, attempts to reduce his/her skin physiological melanin pigmentation. Users apply on the body, usually without medical supervision, in a sustained and prolonged manner, depigmenting compounds, single or in mixtures.<p>This quite harmful practice is documented in several sub-Saharan African countries (Senegal, Mali, Togo, Nigeria…) and in other continents. The absence of Rwandese data prompted us to conduct a study of the practices of voluntary depigmentation in the capital, Kigali.<p>In Rwanda, some plants were used during important ceremonies like wedding (marriage) especially by women and girls to lighten their skin. Fair skin is actually considered as a beauty criterion in some African traditions.<p>We conducted an ethnobotanical survey of 61 Rwandan traditional healers to identify the plants that were used before the introduction of modern cosmetics to "beautify" (lighten) the skin in order to check wether these plants could interfere with the production of melanin.<p>Our survey allowed us to identify and collect 28 species, of which 5 were selected (retained) for their higher percentage of citation by traditional healers [Brillantaisia cicatricosa LINDAU; Chenopodium ugandae (Aellen) Aellen ;Dolichopentas longiflora Oliv. Protea madiensis Oliv. subsp. madiensis and Sesamum angolense Welw.]. These five species have been used for our laboratory study.<p><p>Extracts of increasing polarities were prepared from the five plants and tested for their ability to modulate melanogenesis and tyrosinase (the key enzyme of melanogenesis) in a series of models: (i) human tyrosinase in total extracts from normal melanocytes; (ii) malignant melanocytes in culture (in order to assess the global effect of plant extracts on melanogenesis); (iii) mushroom tyrosinase in solution and on TLC plate; and finally (iv) tyrosine hydroxylase activity of the enzyme.<p>Two ethyl acetate extracts of Protea madiensis Oliv. and of Sesamum angolense Welw have been selected according to their respective inhibitory and activating effect on mushroom tyrosinase. These two extracts were fractionated to isolate and identify active compounds. Three compounds have been isolated from Protea madiensis (2-tridecanone, oleic acid and β-sitosterol). The 2-tridecanone and the oleic acid showed an inhibition of mushroom tyrosinase on TLC and human tyrosinase in cellular extracts. In addition, 2-tridecanone showed an inhibition of the tyrosine hydroxylase activity. β-sitostérol showed no effect on our models but has been identified, in other studies, as a tyrosinase inhibitor. From the ethyl acetate extract of Sesamum angolense, we isolated ursolic acid which increases the mushroom tyrosinase activity on TLC.<p>The ethnobotanical survey allowed us to (state) notice that Rwandan flora contains plants that have interesting cosmetic properties and could be an alternative to the use of harmful depigmenting products which are sold on Rwandese markets.<p>The survey conducted in Kigali city indicates that 27 % of surveyed persons are conscious users of depigmenting products. This percentage seems very high so that measures should be taken to raise awareness about the involved risks and of the existence of traditional medicines with such depigmenting effects. These measures should be accompanied (combined) with the search for natural compounds with depigmenting effect in the hope to identify actives that would be weakly or even non toxic at all.<p>The study of the pigmentation modulation by five selected plant extracts allowed to confirm the information obtained from traditional healers. It also indicates that, apart from an inhibitory effect, some of our plant extracts also contain melanogenesis activators that could be further exploited for tanning, an aspiration of fair-skinned individuals.<p>The isolation and identification of molecules from two plants extracts led us to conclude that our “bioguidance” method performs adequately. Nevertheless, some dereplication measures should be implemented to avoid spending time on isolating already known molecules. <p><p> <p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished