Le coiffage pulpaire direct, dans des situations pathologiques critiques où la vitalité de la dent est menacée, vise à stimuler le potentiel de cicatrisation de la pulpe et induire une régénération dentinaire. Afin d'optimiser cette thérapeutique, le Laboratoire IMEB-ERT 30 a développé en partenariat avec la société SEPTODONT un nouveau matériau, le Biodentine™. Ce ciment, composé essentiellement de silicate tricalcique, possède des qualités physiques permettant son utilisation comme substitut dentinaire. Le premier objectif de notre travail a été d'évaluer les propriétés biologiques du Biodentine™ et ses interactions avec les cellules cibles en culture. La bioactivité du nouveau ciment a ensuite été étudiée à l'aide du modèle de culture de dents entières humaines ex vivo. Ce modèle expérimental original a été mis au point dans notre laboratoire et permet d'étudier les phases précoces de la régénération dentinaire lors du coiffage direct. Grâce à ce modèle, nous avons démontré l'activation, la prolifération et la migration de cellules progénitrices pulpaires périvasculaires en réponse à une lésion cavitaire profonde. Le coiffage direct avec les ciments de silicates tricalciques a induit la formation de foyers minéralisés à proximité de la lésion. La caractérisation moléculaire de ces foyers a montré qu'il s'agit d'une forme de dentine réparatrice synthétisée par des cellules odontoblast-like. Le deuxième objectif de notre travail a été d'étudier l'effet du nouveau biomatériau sur la sécrétion de certains facteurs de croissances, impliqués dans les phases précoces de la cicatrisation pulpaire, et de le comparer à celui d'autres matériaux de coiffage. / The objective of direct pulp capping is to stimulate the pulp healing potential and to induce dentin regeneration. This is of prime importance in critical pathologic situations compromising the tooth vitality. To improve the outcome of this treatment, the IMEB-ERT30 Laboratory, in collaboration with the SEPTODONT Company, has developed a new restorative material called Biodentine™. This cement, essentially composed of tricalcium silicates, has the required physical properties to be used as a dentin substitute. The first aim of this work was to evaluate the biological properties of Biodentine™ and its interactions with the target cells in cell culture. Then, the bioactivity of the new cement was studied using an entire human tooth culture model ex vivo. This original experimental model, developed in our Laboratory, is suitable in studying the early steps of dentin regeneration after direct pulp capping. With this model, we demonstrated the activation, proliferation, and migration of perivascular pulpal progenitor cells in response to pulp injury. The direct pulp capping with tricalcium silicate cements induced mineral foci formation in the vicinity of the pulp lesion. Molecular characterization of these foci confirmed it was of a reparative dentin type produced by odontoblast-like cells. The second objective of our work was to study the effect of the new cement on the secretion of some growth factors involved in the early steps of pulp wound healing, and compare it to that of other pulp capping materials. The results demonstrated an up-regulation of b-FGF, VEGF and PDGF-AB secretion in response to the target cells injuries, suggesting a stimulation of angiogenesis.

Le lumicanne est un petit protéoglycanne riche en leucine de la matrice extracellulaire impliqué, entre autre, dans le contrôle de l'angiogenèse, en particulier l'angiogenèse tumorale. Nous avons déjà démontré que le lumicanne inhibe la progression du mélanome in vivo. Le mécanisme d'action anti-tumoral du lumicanne a été partiellement décrit dans notre laboratoire. L'intégrine alpha2beta1 a été caractérisée comme un récepteur direct du lumicanne. Dans l'étude que nous présentons, nous avons décrit le rôle du lumicanne dans le contrôle de la transition des cellules souche mésenchymateuse (CSM) en cellules progénitrices endothéliales, pouvant contribuer à l'angiogenèse tumorale. Nous avons montré que le lumicanne inhibe spécifiquement la migration et l'invasion des CSM par une diminution de l'expression et de l'activité de la MMP-14. De plus, nous avons démontré que le lumicanne est un inhibiteur compétitif de la MMP-14. Il se lie directement au domaine catalytique de l'enzyme avec une affinité modérée (KD=275,4±16.12nM). De plus, nous avons démontré que le lumicanne diminue la phosphorylation de récepteurs (EGFR, Mer, EphB2, EphB6, ROR) et de protéines kinases (AKT, GSK3 beta et p130CAS). Une diminution de l'expression de la beta-caténine a également été détectée.Notre équipe a précédemment identifié une séquence de 17aa dans la protéine de cœur du lumicanne, la lumcorine, qui est capable de reproduire l'effet anti-migratoire du lumicanne. Nous proposons ici deux mécanismes d'action de la lumcorine: une inhibition de la phosphorylation de FAK et une diminution de l'activité de la MMP-14. De plus, nous avons identifié, au sein de la séquence de la lumcorine, un peptide court de 10aa (L9M) qui est capable de reproduire l'effet anti-tumoral de la lumcorine. Nous montrons que le peptide cyclique L9M diminue la croissance tumorale in vivo.Nos travaux permettent donc de mieux comprendre les mécanismes impliqués dans l'effet anti-tumoral du lumicanne et mettent en évidence de nouveaux peptides prometteurs pour des applications anticancéreuses. / Lumican is a small leucine-rich proteoglycan of the extracellular matrix involved in the control of angiogenesis, particularly tumor angiogenesis. We have previously demonstrated that lumican inhibits melanoma progression in vivo. The anti-tumor mechanism of action of lumican was partially described in our laboratory. The alpha2beta1 integrin was characterized as a direct receptor of lumican. In the present studies, we first described the role of lumican in the control of Mesenchymal Stem Cells (MSC) transition to functional Endothelial Progenitor Cells (EPC), which can contribute to tumor angiogenesis. We showed that lumican inhibits specifically the migration and invasion of MSC by decreasing the expression and activity of MMP-14. Moreover, we demonstrated that lumican reduces the activity of MMP-14 in melanoma cells. We next showed that lumican directly inhibits MMP-14 activity as a competitive inhibitor which binds to the catalytic domain of the enzyme with moderate affinity (KD=275.4±16.12nM). Moreover, we demonstrated that lumican decreases the phosphorylation of some cell surface receptors (EGFR, Mer, EphB2, EphB6, ROR), some kinases (AKT, GSK3 beta and p130CAS) and alters the expression of beta-catenin.Previous works from our laboratory identified a sequence of 17aa within the leucine-rich repeat 9, named lumcorin, which was able to reproduce the anti-migratory effect of lumican. Here, we propose two mechanisms of action of lumcorin: inhibition of phosphorylation of FAK and a decrease of the MMP-14 activity. We also identified in the sequence of lumcorin a short 10aa peptide (L9M) which was capable to reproduce the anti-tumor effect of lumcorin. In addition, the cyclic peptide L9M was demonstrated to reduce tumor growth in vivo.Altogether, our results help to better understand the mechanisms involved in the anti-tumor effect of lumican and identify lumican-derived peptides which could have potential anti-cancer applications.

Les anévrysmes de l'aorte abdominale (AAAs) sont des dilatations artérielles qui exposent le patient au décès par rupture. Ils sont caractérisés notamment par la perte de la monocouche de cellules endothéliales et son remplacement par un épais thrombus mural. Cependant, le lienentre l'accroissement du diamètre anévrysmal, la présence d'un thrombus et la perte en cellules endothéliales reste inexploré. Notre hypothèse est que la perte de l'endothélium contribue au développement des AAAs et que sa restauration par thérapie cellulaire permettrait de stabiliser les AAAs. In vivo, la réparation endothéliale implique le recrutement des cellules endothéliales adjacentes mais également des cellules progénitrices endothéliales (EPCs). Chez l'homme, le nombre et l’activité fonctionnelle des EPCs sont inversement corrélés aux facteurs de risque cardiovasculaire, et très peu de données sont disponibles sur l’activité fonctionnelle des EPCs issues de patients porteurs d'AAA. La présence du thrombus pourrait ainsi diminuer le nombre et les propriétés cicatricielles des EPCs issues de patients porteurs d'AAA. / Summary not transmitted

Die Homöostase wird maßgeblich durch die Niere, bestehend aus Millionen funktioneller Untereinheiten, den Nephronen, aufrechtherhalten. Chronisch geschädigte Nephrone führen zur Entwicklung einer terminalen Nierenerkrankung (TNE). Die Erzeugung renaler Zellen aus humanen pluripotenten Stammzellen (hPSCs) stellt eine vielversprechende Strategie zur regenerativen Therapie und Behandlung von TNE dar. In der vorliegenden Arbeit wurde ein Protokoll zur Differenzierung von renalen Vorläufern (RV) aus hPSCs entwickelt, welches nephronale Zelltypen und Strukturen in vitro und ex vivo erzeugte. Eine selektierte Kombination von Faktoren wurde in diesem 8-Tage-Protokoll genutzt, um die schrittweise Differenzierung der hPSCs zu lenken, indem die embryonale Organogenese der Niere abgebildet wurde. Am Tag 6 der Differenzierung konnten SIX2+/CITED1+ Zellen des metanephrischen Mesenchyms und HOXB7+/GRHL2+ Zellen, welche auf Vorläufer der Ureterknospe hindeuten, nachgewiesen werden. Diese entwickelten sich am Tag 8 weiter zu LGR5+/JAG1+/WT1+ renalen Vesikelzellen. Weiterführende Kultivierung in drei verschiedenen induktiven Medien führte zu WT1+/PODXL+/SYNPO+ Podozytenvorläufern, PDGFRß+/DESMIN+/αSMA+ Mesangialzellen und epithelialen Zellen des proximalen und distalen Tubulus sowie des Sammelrohrs. Außerdem bildeten die Tag-8-Vorläuferzellen spontan 3D renale Organoide aus. Die RV induzierten tubuläre Strukturen an einer Luft-Flüssigkeits-Grenzfläche und integrierten sich in embryonale Nierenaggregate. Zusammenfassend konnte demnach ein Protokoll entwickelt werden, welches entstehenden Nephronen ähnliche RV generierte, die innerhalb von 14 Tagen in spezialisierte nephronale Zelltypen differenzierten. Diese einfache Methode, um renale Zellen aus einem gemeinsamen Vorläuferpool in einer 2D -Kultur zu erzeugen, schafft die Grundlage für eine Produktion im größeren Maßstab, sowie für Modellsysteme in toxikologischen Untersuchungen oder Zelltherapien. / Kidneys are the central organ for homeostasis for our body systems and composed of around a million functional units, the nephrons. Chronically damaged nephrons deteriorate progressively towards end stage renal disease (ESRD), owing to the limited regenerative capacity of adult mammalian kidneys. The generation of renal cells from human pluripotent stem cells (hPSCs) is a promising strategy to develop regenerative therapies for ESRD. In this study, we established a protocol to differentiate hPSCs to renal progenitors (RP), capable of producing nephronal cell types and structures in vitro and ex vivo. An effective combination of factors obtained after intensive screening, was used to create an 8-day-protocol that steered hPSCs to the renal lineage by a step-wise process outlining the embryonic milestones in kidney organogenesis. Six days after growth factor treatment, a mixture of SIX2+/CITED1+ cells representing metanephric mesenchyme and an HOXB7+/GRHL2+ population indicative of ureteric bud progenitors was obtained that developed into LGR5+/JAG1+/WT1+ renal vesicle cells by the day 8. Prolonged cultivation of these day 8 cells in three inductive media resulted in generation of WT1+/PODXL+/SYNPO+ podocyte-precursors, PDGFRß+/DESMIN+/αSMA+-mesangial cells and fractions of proximal, distal and collecting duct tubular epithelial cells in vitro. Moreover, day 8 cells differentiate spontaneously into renal organoids in culture. The hPSC-derived RP gave rise to tubular structures upon culture as a pellet in air-liquid interface and integrated into embryonic kidney re-aggregations. Thus, we demonstrate that our protocol generates RP reminiscent of nascent nephrons, which can be coaxed into specialized nephronal cell types in vitro after 14 days from hPSCs. This simple and rapid method to produce renal cells from a common precursor pool in 2D culture provides the basis for scaled-up production of tailored renal cell types, applicable for drug testing or cell therapies.

Em 2006, Takahashi e colaboradores demonstraram ser possível a obtenção de células-tronco pluripotentes por indução gênica (induced pluripotent stem cells ou iPSCs). Desde o surgimento desta tecnologia diversos modelos animais foram gerados, ampliando as possibilidades de seu uso na pesquisa, como por exemplo, na criação de modelos para doenças genéticas humanas como esclerose lateral amiotrófica, autismo, esquizofrenia, doença de Parkinson e Alzheimer, além do aprimoramento de características relevantes para produção animal. O modelo suíno é considerado vantajoso sobre os outros modelos animais principalmente pela criação já bem estabelecida e similaridades fisiológicas com os humanos. O intuito deste projeto foi reprogramar fibroblastos embrionários suínos através do sistema integrativo à iPSCs, para então diferenciá-las em células progenitoras neurais (neural progenitor cells, NPCs). Para isso, os fibroblastos foram transduzidos com vetores contendo sequencias humanas ou murinas dos genes OCT4, SOX2, c-Myc e KLF4 (hOSKM ou mOSKM) para formação das iPSCs. Estas foram caracterizadas quanto a morfologia, presença de fosfatase alcalina, a expressão dos genes exógenos e endógenos (OSKM, HS OCT4, OCT4, NANOG) através de imunofluorescência e RT-qPCR e formação de corpos embrióides. Então foram submetidas durante 14 dias ao meio de indução neural sob matriz extracelular comercial, gerando células potencialmente similares às NPCs. Estas foram caracterizadas morfologicamente, por imunofluorescência das proteínas NESTINA, BETA TUBULINA III e VIMENTINA, além da expressão de NESTINA e GFAP por RT-qPCR. Foram produzidas com sucesso 3 linhagens de iPSC em diferentes estágios de reprogramação e células positivas para todos os marcadores neurais testados. Os resultados apresentados deverão contribuir para a utilização do modelo suíno em futuros estudos voltados à medicina regenerativa e translacional. / In 2006, Takahashi and collaborators reported the induction into pluripotency of somatic cells (induced pluripotent stem cells, iPSCs). Since then, this technique has much been developed; many animal models have been created opening a new series of opportunities in research. They enable the creation of models for human genetic diseases, for example, amyotrophic lateral sclerosis, autism, schizophrenia, Parkinson´s disease, Alzheimer´s disease and the enhancement of relevant characteristics in agriculture. The swine model is considered to present many advantages over others, including the well-known production and maintenance and physiological similarities to humans. The aim of this project was to reprogram porcine embryonic fibroblasts (pEF) into iPSCs using the lentiviral integrative system, followed by its differentiation into neural progenitor cells (NPCs). The cells were reprogrammed using vector containing either the human sequences (hOSKM) or the mouse sequences (mOSKM) for the OCT4, SOX2, c-Myc and KLF4 genes to form the iPSCs. They were characterized regarding the presence of the Alkaline Phosphatase enzyme, expression of exogenous and endogenous genes (OSKM, HS OCT4, OCT4, NANOG) through immunofluorescence and RT-qPCR, and embryoid body formation. Then, the cells were cultured with neural induction media for 14 days in commercial extracellular matrix, generating cells potentially like NPCs. Those were characterized regarding their morphology, immunofluorescence for NESTINA, BETA TUBULIN III and VIMENTINA and gene expression of NESTINA and GFAP. iPSCs were successfully reprogramed, generating 3 cell lines at different stages of reprograming and cells positive for all the neural markers tested were produced. The results shown will contribute to the use of the porcine model in future regenerative and translational medicine research.

O treinamento físico aeróbio (TF) tem sido utilizado como um importante tratamento não farmacológico da hipertensão arterial (HA), uma vez que ele corrige a rarefação microvascular e reduz a pressão arterial; entretanto, os mecanismos envolvidos são pouco conhecidos. Investigamos se o número e a capacidade funcional das células progenitoras endoteliais (CPE) derivadas da medula óssea, sabidamente diminuídas na HA, melhoram pós TF, potencialmente contribuindo para a neovascularização e regressão da doença. O efeito do TF sobre a pressão arterial, freqüência cardíaca, tolerância ao esforço, consumo de oxigênio (VO2), morfologia e bioquímica da musculatura esquelética foram estudados em ratos espontaneamente hipertensos (SHR, n=28) e Wistar Kyoto (WKY, n=28) com 12 semanas de vida e divididos em 4 grupos: SHR, SHR treinado (SHR-T), WKY e WKY Treinado (WKY-T). O TF promoveu redução da pressão arterial em SHR e bradicardia de repouso acompanhado por um aumento da atividade da citrato sintase muscular, tolerância ao esforço e VO2 nos grupos de animais treinados. Concomitantemente, o TF corrigiu a alteração na distribuição dos tipos de fibra muscular e a rarefação capilar em SHR, mediado em grande parte por um aumento nos níveis protéicos periféricos de VEGF, VEGFR2, eNOS e a desativação das vias de apoptose. O número de CPE (CD34+/Flk1+) no sangue periférico (SP) analisadas por FACS foram aumentadas 115% no grupo WKY-T em comparação ao grupo controle. Em contraste, o grupo SHR reduziu 39% o número de CPE, entretanto o TF normalizou os níveis no grupo SHR-T. Resultado similar foi encontrado na quantificação das CPE na medula óssea (MO) avaliadas por células duplamente positivas para Di-acLDL e Lectina-FITC. A senescência das CPE na MO foi aumentada 126% no grupo SHR vs. WKY, e o TF foi eficiente em reduzir 72% este processo no grupo SHR-T. Além disso, os ensaios funcionais avaliados pelo número de unidades formadoras de colônia mostraram um aumento de 40% na MO e 70% no SP de WKY-T vs. WKY. Em contraste, a HA reduziu 35% na MO e 45% no SP este número de colônias vs. WKY, porém o TF corrigiu esta disfunção das CPE na HA. De fato, o TF recuperou a falha na formação de tubos como capilares sobre matrigel na HA. Os resultados demonstram que o remodelamento vascular acompanhado pela redução da pressão arterial induzido pelo TF na HA ocorreram em sinergia com a recuperação do número e das propriedades funcionais das CPE da MO e SP, bem como de seus fatores mobilizadores e angiogênicos. Estes resultados sugerem que o TF pode participar do reparo vascular por meio da ação das CPE, promovendo a revascularização periférica. Assim, há perspectiva do potencial terapêutico das CPE no tratamento da HA pós TF. / Aerobic exercise training (ET) has been established as an important non-pharmacological treatment for hypertension, since it counteracts microvascular rarefaction and decreased blood pressure; however, underlying mechanisms remain to be further determined. We investigated for the first time if the endothelial progenitor cells (EPC) number and the functional capacity, impaired in hypertension; are improved after ET potentially contributing to neovascularization and disease regression. The effect of ET on blood pressure, heart rate, exercise tolerance, peak VO2 and skeletal muscle morphology and biochemistry was studied in twelve-week old male Spontaneously Hypertensive Rats (SHR, n=28) and Wistar Kyoto (WKY, n=28) assigned into 4 groups: SHR, trained SHR (SHR-T), WKY and trained WKY (WKY-T). The ET promoted a decrease in blood pressure in SHR and resting bradycardia, an increase in exercise tolerance, peak VO2 and citrate synthase activity in trained groups. In parallel, the ET repaired the skeletal muscle fiber type shift and capillary rarefaction in SHR, at least partly, by enhancing protein levels of VEGF, VEGFR-2, eNOS and deactivated apoptosis pathway. Numbers of EPC (CD34+/Flk1+) in the peripheral blood (PB) quantified by FACS analysis were enhanced 115% in WKY-T of control levels. In contrast, the SHR group decreased 39%, but ET normalized in the SHR-T. Similar results were found in the EPC quantification of the bone marrow (BM) by double positive cells to Di-acLDL and Lectin-FITC. BM-EPC senescence was increased 126% in SHR and this process was reduced 72% by ET. Moreover, EPC functional assay by colony-forming units showed an increase of 40% to BM and 70% to PB in WKY-T of control levels. In contrast, the SHR group reduced 35% to BM and 45% to PB; however the ET repaired EPC dysfunction in hypertension. In fact, the ET corrected failure in the capillary-like tube formation on matrigel. The present findings reveal that the vascular remodeling accompanied by reduction of blood pressure induced by ET occurs in synergy with the restoration of the BM and PB- EPC number and functional properties, as well as of their mobilizing and angiogenic factors. These results suggest that the ET can participate in the vascular repair by means of the EPC, promoting the peripheral revascularization in hypertension. In this way, there is perspective of therapeutic potential of the EPC in treatment of hypertension after ET.

Mit der vorliegenden Habilitationsschrift habe ich den Versuch unternommen, die beiden Themenkomplexe meiner bisherigen wissenschaftlichen Tätigkeit als Beispiele für die Rolle von Modellen in der klinischen Forschung zu verwenden. Den Ansto§ dazu gaben Diskrepanzen, die mir in der Auseinandersetzung mit eigenen Ergebnissen und Beobachtungen im Umfeld dieser Themenkomplexe aufgefallen sind: der Rolle kontaminierender Tumorzellen in der Hochdosistherapie maligner Tumoren einerseits und dem Enzym Dipeptidylpeptidase IV (DPP IV) andererseits. Die beobachteten Diskrepanzen sind Ausdruck konkurrierender pathophysiologischer oder therapeutischer Modelle, und die Präferenz eines bestimmten Modells scheint nicht rein rational erklärbar. Welche Faktoren tragen jedoch zur Entscheidung für oder gegen ein bestimmtes Modell bei? Ich möchte den Umgang mit wissenschaftlichen Modellen anhand der genannten Themenkomplexe aus meiner Sicht erörtern. Anschlie§end soll ein Entwurf skizziert werden, in dem die der Entscheidung für oder gegen ein therapeutisches Modell zugrundeliegende Motivationslage besser verständlich wird und die Intentionalität klinischer Forschung auf den Patienten hin berücksichtigt. / In the thesis presented here, I have taken the challenge to use the topics of my scientific work to discuss the role that models appear to exert in clinical science. This decision arose from discrepancies that became evident in the comparative assessment of my own studies in relation with the surrounding scientific context: the role of tumor cells contaminating peripheral blood or progenitor cell harvests as part of a high-dose chemotherapy regimen on the one hand, and the enzyme dipeptidyl peptidase IV (DPP IV) on the other. The observed discrepancies appear to result from competing pathophysiological or therapeutic models, and the preference or rejection of one model apparently cannot be explained solely by rational factors. I will discuss the application of models in the context of the topics which my scientific work has been focusing on, and I will develop a draft proposal which will render the individual motivational status underlying the decision in favor of or against a distinct model easier to understand, with attention to the intentionality of clinical research towards the patient.

Les syndromes myélodysplasiques (SMD) forment un groupe de pathologies clonales de la cellule souche hématopoïétique (CSH) caractérisées par une hématopoïèse inefficace. La présence d’au moins une anomalie génétique (anomalie cytogénétique ou mutation somatique) est observée dans plus de 90% des cas. Ainsi, plusieurs clones moléculaires pouvaient coexister au moment du diagnostic de la maladie. Dans les SMD avec délétion du chromosome 5 (del(5q)), il a récemment été montré que les anomalies étaient présentes dès le stade de la CSH. Dans les SMD, la pénétrance des anomalies génétiques décrites est incomplète. De plus, peu de choses sont actuellement connues sur l’ordre d’apparition des mutations et leur impact fonctionnel sur les différents clones moléculaires dans le cas des SMD non-del(5q). Grâce au séquençage d’exome entier (WES) de patients ne présentant aucune mutation dans les gènes décrits dans les SMD, nous avons décrit l’existence de mutations dans les gènes BCOR et BCORL1, chez respectivement 4,2% et 0,8% des patients. Les mutations du gène BCOR arrivent tardivement au cours de l’évolution de la maladie et affectent le pronostic des patients. Des approches à l’échelle unicellulaire nous ont également permis d’observer que la majeure partie des mutations identifiées chez les patients sont retrouvées dès le stade CD34+CD38-. Chez les patients, plusieurs clones moléculaires coexistent à ce stade. De plus, les mutations des gènes de l’épissage et de la régulation épigénétique sont fréquemment acquises en premier dans les cellules hématopoïétiques les plus immatures des patients porteurs de SMD. Nous avons observé que certaines mutations, acquises secondairement, sont réparties inégalement dans les différents compartiments hématopoïétiques et peuvent avoir un impact sur la différenciation hématopoïétique. Enfin, nous montrons que la répartition des clones moléculaires évolue au cours du temps. En réponse au traitement par Lenalidomide, on observe également une évolution rapide de l’architecture clonale qui peut être liée au statut de réponse des patients. Ces résultats tendent à confirmer l’hétérogénéité génétique mais aussi fonctionnelle des SMD. Nous avons pu identifier de nouvelles mutations impliquées secondairement dans la physiopathologie des SMD. Il existe une dominance clonale précoce dans les SMD du fait de l’acquisition de toutes les mutations dans les cellules hématopoïétiques immatures. Cependant, les différentes populations hématopoïétiques peuvent présenter des génotypes différents. Enfin cette architecture est variable au cours de l’évolution de la maladie. / Myelodysplastic syndromes (MDS) are a group of clonal disorders of the hematopoietic stem cell (HSC) characterized by ineffective hematopoiesis. At least one genetic abnormality (cytogenetic abnormality or somatic mutation) is observed in more than 90% of cases. Thus, it has been observed several molecular clones which could coexist at diagnosis of the disease. In MDS with deletion of chromosome 5 (del (5q)), it has recently been shown that defects were present in the HSC. In MDS, the penetrance of genetic abnormalities described is incomplete. In addition, little is currently known about the order of appearance of mutations and their functional impact on different molecular clones in the case of non-del (5q) MDS. Through the whole exome sequencing (WES) of patients without mutation in the genes described in MDS, we described the existence of mutations in genes BCOR and BCORL1, in respectively 4.2% and 0.8% of patients. Mutations in the gene BCOR were acquired lately during the course of the disease and affect the prognosis of patients. Approaches at the single cell level have also allowed us to observe that most of the mutations identified in patients are found at the immature differentiation stage CD34+CD38-. In patients, several molecular clones could coexist at this stage. In addition, mutations in gene splicing and epigenetic regulation are frequently first acquired in the most immature hematopoietic cells of MDS patients. We found that certain mutations, acquired in a second time, are distributed unevenly in different hematopoietic compartment and may have an impact on hematopoietic differentiation. Finally, we showed that the distribution of molecular clones evolves over time. In response to treatment with Lenalidomide, it has also been observed a rapid evolution of clonal architecture that can be linked to patient response status. These results tend to confirm the genetic but also functional heterogeneity in MDS. We have identified new mutations involved in the pathogenesis of MDS. We observed an early clonal dominance in MDS because of the acquisition of all mutations in immature hematopoietic cells. However, different hematopoietic populations can have different genotype. Finally, the architecture of mutations could be modifying during the course of the disease.

Das erwachsene Gehirn enthält neuronale, multipotente Stammzellen, aus denen in den beiden bekannten neurogenen Regionen des Gehirn, im Hippocampus und im olfaktorischen System, neue Nervenzellen hervorgehen. Aus Transplantationsstudien und anderen Untersuchungen weiß man, daß es die zelluläre Umgebung ist, die die neurogene Permissivität und damit die Entwicklung einer reifen neuen Nervenzelle aus einer Stamm- oder Vorläuferzelle, bestimmt. Die Schlüsselfrage lautet daher: Was macht eine neurogene Region neurogen? Neurogenität ist mehr als die Präsenz von neuralen Stammzellen. Die aktivitätsabhängige Regulation adulter hippocampaler Neurogenese stellt eine physiologische, positive Modulation von Neurogenität im erwachsenen Gehirn dar. Aktivitätsabhängige Regulation adulter hippocampaler Neurogenese ist vielstufig und kein An/Aus-Phänomen. Die unterschiedlichen Stufen der Regulation unterliegen unterschiedlicher genetischer Determination und unterschiedlicher Empfindlichkeit auf aktivitätsabhängige Stimuli. Die Steuerung des Überlebens neugeborener Zellen stellt möglicherweise den entscheidenden Schritt auf dem Weg zu einem neuen Neuron dar. Die aktivitätsabhängige Selektion durch eine überlebensfördernde Wirkung rekrutiert jedoch aus einem Pool proliferierender Vorläuferzellen, die das neurogene Potential darstellen. Die subtile Regulation adulter hippocampaler Neurogenese durch funktionsabhängige Stimuli legt eine Relevanz für hippocampale Funktion, insbesondere Lern- und Gedächtnisvorgänge nahe. Entsprechend muß aber auch eine Bedeutung für hippocampale Pathologie diskutiert werden. Das Verständnis darüber, wie Neurogenität funktions- und aktivitätsabhängig modulierbar ist, ist von größter Relevanz für die Frage, ob und wie sich Neurogenese aus ruhenden neuronalen Stamm- und Vorläuferzellen auch außerhalb neurogener Regionen induzieren und in therapeutischer Absicht nutzen läßt. / The adult brain contains neuronal, multipotent stem cells. In two neurogenic regions of the adult brain, hippocampus and olfactory system, new neurons are generated from these stem cells. From transplantation studies and other investigations it is known that the cellular microenvironment provides the neurogenic permissiveness and determines the development of a mature new neuron from a stem or progenitor cell. Thus, the key question is, what defines a neurogenic region as neurogenic, if it is not the presence of neural stem cells alone. The activity-dependent regulation of adult hippocampal neurogenesis represents a physiologic and positive modulation of neurogenic permissiveness in the adult brain. Activity-dependent regulation of adult hippocampal neurogenesis occurs on multiple steps and is not an on/off phenomenon. The different levels of regulation are differentially influenced by genetic determination and different susceptibility to activity-dependent stimuli. The regulation of the survival of a newly generated cells might be the key step in the development of a new neuron. The activity-dependent recruitment of new neurons by means of a survival-promoting effect acts upon a pool of proliferating progenitor cells, which represent the neurogenic potential. The subtle regulation of adult neurogenesis by functional stimuli suggests a relevance of adult hippocampal neurogenesis for hippocampal function, in particular learning and memory. Accordingly, a potential relevance for hippocampal pathology has to be considered. Insights on how neurogenic permissiveness can be modulated in response to functional stimuli has important implications for the question, if and how neurogenesis from quiescent neuronal stem or progenitor cells can be induced inside and outside of neurogenic regions of the adult brain and can be used for therapeutic purposes.

Introdução. As células progenitoras hematopoéticas (CPH) têm sido utilizadas no tratamento de algumas doenças malignas hematológicas e de desordens hematopoéticas. Dentre estas células estão as CPHs provenientes de cordão umbilical e placentário (SCUP). Para coleta e armazenamento destas células foram criados os Bancos de Sangue de Cordão Umbilical e Placentário (BSCUP). No Brasil existe a rede BrasilCord e o BSCUP do Hemocentro de Ribeirão Preto é um dos que compõem esta rede. Objetivo. Avaliar a viabilidade financeira de um banco de sangue de cordão umbilical e placentário público comparando os custos de seus procedimentos com os valores ressarcidos definidos pela tabela SUS. Metodologia: Este estudo utilizou a metodologia de Custeio Baseado em Atividades (Activity-Based Costing - ABC), que procura reduzir as distorções provocadas pelo rateio arbitrário dos custos indiretos utilizando direcionadores de custos. Foram avaliados os custos diretos e indiretos da coleta, transporte, processamento, testagem e criopreservação de CPH proveniente de SCUP no primeiro semestre de 2015. Para os custos indiretos foram definidos os direcionadores de custo. Resultados Os resultados mostram que o BSCUP do Hemocentro de Ribeirão Preto foi deficitário no primeiro semestre de 2015. O déficit apurado por unidade foi de R$1.155,69 para o processamento semiautomatizado e R$1.703,78 para o processamento automatizado. O déficit total no período foi de R$ 100.376,50 quando 50 unidades foram processadas utilizando o método semiautomatizado e 25 pelo método automatizado. Conclusão. O valor ressarcido pelo SUS não cobre os gastos do BSCUP do Hemocentro de Ribeirão Preto. Isso pode ser atribuído a várias causas como: sistemática de pagamento pelo SUS apenas pelo produto criopreservado, elevados índices de rejeição das doadoras na maternidade e de descarte das unidades coletadas, embora este último seja menor que o descrito na literatura, e o custo do armazenamento em longo prazo. Os custos do BSCUP são menores que os descritos na literatura e poderiam ser reduzidos com melhorias de processos de gestão e aumento do número de unidades criopreservadas, bem como, por meio de descentralização de coletas para processamento centralizado. / Introduction. Hematopoietic progenitor cells (HPC) are being used in some hematologic malignancies and hematopoietic disorders treatment. Among these cells are the HPCs from umbilical blood cord (UCB). Umbilical cord blood banks were created to collect and store these cells. In Brazil there is a net called Brasilcord and the umbilical cord blood bank (UCBB)of the Hemocentro de Ribeirão Preto belongs to this net. Objective. To evaluate the financial viability of a public umbilical cord blood bank and compare the costs from its procedure with the values reimbursed defined by the SUS table. Methodology.This study used the ActivityBased Costing, which tries to reduce distortions caused by arbitrary apportionment of indirect costs using cost drivers. Direct and indirect costs of collection, transport, processing, testing and cryopreservation of HPCs from umbilical cord blood were evaluated. Cost drivers were defined for indirect costs. Results. The results showed that there was a deficit in the UCBB of the Hemocentro de Ribeirão Preto in the first semester of 2015. The deficit was R$1.155,69 when the unit was processed by the semi-automated method and R$1.703,78 when it was processed by an automated method. The total deficit in the period was R$100.376,50 as 50 units were processed by a semi-automated method and 25 by an automated method. Conclusion. The amount reimbursed by SUS does not cover the UCBB of Hemocentro de Ribeirão Preto expenses. This can be attributed to several causes such as: systematic of reimbursement used by SUS that only cryopreserved units are payed, high percentage of deferral in the maternity and of discard of collected units, although this latter is smaller than that described in the literature and the cost of storage of the units for long periods. The costs of UCBB are lower than that described in the literature and could be reduced with improvements in managing process and increase the number of cryopreserved units as well as decentralization of collections for centralizes processing.