Made available in DSpace on 2014-12-17T15:32:29Z (GMT). No. of bitstreams: 1 CassianoFWN_TESE.pdf: 3914655 bytes, checksum: c6fb9bd86bba433eb6a5a047a9337861 (MD5) Previous issue date: 2010-09-23 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The odontogenic keratocysts are distinguished from other odontogenic cystic lesions by their potentially aggressive clinical behavior and association, in some cases, with Gorlin syndrome. Studies have suggested that syndrome keratocysts, in comparison with sporadic lesions, have higher growth and infiltration capacity and higher recurrence tendency. The aim of this study was to analyze, by means of immunohistochemistry, the expressions of receptor activator of nuclear factor ?B ligand (RANKL) and osteoprotegerin (OPG), the angiogenic index (CD34) and the presence of myofibroblasts (?-SMA) in primary and recurrent sporadic keratocysts and in keratocysts associated with Gorlin syndrome. The sample was composed by 30 sporadic keratocysts (22 primary and 8 recurrent) and 22 syndrome keratocysts. In the epithelium and in the fibrous capsule of the lesions, the immunoexpression of RANKL and OPG was evaluated by determination of the percentage of positive cells, according to the following scores: 0 (less than 10% of positive cells), 1 (11% - 50% of positive cells), 2 (51% - 75% of positive cells) and 3 (more than 76% of positive cells). In addition, cases were classified according to the RANKL score/ OPG score ratio, as follows: RANKL > OPG, RANKL < OPG, and RANKL = OPG. The angiogenic index was analyzed by counting the microvessels immunoreactive to anti-CD34 antibody in 5 fields (200?). The analysis of myofibroblasts was performed by counting the cells immunoreactive to anti-?-SMA antibody in 10 fields (400?). The analysis of the expressions of RANKL and OPG in the epithelial lining and in the fibrous capsule did not reveal significant differences between groups (p > 0.05). Regarding the RANKL/ OPG ratio in the epithelial lining, most sporadic primary (54.5%) and syndrome lesions (59.1%) showed RANKL < OPG ratio and RANKL = OPG ratio, respectively (p > 0.05). With respect to the RANKL/ OPG ratio in the fibrous capsule, the majority of sporadic primary (81.8%) and sporadic recurrent lesions (75.0%) and most syndrome lesions (45.5%) showed RANKL = OPG ratio (p > 0.05). The mean number of microvessels was 69.2 in sporadic primary lesions, 67.6 in recurrent lesions, and 71.6 in syndrome lesions, with no significant differences between groups (p > 0.05). The mean number of myofibroblasts was 34.4 in sporadic primary lesions, 29.3 in recurrent lesions, and 33.7 in syndrome lesions, with no significant differences between groups (p > 0.05). In conclusion, the results of the present study suggest that the differences in the biological behavior between sporadic keratocysts and keratocysts associated with Gorlin syndrome may not be related to the expressions of RANKL and OPG, the RANKL/ OPG ratio, the angiogenic index or the number of myofibroblasts in these lesions / Os ceratocistos odontog?nicos se destacam em rela??o a outras les?es c?sticas odontog?nicas pelo comportamento cl?nico potencialmente agressivo e por se apresentarem associados, em alguns casos, ? s?ndrome de Gorlin. Estudos t?m sugerido que os ceratocistos sindr?micos, em compara??o ?s les?es isoladas, possuem maior capacidade de crescimento e infiltra??o e maior tend?ncia ? recorr?ncia. O objetivo do presente trabalho consistiu em analisar, por meio de imuno-histoqu?mica, as express?es do ligante do receptor ativador do fator nuclear ?B (RANKL) e da osteoprotegerina (OPG), o ?ndice angiog?nico (CD34) e a presen?a de miofibroblastos (?-SMA), em ceratocistos isolados prim?rios e recorrentes e ceratocistos associados ? s?ndrome de Gorlin. A amostra foi composta por 30 ceratocistos isolados (22 prim?rios e 8 recorrentes) e 22 ceratocistos sindr?micos. A express?o de RANKL e OPG foi avaliada no epit?lio e na c?psula fibrosa das les?es, estabelecendo-se o percentual de c?lulas imunopositivas, de acordo com os escores: 0 (? 10% das c?lulas positivas), 1 (11% - 50% das c?lulas positivas), 2 (51% - 75% das c?lulas positivas) e 3 (? 76% das c?lulas positivas). Al?m disso, os casos foram categorizados, segundo a propor??o RANKL/ OPG, em: RANKL > OPG, RANKL < OPG e RANKL = OPG. O ?ndice angiog?nico foi analisado por meio da contagem dos microvasos imunomarcados pelo anticorpo anti-CD34, em 5 campos (200?). Para a avalia??o dos miofibroblastos, foram quantificadas as c?lulas imunorreativas ao anticorpo anti-?-SMA, em 10 campos (400?). A an?lise das express?es de RANKL e OPG, no revestimento epitelial e na c?psula fibrosa, n?o revelou diferen?as significativas entre os grupos (p > 0,05). Em rela??o ? propor??o RANKL/ OPG no revestimento epitelial, grande parte das les?es isoladas prim?rias (54,5%) e sindr?micas (59,1%) exibiu propor??o RANKL < OPG e propor??o RANKL = OPG, respectivamente (p > 0,05). Em rela??o ? propor??o RANKL/ OPG na c?psula fibrosa, a maioria das les?es isoladas prim?rias (81,8%) e isoladas recorrentes (75,0%) e grande parte das les?es associadas ? s?ndrome de Gorlin (45,5%) revelaram propor??o RANKL = OPG (p > 0,05). O n?mero m?dio de microvasos foi de 69,2 nas les?es isoladas prim?rias, 67,6 nas les?es recorrentes e 71,6 nas les?es sindr?micas, sem diferen?as significativas entre os grupos (p > 0,05). A an?lise dos miofibroblastos revelou valores m?dios de 34,4 nas les?es isoladas prim?rias, 29,3 nas les?es recorrentes e 33,7 nas les?es sindr?micas, sem diferen?as significativas entre os grupos (p > 0,05). Em conclus?o, os resultados do presente estudo sugerem que as diferen?as no comportamento biol?gico entre ceratocistos isolados e associados ? s?ndrome de Gorlin podem n?o estar relacionadas ?s express?es de RANKL e OPG, ? propor??o RANKL/ OPG, ao ?ndice angiog?nico ou ? quantidade de miofibroblastos presentes nas les?es

O crescimento e a progressão tumorais estão associados à indução da angiogênese. O objetivo deste estudo foi avaliar a imunoexpressão do VEGF e a densidade de microvasos em adenocarcinomas gástricos. Espécimes cirúrgicos de 89 pacientes submetidos à ressecção gástrica com dissecção linfonodal a D2 foram analisados quanto à densidade microvascular tumoral. Testes imunohistoquímicos foram realizados utilizando-se os anticorpos CD31, CD34, Fator VIII e VEGF através do método da streptavidina-biotina. Os vasos foram contados nas áreas de maior vascularização tumoral e o VEGF foi graduado de 0 a 3, de acordo com a intensidade da imunocoloração. Os resultados imunohistoquímicos foram comparados com os dados patológicos e de extensão loco-regional da doença. Sessenta pacientes (67,4%) eram do sexo masculino e a média de idade foi 59,9 (±13,8) anos. Os tumores foram classificados em tipo intestinal em 62 casos (69,7%) e em difuso em 27 (30%). Onze pacientes (12,4%) apresentaram tumores precoces. A densidade microvascular apresentou média de 67,8 (±31,5) para o anticorpo CD31 e 94,2 (±39,0) para o CD34. A média do número de vasos corados pelo Fator VIII foi 9,2 (±8,2). Houve uma correlação entre os resultados imunohistoquímicos para CD31 e CD34, com r=0,57 e p=0,01. Segundo o VEGF os tumores foram: grau 0 = 16 (18%), I = 48 (53,9%), II = 16 (18%) e III = 9 (10,1%). Não houve associação entre a DMV e os dados patológicos: tipo histológico, grau de diferenciação, tamanho do tumor, invasão da parede gástrica, metástases linfonodais e metástase à distância. A imunoexpressão do VEGF foi associada com alta DMV (p=0,03) e com o estádio tumoral - TNM (p=0,003). Os resultados deste estudo permitiram concluir que a coloração imunohistoquímica com o Fator VIII não é segura como um marcador de células endoteliais; que os anticorpos CD31 e CD34 foram úteis na avaliação da DMV; e que a imunoexpressão do VEGF pode identificar um comportamento biológico mais agressivo / Tumor growth and progression, particularly in aggressive and malignant tumors, are associated with the induction of angiogenesis. The aim of this study was to evaluate the role of VEGF immunoexpression and microvascular density in gastric adenocarcinomas. Specimens from eightynine patients who underwent gastric resection with DII lymph node dissection were analyzed regarding microvascular density of the tumors. Immunohistochemistry for CD31, CD34, Factor VIII and VEGF were performed using the streptavidin-biotin-method. The vessels were counted in a hot spot area of the tumors and VEGF was categorized as grade 0 to III, according to the intensity. Immunohistochemical results were compared to pathological data and loco-regional extension of the disease. In the results of 89 patients, sixty (67.4%) were men, and the mean age was 59.9 ± 13.8 years. The tumors were classified as intestinal type in 62 (69.7%) and diffuse in 27 (30.3%). Eleven (12.4%) were early gastric tumors. Microvascular density showed a mean of 67.8 (± 31.5) for CD31 and 94.2 (± 39) for CD34 vessels. The mean number of vessels revealed by the antibody Fator VIII was 9.2 (± 8.2). There was a correlation between immunohistochemistry for CD31 and CD34, r = 0.57, p < 0.01. The tumors were classified for VEGF as: grade 0 = 16 (18%); I = 48 (53,9%); II = 16 (18%) and III = 9 (10,1%). There was no association between immunohistochemistry and pathological data including: histologic type, grade of differentiation, tumor size, tumor depth, lymph node metastasis and distant metastasis. However, VEGF immunoexpression was associated with high microvascular density (p = 0,03) and there was an association between the immunoexpression of VEGF and the tumor stage - TNM (p = 0,003). It was concluded that immunohistochemical staining for anti-Factor VIII was not reliable as a microvascular density marker; CD31 and CD34 were useful to demonstrate microvascular density and VEGF immunoexpression may identify tumors with more aggressive biological behavior

La greffe de cellules souches hématopoïétiques est parfois le seul traitement efficace contre les cancers hématologiques ainsi que plusieurs autres désordres reliés au système hématopoïétique. La greffe autologue est souvent le traitement de choix pour les patients atteints de lymphome ou de myélome. Dans ce cas, les cellules souches hématopoïétiques (CSH) du patient sont récoltées et congelées. Le patient subit ensuite des traitements de chimiothérapie et/ou radiothérapie qui éliminent les cellules malignes, mais détruisent aussi son système hématopoïétique. Ce dernier sera ensuite reconstitué par la greffe de CSH. Ces traitements ont pour conséquence de plonger le patient en état d’aplasie pour une période variant de 2 à 4 semaines. La thrombocytopénie (faible taux de plaquettes) est une complication majeure nécessitant des transfusions plaquettaires répétées et associée à une augmentation de la mortalité hémorragique post-transplantation. Il serait particulièrement intéressant de développer une thérapie accélérant la reconstitution des mégacaryocytes (MK), ce qui aurait pour effet de raccourcir la période de thrombopénie et donc de diminuer les besoins transfusionnels en plaquettes et potentiellement augmenter la survie. HOXB4 est un facteur de transcription qui a déjà démontré sa capacité à expandre les CSH et les progéniteurs multipotents (CFU-GEMM) donnant naissance aux MK. Il est donc un bon candidat pour l’expansion des progéniteurs MK. Comme la protéine HoxB4 a par contre une courte demi-vie (~1.1h), des protéines HoxB4 de deuxième génération avec une plus grande stabilité intracellulaire ont été créées (1423 (HoxB4L7A), 1426 (HoxB4Y23A) et 1427 (HoxB4Y28A)). Nous avons donc étudié la capacité d’HoxB4 sauvage et de deuxième génération à expandre les CSH, ainsi que les MK donnant naissance aux plaquettes. La surexpression rétrovirale de ces protéines HoxB4Y23A et HoxB4Y28A conduit à une expansion des progéniteurs MK murins in vitro supérieure à HoxB4-wt, 1423 et au contrôle GFP. La reconstitution plaquettaire in vivo dans un modèle murin a ensuite été évaluée par des transplantations primaires et secondaires. Les résultats révèlent que la surexpression rétrovirale des différents HoxB4 n’apporte pas de bénéfice significatif à la reconstitution plaquettaire des souris. Lorsque cultivées dans un milieu favorisant la différenciation mégacaryocytaire, le traitement de cellules CD34+ dérivées du sang de cordon ombilical avec les protéines recombinantes TATHoxB4WT ou de seconde génération n’a pas augmenté la production plaquettaire. Par contre, de manière intéressante, les cellules CD34+ provenant de sang mobilisé de patients atteints de myélome et mises en culture dans un milieu favorisant l’expansion des CSH ont montré des différences significatives dans la différenciation des progéniteurs MK en présence de la protéine recombinante TATHoxB4. La protéine HOXB4 possède donc un avenir prometteur quant à une amélioration de l’état thrombocytopénique chez les patients. / Haematopoietic stem cell (HSC) transplantation is the most efficient treatment against a number of cancers or other disorders of the hematologic system. Prior to HSC transplantation, patients are exposed to high doses of radiotherapy and/or chemotherapy to eliminate malignant cells. However, these treatments result in a state of aplasia, particularly in thrombocytopenia, which is characterised by very low blood platelet counts. Platelets produced by megakaryocytes (MK) are essential components of the blood system and play a critical role in the prevention of bleeding. Thus a low platelet blood level is a major complication and contributes significantly to transplant related mortality. At present, regular infusion of platelets isolated from healthy donors is the treatment of choice for thrombocytopenia. However, this is cumbersome for patients as well as donors and, in many instances results in platelet refractoriness due to the generation of auto-antibodies against disparate HLA molecules expressed on donor platelets. Therefore, the development of strategies to accelerate MK production and thus platelet reconstitution post HSC transplant would represent a major advance. It has already been shown that HoxB4 expands HSC and multipotent progenitors (CFU-GEMM) that give rise to megakaryocytes (MK). Thus HoxB4 is a great candidate for in vitro MK progenitor expansion. However, the short half-life of HoxB4 protein prompted us to generate a second generation of HoxB4 proteins with greater intracellular stability. We therefore studied the capacity of wild type (WT) and HoxB4 with 3 substitutions (1423 (HoxB4L7A), 1426 (HoxB4Y23A) and 1427 (HoxB4Y28A) resulting in a longer protein half-life to expand HSC as well as MK progenitors. Retroviral-mediated expression of HoxB4Y23A and HoxB4Y28A proteins showed a greater expansion of murine MK progenitors, in comparison with HoxB4WT or HoxB4L7A proteins or GFP control. We also evaluated the ability of HSC expressing second generation HoxB4 to generate platelets in a murine model. Our results show that retroviral-mediated transduction of second generation HoxB4 in murine HSC does not provide a significant advantage over HoxB4WT in platelet reconstitution in mice. Interestingly, treatment of CD34+ cells derived from cord blood showed only marginal effect of HoxB4WT or second generation HoxB4 soluble recombinant proteins when cultured under conditions optimized for megakaryocyte differentiation. Unexpectedly, CD34+ cells derived from mobilized peripheral blood of myeloma patients showed a significant increase in MK progenitor differentiation in the presence of TAT-HoxB4WT when cultured in expansion medium for HSC. Thus, HoxB4 holds promise in autologous HSC transplantation for the treatment of thrombocytopenic patients.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-09-30T18:51:57Z No. of bitstreams: 2 Dissertação- Maria Helena Tavares Vilela-2013.pdf: 2276556 bytes, checksum: 772ad6ab585b17765e734a42bc50eb7e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-09-30T19:29:06Z (GMT) No. of bitstreams: 2 Dissertação- Maria Helena Tavares Vilela-2013.pdf: 2276556 bytes, checksum: 772ad6ab585b17765e734a42bc50eb7e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-09-30T19:29:06Z (GMT). No. of bitstreams: 2 Dissertação- Maria Helena Tavares Vilela-2013.pdf: 2276556 bytes, checksum: 772ad6ab585b17765e734a42bc50eb7e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-10-09 / The proper management of breast phyllodes tumors (PTs) remains challenging, due to the difficulty of the correct preoperative diagnosis. The aim of this study was to evaluate the usefulness of the Ki-67, CD10, CD34, p53, CD117 and the number of mast cells for the differential diagnosis between benign PTs and cellular fibroadenomas (CFs), and also at the grading of PTs. 51 primary PTs and 14 FCs were examined by immunohistochemistry (IH). Through the evaluation of the expression of CD117, greater epithelial expression was found at the FCs and an increased number of mast cells in benign TFs. The stromal expression of the Ki-67, CD10, CD34 and p53 showed relevance to the grading of PTs. / O manejo adequado dos tumores filóides (TFs) mamários continua desafiador, devido à dificuldade do diagnóstico pré-operatório correto. O objetivo deste estudo foi avaliar a utilidade do Ki-67, do CD34, do CD10, do CD117, da p53 e do número de mastócitos no diagnóstico diferencial entre os TFs benignos e fibroadenomas celulares (FCs), bem como na graduação dos TFs. Foram examinados 51 TFs primários e 14 FCs por imunohistoquímica (IH). Na marcação pelo CD117, houve maior expressão epitelial nos FCs e maior número de mastócitos nos TFs benignos. A expressão estromal do Ki-67, da p53, do CD10 e CD34 mostrou-se significante na graduação dosTFs.

The first definitive haematopoietic stem cells (HSCs) produced during embryonic development are generated from a specialised subset of endothelial cells known as haemogenic endothelium. Recently, it was reported that Gata3 plays a dual role in the development of sympathetic nervous system and haematopoietic system. In fact, Gata3 has proven to be crucial for the production of HSCs through regulation of catecholamine production from the co-developing sympathetic nervous system. Also, it was recently shown that Gata3 is expressed in the haemogenic endothelium and haematopoietic progenitor cells. Here, I will specifically examine the role of Gata3 in the production of HSCs; if it is expressed and plays a role in the precursors from which HSCs arise. Using a Gata3-GFP reporter mouse line, we found that Gata3 is expressed in various cell types in the HSCs microenvironment, including mesenchymal cells, endothelial cells, haematopoietic cells and sympathetic nervous system, and this expression was stage dependant. In the endothelial cells, we have found that the haemogenic endothelium activity is enriched in Gata3 expressing cells. Within the haematopoietic cells, we have found that Gata3 marks a specific stage along the developmental pathway towards the generation of definitive haematopoietic stem cells, and that Gata3 expressing haematopoietic cells are enriched for the most immature and stem cell like progenitors. Moreover, Gata3 will be specifically knocked out in haemogenic endothelial cells to determine whether it plays an essential role in the production of HSCs from the endothelium using the Vec-Cre system. We found that Gata3 within the haemogenic endothelium plays a major role in haematopoietic progenitors formation, and possibly haematopoietic stem cell formation. Finally, we used molecular assay (RNA seq) to identify the role of Gata3 in the haematopoietic stem cell microenvironment and found that Gata3 plays a major role in the development and differentiation of various cells and systems, and implicated Gata3 as cell cycle regulator. In summary, we found that Gata3 expressing cells is enriched for haemogenic endothelium, crucial for the haematopoietic progenitors formation, plays and important role in endothelial to haematopoietic transition, and plays a key developmental role in both haematopoietic stem cell and its microenvironment.

Abstract Uterine leiomyomas are the most common benign tumours in females. They are myometrial neoplasms, may present single or multiple, and may be located in various sites of the uterus. Leiomyomas distort the uterine cavity and the uterus itself, causing abnormal vaginal bleeding, reduced fertility and also pelvic pressure and pain symptoms. The aim of this study was to elaborate current knowledge on familial uterine leiomyomas and to explore the possible association between uterine leiomyoma and cardiovascular disease risk factors, and also the association between leiomyomas and endometriosis. The natural history of familial uterine leiomyoma study showed significant differences between familial and non-familial leiomyoma cases, familial cases having more severe clinical characteristics. They presented with multiple uterine leiomyomas and were more often symptomatic. They were also diagnosed at a younger age. The prevalence study on uterine leiomyomas and endometriosis offered confirmation of an association between the diseases. Uterine leiomyomas and endometriosis seem to decrease female fertility independently of each other. Uterine leiomyomas related to the hereditary leiomyomatosis and renal cell cancer (HLRCC) tumour syndrome were studied in regard to their clinical characteristics and immunophenotype. The study provided evidence that women with HLRCC may be identified through distinct leiomyoma clinical characteristics, and routine-use IHC of CD34 and Bcl-2. Distinguishing these leiomyoma cases from sporadic ones may identify families affected by fumarate hydratase (fumarase, FH) mutation. Uterine leiomyoma and cardiovascular disease risk factors were studied in The Northern Finland Birth Cohort 1966 (NFBC1966). The study showed an association between leiomyomas and raised cardiovascular disease risk factors, serum lipids and metabolic syndrome in particular. These findings may suggest that there are shared predisposing factors underlying both uterine leiomyomas and adverse metabolic and cardiac disease risks, or that metabolic factors have a role in biological mechanisms underlying leiomyoma development. This study provides novel information on clinical characteristics of familial uterine leiomyomas and on the immunophenotype of HLRCC-related leiomyomas. The study also offers significant confirmation of associations between uterine leiomyomas and both endometriosis and several CVD risk factors. / Tiivistelmä Kohdun leiomyoomat ovat naisten yleisin hyvänlaatuinen kasvain. Ne ovat myometriumin neoplastisia muutoksia ja ne ilmenevät joko yksittäisinä tai monilukuisina, ja ne voivat sijaita missä tahansa kohdun myometriumia. Leiomyoomat muuttavat kohdun ja kohtuontelon säännöllistä muotoa. Lisäksi ne aiheuttavat vuotohäiriöitä, alentunutta hedelmällisyyttä, ja lantion alueen painetta ja kipua. Tämän tutkimuksen tavoitteena oli laajentaa nykyistä tietämystä suvuittain esiintyvistä kohdun leiomyoomista ja selvittää mahdollista leiomyoomien ja kardiovaskulaaritautiriskin assosiaatiota, ja lisäksi selvittää leiomyoomien ja endometrioosin assosiaatiota. Suvuittain esiintyvien kohdun leiomyoomien taudinkulkua selvittävässä tutkimuksessa osoitettiin merkittäviä eroja suvuittain ja ei-suvuittain esiintyvien leiomyoomien välillä. Suvuittain esiintyvien leiomyoomien kliininen taudinkuva oli vaikeampi, leiomyoomia oli kohdussa useampia ja ne aiheuttivat useammin oireita ja lisäksi ne diagnosoitiin nuoremmalla iällä. Kohdun leiomyoomien ja endometrioosin yleisyyttä selvittävä tutkimus antoi lisävahvistusta sille havainnolle, että nämä taudit assosioivat keskenään. Tutkimustuloksen mukaan leiomyoomat ja endometrioosi vähentävät naisen hedelmällisyyttä toisistaan riippumatta. Perinnöllinen kohdun leiomyomatoosi ja munuaissyöpä (hereditary leiomyomatosis and renal cell cancer, HLRCC) -kasvainoireyhtymään liittyvän kohdun leiomyoomia selvittävän tutkimuksen tuloksien mukaan HLRCC-naisten kohdun leiomyoomien kliiniset ominaisuudet poikkeavat satunnaisesti esiintyvien leiomyoomien ominaisuuksista. Naisella HLRCC voitaisiinkin tunnistaa näiden poikkeavien ominaisuuksien perusteella, sekä immunohistokemiallisilla värjäyksillä CD34 ja Bcl-2. Fumaraattihydrataasi (fumaraasi, FH) -geenin mutaatiota kantava suku voitaisiin siten tunnistaa yksittäisen HLRCC leiomyoomatapauksen avulla. Pohjois-Suomen syntymäkohortti 1966 (Northern Finland Birth Cohort 1966, NFBC1966) tutkittiin kohdun leiomyoomia ja kardiovaskulaarisairauden riskitekijöitä. Tutkimustuloksien perusteella kohdun leiomyoomat assosioivat koholla olevien kardiovaskulaarisairauden riskien kanssa, erityisesti seerumin lipidien ja metabolisen syndrooman suhteen. Näiden tutkimustulosten perusteella voidaan esittää, että leiomyoomien ja terveydelle epäedullisen metabolian ja kardiovaskulaaritaudin riskien taustalla on mahdollisesti joitain yhteisiä altistavia tekijöitä, tai että metabolisilla tekijöillä on rooli kohdun leiomyoomien tautimekanismissa. Tämä tutkimus on tuottanut uutta tietoa suvuittain esiintyvien kohdun leiomyoomien kliinisestä taudinkuvasta ja HLRCC:n liittyvien leiomyoomien immunofenotyypistä. Lisäksi tämä tutkimus esittää lisävahvistusta kohdun leiomyoomien ja endometrioosin assosiaatiolle sekä useille kardiovaskulaaririskitekijöille.

Multipotent mesenchymal stromal cells (MSC) were first discovered in bone marrow and can be isolated from “virtually all organs”. They could participate in tissue maintenance and self-renewing process. They are able to adhere to plastic surfaces and acquire a fibroblastic shape when isolated. They are characterized by a particular phenotype and are able to differentiate into several cell types if cultivated in a specific induction medium. These characteristics were defined on MSC in culture and do not represent how they may be in situ.MSC present particular properties. They can secrete growth factors and several cytokines that give them a trophic activity on one hand and the ability to modulate the immune system on the other hand. They are also able to differentiate. These different properties make them an attractive candidate for cell therapy.MSC are already the focus of several pre-clinical and clinical studies. Nevertheless, the results of these studies are difficult to interpret due to limited understanding of their basic biology. MSC are poorly defined in situ and are heterogeneous. Their heterogeneity is dictated by their tissue of origin and cell preparation. To date, there is no standard protocol for MSC isolation and culture. This leads to numerous questions regarding patient safety, and these questions require answers.The first part of the work deals with the methods used to optimize the extraction of MSC and purification from adipose tissue, one of the main sources of autologous MSC with bone marrow. Classical methods require an enzymatic digestion step. The enzyme used and the duration of adipose tissue digestion time can induce cellular alterations and modify cell functions. Moreover, the addition of a xenobiotic increases the risk of contamination and complicates the monitoring of good manufacturing practices (GMP). We propose a method that does not require this enzymatic digestion step while being easier, safer, faster, gentler and less expensive. Compared to the classical enzymatic method, our method yields an equivalent number of MSC from adipose tissue while preserving their properties.The second part of this work focuses on the characterization of the MSC subpopulations from adipose tissue and compares them to those from bone marrow, which are the historical gold standard. The study made it possible to deepen the knowledge of MSC surface markers in situ from these 2 sources. It also evaluated the various properties of the isolated subpopulations thanks to the cell surface markers CD271, SUSD2, MSCA-1, CD44 and CD34. We showed that MSC from bone marrow express MSCA-1, CD271 and SUSD2 markers in situ. We also found that a population clearly positive for the CD34 does exist in situ with different properties compared to those of the unselected populations or the negative counterpart. 2 populations that are negative and positive for CD44 also exist with similar properties.In contrast to bone marrow MSC, only one selection was able to effectively isolate MSC from adipose tissue by a positive selection based on the expression of CD34. We also isolated a CD271+ population but only from lipoaspirate samples and not from abdominoplasty samples. Collectively, our results suggest that MSCA-1 seems to be the best marker through which to isolate MSC from bone marrow and that CD34 is the only marker able to positively isolate cells from adipose tissue. Thus, we show that the MSC from the different sources share similar properties although they have specific characteristics. The choice of the source and of the marker with which to isolate a particular subpopulation is important depending on their intended clinical use. / Les cellules stromales mésenchymateuses multipotentes (CSM) ont été mises en évidence dans la moelle osseuse et peuvent être isolées de « virtuellement tous les organes ». Elles participeraient à la maintenance et au renouvellement des tissus. Une fois isolées, elles sont capables d’adhérer à des surfaces en plastique en prenant une forme fibroblastique. Elles sont caractérisées par un phénotype particulier et peuvent se différencier en divers types cellulaires lorsque cultivées dans un milieu d’induction spécifique. Ces caractéristiques ont été définies sur les CSM en culture et ne reflètent pas forcément ce qui se passe in situ.Les CSM présentent des propriétés particulières. Elles peuvent sécréter des facteurs de croissance ainsi que de nombreuses cytokines qui leur permettent d’une part d’avoir une activité trophique et d’autre part de moduler le système immunitaire. Elles sont aussi capables de se différencier. Ces différentes propriétés les rendent particulièrement attractives pour la thérapie cellulaire.Les CSM font déjà l’objet de nombreuses études pré-cliniques et cliniques dont les résultats sont difficilement interprétables car nous n’avons à l’heure actuelle qu’une compréhension limitée de leur biologie de base. Les CSM sont encore mal définies in situ et sont hétérogènes. Cette hétérogénéité provient de leur différence d’origine et de leur préparation cellulaire :il n’existe aucune standardisation des protocoles d’isolation et de culture. Cette hétérogénéité entraine de nombreuses questions relatives à la sécurité du patient qui doivent être élucidées.La première partie de ce travail cherche à optimiser les méthodes d’extraction et de purification des CSM du tissu adipeux humain, la principale source de CSM autologues avec la moelle osseuse. Les méthodes classiques requièrent une étape de digestion enzymatique dont l’enzyme utilisée et le temps de digestion du tissu adipeux peuvent induire des altérations cellulaires et modifier leurs fonctions. De plus, l’adjonction de xénobiotiques augmente le risque de contamination et complique le suivi des bonnes pratiques de fabrication (BPF). Nous proposons une méthode qui s’affranchit de cette étape de digestion enzymatique tout en étant plus facile, plus sûre, plus rapide, moins chère et moins traumatisante pour les cellules. Elle permet d’obtenir un nombre tout aussi important de CSM du tissu adipeux que la méthode enzymatique classique en préservant leurs propriétés.La deuxième partie de ce travail vise à caractériser les sous populations de CSM du tissu adipeux humain en les comparant à celles de la moelle osseuse, source de référence historique. Cette étude a permis d’approfondir la connaissance des marqueurs de surface des CSM de ces 2 sources in situ, tout en évaluant les différentes propriétés des sous-populations isolées grâce aux marqueurs de surface CD271, SUSD2, MSCA-1, CD44 et CD34. Nous avons montré que les CSM de la moelle osseuse expriment les marqueurs MSCA-1, CD271 et SUSD2 in situ et qu’il existait une sous-population clairement positive pour le CD34 avec des propriétés différentes de celles de la population non sélectionnée ou négative pour ce marqueur. Il existe aussi 2 sous-populations positive et négative pour le CD44 avec des propriétés similaires.Contrairement aux CSM de la moelle osseuse, une seule sélection a permis d’isoler efficacement les CSM du tissu adipeux par une sélection positive sur base de l’expression du CD34. Nous avons pu aussi isoler une population CD271+ mais seulement des prélèvements de lipoaspirations et non des abdominoplasties.Au vu de nos résultats, MSCA-1 semble le meilleur marqueur pour isoler les CSM de la moelle osseuse tandis que le CD34 est le seul marqueur capable d’isoler positivement celles du tissu adipeux. Ainsi, nous montrons que les CSM issues de différentes sources partagent des propriétés similaires avec cependant des caractéristiques propres. Le choix de la source et du marqueur pour isoler une sous-population sont donc importants en fonction de leur utilité clinique envisagée. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

De nos jours, la greffe de cellules souches hématopoïétiques (GCSH) de type allogénique est le traitement standard pour de nombreuses hémopathies malignes, et ce, en dépit des taux élevés de complications liées à la transplantation, telles que les infections, les rechutes et la maladie du greffon contre l’hôte (GVH), associées à cette procédure. Depuis les deux dernières décennies, plusieurs stratégies visant à contrôler la maladie du GVH ont émergé et incluent la déplétion partielle et la manipulation ex vivo des cellules T du greffon. En revanche, ces stratégies peuvent entrainer de sévères déficits immunitaires post-greffe. En comparaison avec les autres sources de cellules souches, une faible incidence de la maladie du GVH et de rechute est observée chez les patients recevant une greffe de sang de cordon ombilical (SC). En contrepartie, ce type de greffe engendre des retards dans la restauration immunitaire, rendant ainsi les patients plus susceptibles aux agents pathogènes. À l’inverse, une augmentation plus importante de la production de cellules T naïves, d’émigrants thymiques récents et de l’abondance des clonotypes des cellules T fut détectée chez les patients ayant reçu une GCSH dérivée du sang d’un seul cordon traité avec UM171, une molécule utilisée pour l'amplification ex vivo des CSHs dérivés de SC, relativement aux patients ayant reçu une GCSH standard. Dans ce mémoire, nous essaierons de comprendre le mécanisme moléculaire sous-jacent à la restauration immunitaire chez les patients transplantés avec un greffon de SC traité avec UM171, nous tenterons de déceler des progéniteurs lymphoïdes au sein des cellules de SC traitées avec UM171 et nous essaierons d’identifier un marqueur de surface qui pourrait être utilisé pour enrichir les progéniteurs lymphoïdes. Nos résultats démontrent la présence de deux "clusters" lymphoïdes, PCL et LMPP, qui expriment potentiellement CD10, et sont plus nombreux dans les échantillons de SC CD34+ traités avec UM171 comparativement aux contrôles. En utilisant les marqueurs de surface CD10 et CD45RA, nous avons pu identifier deux populations CD34+ (CD10medCD45RAmed/lo et CD10hiCD45RAhi) qui sont multipliées dans les cultures traitées avec UM171 relativement aux contrôles. En outre, nous démontrons également que seules les cellules CD45RA- peuvent générer les deux populations CD10+ in vitro, ce qui suggère que les cellules CD10medCD45RAmed/lo définissent une population plus primitive que les cellules CD10hiCD45RAhi. De surcroit, nous proposons que la population CD10medCD45RAmed/lo pourrait contenir des LMPPs qui se différencieront en PCLs inclus dans la population CD10hiCD45RAhi. D'un point de vue fonctionnel, nous observons un développement et une maturation des lymphocytes T nettement plus rapide pour la population CD10+CD34+ relativement à la population CD34+ totale à 3 et 6 semaines, ce qui suggère que CD10 pourrait être utilisé comme marqueur de progéniteurs lymphoïdes. Par ailleurs, nous détectons également un pourcentage plus élevé de cellules ayant un phénotype ILC dans la population CD10+CD34+ comparativement à la population CD34+ totale, ce qui suggère que la population CD10+CD34+ peut potentiellement contenir des progéniteurs lymphoïdes capables de produire différents types de cellules immunitaires. D’autre part, la population CD45RA+ produit plus rapidement des lymphocytes T contrairement à la population CD45RA-. En conséquence, nous proposons que la population CD45RA- contient des cellules primitives (ex. CSH) qui à leur tour génèrent des progéniteurs multipotents contenus dans la population CD10+CD34+ (ex. LMPP) et qui éventuellement produiront des progéniteurs à potentiel plus restreint inclus dans la population CD45RA+ (ex. CLP). En somme, ce mémoire identifie pour la première fois une population CD10+CD34+ présente dans le greffon traité avec UM171 avec un potentiel T et produisant potentiellement des ILCs. L’un des problèmes majeurs liés à la GCSH dérivée du SC est une restauration immunitaire retardée; par conséquent, optimiser l’infusion de cellules CD34- en augmentant le nombre de progéniteurs lymphoïdes pourrait considérablement aider à stimuler la restauration immunitaire chez les patients recevant une GCSH dérivée de SC. / Currently, allogeneic hematopoietic stem cell transplantation is a standard treatment for many hematological malignancies, although high rates of transplant-related complications such as infections, relapse, and GVHD has constrained the implementation of HSCT. Strategies have emerged to manage GVHD and include partial T-cell depletion and ex vivo manipulation of donor T cells, albeit they have adverse effects on post-transplantation immune recovery and can cause profound immunodeficiency. In comparison to other stem cell sources, a lower incidence of chronic GVHD and relapse is reported in patient receiving cord blood (CB) transplant, although they also display an enhanced susceptibility to pathogens caused by an ineffective T-cell reconstitution. In contrast, we reported a greater increase in naïve T cells production, recent thymic emigrants and T cell clonotype from 3 months to 6 and 12 months in patient transplanted with a single CB graft expanded with UM171, a small molecule used for the ex vivo expansion of CB HSCs, as compared to counterpart patients receiving unmanipulated CB graft. In this master research, we aimed to understand the molecular mechanism underlying T-cell reconstitution in patients transplanted with UM171- expanded CB graft. We tried to identify lymphoid progenitors within the highly heterogeneous CD34+ CB cells treated with UM171 using Cite-Seq, find a surface marker that can be used to enrich for early lymphoid progenitors using flow cytometry, and assess their lineage potential using artificial thymic organoids. Our results highlight the presence of two lymphoid clusters, CLP and LMPP, expressing CD10. The LMPP cluster is about 6 fold expanded in UM171-treated cells as compared to uncultured and DMSO-treated cells. Using the marker CD10 and CD45RA we identify two CD34+ populations (CD10medCD45RAmed/lo and CD10hiCD45RAhi) that are significantly expanded in CD34+ CB cells cultured in the presence of UM171 in contrast to uncultured CD34+ CB cells and DMSO-supplemented cultures. Furthermore, we demonstrate that only the CD45RA-negative cells can give rise to both CD10-positive subsets in vitro, suggesting that CD10medCD45RAmed/lo cells define a subset with a more primitive phenotype than CD10hiCD45RAhi. We propose that the CD10medCD45RAmed/lo subset could contain LMPPs that will commit to CLPs contained within the CD10hiCD45RAhi subset. Functionally, we compared T-cell development and maturation of the CD10+CD34+ subset (including CD10medCD45RAmed/lo and CD10hiCD45RAhi) to the CD45RA+CD34+, CD45RA-CD34+ and total CD34+ subsets and denote a faster T cell development and maturation at 3 and 6 weeks within the CD10-positive subset in contrast to the total CD34+ subset, suggesting that CD10 can be used as a marker of lymphoid precursors in UM171 cultures. We also observe a higher percentage of ILC-like cells within the CD10-positive subset as compared to total CD34+ cells suggesting that the CD10- positive subset potentially contains lymphoid precursors with multilineage capacity. Faster T cell development is observed for the CD45RA+ subset whereas CD45RA- cells display the slowest T cell development, hence we propose that the CD45RA- subset contains primitive cells (e.g. HSC) that segregate into lineage-biased multipotent progenitors enriched in the CD10-positive subset (e.g. LMPP) that will give rise to lineage-restricted precursors that are CD45RA+ (e.g. CLP). In sum, our findings represent the first identification of a CD10-positive population within the UM171- expanded graft that has T potential and could potentially produce ILCs. Delayed immune reconstitution is a major obstacle impeding the implementation of CB HSCT; therefore optimizing infusion of CD3+ donor cells by enriching for lymphoid precursors could help boost T-cell reconstitution following allogeneic HSCT.

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Human cytomegalovirus (HCMV) is a widespread human herpesvirus pathogen and prototypical member of the β-herpesvirus subfamily. Like all herpesviruses, the virus establishes a lifelong latent infection following host exposure, which has the potential to reactivate periodically and contribute to recurrent disease processes. In individuals with weak or compromised immune systems, such reactivation can lead to profound pathology. Understanding how latent infections are maintained is important for uncovering how HCMV causes disease. The study of viral genes that are expressed during latent infection grants insight into how latency is regulated and how it could be therapeutically targeted. To that end, this project has sought to evaluate the functional significance of one such viral gene termed LUNA in the context of latency. In models of experimental latent infection based on primary myeloid cells, levels of viral gene transcription were found to be significantly reduced following infection with LUNA deletion mutant viruses, consistent with corresponding observable changes in post-translational histone modifications over the viral promoters of latency-associated genes. Additionally, using luciferase reporter systems, latency-associated viral gene promoters became activated in response to the expression of wild-type LUNA. Together, these findings argue for a role of LUNA in regulating viral gene expression during latent HCMV infection. One possible mechanism by which LUNA may fulfil its role is by targeting cellular ND10 structures, known intrinsic inhibitors of herpesvirus gene expression, for disruption. In support of this, latently infected cells were found to be devoid of ND10, a phenotype that was recapitulated by the direct expression of wild-type LUNA. Furthermore, mutation studies confirmed the identification of a novel deSUMOylase activity encoded by LUNA that was responsible for mediating ND10 disruption. Use of a catalytically inactive LUNA mutant in transcriptional analyses of latent infection also generated similar results as with the LUNA deletion viruses. Overall, these data support the hypothesis that LUNA serves as an important regulator of viral gene expression during latency, which is likely linked to its ability to target ND10 structures for disruption, thus raising the possibility that inhibition of deSUMOylation may serve as a novel therapeutic strategy to target latent HCMV infection.