O complexo da esclerose tuberosa (TSC) é um transtorno clínico, com expressividade variável, caracterizado por hamartomas que podem ocorrer em diferentes órgãos. Tem herança autossômica dominante e é devido a mutações em um de dois genes supressores de tumor, TSC1 ou TSC2. Estes codificam para as proteínas hamartina e tuberina, respectivamente, que se associam formando um complexo macromolecular que regula funções como proliferação, diferenciação, crescimento e migração celular. As lesões cerebrais podem ser muito graves em pacientes com TSC e caracterizam-se por nódulos subependimários (SEN), astrocitomas subependimários de células gigantes (SEGA), tuberosidades corticais e heterotopias neuronais, podendo relacionar-se clinicamente à epilepsia refratária à terapia medicamentosa, deficiência intelectual, desordens do comportamento e hidrocefalia. O potencial de crescimento de SEGA até os 21 anos de idade dos pacientes exige acompanhamento periódico por exame de imagem e condutas clínicas ou cirúrgicas, conforme indicação médica. As lesões subependimárias têm sido explicadas por déficits de controle da proliferação, crescimento e diferenciação de precursores neuro-gliais na zona subventricular telencefálica. Embora a capacidade da tuberina em inibir a proliferação celular pela repressão do alvo da rapamicina em mamíferos (mTOR) esteja bem documentada, outros aspectos celulares do desenvolvimento de SEGA ainda não foram examinados. Assim, é importante estabelecer um sistema in vitro para o estudo de células da zona subventricular e testá-lo na análise das proteínas hamartina e tuberina. Neste sentido, o cultivo de neuroesferas em suspensão é muito apropriado. Neste estudo, buscamos relacionar a expressão e distribuição subcelular da hamartina e tuberina à capacidade proliferativa e de diferenciação das células de neuroesferas cultivadas in vitro a partir da dissociação da vesícula telencefálica de embriões de ratos normais. Analisamos a expressão e distribuição subcelular da hamartina e tuberina por imunofluorescência indireta em células entre a primeira e a quarta passagens das neuroesferas, sincronizadas nas fases G1 ou S do ciclo celular e após a reentrada no ciclo celular, através da incorporação de 5-bromo-2\'-desoxiuridina (BrdU) e imunofluorescência com anticorpo anti-BrdU. Em geral, células de neuroesferas apresentaram baixa colocalização entre hamartina e tuberina in vitro. A expressão da tuberina foi elevada em basicamente todas as células das esferas e fases do ciclo celular; ao contrário, a hamartina apresentou-se principalmente nas células da periferia das esferas. A colocalização entre hamartina e tuberina foi observada em células mais periféricas das esferas, sobretudo no citoplasma e, em G1, no núcleo celular. A proteína rheb, que conhecidamente interage diretamente com a tuberina, apresentou distribuição subcelular muito semelhante à desta. Ao carenciamento das células visando à parada do ciclo celular na transição G1/S, tuberina distribuiu-se ao núcleo celular em quase todas as células avaliadas e, de forma menos frequente, a hamartina também. À reentrada no ciclo celular pelo reacréscimo dos fatores de crescimento, avaliaram-se células com incorporação de BrdU ao seu núcleo celular, após 72 e 96 horas. Nestas, tuberina mostrou-se novamente no citoplasma de forma preponderante e hamartina manteve-se citoplasmática, em geral subjacente à membrana plasmática, em níveis mais baixos. Os grupos cujas células reciclaram por 72 ou 96 horas diferiram quanto ao aumento significativo da expressão da hamartina em células proliferativas no último. À diferenciação neuronal, aumentaram-se os níveis de expressão de hamartina observáveis à imunofluorescência indireta, tornando-se equivalentes àqueles da tuberina. Concluímos que as células de neuroesferas cultivadas em suspensão apresentam-se como um sistema apropriado ao estudo da distribuição das proteínas hamartina e tuberina e sua relação com o ciclo celular / The tuberous sclerosis complex (TSC) is a clinical disorder with variable expressivity, characterized by hamartomas that can occur in different organs. It has autosomal dominant inheritance and is due to mutations in one of two tumor suppressor genes, TSC1 or TSC2. These encode for the proteins hamartin and tuberin, respectively, which are associated in a macromolecular complex which functions as a regulator of cell proliferation, differentiation, growth and migration. TSC brain lesions may be severe and are characterized by subependymal nodules (SEN), subependymal giant cell astrocytomas (SEGA), neuronal heterotopias and cortical tubers, and may be clinically related to refractory epilepsy, intellectual disability, behavioral disorders and hydrocephaly. The growth potential of SEGA up to 21 years of age in TSC patients requires regular monitoring by imaging. Clinical and surgical interventions may be medically indicated. Subependymal lesions have been explained by deficient control of proliferation, growth and differentiation of neuro-glial progenitors from the telencephalic subventricular zone. While tuberin ability to inhibit cell proliferation by repressing the mammalian target of rapamycin (mTOR) has been well documented, other cell aspects of SEGA development have not been thoroughly examined. Therefore, it is important to establish conditions for an in vitro system to study the cells from the subventricular zone and to test its suitability for the study of the TSC proteins. In this regard, the neurosphere suspension culture is very appropriate. We evaluated the expression and subcellular distribution of hamartin and tuberin in relation to the proliferation and differentiation capability of neurosphere cells derived in vitro from the dissociation of the telencephalic vesicle of normal E14 rat embryos. These analyses were performed by indirect immunofluorescence in cells from first through fourth passages of neurospheres, synchronized in G1 or S phases of the cell cycle, and after reentry into the cell cycle by the addition of 5-brome-2\'-desoxyuridine (BrdU) and immunolabeling with anti-BrdU antibody. In general, neurosphere cells presented low colocalization between hamartin and tuberin in vitro. Tuberin expression was relatively high in basically all neurosphere cells and cell cycle phases, whereas hamartin distributed mainly to cells from the periphery of the spheres. In these cells, hamartin and tuberin colocalization was evident mostly in the cytoplasm and, in G1, also in the cell nucleus. Rheb, which is known to interact directly with tuberin, had subcellular distribution very similar to tuberin. Cell starvation indicating cell cycle arrest at G1/S redistributed tuberin to the cell nucleus in virtually all cells examined, what was accompanied by nuclear location of hamartin in a small subset of cells. When cells were allowed to reenter cell cycle by adding growth factors, we evaluated BrdU-labeled nuclei 72 and 96 hours later. In the two groups, tuberin was shown to move back to the cytoplasm as well as hamartin, which apparently maintained its lower expression levels distribution underneath the plasma membrane. Group of cells that recycled for 96 hours had significantly more expression of hamartin than those cells that cycled for only 72 hours. After neuronal differentiation, hamartin expression levels observed by immunofluorescence were similar to those of tuberin. We conclude that neurosphere cells cultured in suspension showed to be an appropriate cell system to study hamartin and tuberin distribution in respect to the cell cycle

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.

Submitted by Biblioteca do Instituto Biomédico BIB (uffbib@gmail.com) on 2017-06-05T19:47:11Z No. of bitstreams: 1 Helena Naly Miguens Rocha.pdf: 1130470 bytes, checksum: 43146823dc28af52b34aa41dd863ff95 (MD5) / Made available in DSpace on 2017-06-05T19:47:11Z (GMT). No. of bitstreams: 1 Helena Naly Miguens Rocha.pdf: 1130470 bytes, checksum: 43146823dc28af52b34aa41dd863ff95 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Mecanismos de ativação e reparo endoteliais em resposta ao fluxo sanguíneo retrógrado (FSR) exacerbado ainda não foram completamente elucidados, nem em condições fisiológicas, nem na hipertensão arterial sistêmica (HAS). O objetivo deste estudo foi determinar os efeitos do FSR exacerbado sobre biomarcadores endoteliaisem indivíduos saudáveis e com HAS. Oito homens saudáveis (grupo CT; 36±3) e oito pacientes com HAS(grupo HAS;39±5) foram submetidos a manobra de indução de FSR em um dos braços, através da insuflação de dois manguitos, um no antebraço a 75mmHg e outro manguito próximo ao ombro a 40 mmHg, por 30 minutos. A avaliação do fluxo sanguíneo (ultrassom vascular) e a coleta de sangue foram realizadas no momento basal e no 30º minuto de manobra em ambos os braços (contralateral e ipsilateral). Ativação endotelial, micropartículas endoteliais (MPE) e células progenitoras endoteliais (CPE) foram mensuradas por citometria de fluxo. Nitrito foi mensurado por NOA Sievers. Em condições basais, fluxo sanguíneo médio, condutância vascular, taxa de cisalhamento média (p<0,01) e MPE (p=0,03) foram maiores no grupo HAS quando comparado ao grupo CT. Níveis basais de CPE estavam reduzidos no grupo HAS, permanecendo assim durante a manobra (p<0,01). Ambos os grupos apresentaram redução no fluxo sanguíneo médio e na condutância vascular (p≤0,01), bem como aumento na taxa de cisalhamento retrógrado (p<0,01) e no índice de cisalhamento oscilatório (p<0,01) durante a manobra. Somente o grupo HAS aumentou o número de MPE (p=0,02) e a ativação endotelial (p=0,04) durante a manobra. A razão MPE/CPE foi maior em ambos os momentos no grupo HAS (p<0,02). A resposta dos níveis séricos de nitrito a manobra foi menor no grupo HAS (p=0,03). Conclui-se que pacientes com HAS apresentam um quadro subclínico de disfunção endotelial com comprometimento no reparo vascular, o que foi agravado pela indução de FSR. / Endothelial activation and repair mechanism in response to increased retrograde blood flow (RBF) have not beenfully elucidated, neither in physiological conditions nor in hypertension. We aimed to determinethe effects of increased RBF on endothelial biomarkers in healthy individuals and hypertensive patients. Eight healthy subjects (CT group; 36±3) and eight hypertensive men (HT group; 39±5) underwent a maneuver to increase RBF, using two pneumatic cuffs: one in the forearm, inflated to 75 mmHg and one near the shoulder, inflated to 40 mmHg, for 30 minutes.Blood flow measures (ultrasound doppler) and blood samples were obtained at baseline and during the last minute of the maneuver from both arms (ipsilateral and contralateral). Endothelial activation, endothelial microparticle (EMP) and endothelial progenitor cell (EPC) were measured by flow cytometry. Nitrite was measured through NOA Sievers.At baseline, mean blood flow, vascular conductance, mean shear rate (p<0.01) and EMP (p=0.03) were higher in HT group than CT group. Baseline EPCs levels were reduced in HT group, which was sustained during the maneuver (p<0.01). Both groups presented decreased mean blood flow and vascular conductance (p<0.01), along with increased retrograde shear rateand oscillatory shear index (p<0.01), during the maneuver. Only the HT group showed increased EMP (p=0.02) and endothelial activation levels (p=0.04). EMP/EPC was higher in HT group in both moments (p<0.02).Nitrite levels in response to the maneuver were lower in HT group (p=0.03).Hypertensive patients present a subclinical endothelial dysfunction along with impaired endothelial repair, which was worsened by the RBF induction.

A. Background<p><p>Skin wound healing (WH) is a dynamic and extremely determinate process of cellular, humoral and molecular mechanisms which begins directly after wounding and can last for years. WH is described as is an intricate process in which the skin (or another organ-tissue) repairs itself after injury. The process of skin WH occurs through the actions of an interplay of cells, growth factors and cytokines leading to wound closure.<p><p>WH occurs in three precisely and highly programmed phases: the inflammatory phase (day 0 to day 7) followed by the proliferative phase or vasculogenic phase (day 7 to day 21) and finally the remodeling phase (2 days - up to 2 years). For a successful healing, all three phases must occur in the proper sequence and time frame.<p><p>Many factors can interfere with one or more phases of the WH process, thus causing improper or impaired healing. The proliferation phase, in particular, requires the participation of various cells types such as fibroblasts, endothelial cells (ECs) and endothelial progenitor cells (EPCs), to produce a healthy well-vascularized granulation tissue for epithelization and wound closure.<p><p>A.1 Wound Healing And Obesity<p><p>In 2008, over 1.4 billion adults, 20 and older, were overweight. Of these, obesity has been shown to affect over 500 million people (OMS website). Moreover, the prevalence of obesity continues to rise, and by 2018, it is estimated that obesity will cost $ 347 billion annually.<p><p>Each year, in the US, approximately 33 million overweight and obese patients undergo surgery. Obesity causes a number of known health problems and increased post-surgical complications such as wound infection, dehiscence, hematoma and seroma. Surgeons anecdotally report WH complications among obese patients; however, little research has been conducted to investigate the mechanisms mediating impaired obesity-related WH. <p><p>Some previous work on diabetic patients and diabetic mice showed an imbalance between pro-oxydant and anti-oxydant genes as well as impaired EPCs proliferation and tube formation during the WH process. More then a hundred cytologic factors have been found to impair WH in the type 2 diabetic patient. It is a very complex and multifactorial problem involving decreased growth factors secretion, impaired keratinocyte and fibroblast functions, impaired EPs function, alteration of the macrophage function and granulation tissue synthesis, etc. <p><p>Based on these findings and because obesity is associated with the development of type 2 diabetes, we hypothetize that, impaired balance between pro-apoptotic/anti-apoptotic and pro- oxydant /anti-oxydant genes is involved in impaired WH. Furthermore, we hypothetize that impaired EPCs function leads to the perturbation of the proliferation phase of obesity impaired WH.<p><p>A.2. Wound Healing and Age<p><p>The world population is aging; by 2030, nearly 20% of Americans, (± 72 million people), will be 65 years old and older. In 2010, 17% of the European population was over the age of 65. By 2060, it is projected that the share of those aged 65 and over will rise to 30%, accounting for more then 150 million people. (ec.europa.eu) These aging subjects undergo an increasing number of surgical procedures: in the past two decades, the percentage of surgeries in patients over 65 has doubled to nearly 40%.<p>As a corollary, it is well established knowledge that elderly WH is impaired. However, little is known about the underlying mechanisms of age-related impaired WH.<p><p>As previously mentioned, adult BM-derived EPCs contribute to peripheral tissue repair and regeneration. In light of the abundant literature suggesting that neovascularization is impaired in the elderly, we characterize a novel model of senile cutaneous WH and investigate the role that vasculogenesis plays in the pathogenesis of age related impaired WH.<p>Aged mice colonies have traditionally been the model for aged small mammalian research, however, the ability to use a readily-available transgenic mouse model with features of accelerated aging would aid in the exploration of targeted therapies and a great number of age-related investigations.<p><p>We hypothesize that the Hutchinson-Gilford Progeria Syndrome (HGPS) Zmpste24 deficient (Zmpste24-/-) mouse mimics physiological ageing and can be used as a novel model for the study of senescent WH. We further hypothetized that impaired balance between pro-apoptotic/anti-apoptotic and pro-oxydant /anti-oxydant genes as well as impaired EPCs function are responsible for the impairment of the proliferative phase, leading to overall impaired WH.<p><p>A.3 Aims<p><p>Recently, a great deal of research has been directed at understanding the critical factors inducing poorly healing wounds. However, a lot remains unclear.<p><p>It is now well accepted that new blood vessel formation occurs not only by angiogenesis (blood vessels formation from a preexisting network of capillaries), but also by vasculogenesis (blood vessels formation from BM SCs recruitment) and that EPCs contribute to as much as 25% of new blood vessels formed in healing tissues4. They are mobilized from the BM in response to injury and production of local cytokines, are incorporate into wounds and play an integral role in systemic tissue repair. <p><p>Based on this finding, we hypothesized that co-morbidities related impaired WH may be due, in part, to decreased EPCs number, migration/homing, and/or function resulting in impaired vasculogenesis. Because age and/or obesity have been shown to be one of the most common predictors of altered WH, we decided to focus on these two parameters.<p><p>Following a bedside to bench approach the purpose of this work was to 1) develop coherent and translatable models of co-morbidity digging in the physiologic/pathologic mechanisms underlying altered healing in obese and senile mice; 2) develop targeted therapeutics to improve impaired WH.<p><p>B. Material and Methods<p><p>B.1 Human Model<p><p>Since obesity impairs WH and BM EPCs are important for tissue repair, we hypothesize that obesity- impaired WH is due, in part, to impaired EPCs mobilization, trafficking, and function. Peripheral blood was obtained from non diabetic, obese (BMI > 30, n = 25), and non obese (BMI < 30, n = 17) subjects. Peripheral blood human EPCs were isolated, quantified, and functionally assessed.<p>As for aged impaired WH, EPCs of aged subjects have already been found to have decreased adhesion, migration and proliferative properties as well as being decreased in number in elderly patients undergoing surgery compared to younger patients.<p><p>B.2. Mice Models<p><p>Two models of WH were developed and characterized.<p>In order to isolate the effect of obesity on EPCs and WH, OB non-diabetic female TallyHo/JngJ mouse were selected (Female mice don’t express hyperglycemia and hyperinsulinemia). Female SWR/J non-OB mice were used as control mice. In order to limit variables, TallyHO/JngJ obese mice were selected over other OB mice that exhibit a polygenic type of obesity (Jackson Laboratory Website). By selecting this mouse model, we have excluded in our selection of the ideal model common confounding factors such as hyperglycemia, hyperinsulinemia, immune disorders.<p><p>Zmpste24 is a metalloproteinase involved in the maturation of lamin A (LmnA), an essential component of the nuclear envelope. When Zmpste24 or LmnA are knocked-out, mice exhibit profound nuclear architectural abnormalities and histopathological defects that phenocopy an accelerated aging process. Of crucial importance, the lamin-A dependent nuclear alterations seen in Zmpste24-deficient mice have also been found in human physiological aging. We defined the utilization of the Hutchinson-Gilford Progeria Syndrome (HGPS) Zmpste24 deficient (Zmpste24- /-) mouse as a novel model for the study of senescent WH (controls used were C57BL/6J mice).<p><p>B.3. Wounding Model and Data Collection<p><p>All mice group underwent wounding using a stented wound model developed in our laboratory and previously published. Briefly, paired 6-mm circular, full-thickness wounds extending through the panniculus carnosus were made on the dorsal skin of the mouse. An O-ring, 12-mm splint made of silicone sheeting was then sutured to the skin around the wound. To minimize wound contraction and reliably recapitulated the granulation and re-epithelialization seen in human WH by secondary intention. Time to wound closure was measured using standardized digital photographs taken on days 0, 7, 14, and 21. Wound closure was calculated as a percentage of the original wound.<p><p>For each model, EPCs were harvested, quantified by flow-cytometry and their function tested. Wounds were harvested at various time points and RNA, DNA and protein analysis were conducted. Finally immunohistochemistry to assess epidermal thickness, vascularity and WH were also realized.<p><p>In a second step, after characterization of the models, local (using targeted siRNA gel) and systemic therapies (using AMD3100, a PC mobilizer) were applied on the wounds and compared to controls. WH was monitored. We conducted the previously mentioned analysis (RT-PCR, ELISA and DNA analysis) on the harvested samples.<p><p>All values are expressed as a mean ± standard error of mean (SEM). The number of mice per treatment group was determined using G*Power (G*Power©, Melbourne, Australia) to provide a power greater than 0.80. Student T test was realized to compare two groups among each other.<p><p>C. Results<p><p>C.1. Human EPCs Have Impaired Function<p><p>There was no difference in the number of baseline circulating human EPCs in non-diabetic OB and non-OB<p>subjects, but EPCs from OB subjects had impaired adhesion (p<0.05), migration (p<0.01), and proliferation (p<0.001).<p><p>C.2. Obesity and Wound Healing<p><p>TallyHo/JgnJ OB mice demonstrated significantly impaired healing when compared to SWR/J control mice. They healed at an average of 28 ± 2 days (p<0.05). Post-wounding circulating EPCs were quantified and wounds were analyzed. Circulating EPCs recruitment is impaired in wounded TallyHo/JngJ mice and their wounds shown significantly decreased new blood vessel formation through decreased HIF-1α/SDF-1α signaling (p<0.05). Their wounds are characterized by increased apoptosis, increased DNA damage and impaired pro-/anti-oxydant balance. Immunonistochemistry and histology showed decreased vascular vessels in TallyHo/JngJ wounds and thinner epidermal thickness.<p><p>In the local treatment phase, local p53 silencing consistently improved WH to a nearly normal healing time (wounds healed in 18 ± 2 days, p<0.05). sip53 treatment showed a significant decrease in pro-apoptotic markers (p53, Bax, PUMA p<0.05) and a significant increase in angiogenic markers (VEGF, SDF-1α, HIF-1α) with increased blood vessel formation and decreased DNA damage.<p><p>C.3. Age and Wound Healing<p><p>In these experiments, we show that not only is Zmpste24-/- WH impaired when compared to C57BL/6J mice (Zmpste24-/- mice healed at average 40 days ± 2 days p<0.05) at all time points but that they also showed decreased vascularity and proliferation in the wound bed (p<0.05).<p><p>Histological analysis was performed utilizing hematoxylin and eosin staining to assess epidermal thickness, CD31 immunofluorescence to assess vascular density, p53 and caspase 3 to assess apoptosis, 8’OHdG staining to assess DNA damage and PCNA to assess proliferation. Epidermal thickness was significantly decreased in Zmpste24-/- animals compared to WT as well as vascular density, and proliferation in Zmpste24-/- wound tissue (p<0.05). <p><p>Circulating vasculogenic EPCs recruitment was impaired in Zmpste24-/- mice and their wounds showed significantly decreased new blood vessel formation through decreased HIF-1α/SDF-1α signaling (p<0.05). Zmpste24-/- wounds are characterized by increased apoptosis and an abnormal rise in ROS.<p>In the treatment phase, local p53 silencing consistently improved healing by more then a two fold (18 ± 2 days). VEGF production was significantly increased and pro-apoptotic factors were significantly downregulated in siRNA-treated Zmpste24-/- mice (p<0.05). DNA damage due to ROS production was also shown to be significantly decreased following treatment. Our results suggest a vasculogenic dysfunction in wound closure and showed that the specific knock down of p53 significantly improves WH.<p><p>Because EPCs showed impaired function, lower peripheric blood counts and impaired SDF-1α/HIF-1α signaling, we hypothesized that improving their mobilization by using a progenitor cell mobilizer, AMD3100, known to mobilize SCs from the BM, in a systemic treatment phase will improve WH. Peripheral blood counts were significantly increased and time to wound closure significantly decreased (20 days ± 2, p<0.05). Vasculogenic markers and anti- apoptotic molecules were upregulated compare to non-treated animals.<p><p>D. Conclusions<p><p>Obesity impaired wound closure is a complex problem with many contributory factors. Our results suggest that obesity impairs the BM-derived EPCs response to peripheral injury and this, in turn, impairs wound closure. This impairment is associated with decreased new blood vessel formation and increased DNA damage leading to an increase in the p53 pathway. We also demonstrate that targeted siRNA therapy can partially rescue impaired WH due to obesity. Based on these results we support the encouraging argument that, WH and closure has the potential be improved through specific local and systemic therapies in vivo in our rodent model and that further studies are needed to support this in a clinical environment.<p><p>Impaired WH due to ageing is a complex phenomenon that is partially understood. We demonstrate that the Zmpste24-/- transgenic knockout mouse provides a model for age-related WH investigation. Zmpste24-/- animals heals their wounds with significant delays, showed impaired EPCs mobilization following wounding through an impaired HIF-1α/SDF-1α pathway and increased apoptosis. Furthermore, WH can be improved through specific local siRNA therapy and systemic stem cell mobilization therapies.<p><p>Our results suggest strong similar patterns between obesity and ageing in the way they mediate WH impairments trough (premature) ageing. Our encouraging endeavor to bring WH back to baseline in these diseased models underlines the possibility to reverse the microenvironment alterations and improves EPCs contribution to the WH process. Because EPCs are involved in virtually every tissue repair process happening in the human body, we hope that this work will lead the way for new research in various fields in medicine to improve wound care and quality of life of patients. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

L’endothélium joue un rôle primordial dans le développement et le maintien des multiples fonctions vasculaires. Il est ainsi largement impliqué dans des situations pathologiques comme les maladies cardio-vasculaires. La description de marqueurs endothéliaux circulants a permis une exploration non invasive de l'endothélium. Notre équipe s’est intéressée principalement aux cellules endothéliales circulantes (CEC), dont le taux reflète la lésion ou l’activation de l’endothélium, et aux progéniteurs endothéliaux circulants (PEC), marqueurs de régénération endothéliale. La découverte en 1997 par Asahara de la présence chez l’adulte de ces PEC, participant à la formation de nouveaux vaisseaux par vasculogenèse, a ouvert de nouvelles perspectives, notamment pour la thérapie cellulaire des pathologies ischémiques. Ce travail a consisté à développer les méthodes d’étude de ces cellules dans plusieurs contextes. Tout d’abord, nous avons exploré l’utilité de ces marqueurs dans la physiopathologie de l’hypertension artérielle pulmonaire (HTAP). Puis nous avons analysé le potentiel de mobilisation des progéniteurs endothéliaux à partir de la paroi vasculaire lors d’une ischémie locale chez des volontaires sains dans le cadre du développement d’un produit de thérapie cellulaire autologue. Une partie de ce projet a été de mettre en place et d’optimiser les techniques d’étude de ces marqueurs. Les CEC ont été quantifiées par immunoséparation magnétique (IMS), technique mise au point en 1992 (Dignat-George 1992) et transférée dans notre laboratoire. La quantification des PEC a été réalisée par cytométrie en flux et par culture cellulaire. En culture, deux types de PEC sont décrits : les PEC précoces, dont l’origine est monocytaire et pour lesquels la culture est déjà standardisée, et les « Endothelial Colony Forming Cells » (ECFC), seules cellules présentant des caractéristiques de cellules endothéliales progénitrices et pouvant être proposées comme produit de thérapie cellulaire. Nous avons optimisé la quantification des ECFC en culture en étudiant l’effet de diverses matrices et de la densité d’ensemencement des cellules mononucléées issues du sang total sur l’obtention de ces cellules et leurs propriétés angiogènes. La dysfonction endothéliale a été décrite comme un élément central dans le développement de l’HTAP dont le diagnostic repose sur la mesure de la pression artérielle pulmonaire par cathétérisme cardiaque droit. En l’absence de marqueur biologique non invasif dans cette maladie, nous avons quantifié les CEC et les progéniteurs circulants dans deux études. Une étude réalisée chez des patients adultes a montré une augmentation spécifique des CEC dans l’HTAP et non dans l’hypertension pulmonaire thromboembolique chronique. Ainsi les CEC semblent être le reflet des lésions endothéliales pulmonaires et non de la sévérité clinique des patients. L’autre étude a montré l’intérêt de la quantification des CEC dans la prise en charge thérapeutique des enfants souffrant d’HTAP secondaire à une cardiopathie congénitale, dont les formes irréversibles présentaient des taux élevés de CEC. Nous avons ainsi défini un nouveau marqueur non invasif à utilité diagnostique et pronostique. Les PEC sont des cellules rares dans le sang circulant, difficiles à expandre, et dont les essais de mobilisation médullaire se sont révélés insuffisants. L’hypothèse récente d’une réserve vasculaire des progéniteurs endothéliaux nous a conduits à étudier l’effet d’un processus d’ischémie locale sur la mobilisation de ces cellules chez des volontaires sains. Deux groupes d'âge ont été inclus afin d'évaluer l'impact du vieillissement sur la méthode de mobilisation étudiée. Malgré un effet de cette ischémie sur la dilatation endothéliale cette méthode n’a pas permis de mobiliser significativement les PEC issus de la paroi endothéliale, quel que soit l'âge des sujets. A l’inverse, l’hypoxie a eu un effet délétère sur les capacités angiogènes des ECFC. / The endothelium plays a key role in the development and the homeostasis of vascular functions. It is also well involved in pathological situations like cardiovascular diseases. Thanks to the description of circulating endothelial markers, non invasive study of the endothelium is now possible. Our group was particularly interested in circulating endothelial cells (CECs), the level of which reflects an endothelial activation or lesion, and to circulating endothelial progenitors cells (EPCs), markers of endothelial repair. EPC description by Asahara in 1997 in adult blood, involved in new blood vessel formation by vasculogenesis, offered new perspectives, specially for cell therapy in ischemic diseases. This work consisted in the development of methods to study these markers in different contexts. First, we explored the interest of these markers in the physiopathology of pulmonary arterial hypertension (PAH). Then we evaluated endothelial progenitors mobilization from the vascular wall by a local ischemia process in healthy volunteers, in the perspective of an autologous cell therapy product development. One part of this project was the implementation and optimization of the methods to study CEC and EPC. CEC were quantified by magnetic immunoseparation. This technique was developped in 1992 by F. Dignat-George's group and transferred in our laboratory. EPC were quantified by flow cytometry and cell culture. Two types of EPC are described in culture: the early EPC, which originate from monocyte lineage and which culture is standardized, and the « Endothelial Colony Forming Cells » (ECFC), the only cells presenting endothelial progenitor cell properties and which use as a cell therapy product can be considered. ECFC quantification by culture was optimized by assessment of the impact of diverse matrices and seeding concentrations of mononuclear cells isolated from whole blood, on ECFC commitment and their angiogenic properties. Endothelial dysfunction was described as a central element in the development of PAH, which diagnosis is based on the use of right heart catheterization. Due to the lack of noninvasive marker for this disease, CEC and circulating progenitors were quantified in two studies. One of them realized in adult patients showed a specific increase of CEC in PAH and not in post-embolic PH. CEC would then reflect the presence of specific endothelial lesions and not the clinical state of the patients. The other study demonstrated the interest of CEC quantification in the therapeutic care of children with PAH secondary to congenital heart disease, for whom patients in irreversible state had a higher level of CEC. We then defined a new noninvasive biomarker.that can be used for the diagnosis and prognosis of PAH. EPC are rare events in whole blood, difficult to expand and for which, mobilization protocols revealed insufficient. The recent hypothesis of a vascular reservoir for endothelial progenitor led us to study the effect of a local ischemia procedure on the mobilization of these cells in healthy volunteers. Two age groups were included to assess the impact of aging on this procedure. Despite a significant endothelial dilation with the local ischemia, no EPC were mobilized, whatever the age group. Ischemia even altered ECFC angiogenic properties.

Type 1 diabetes develops through the progressive destruction of the insulin-producing beta-cells. Regeneration or replacement of beta-cells is therefore needed to restore normal glucose homeostasis. Presently, normoglycemia can be achieved by the transplantation of whole pancreas or isolated islets of Langerhans. Islet transplantation can be performed through a simple laparoscopic procedure, but the long-term graft survival is low due to poor revascularization and early cell death. This thesis examined the possibility of using different auxiliary cells (Schwann cells, endothelial progenitor cells, and neural crest stem cells) to improve the engraftment and function of endogenous and transplanted islets. Co-transplantation of Schwann cells with islets improved islet graft function early after transplantation, and caused an increased islet mass at one month posttransplantation. However, the vascular densities of these grafts were decreased, which also related to an impaired graft function. Islet grafts containing endothelial progenitor cells had a superior vascular density, with functional chimeric blood vessels and substantially higher blood perfusion and oxygen tension than control transplants. By culturing and transplanting islets together with neural crest stem cells it was found that islets exposed to these cells had a higher beta-cell proliferation compared with control islets. At one month posttransplantation, the grafts with neural crest stem cells also had a superior vascular- and neural density. The potential of intracardially injected neural crest stem cells to home to the pancreas and ameliorate hyperglycemia in diabetic mice was investigated. During a three-week period after such cell treatment blood glucose concentrations decreased, but were not fully normalized. Neural crest stem cells were present in more than 10% of the pancreatic islets at two days postinjection, at which time the beta-cell proliferation was markedly increased when compared with islets of saline-treated diabetic animals. Three weeks later, a doubled beta-cell mass was observed in animals receiving neural crest stem cells. In summary, islets can easily be transplanted together with different auxiliary cells. Some of these cells provide the possibility of improving vascular- and neural engraftment, as well as beta-cell growth and survival. Systemic administration of neural crest stem cells holds the potential of regenerating the endogenous beta-cells.

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.

The epidermis is the first barrier of protection of living organisms against external attacks. It is constantly renewed throughout life, through a process called "homeostasis", which ensures that every cell lost on its surface is replaced by new ones. Recent studies have shown that this balance is ensured by a hierarchy of stem cells (SC) and progenitors that perform 3 types of cell divisions, each having a fixed probability. Although the epidermis has been extensively studied during homeostasis, little is known about the cellular dynamics taking place when the epidermis must expand its surface. Are these probabilities of division immutable or can they change? In this project, we focused on two conditions of epidermal expansion: postnatal growth and wound healing. Using the mouse tail epidermis as a model, we show that the re-epithelialization after a wound is achieved via the formation of two transient compartments that are spatially and molecularly distinct :a leading edge and a proliferative hub. We show that the leading edge cells have a specific transcriptional signature that is independent of their quiescent state and we propose new markers not previously described. Using the technique of "lineage tracing", coupled with clonal analysis and mathematical modeling, we highlight the proliferation dynamics of SCs and progenitors during healing. We show that different populations of cells residing in different compartments, the hair follicle infundibulum and the interfollicular epidermis, acquire a similar dynamics and re-activate their SC while the progenitors increase their rate of proliferation without changing their division probabilities. This similar proliferation dynamics in two compartments of the epidermis suggests that division probabilities are not dictated by the cell of origin. Interestingly, cell dynamics is different during postnatal growth. Using lineage tracing, clonal analysis and single-cell transcriptional analysis, we demonstrate that the post-natal epidermis is composed of a homogeneous population of equipotent progenitors which ensure a harmonious tissue growth through a constant imbalance towards self-renewing divisions and an ever decreasing proliferation rate. On the other hand, we show that basal cells in the adult epidermis display a greater molecular heterogeneity and that this heterogeneity is acquired progressively at the end of growth. Finally, by coupling in vivo measurements and in vitro micro-patterning experiments, we show that the orientation of cell division of equipotent progenitors is locally influenced by the alignment of the collagen fibers of the underlying dermis. These data suggest that SC specification occurs late in postnatal development and that proliferation dynamics are not immutable and could therefore be influenced by extrinsic factors. / L’épiderme est la première barrière de protection des organismes vivants contre des attaques extérieures. Il est constamment renouvelé au cours de la vie, via un processus appelé « homeostasie », qui assure que chaque cellule perdue à sa surface soit remplacée par de nouvelles. Des études récentes ont montré que cet équilibre était assuré par une hiérarchie de cellules souches (CS) et de progéniteurs qui réalisent 3 types de divisions cellulaires, chaque type de division ayant une probabilité fixe. Bien que l’épiderme ait été intensivement étudié durant l’homeostasie, peu de choses sont connues concernant la dynamique cellulaire prenant place lors de phénomènes où l’épiderme doit grandir. Ces probabilités de division sont-elles immuables ou peuvent-elles au contraire changer ?Dans ce projet, nous nous sommes intéressés à deux conditions d’expansion de l’épiderme :la croissance post-natale et la cicatrisation des plaies. En utilisant l’épiderme de la queue de souris comme modèle, nous montrons que la ré-épithélialisation d’une plaie est réalisée via la formation de deux compartiments cellulaires transitoires distincts spatialement et du point de vue moléculaire :un front de migration et un centre prolifératif. Nous montrons que les cellules du front de migration ont une signature transcriptionnelle spécifique qui est indépendante de leur état de quiescence et proposons de nouveaux marqueurs non décrits auparavant. En utilisant la technique du « lineage tracing », couplée à une analyse clonale et à de la modélisation mathématique, nous mettons en évidence la dynamique de prolifération des CS et des progéniteurs lors de la cicatrisation. Nous montrons que différentes populations de cellules résidant dans des compartiments différents, l’infundibulum du follicule pileux et l’épiderme interfolliculaire, acquièrent une dynamique similaire et ré-activent leur CS tandis que les progéniteurs augmentent leur taux de prolifération sans changer leur probabilité de division. Cette dynamique de prolifération similaire dans deux compartiments de l’épiderme suggère que les probabilités de divisions ne sont pas dictées par la cellule d’origine. De façon intéressante, la dynamique cellulaire est par contre différente durant la croissance post-natale. En utilisant le lineage tracing, l’analyse clonale et des analyses transcriptionnelles sur cellule unique, nous démontrons que l’épiderme post-natal est composé d’une population homogène de progéniteurs équipotents qui présentent un constant déséquilibre envers des divisions d’auto-renouvèlement et un taux de prolifération décroissant, assurant une croissance harmonieuse de l’épiderme. En revanche, les cellules basales de l’épiderme adulte montrent une plus grande hétérogénéité moléculaire et cet hétérogénéité est acquise progressivement à la fin de la croissance. Enfin, en couplant des mesures in vivo et des expériences de micro-patterning in vitro, nous montrons que l’orientation de la division cellulaire des progéniteurs équipotents est localement influencée par l’alignement des fibres de collagène du derme sous-jacent. Ces données suggèrent que la spécification des CS survient tardivement au cours du développement post-natal et que la dynamique de prolifération n’est pas immuable et pourraient donc être influencée par des facteurs extrinsèques. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

Le tissu adipeux (TA) est réparti dans tout le corps en différents dépôts. Il existe deux types distincts aux fonctions biens spécifiques : le tissu adipeux blanc sert de réservoir énergétique et stocke les lipides et le tissu adipeux brun permet la thermogénèse. Par ses fonctionnalités et son pouvoir endocrine, le TA assure le maintien de l’homéostasie énergétique. De graves désordres métaboliques résultent d’une surabondance retrouvée au cours de l’obésité ou lors d’un remodelage dans les lipodystrophies. Certaines ont une origine génétique, d’autres sont induites par des médicaments comme les inhibiteurs de la protéase (IP) du VIH administrés dans la thérapie antirétrovirale. Le pool de progéniteur adipeux (PA) présent dans chaque dépôt est essentiel au maintien de ce tissu car il permet de renouveler le stock d’adipocytes. Nous avons caractérisé et identifié de nouveaux gènes impliqués dans la boucle autocrine/paracrine de l’activineA qui est responsable de l’auto-renouvellement du pool de PA dont IER3. Son expression augmente chez les patients obèses et diminue sous traitement par les IP. La modélisation des différents dépôts montre que les IP inhibent préférentiellement l’auto-renouvellement ou la différenciation adipocytaire des PA en fonction de leur localisation. Les lipodystrophies induites par la thérapie antirétrovirale auraient des causes multifactorielles. Enfin, nos résultats révèlent que les IP diminuent drastiquement et sélectivement la production d’adipocytes bruns. Ces effets doivent être considérés dans un contexte de développement inopportun du tissu adipeux brun afin de corriger des désordres métaboliques associés à certaines pathologies. / The adipose tissue (AT) is distributed throughout the body in different depots. There are two distinct types with specific functions: the white adipose tissue is used as an energetic reservoir and stores the lipids whereas the brown adipose tissue allows the thermogenesis. By its functionalities and its endocrinal capacity, the AT ensures the energetic homeostasis maintenance. Severe metabolic disorders result from an excess found during obesity or a remodelling in the lipodystrophies. Some of them have a genetic origin, the others are induced by drugs such as the HIV-protease inhibitors (PI) administered in the antiretroviral therapy against HIV. The adipose progenitor (AP) pool present in each depot is necessary for the maintenance of this tissue because it allows to renew the adipocyte stock. We characterized and identified new genes involved in the autocrine/paracrine Activin A loop which is responsible for AP pool self-renewal of whom is IER3. Its expression increases in obese patients and decreases under PI treatment. The modelling of the different depots shows that PI inhibit preferentially PA self-renewal or adipose differentiation depending on their localisation. Thus, lipodystrophies induced by antiretroviral therapy would have multifactorial causes. Finally, our results reveal PI dramatically and selectively reduce the brown adipocyte production. These effects should be considered in the context of inappropriate brown adipose tissue development in order to correct metabolic disorders associated to some pathologies.

The influence of immunosuppressive drugs on neural stem/progenitor cell fate in vitro.