No description available.

Pulmonary arterial hypertension (PAH) is a devastating lung disease characterized by arterial pruning, occlusive vascular remodeling, and inflammation contributing to increased pulmonary vascular resistance with resultant right heart failure. Endothelial cell (EC) injury and apoptosis are commonly considered triggers for PAH, the mechanisms leading from injury to complex arterial remodeling are incompletely understood. While current therapies can improving symptoms, with the exception of parenteral prostacyclin, they do not significantly prolong transplant free survival. As well, there are no therapies that can regenerate the damaged lung short of transplantation. In this project, I sought to both advance the understanding of disease pathogenesis and explore regenerative therapeutic options for PAH. To this end, I first employed single cell RNA sequencing (scRNA-seq) at multiple time points during the Sugen 5416 (SU) – chronic hypoxia (CH) model of PAH, to provide new insights into PAH pathogenesis both during onset and progression of disease. We also employed microCT analysis to visualize and quantify the arterial pruning associated with PH and found significant loss up to 65% of the healthy arteriolar volume in this model. Through scRNA-seq analysis performed at four timepoints spanning the onset and progression of disease, two disease-specific EC cell types emerged as key drivers of PAH pathogenesis. The first was the emergence of capillary ECs with a de-differentiated gene expression profile, which we termed dedifferentiated capillary (dCap) ECs, with enrichment for the Cd74 gene. Interestingly, RNA velocity analysis suggested that these cells may be undergoing endothelial to mesenchymal transition during PAH development. At later times, a second arterial EC population became apparent, which we termed activated arterial ECs (aAECs), since it uniquely exhibited persistently elevated levels of differential gene expression consistent with a migratory, invasive and proliferative state. Interestingly, the aAECs together with the smooth muscle (SM)-like pericytes, a population which was also greatly expanded in PAH, expressed Tm4sf1, a gene previously associated with a number of cancers and abnormal cell growth. Furthermore, by immunostaining, TM4SF1 was found to be spatially localized to sites of complex and occlusive arterial remodeling, associated with both endothelial cells and pericytes in these lesions, suggesting an important role for the aAECs and SM-like pericytes in arterial remodeling and PH progression. Together, these findings suggest that aAECs, dCap ECs, and SM-like pericytes are emerging cell populations responsible for lung arterial remodeling in PAH, which drives disease progression, and that TM4SF1 may be a novel therapeutic target for this disease. As a first step in trying to develop approaches to regenerate lung arterial bed that is lost in PAH, we investigated the potential role of endothelial colony forming cells (ECFCs) and mesenchymal stromal cell (MSC) derived extracellular vesicles (EVs) as novel therapeutics, on the premise that these stem/progenitor cells would stimulate lung regeneration by mainly paracrine mechanisms. Additionally, we used biomaterials to microencapsulate cells and EVs to improve their local delivery and retention. While ECFCs were found to be ineffective in treating the monocrotaline model on their own, they were poorly retained in the lung and microencapsulation of ECFCs led to enhanced lung delivery within the first 72 hours, with resultant hemodynamic improvements in this model of PAH. MSCs are well known to be immunomodulatory and proangiogenic, largely acting through paracrine mechanisms, including by the release of EVs. Yet, following intravenous administration, nano sized EVs are rapidly cleared from circulation, potentially limiting their therapeutic potential. I adapted our microencapsulation strategy for EVs, and demonstrated significantly greater retention of microgel-loaded EVs were within the lung, resulting in enhanced local cell uptake. Interestingly, the hydrogel used for microencapsulation induced a local immune response which made it unsuitable for testing any potential therapeutic benefits of MSC-EVs in this study. Nonetheless, this work demonstrated proof-of-principle for the utility of microencapsulation as a strategy to enhance EV lung delivery. Overall, this work has identified novel lung cell populations (aAECs, dCap ECs, SM-like pericytes) driving arterial remodeling associated with PH progression, demonstrated the potential of ECFCs as a regenerative cell for the treatment of PAH, and illustrated the utility of microencapsulation as a tool to enhance lung targeting of both cells and EVs.

<p><strong>Background:</strong> Heart failure (HF) constitutes an important growing medical and economic problem with high prevalence and mortality. Prognosis assessment remains a challenge because of the dynamic nature of HF and the existence of some unexplained variation in outcomes. Our objective was to refine the process of prognostic assessment in current HF patients.</p> <p><strong>Methods:</strong> We conducted a systematic review to identify existing risk prediction models in ambulatory HF patients, a meta-analysis to identify mortality predictors in HF patients treated with an implantable cardioverter defibrillator (ICD), a retrospective cohort study to validate a new model, the HF Meta-Score, derived from the results of the meta-analysis and a cross-sectional and prospective cohort study to evaluate whether circulating progenitor cells (CPCs) are associated with functional capacity and mortality in ambulatory HF patients.</p> <p><strong>Results:</strong> We identified 20 risk prediction models in ambulatory HF patients; only five were externally validated showing limited discrimination and calibration. The two most validated models were derived from HF cohorts from the 1990s and reported limited performance in ICD patients. In a meta-analysis, we identified that age, baseline renal function, history of heart failure, chronic obstructive pulmonary disease, diabetes, peripheral vascular disease, left ventricular ejection fraction, NYHA class, atrial fibrillation, wide QRS and the occurrence of appropriate or inappropriate ICD shocks were independent mortality predictors. Some of these predictors were omitted in previously identified models. From the results of the meta-analysis, we developed the HF Meta-Score that showed better performance that an existing model. We observed that CPCs were independently associated with functional capacity and outcomes in ambulatory HF patients.</p> <p><strong>Conclusions:</strong> These results open many pathways to further refine the prognostic assessment in ambulatory HF patients. The HF Meta-Score is a promising score. The clinical utility of the HF Meta-Score and of the incorporation of new predictive factors, such as CPCs, needs to be tested.</p> / Doctor of Philosophy (PhD)

No / A neurological disorder is any disorder or abnormality in the nervous system. Among different neurological disorders, Alzheimer’s disease (AD) is recognized as the sixth leading cause of death globally. Considerable research has been conducted to find pioneer treatments for this devastating disorder among which cell therapy has attracted remarkable attentions over the last decade. Up to now, targeted differentiation into specific desirable cell types has remained a major obstacle to clinical application of cell therapy. Also, potential risks including uncontrolled growth of stem cells could be disastrous. In our novel protocol, we used basal forebrain cholinergic progenitor cells (BFCN) derived from human chorion-derived mesenchymal stem cells (hC-MSCs) which made it possible to obtain high-quality population of cholinergic neurons and in vivo in much shorter time period than previous established methods. Remarkably, the transplanted progenitors fully differentiated to cholinergic neurons which in turn integrated in higher cortical networks of host brains, resulting in significant improvement in cognitive assessments. This method may have profound implications in cell therapies for any other neurodegenerative disorders. / This work was carried outwithin the framework of a collaborative project (Project Grant No. 94-02-30-25922) by the School of Medicine, Iran University of Medical Sciences, (Project Grant No. REP209) council for stem cell sciences and technologies (Presidency of the Islamic Republic of Iran, vice-presidency for science and technology), and Iran National Science Foundation (INSF).

Yes / The maintenance of a proper nuclear architecture and 3D organization of the genes, enhancer elements and transcription machinery plays an essential role in tissue development and regeneration. Here we show that in the developing skin, epidermal progenitor cells of mice lacking p63 transcription factor display alterations in the nuclear shape accompanied by marked decrease in expression of several nuclear envelop-associated components (Lamin B1, Lamin A/C, SUN1, Nesprin-3, Plectin) compared to controls. Furthermore, ChIP-qPCR assay showed enrichment of p63 on Sun1, Syne3 and Plec promoters, suggesting them as p63 targets. Alterations in the nuclei shape and expression of nuclear envelope-associated proteins were accompanied by altered distribution patterns of the repressive histone marks H3K27me3, H3K9me3 and heterochromatin protein 1- alpha in p63-null keratinocytes. These changes were also accompanied by downregulation of the transcriptional activity and relocation of the keratinocyte-specific gene loci away from the sites of active transcription towards the heterochromatin-enriched repressive nuclear compartments in p63-null cells. These data demonstrate functional links between the nuclear envelope organization, chromatin architecture and gene expression in keratinocytes and suggest nuclear envelope-associated genes as important targets mediating p63-regulated gene expression programme in the epidermis.

Yes / Expression of the transmembrane NG2 chondroitin sulphate proteoglycan (CSPG) defines a distinct population of NG2-glia. NG2-glia serve as a regenerative pool of oligodendrocyte progenitor cells in the adult central nervous system (CNS), which is important for demyelinating diseases such as multiple sclerosis, and are a major component of the glial scar that inhibits axon regeneration after CNS injury. In addition, NG2-glia form unique neuron–glial synapses with unresolved functions. However, to date it has proven difficult to study the importance of NG2-glia in any of these functions using conventional transgenic NG2 ‘knockout’ mice. To overcome this, we aimed to determine whether NG2-glia can be targeted using an immunotoxin approach. We demonstrate that incubation in primary anti-NG2 antibody in combination with secondary saporin-conjugated antibody selectively kills NG2-expressing cells in vitro. In addition, we provide evidence that the same protocol induces the loss of NG2-glia without affecting astrocyte or neuronal numbers in cerebellar brain slices from postnatal mice. This study shows that targeting the NG2 CSPG with immunotoxins is an effective and selective means for killing NG2-glia, which has important implications for studying the functions of these enigmatic cells both in the normal CNS, and in demyelination and degeneration.

Qualificar as primeiras unidades de Sangue de Cordão Umbilical e Placentá- rio (SCUP) criopreservadas no Banco de Sangue Umbilical e placentário da Fundação de Hematologia e Hemoterapia de Pernambuco (BSCUP/HEMOPE), no período de dezembro de 2014 a junho de 2017. Justificativa: O sangue do cordão é rico em células progenitoras hematopoéticas (CPH), utilizado para transplante e tratamento de patologias benignas e malignas. Avaliar as primeiras unidades coletadas no BSCUP/ HEMOPE será de grande valia para sua caracterização, planejamento de ações de melhoria do processo, contribuindo para o aumento da eficácia dos transplantes. Métodos: Foram triadas gestantes sem histórico de diagnóstico para doenças transmissíveis pelo sangue, com gestação terminada em recém-nascido (RN) vivo; caracterizadas através das variáveis sociodemográficas: idade, idade gestacional, antecedentes obstétricos e cor da pele dos RN foram caracterizados pelas variáveis explicativas: Gênero e Peso; as amostras das mães foram coletadas no dia do parto ou em até 48h após. O tempo entre o término da coleta e o início da criopreservação da unidade das CPH não excedeu 48 horas. As unidades de SCUP foram avaliadas quanto ao volume, total de células nucleadas (TCN), quantificação de células CD 34+, contagem de eritroblastos viabilidade celular e contaminação microbiológica. Neste estudo foi avaliada a variável \"unidades adequadas ao uso em relação ao peso do receptor, tomando com o base o TCN e o peso de um provável receptor, assim categorizados: unidades <12,5 x10e8 para peso 12,5 para peso >50kg; Resultados: Volume Inicial (ml):(min.50,0; max.166,80); Volume final(mL): (min.19,07;max.21,75); TCN pré (x108): (min.6,0;max.27,80);TCN pós(x108): (min.5,0; max.22,6); Recuperação Celular (%): (min.0,67; max.39,50); CD34+(x106):(min.0,67;max.39,50);Viabilidade(%): (min.71,35max.100); Conclusão: Das 113 unidades armazenadas, 89 (78,76%) atendem a receptores < 50kg (crianças) e 24 (21,24%) atendem a receptores >50k (adultos). O inventário apresentou resultados em conformidades com o especificado na legislação vigente, portanto, qualificadas para atender à demanda transfusional de transplantes de CPH. / To qualify the first units of cryopreserved Umbilical Cord and Placental Blood (SCUP) in the Umbilical and Placental Blood Bank of the Hematology and Hemotherapy Foundation of the State of Pernambuco (BSCUP / HEMOPE), from December 2014 to June 2017 Rationale: Cord blood is rich in hematopoietic progenitor cells (HPC), used for transplantation and treatment of benign and malignant pathologies. Evaluating the first units collected in the BSCUP / HEMOPE will be of great value for its characterization, planning of actions to improve the process, contributing to increase the efficiency of transplants. Methods: Pregnant women with no history of diagnosis for blood-borne diseases were screened, with gestation terminated in live newborn (NB); characterized by the sociodemographic variables: age, gestational age, obstetric history and skin color, NB were characterized by the explanatory variables: Gender and Weight; the samples of the mothers were collected on the day of delivery or within 48 hours after delivery. The time between the end of the collection and the beginning of the cryopreservation of the MHC unit did not exceed 48 hours. SCUP units were evaluated for volume, total nucleated cells (TNC), CD34 + cell count, cell viability erythroblast counts, and microbiological contamination. In this study, the variable \"units suitable for use in relation to the weight of the receptor\" was evaluated, taking as base the TNC and the weight of a probable receiver, as follows: units <12.5 x10e8 for weight 12 , 5 for weight> 50 kg; Results: Initial volume (ml): (min.50.0, max.166.80); Final volume (mL): (min.19.07, max.21.75); TCN pre (x108): (min.6,0; max.27,80); TCN post (x108): (min.5,0; max.22,6); Cellular Recovery (%): (min.0.67, max.39,50); CD34 + (x106): (min.0.67, max.39.50); Viability (%): (min.71,35max.100); : Of the 113 stored units, 89 (78.76%) attend to receptors <50kg (children) and 24 (21.24%) attend receptors> 50k (adults). Conclusion: The inventory presented results in compliance with that specified in current legislation, therefore, qualified to meet the transfusional demand for HPC transplants.

Background: Receptor tyrosine kinase, c-Kit (CD117) plays a pivotal role in the maintenance and expansion of hematopoietic stem/progenitor cells (HSPCs). Additionally, over-expression and/or mutational activation of c-Kit have been implicated in numerous malignant diseases including acute myeloid leukemia. However, the translational regulation of c-Kit expression remains largely unknown. Methods and results: We demonstrated that loss of Pim1 led to specific down-regulation of c-Kit expression in HSPCs of Pim1(-/-)mice and Pim1(-/-)2(-/-)3(-/-) triple knockout (TKO) mice, and resulted in attenuated ERK and STAT3 signaling in response to stimulation with stem cell factor. Transduction of c-Kit restored the defects in colony forming capacity seen in HSPCs from Pim1 (-/-) and TKO mice. Pharmacologic inhibition and genetic modification studies using human megakaryoblastic leukemia cells confirmed the regulation of c-Kit expression by Pim1 kinase: i.e., Pim1-specific shRNA knockdown down-regulated the expression of c-Kit whereas overexpression of Pim1 up-regulated the expression of c-Kit. Mechanistically, inhibition or knockout of Pim1 kinase did not affect the transcription of c-Kit gene. Pim1 kinase enhanced c-Kit S-35 methionine labeling and increased the incorporation of c-Kit mRNAs into the polysomes and monosomes, demonstrating that Pim1 kinase regulates c-Kit expression at the translational level. Conclusions: Our study provides the first evidence that Pim1 regulates c-Kit gene translation and has important implications in hematopoietic stem cell transplantation and cancer treatment.

No description available.

Traumatic brain injury (TBI) caused by traffic and fall accidents, sports-related injuries and violence commonly results in life-changing disabilities. Cognitive impairments following TBI may be due to disruption of axons, stretched by the acceleration/deceleration forces of the initial impact, and their surrounding myelin in neuronal networks. The primary injury, which also results in death to neuronal and glial cells, is followed by a cascade of secondary injury mechanisms including a complex inflammatory response that will exacerbate the white matter injury. Axons are supported and protected by the ensheathing myelin, ensuring fast conduction velocity. Myelin is produced by oligodendrocytes (OLs), a cell type vulnerable to many of the molecular processes, including several inflammatory mediators, elicited by TBI. Since one OL extends processes to several axons, the protection of OLs is an important therapeutic target post-TBI.  During development, OLs mature from oligodendrocyte progenitor cells (OPCs), also present in the adult brain. The aim of this thesis was to investigate white matter pathology, with a specific focus on the OL population, in experimental and clinical TBI. Since the inflammatory response may contribute to OL cell death and OPC proliferation, neutralization of interleukin-1β (IL-1β) was investigated. The lateral and central fluid percussion injury models were used in mice and rats where memory, learning and complex behaviors were investigated by two functional tests. Brain tissue, surgically resected due to life-threatening brain swelling or hemorrhage, from TBI patients was also investigated. Axonal injury, myelin damage, microglia alterations and OPCs and OL cell death were investigated by immunohistochemical techniques. In focal and diffuse experimental TBI, OL cell death was observed in important white matter tracts. OL cell death was accompanied by myelin damage, axonal injury and presence of microglia as well as an increased number of OPCs in both the experimental and human setting. OPCs were found to proliferate in diffuse TBI in mice where both complex behavioral changes and impaired memory were observed. Neutralization of IL-1β normalized and improved these behavioral alterations and also lead to a preserved number of mature OLs although without influencing OPC proliferation. The results provided in this thesis indicate that white matter pathology is a key component of the pathophysiology of TBI. The OPC proliferation may influence regeneration post-injury and might be an important future therapeutic targets for TBI. The present studies also suggest that treatment strategies targeting neuroinflammation may positively influence behavioral outcome and OL cell death in TBI. / <p>(Faculty of Medicine)</p>