Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-07-11T17:13:30Z No. of bitstreams: 1 HugoCostaNeto_DISSERT.pdf: 13162279 bytes, checksum: 29adac7972f3d85035eb3c3d51febbdb (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-07-14T21:07:41Z (GMT) No. of bitstreams: 1 HugoCostaNeto_DISSERT.pdf: 13162279 bytes, checksum: 29adac7972f3d85035eb3c3d51febbdb (MD5) / Made available in DSpace on 2016-07-14T21:07:41Z (GMT). No. of bitstreams: 1 HugoCostaNeto_DISSERT.pdf: 13162279 bytes, checksum: 29adac7972f3d85035eb3c3d51febbdb (MD5) Previous issue date: 2016-02-22 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / Dentre os cistos odontog?nicos comumente encontrados na pr?tica cl?nica odontol?gica, os cistos radiculares (CRs) e os cistos dent?geros (CDs) representam conjuntamente os mais frequentes cistos dos ossos gn?ticos. Os cistos odontog?nicos possuem origem inflamat?ria ou de desenvolvimento. No entanto, altera??es inflamat?rias secund?rias podem ser vistas nos ?ltimos. Alguns estudos t?m identificado os mast?citos nessas les?es c?sticas e sua poss?vel rela??o com a angiog?nese. Nesta perspectiva, a presente pesquisa objetivou avaliar e comparar a express?o imunoistoqu?mica do CD34 e da triptase em CDs inflamados e CRs e verificar se os mast?citos influenciam na angiog?nese destas les?es. Para tanto, foram selecionados 20 casos de CDs inflamados e 20 casos de CRs para serem submetidos ? an?lise morfol?gica e imunoistoqu?mica. A imunomarca??o de cada caso foi avaliada de forma quantitativa. Ap?s a identifica??o das ?reas de maior imunorreatividade, foram analisadas a densidade microvascular (DMV), a ?rea microvascular (AMV) e o per?metro microvascular (PMV) mensurados atrav?s da imunoexpress?o do CD34 e a densidade dos mast?citos (DMC) mensurada por meio da imunoexpress?o da triptase, realizadas nas mesmas ?reas dos consecutivos campos representativos de cada caso. A an?lise estat?stica foi realizada atrav?s dos testes de Mann-Whitney, Qui-quadrado de Pearson, Exato de Fisher e Correla??o de Spearman (r), com n?vel de signific?ncia estabelecido em 5% (p < 0,05). Os resultados demonstram diferen?as estatisticamente significativas entre as les?es c?sticas supracitadas em rela??o ? avalia??o da DMC (p < 0,001). Al?m disso, a an?lise da DMV revelou diferen?as estatisticamente significativas entre as les?es c?sticas (p = 0,007) e tamb?m no que se refere ? intensidade do infiltrado inflamat?rio (p = 0,021). Por fim, observou-se nos casos de CDs inflamados, moderada correla??o positiva entre a DMC e a AMV (r = 0,660; p = 0,002), assim como moderada correla??o positiva entre a DMC e o PMV (r = 0,634; p = 0,003). Face ao exposto, pode-se concluir que os mast?citos participam em diferentes etapas da angiog?nese associada ? inflama??o dos CRs e CDs.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-07-11T17:13:31Z No. of bitstreams: 1 PatriciaGuerraPeixeGoncalves_DISSERT.pdf: 943209 bytes, checksum: 858cf53a3d375685e3e69be1d38caba2 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-07-15T20:21:04Z (GMT) No. of bitstreams: 1 PatriciaGuerraPeixeGoncalves_DISSERT.pdf: 943209 bytes, checksum: 858cf53a3d375685e3e69be1d38caba2 (MD5) / Made available in DSpace on 2016-07-15T20:21:04Z (GMT). No. of bitstreams: 1 PatriciaGuerraPeixeGoncalves_DISSERT.pdf: 943209 bytes, checksum: 858cf53a3d375685e3e69be1d38caba2 (MD5) Previous issue date: 2016-02-22 / A angiog?nese e a linfangiog?nese s?o altera??es tamb?m decorrentes da inflama??o gengival provocada por microrganismos presentes no biofilme dental, bem como pela a migra??o de c?lulas de defesa e secre??o de mediadores inflamat?rios no local da agress?o. Este estudo teve por objetivo avaliar a angiog?nese e linfangiog?nese em 90 esp?cimes de bi?psias de tecido gengival clinicamente saud?vel, com gengivite e com periodontite cr?nicas. Os cortes histol?gicos foram avaliados pela colora??o de hematoxilina e eosina e pela t?cnica de imunoistoqu?mica atrav?s da imunomarca??o de CD34 e podoplanina, para avaliar, respectivamente, o ?ndice angiog?nico e linfangiog?nico, por meio da t?cnica de contagem microvascular. Os resultados mostraram que h? correla??o entre os ?ndices (p=0,030), por?m, mostrou que na periodontite h? menos n?meros de vasos linf?ticos do que no tecido gengival clinicamente saud?vel (p=0,016). A podoplanina mostrou marca??o no epit?lio e que h? rela??o da intensidade de marca??o com a intensidade do infiltrado inflamat?rio, sendo mais intensa a marca??o na presen?a de infiltrado inflamat?rio severo (p=0,033). Concluiu-se neste estudo que h? menor n?mero de vasos sangu?neos na periodontite em compara??o com a gengiva clinicamente saud?vel. As sinaliza??es presentes no processo inflamat?rio, bem como o real papel da vasculatura sangu?nea e linf?tica gengival ainda n?o est?o totalmente elucidadas / Angiogenesis and lymphangiogenesis are changes that occur due to gingival inflammation caused by microorganisms present in the biofilm, as well as the migration of immune cells and secretion of mediators in the aggressed site. This study aimed to research angiogenesis and lymphangiogenesis in 90 specimens of clinically healthy, with gingivitis and chronic periodontitis gingival tissue biopsies. The histological sections were evaluated by hematoxylin and eosin and the immunohistochemical technique through immunostaining for CD34 and podoplanin. To evaluate the angiogenic and lymphangiogenic indexes we performed a microvessel counting technique. The results showed that there is a correlation between the indexes (p = 0.030), however, we observed that periodontitis showed less lymphatic vessels than clinically healthy gingival tissue (p = 0.016). Podoplanin showed positive staining in the basal layers of the epithelium, and we observed a relationship between immunostaining intensity and the intensity of inflammatory infiltrate, with more intense staining in the presence of severe inflammatory infiltrate (p = 0.033). For this study, we concluded that there are fewer blood vessels in periodontitis compared with clinically healthy gingiva. The signaling present in the inflammatory process and the actual role of gingival blood and lymphatic vasculature are not fully understood, with further studies on angiogenesis and lymphangiogenesis being suggested.

Background and objectives: In 2006 Shinya Yamanaka successfully reprogrammed mouse fibroblasts back to an embryonic stem cell-like state (called induced pluripotent cells, iPS cells) using retrovirus to introduce four genes that encode critical transcription factor proteins (Oct4, Sox2, KLF4, and c-Myc). This ability to reprogram has promising future applications in clinical and biomedical research for study of diseases, development of candidate drugs and to support therapeutic treatments in regenerative medicine. However, the clinical applications have to meet GMP requirements without the risk of insertional mutagenesis associated with retrovirus. Chromatin modifying agents are widely used in many protocols to generate iPS cells and culture of blood CD34+ cells with chromatin-modifying agents can lead to an increase in marrow repopulating cells and in the case of valproic acid increased erythroid cell colony formation. We undertook research to help understand what effects these reagents have on mobilised peripheral blood (mPB) CD34+ cells and optimised the expansion medium protocol to facilitate reprogramming work. This project aims to utilize peripheral blood mononuclear cells (MNC), one of the most easily accessible tissues to generate iPS cells using an efficient non-viral, feeder cell free methodology, with the ultimate goal of moving this methodology towards clinical use. Materials and Methods: G-CSF mobilised peripheral blood, buffy coat, cord blood and fetal liver were obtained from patients and donors under informed consent and ethics committee approval. Haematopoietic stem/progenitor cells CD34+ or CD133+) isolated by magnetic separation were flow cytometry sorted into CD34+/CD133+, CD34+/CD133-, and CD34-/CD133+ sub-populations and their lineage potential were assessed in colony forming unit assays. The effect of epigenetic modifiers valproic acid and 5-aza-2-deoxycytidine used singly or in combination with each other and with IL3 on phenotype and lineage potential of cultured CD34+ cells from mobilised peripheral blood were assessed by flow cytometry and colony-forming unit assays. Prior to reprogramming mononuclear cells from peripheral blood or CD34+ cells from blood were expanded in culture medium supplemented with stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (Flt3L) and Interleukin- 3 (IL-3) for several days. Actively proliferating cells were reprogrammed by electroporation using episomal vectors with an oriP/EBNA-1 backbone to deliver five reprogramming genes, Oct4, Sox2, Lin28, L-Myc, and Klf4. Electroporated cells were seeded onto matrigel coated plates immediately after transfection or were reseeded after three days’ culture. Subsequently, cells were cultured in specific medium on different days. When iPS colonies appeared, they were picked and cultured as for ES cells. Once established, iPS cell lines were immunophenotyped using flow cytometry and immunofluorescence and their potential to differentiate into the three germ layers was assessed in vitro. Results and Conclusion: The largest subpopulation of CD34+ cells was CD34+/CD133+ population which was essentially committed to myeloid colony production, while much smaller CD34+/CD133- subpopulation had a greater capacity to generate erythroid colonies. Optimised cytokine cocktail for expansion of CD34+ cells included IL-3, important in improving expansion and maintaining functionality of CD34+ cells. The optimised cytokine cocktail comprised 100 ng/ml SCF, 10 ng/ml Flt3L, and 20 ng/ml IL-3, which maintained CD34+ cells and MNC in an active proliferating state. In addition, valproic acid and IL3 were found to act synergistically, to increase the numbers of CD34+/CD36+ positive cells. However, we found that an apparent increase in red cell colony formation actually resulted from a decrease in white cell colonies, so no overall increase in red cell colonies was seen when equivalent numbers of CD34+ cells were plated. Proliferating MNC maintained in optimised cytokine cocktail were amenable to electroporation for the effective delivery of episomal transcription factors (Oct4, Sox2, Klf4, L-Myc, and Lin28) within a backbone of oriP/EBNA-1. We successfully developed an efficient and simple method for reprogramming MNC from fresh or frozen samples to generate induced pluripotent cells using episomal vectors in a feeder-free system without any requirement for small molecules and the highest reprogramming efficiency is 0.033% (65 colonies from 2 ◊ 105 seeding MNC). The cytokine cocktail and reprogramming methods work better in CD34+ cells from cord blood or fetal liver, and we obtained 148 iPS colonies from 105 seeding cells (0.148%) at most. In addition, fibroblasts from adult and fetal liver can be successfully reprogrammed using the same reprogramming method. The use of episomal vectors with an oriP/EBNA-1 backbone to deliver reprogramming genes, and efficient electroporation were the most important factors in efficiency of the reprogramming process. In addition, it is pivotal to initiate transfection when cells are actively proliferating. The iPS cell lines we generated maintained the successful expression of ES markers including Oct4, Nanog, SSEA3. SSEA4, TRA-1-60 and TRA-1-81, and had the capacity to successfully differentiate into cell types of ectoderm, mesoderm and endoderm layers in vitro.

Vascular disease is the leading cause of mortality and morbidity in the western world, and account for the 1 of every 3 death’s in the US, but a cure for vascular disease is yet to be realized. Hematopoietic stem progenitor cells (HSPCs) are mobilized from bone marrow and have the innate propensity to accelerate vascular repair by reendothelialization and revascularization of ischemic areas. The vasoreparative ability of HSPCs is largely due to their capacity to home to the areas of hypoxia and their sensitivity to hypoxia plays a critical role in the vasoreparative functions of these cells. The discovery of vasoreparative potential of HSPCs resulted in a breakthrough approach of cell-based therapies for the treatment of ischemic vascular diseases. However, success of this approach is essentially dependent on the number of cells that could be collected from an individual. Therefore, novel mechanism-based strategies are needed to enhance the outcomes of autologous cell-based therapies in poor mobilizers and older adults. Recent evidence of a potential role of the vasoprotective axis of the renin angiotensin system (RAS) in HSPCs functions offers a breakthrough. Angiotensin-(1-7), the primary mediator of the protective functions which acts on Mas receptor (MasR), is generated by angiotensin converting enzyme-2 (ACE2). In this study, we tested the effects of hypoxia on stimulation of vasoreparative potential of HSPCs and in upregulation of ACE2 and MasR. Importantly, we delineated the molecular mechanism of hypoxic exposure in regulation of ACE2 and MasR in a HIF1α- dependent manner and hypoxic exposure induced shedding of the membrane bound ACE2 in HSPCs. We used luciferase, a reporter assay, cell-based assays, gene/protein expression studies and pharmacological strategies in human and mouse HSPCs to test our hypotheses. To verify the biological significance of hypoxia, we performed in vivo studies in mice and humans, which recapitulated the in vitro observations on vascular protective axis of RAS in HSPCs. Collectively, these studies provided mechanistic insights into hypoxic regulation of vascular protective axis of RAS in HSPCs and also provided compelling evidence for the clinical use of hypoxia as a promising approach for enhancing the vasoreparative outcomes of cell-based therapies. / American Heart Association grant, 13SDG16960025 / National Institutes of Health, National institute of Aging (NIA), 1R01AG056881

The human bone marrow vasculature constitutively expresses both E-selectin and P-selectin where they interact with the cell-surface glycan moiety, sialyl Lewis x, on circulating hematopoietic stem/progenitor cells (HSPCs) to mediate the essential tethering/rolling step. Although several E-selectin glycoprotein ligands (E-selLs) have been identified, the importance of each E-selL on human HSPCs is debatable and requires additional methodologies to advance their specific involvement. The first objective was to fill the knowledge gap in the in vitro characterization of the mechanisms used by selectins to mediate the initial step in the HSPCs homing by developing a real time immunoprecipitation-based assay on a surface plasmon resonance chip. This novel assay bypass the difficulties of purifying ligands, enables the use of natively glycosylated forms of selectin ligands from any model cell of interest and study its binding affinities under flow. We provide the first comprehensive quantitative binding kinetics of two well-documented ligands, CD44 and PSGL-1, with E-selectin. Both ligands bind monomeric E-selectin transiently with fast on- and off-rates while they bind dimeric E-selectin with remarkably slow on- and off-rates with the on-rate, but not the off-rate, is dependent on salt concentration. Thus, suggest a mechanism through which monomeric selectins mediate initial fast-on and -off binding to capture the circulating cells out of shear-flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to slow rolling significantly. The second objective is to fully identify and characterize E/P-selectin ligand candidates expressed on CD34+ HSPCs which cause enhanced migration after intravenous transplantation compared to their CD34- counterparts. CD34 is widely recognized marker of human HSPCs but its natural ligand and function on these cells remain elusive. Proteomics identified CD34 as an E-selL candidate on human HSPCs, whose binding to E-selectin was confirmed using some static and flow-based assays. E-selectin binds to CD34 with an affinity comparable to the well-described E-selLs CD44/HCELL and PSGL-1. CD34 knockdown resulted in faster-rolling velocities compared to control cells especially at and above three dyne/cm2. CD34 is the first selectin ligand since PSGL-1 reported to bind E-/P-/L-selectins and likely plays a key role in directing the migration of human HSPCs to the bone marrow.

CD26/DPPIV is a dipeptidyl peptidase that cleaves and destroys a variety of substrates such as the chemokine SDF-1α, a chemokine expressed along bone marrow endothelium, which is essential for the recruitment of hematopoietic stem cells (HSCs) via binding with its receptor CXCR4 to the bone marrow. Thus, CD26 is thought to interfere with the second step, chemokine/chemokine receptor interactions, of the cellular migration paradigm. To further study the role of CD26 in the migration of HSCs, we screened several human leukemic cell lines to find a model cell line that expresses active CD26 and discovered that the pro-monocytic cell line, U937 was optimal for this purpose. U937 cells were used to optimize a variety of assays including an CD26 activity assay and transwell migration assay with and without the use of a CD26 inhibitor, Diprotin A. Then, we isolated short-term and long-term HSCs from the bone marrow of C57BL/6N mice using a combination of surface markers and a fluorescence-activated cell sorter. The expression levels of Step 2’s homing molecules were measured by FACS in both fractions of HSCs. Interestingly, we detected differences in the expression of CD26 between these two populations that may help explain the inability of long-term HSCs to migrate to the bone marrow. Thus, through the use of a CD26 inhibitor the long-term HSCS migration to the bone marrow could be enhanced, leading to a prolonged and efficient stem cell engraftment activity. Such studies are could help develop protocols to improve stem cell engraftment for patients suffering from hematological diseases such as leukemia.

Stem cell transplant is often considered the last hope for the survival for many cancer patients. The CD34+ cell content of a collection of stem cells has appeared as the most reliable indicator of the quantity of desired cells in a peripheral blood stem cell harvest and is used as a surrogate measure of the sample quality. Factors predicting the yield of CD34+ cells in a collection are not yet fully understood. Throughout the literature, there has been conflicting evidence with regards to age, gender, disease status, and prior radiation. In addition to the factors that have already been explored, we are interested in finding a cancer-chemotherapy interaction and to develop a predictive model to better identify which patients will be good candidates for this procedure. Because the amount of CD34+ cells is highly skewed, most traditional statistical methods are inappropriate without some transformation. A Bayesian generalized regression model was used to explain the variation of CD34+ collected from the sample by the cancer chemotherapy interaction. Missing data was modeled as unknown parameters to include the entire data set in the analysis. Posterior estimates are obtained using Markov chain methods. Posterior distributions identified weight and gender as well as some cancer-chemotherapy interactions as significant factors. Predictive posterior distributions can be used to identify which patients are good candidates for this procedure.

Vitamin-C (Vit-C) has been shown to modulate hematopoietic stem cells and leukemia stem cell frequency in-vivo. Herein, Vit-C analogue, L-ascorbic acid 2-phosphate (AA2P), was investigated as a new potential HSC expansion agonist. Cord blood CD34+ cells were expanded in cultures with or without AA2P. AA2P induced a 2-fold increase in the expansion of stem and progenitor subsets including lymphoid-primed multi-potential progenitors (p<0.05, n=3) and functional colony forming progenitors. The functional properties of AA2P grafts was evaluated with a xenotransplant model. Superior platelet levels in the periphery (p<0.05) and human bone marrow engraftment (median 75% hCD45+ cells for AA2P Vs. 48% for PBS control at week-22, n=3, p<0.05) was detected in AA2P cohorts Vs. control. In summary, my results demonstrate that AA2P is a new stem and progenitor expansion agonist with AA2P-expanded stem and progenitor cells capable of increased engraftment and higher platelet recovery. These findings may aid to overcome cord blood limitations; thereby, improving clinical relevance.

No description available.

<p>The recent and dramatic rise in allergic disease, coupled with the manifestation of the disease within the first years of life, suggests that <em>in utero</em> events are likely critically important to the inception of allergy. Epidemiological and experimental evidence suggest that both genetic predisposition and prenatal environmental exposures (e.g., <em>in utero</em> microbial exposures) play a role in modulating neonatal immunity and subsequent development of allergy. Of relevance to the work in this thesis, reports suggest that bacterial agents can directly alter myelopoiesis and, in connection to allergy, we have previously shown that cord blood (CB) progenitors from high-atopic risk infants demonstrate altered hematopoietic responses. However, whether CB progenitor cell hematopoietic responses are directly altered by microbial stimulation, and what effect maternal atopy has on these responses are unclear. Therefore, this thesis examines the influences of bacterial lipopolysaccharide (LPS) stimulation (innate immunity), maternal atopy, and adaptive immune stimuli (representative of an atopic milieu) on CB progenitor cell eosinophilopoiesis. We show that CB progenitors from healthy, pregnant women respond to LPS through increased eosinophil-basophil (Eo/B) colony forming units (CFU) via the mitogen-activated protein kinase (MAPK) signalling pathway (Chapter 2), whereas the presence of maternal atopy (as defined by skin prick test positivity) is associated with reduced CB CD34⁺ cell LPS-induced Eo/B CFU formation (Chapter 3). To investigate the potential mechanism of reduced eosinophilopoiesis in high-atopic risk infants, CB progenitors stimulated with IL-4 (a surrogate <em>ex vivo</em> for maternal atopy), but not IL-13, demonstrate reduced LPS-induced MAPK activation and Eo/B CFU formation (Chapter 4). This novel work provides insight into mechanisms relating to the influence of maternal atopy and/or potential intrauterine exposures (e.g., prenatal cytokines) on the responsiveness of CB progenitor cells to LPS, which may be of key importance for the development of atopic illnesses. These observations may help in the generation of novel biomarkers and therapeutic targets for childhood atopy.</p> / Doctor of Philosophy (PhD)