Orientadores: Maria Luiza Moretti, Angélica Zaninelli Schreiber / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T08:47:24Z (GMT). No. of bitstreams: 1 Moraes_JoseRenatode_M.pdf: 2487255 bytes, checksum: 4c5a21c13a0b57cf864dbf79cca990fd (MD5) Previous issue date: 2014 / Resumo: Introdução: os fungos do gênero Fusarium são ubíquos no meio ambiente e causam doenças em plantas e aos seres humanos. Objetivos: 1)estabelecer o protocolo para a coleta e o isolamento de Fusarium spp. no ar; 2)isolar fungos do gênero Fusarium do ar da enfermaria de Hematologia e da Unidade de Transplante de Medula Óssea (TMO) no Hospital de Clínicas (HC) da Universidade Estadual de Campinas (UNICAMP) e comparar a relação genética de isolados ambientais de Fusarium spp. com os isolados a partir de hemoculturas de rotina diagnóstica de pacientes hospitalizados durante os anos de 2002 a 2013, 3)identificar os fungos do gênero Fusarium ao nível de complexo de espécies por PCR em tempo real, e ao nível de espécie pelo sequenciamento de fragmento do gene EF1?. Metodologia: foram colhidas amostras de ar nas unidades de TMO (que possui controle de ar através de filtros HEPA e fluxo de ar de pressão positiva) e na Hematologia (unidade sem controle do ar) do HC. De 2006 a 2013 foram identificados 18 isolados de Fusarium spp. em culturas de sangue. A coleta de ar foi realizada através do amostrador de ar BioSamp modelo MBS 1000D (YotsubishiCorp Japão). Um meio de cultura seletivo para Fusarium (Agar Komada com modificações) foi usado na placa de Petri dentro do amostrador de ar. O volume de ar de 1000 mL e 500 mL foram aspirados da Hematologia e enfermarias de TMO, respectivamente. Após o tempo de incubação, as colônias que cresceram nas placas foram avaliadas macroscopicamente e microscopicamente para identificação morfológica. Fusarium spp. foram identificados através de um novo ensaio de PCR em tempo real composto por dois ensaios de PCR em tempo real: um específico para o gênero Fusarium e um ensaio específico para detecção específica do complexo de espécies de Fusarium solani (FSSC). Para confirmar o complexo de espécies de Fusarium, uma região parcial do gene EF-1? foi sequenciado. O alinhamento das sequências foi realizado no programa Clustal W e a análise filogenética utilizou o programa Mega5 com o algorítimo Neighbor-Joininge Bootstrap de 1000 replicatas. Resultado: foram isoladas 108 cepas de Fusarium spp. destes 28% na unidade de TMO e 78% na Hematologia. Os complexos de espécies encontrados foram: 10% FCSC, 21% FIESC, 1% FOSC, 12% FSSC, 51% GFSC e 5% não definido. Na TMO a maior prevalência foi da espécie Fusarium solani (9/30 isolados) e na Hematologia Fusarium verticillioides (25/31 isolados). Não foi possível constatar relevância entre a temperatura e umidade do ar em relação ao aumento ou diminuição de isolados nas coletas. Nove isolados FSSC do ar e 8 de sangue apresentaram BT 99%. Conclusão. Os dados de análise filogenética sugeriram que os isolados de sangue de pacientes e ambientais foram similares sugerindo que o ambiente pôde representar uma fonte potencial de infecção para os pacientes imunodeprimidos / Abstract: Introduction:the fungus Fusarium are ubiquitous in the environment and cause diseases in plants and humans. Objectives: isolate fungi Fusarium in the air of the ward Hematology and Unit of Bone Marrow Transplantation (BMT) at the Hospital de Clinicas (HC), State University of Campinas (UNICAMP), compare the phylogenetic relationship of environmental isolates of Fusarium spp .with isolates from blood cultures of routine diagnosis of hospitalized patients during the years 2002-2013, to establish protocol for the collection and isolation of Fusarium spp. in the air, identify the fungi Fusarium species complex level by real time PCR, and to species level by sequencing the fragment EF1 ? gene. Methodology: air sample was collected in BMT units (which has control of air through HEPA filters and air flow positive pressure) and Hematology (there is not self control air inlet) HC. From 2006 to 2013, 18 samples obtained from blood cultures with the presence of Fusarium spp. were added to study the relationship between degree of molecular clinical isolates isolated from the air. The air sampling was performed using the Bio air sampler Samp MBS model 1000D (Yotsubishi Corp. Japan). A selective medium for Fusarium (Komada Agar changes by Y. Mikami, Chiba University) was used in the petri dish in the air sampler. The air volume of 1000 mL and 500 mL were aspirated Hematology and BMT wards, respectively. After the incubation time, colonies that grew on the plates were evaluated macroscopically and microscopically for morphological identification. Fusarium spp. were identified by PCR of a new system in real time according to Muraosa et al. comprising two PCR assays in real time: the specific assay Fusarium and a specific assay for specific detection of Fusarium solani (FSSC) complex. To confirm the complex of Fusarium species, a partial region of the EF-1? gene was sequenced. The alignment of sequences was performed in Clustal W program and phylogenetic analysis used Mega5 program with the neighbor-joining algorithm and bootstrap 1000 replicates. Results: 108 ceepas Fusarium spp were isolated. 28% of these isolates form the BMT unit and 78% in Hematology. Overall the species complexes ficarm divided as follows: 10% FCSC, 21% FIESC 1% FOSC, FSSC 12%, 51% and 5% GFSC nd not defined). In the BMT was higher prevalence of the species Fusariu solani (9/30 isolates) and the Hematology espéciecom largest presence is Fusarium verticillioides (25/31 isolates). Unable to find relevance between temperature and air humidity in relation to the increase or decrease in collections of isolates. Nine isolates of Fusarium solani air obtained from the same source ancestras 8 Fusarium solani isolates from blood (BT 99%). Conclusion:Fusarium spp were isolated in 108 environmental samples en all evaluated environments. Phylogenetically findings suggest equivalence between environmental isolates and isolates from the blood of patients. The collection protocol set showed satisfactory for the specific isolation of Fusarium result. The findings of Fusarium were grouped into complexes FSSC and FSSC not the technique of real-time PCR and sequencing of the gene EF1? allowed to classify the isolates in the species level / Mestrado / Clinica Medica / Mestre em Ciências

La cystinose est une maladie lysosomale héréditaire due à une mutation du gène CTNS codant pour un transporteur de cystine, cystinosin, et est caractérisée par l’accumulation de cystine dans les organes causant leur dégénération.Si le rein est le premier organe affecté par la maladie, sa pathogénèse n’est pas encore totalement comprise. La recherche de nouveaux partenaires de la cystinosine a révélé une interaction avec un membre de la famille des galactines, galectine-3 (Gal3). L’étude de cette interaction a mis en évidence un nouveau rôle de la cystinosine dans l’inflammation chronique impliquée dans la pathogénèse rénale de la cystinose. Dans les reins du modèle murin de la cystinose (Ctns-/-), l’expression de Gal3 est augmentée et de nombreux infiltrats de cellules inflammatoires sont observés. De plus, l’expression de la cytokine pro-inflammatoire Monocyte Chemoattractant Protein-1 (MCP1) est augmentée dans le sérum des souris Ctns-/-.Au contraire, peu d’infiltrat et un taux normal de MCP1 sont observés dans les souris Ctns-/-Gal3-/-, ainsi qu’une meilleure structure et fonction rénale.Ce travail pourrait permettre la découverte de nouvelles cibles thérapeutiques pour la cystinose.Des études antérieures ont montré que la transplantation de Cellules Souches Hématopoïétiques (CSH) saines peut efficacement traiter la cystinose dans les souris Ctns-/-. Toutefois, dû aux risques liés à une transplantation allogénique, une transplantation autologue de CSH génétiquement modifiées ex vivo pour exprimer une copie fonctionnelle du gène CTNS a été développée.Nous résumons ici les études pharmacologiques et toxicologiques ainsi que le développement du protocole de transduction des cellules humaines qui seront inclus dans une application intitulée« Investigational New Drug » qui sera soumise à la FDA afin de débuter un essai clinique de phase I/II. / Cystinosis is an inherited lysosomal storage disorder caused by mutations in the gene CTNS encoding the cystine transporter cystinosin, and is characterized by accumulation of cystine in the tissues leading to multiorgan degeneration.The kidney is the first organ impacted by cystinosis but the pathogenesis is still not fully understood. The study of new partners of cystinosin revealed an interaction with galectin-3, a member of the galectin’s family. The investigation of this interaction unraveled a new role for cystinosin in chronic inflammation associated with kidney pathology in cystinosis. Indeed, the cystinosis mouse model, Ctns-/ mice, had increased expression of Gal3 and abundant pro-inflammatory infiltrates in their kidney, as well as increased expression of Monocyte Chemoattractant Protein-1 (MCP1), a proinflammatory cytokine, in their serum.In contrast, few infiltrates and normal MCP1 levels were observed in the Ctns-/- Gal3-/- mice, which also demonstrated better kidney structure and function.This study may lead to the discovery of new drug targets for cystinosis treatment.Previous studies showed that wild-type Hematopoietic Stem Cells (HSCs) transplantation had the potential to rescue cystinosis in Ctns-/- mice.However, due to the risks associated with allogeneic transplant, an autologous transplantation of HSCs genetically modified ex vivo to express a functional CTNS gene has been developed in the laboratory. In this work, we summarized the pharmacology and toxicology studies and manufacturing development that will be included in an Investigational New Drug application to be submitted to the FDA to start a phase I/II clinical trial for cystinosis.

Nanoscopic spatial reorganization of selectin ligands, CD44 and PSGL-1, during the initial step of hematopoietic stem/progenitor cell (HSPC) homing, tethering and rolling of migrating cells over E-selectins, has been recently reported. However, the exact spatial distribution of these ligands and their spatial reorganization during the cell rolling on E-selectins are still an open question. The spatiotemporal characterization at the nanoscale level requires high resolution imaging methods. In this study, I quantitatively characterize nanoscopic spatiotemporal behavior of the selectin ligands on the migrating cells to understanding the molecular mechanism of the cell rolling at the nanoscale level by means of a microfluidics-based 3D super-resolution fluorescence microscopy technique. The obtained results suggest that PSGL-1 on the cell shows significant change in the axial distribution on the cell during the cell rolling on E-selectin whereas the spatial distribution of CD44 along the axial direction is not affected significantly by the cell rolling. These findings indicate that each selectin ligand has a distinct contribution to the initial step of the HSPC homing because of their distinct spatial localizations on the cells that regulate at least partly the accessibility of these ligands to the surface E-selectin.

No description available.

The plant homeodomain 6 gene (PHF6) is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL); however, its specific functional role in leukemia development remains to be established. Here, we show that loss of PHF6 is an early mutational event in leukemia transformation. Mechanistically, genetic inactivation of Phf6 in the hematopoietic system enhances hematopoietic stem cell (HSC) long-term self-renewal and hematopoietic recovery after chemotherapy by rendering Phf6 knockout HSCs more quiescent and less prone to stress-induced activation. Consistent with a leukemia-initiating tumor suppressor role, inactivation of Phf6 in hematopoietic progenitors lowers the threshold for the development of NOTCH1-induced T-ALL. Moreover, loss of Phf6 in leukemia lymphoblasts activates a leukemia stem cell transcriptional program and drives enhanced T-ALL leukemia-initiating cell activity. These results implicate Phf6 in the control of HSC homeostasis and long-term self-renewal and support a role for PHF6 loss as a driver of leukemia-initiating cell activity in T-ALL. Phf6 controls HSC homeostasis, leukemia initiation, and T-ALL leukemia-initiating cell self-renewal. These results substantiate a role for PHF6 mutations as early events and drivers of leukemia stem cell activity in the pathogenesis of T-ALL. Further, in the hematopoietic system stem cell aging is characterized by accumulation HSCs with poor self-renewal capacity and myeloid biased differentiation. Despite improved appreciation of the cell intrinsic and cell extrinsic mechanisms driving age-associated HSC functional exhaustion, no interventions have proven effective in delaying HSC aging to date. Here, we show that genetic inactivation of the Phf6 prevents age- associated HSC functional decline. Immunophenotypic and single cell transcriptomics profiling demonstrated markedly decreased accumulation of immunophenotypically-defined HSCs, reduced myeloid bias and decreased upregulation of transcriptional programs associated with stem cell aging in old hematopoietic-specific Phf6 knockout mice. Functionally, Phf6 knockout HSCs from aged mice demonstrated increased hematopoietic reconstitution capacity and preservation of lymphoid differentiation potential. Mechanistically, analysis of long-term HSCs from old Phf6 knockout mice revealed reduced levels of ongoing DNA damage and downregulation of genotoxic stress-induced transcriptional signaturesconducive of HSC aging. These results identify Phf6 as an important epigenetic regulator of HSC aging, whose inactivation counters the functional deterioration of HSC activity induced with age. Microprotein encoding genes are a class of genes which encode poly-peptide gene products comprised by 100 or fewer amino acids. Until recently, many such genes had been considered of low- or no-coding potential given the technical limitations associated with identification of such small proteins. However, recently prominent examples of microprotein encoding genes have been reported with a wide variety of regulatory functions. Therefore, we hypothesized that novel microprotein genes exist within the human genome with oncogenic and tumor suppressive roles. To test this hypothesis, we developed a pipeline for identification of microproteins based on conservation of the open reading frame. Leveraging PLATE-seq to generate a high-dimensional readout in a loss-of-function screen, we then screened for microproteins with potential tumor suppressive or oncogenic function. From this, we identified a brain- specific, 65 amino-acid microprotein encoded in within LINC00617 (TUNAR) which is conserved at the protein level across vertebrates. We experimentally validated the protein-level expression of the TUNAR microprotein. In vitro and in vivo knockout and overexpression experiments demonstrate a role for TUNAR as a tumor suppressor in glioma. Specifically, we show that loss of Tunar in the mouse brain results in lower expression of Fermt1 and genes in the integrin signaling pathway. Consistently, overexpression of TUNAR in human glioblastoma multiforme cell lines significantly impeded cellular migration suggesting a role of Tunar in glioma cell dissemination. Finally, human glioma sequencing and copy number data were mined to determine the prognostic significance of the loss of TUNAR in human gliomas. These analyses demonstrated that copy number loss of TUNAR is associated with poor outcomes in lower grade gliomas and that TUNAR expression and glioma grade are strongly, negatively correlated suggesting that TUNAR likely has tumor suppressive effects in human glioma.

Thesis (MTech (Nursing))--Cape Peninsula University of Technology, 2017. / Stem cell transplantation has become one of the standard methods of treatment for patients with malignant and benign blood disorders. The multidisciplinary team interacting with these patients and their families, must be knowledgeable concerning the appropriate quality health care. The objectives of the study were to explore the knowledge of the members of the health care team in terms of the processes that need to be adhered to with stem cells transplantation, as well as exploring the perceptions amongst the health care team members and their reactions towards patients undergoing stem cell transplantation. An exploratory research design with a qualitative approach was employed. Data collection took place at two stem cell transplant units in the Western Cape, using non-probability purposive sampling technique. The health care team members included a medical doctor, dietician, physiotherapist, social worker, radiographer and nursing staff. Data was collected by face-to-face personal interviews which were transcribed and analysed by using coding and thematic analysis. The majority of the professional participants could identify the processes for stem cell transplantation, which affirmed their knowledge. The non-professional health care team member, could also identify the types of methods and processes of stem cell transplantation. Participants stated that the health care team members had passion for this treatment option. Some participants felt it to be emotionally challenging to work in the environment, especially with paediatric patients and the dying. However, some health care team members could detach themselves emotionally from the patients. The team stated that the stem cell transplanted patients need special care to overcome all challenges experienced, but were positive about treatment. It is evident that management of stem cell transplanted patients is complicated and the health care team members must have knowledge, skills and the appropriate attitude to practice in these units. This study emphasised how vital it is that stem cell transplantation be included in the training programs of the multidisciplinary team. Health care practitioners in the field must stay abreast with stem cell research in order to effectively conduct health promotions for patients and staff. In addition, hematology and transplant awareness campaigns should also be conducted in order to educate society and suggest referrals if necessary.

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.

This article provides a brief review of the pathophysiology of osteoarthritis and the ontogeny of chondrocytes and details how physical exercise improves the health of osteoarthritic joints and enhances the potential of autologous chondrocyte implants, matrix-induced autologous chondrocyte implants, and mesenchymal stem cell implants for the successful treatment of damaged articular cartilage and subchondral bone. In response to exercise, articular chondrocytes increase their production of glycosaminoglycans, bone morphogenic proteins, and anti-inflammatory cytokines and decrease their production of proinflammatory cytokines and matrix-degrading metalloproteinases. These changes are associated with improvements in cartilage organization and reductions in cartilage degeneration. Studies in humans indicate that exercise enhances joint recruitment of bone marrow-derived mesenchymal stem cells and upregulates their expression of osteogenic and chondrogenic genes, osteogenic microRNAs, and osteogenic growth factors. Rodent experiments demonstrate that exercise enhances the osteogenic potential of bone marrow-derived mesenchymal stem cells while diminishing their adipogenic potential, and that exercise done after stem cell implantation may benefit stem cell transplant viability. Physical exercise also exerts a beneficial effect on the skeletal system by decreasing immune cell production of osteoclastogenic cytokines interleukin-1β, tumor necrosis factor-α, and interferon-γ, while increasing their production of antiosteoclastogenic cytokines interleukin-10 and transforming growth factor-β. In conclusion, physical exercise done both by bone marrow-derived mesenchymal stem cell donors and recipients and by autologous chondrocyte donor recipients may improve the outcome of osteochondral regeneration therapy and improve skeletal health by downregulating osteoclastogenic cytokine production and upregulating antiosteoclastogenic cytokine production by circulating immune cells.

Cord blood (CB) has emerged as a significant source of hematopoietic stem cells (HSC) for transplantation. Large distances between collection and processing sites combined with staff availability can lead to long processing delays of CB unit (CBU). Standard agencies limit CBU storage at room temperature (RT) to a maximum of 48 hours from collection to freezing. Slow-engraftment and graft failure are major issues related to CB transplantation. I hypothesized that prolonged storage at RT reduces the engraftment activities of CBU due to the loss in HSC numbers. I set to test my hypothesis by performing serial and limiting-dilution transplantation assays in immunodeficient mice. My results showed that the engraftment activity of CBU was significantly perturbed by prolonged storage (>40 hours) at RT. In line with my hypothesis, the transplantation assays suggested that the engraftment deficit originates from loss in HSC numbers. My findings provide results for CB banks to make an informed decision on how long CBU can be stored at RT before processing. Conversely, CBU must be cryopreserved before use, and loss of function can occur due to osmotic shock and mechanical damage from uncontrolled ice-crystal growth (ice-recrystallization) during freezing and thawing. Current cyroprotectants like dimethyl-sulfoxide fail to inhibit ice-recrystallization. However, a novel class of small ice-recrystallization inhibitor (IRI) molecules (N-aryl-D-aldonamides) have been developed. I hypothesized that supplementation of cryopreservation solution with IRIs will improve the post-thaw viability and engraftment activity of CBU. Herein, I identified two IRIs (IRI 2 and IRI 6) that improved the post-thaw recovery of hematopoietic clonogenic and multipotent progenitors. Moreover, supplementation of CB graft with IRI 2 was beneficial to engraftment and had no negative impact on the differentiation and self-renewal activities of HSCs. Taken together, my results demonstrate for the first time that IRI may be beneficial to the engraftment activity of HSC graft and support further investigation.

Maintenance of the hematopoietic system is dependent upon the proper regulation and orchestrated functions of the hematopoietic stem cell (HSC) pool. A number of extrinsic signaling pathways and intrinsic regulators have been found to regulate HSC processes. However a full understanding of the ability of HSC to balance the processes of self-renewal, quiescence, and lineage specification is not yet clear. We therefore set out to identify novel HSC regulators by comparative gene expression analysis of whole genome transcriptomes generated for long-term (LT)-HSC (Hoechst low/- Lin- Sca1+ cKit+ CD34-), short-term (ST)-HSC (Hoechst low/- Lin- Sca1+ cKit+ CD34+), and non-HSC (Hoechst+) of the bone marrow. These studies identified IGF2 as one of the most differentially expressed genes within LT-HSC, suggesting a potential role for IGF2 in the regulation of HSC. Using a combination of lentiviral-mediated overexpression and knockdown experiments, we found IGF2 to confer enhanced self-renewal in vitro and in vivo. Overexpression of IGF2 resulted in an increased percentage of multi-lineage colonies within colony-forming unit (CFU) assays without affecting lineage specification. In vivo, serial bone marrow transplantation revealed that IGF2 within HSC enhances short-term and long-term donor contribution. Analysis of the expression of key cell cycle regulators revealed that IGF2 induced upregulation of p57 expression specifically within HSC. This upregulation could be attributed to differences in the methylation status of the p57 promoter in HSC compared to other progenitor and mature blood cell populations. p57, a member of the Cip/Kip family of cyclin dependent kinase inhibitors, has recently been shown to be required for the regulation of HSC quiescence and long-term self-renewal. Analysis of bone marrow obtained from primary and secondary transplant recipients showed that overexpression of IGF2 resulted in an increased percentage of quiescent HSC. Treatment of HSC overexpressing IGF2 with LY294002, a PI3K-Akt inhibitor, prevented IGF2-mediated upregulation of p57 expression. These findings demonstrate that within HSC, IGF2 induces p57 expression through activation of the PI3K-Akt pathway to regulate HSC quiescence. We have identified a novel role for IGF2 in HSC function, providing new insights into the biology of HSC and opening potential platforms for the development of better therapies involving HSC-mediated hematopoietic reconstitution.