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Mobilisation, Isolation and Coculture of Haematopoietic Stem CellsJing, Duohui 17 February 2011 (has links) (PDF)
Since decades, hematopoietic stem cell transplantation (HSCT) has become a well established treatment modality for hematological malignancies and non-malignant disorders. Autologous and allogeneic hematopoietic stem cells (HSCs) mobilized into the peripheral blood (PB) have been used as a preferred source of transplantable stem cells1-3. And umbilical cord blood (UCB) has been introduced as a more attractive HSC source for HSCT, because fetal stem cells in UCB are speculated to be more primitive in comparison to adult stem cells. However the limited amount of HSCs is limiting their application for stem cell therapy in clinic. Therefore, people started to utilize extra-embryonic tissue to harvest more fetal stem cells, while people also tried to optimize the clinical protocol to mobilize more adult stem cells out of adult bone marrow. The innovative strategies and feasible procedures were discussed in this thesis.
The axis of the chemokine receptor CXCR4 and its ligand SDF-1 is important for trafficking and homing of HSCs. It has already been demonstrated that the bicyclam AMD3100, a CXCR4 antagonist, in combination with G-CSF is able to induce a significant mobilization of CD34+ cells4. And human placenta is a potent hematopoietic niche containing hematopoietic stem and progenitor cells throughout development5. The homing of HSCs to the placenta is probably also mediated by the expression of SDF-1 as demonstrated for the bone marrow niche. In this study (part 1 of the chapter “Results and discussions”), we utilized AMD3100 to mobilize HSCs from placenta. And we can demonstrate that the CXCR4 antagonist AMD3100 mobilise placenta derived CD34+ cells ex utero already after 30 min of incubation and may further enhance the efficacy of harvesting placenta-derived HSC.
The alpha4 integrin CD49d is involved in migration and homing of hematopoietic stem cells (HSC). Therapeutic application of natalizumab, an anti-CD49d antibody, in patients with multiple sclerosis (MS) has been associated with increased levels of circulating CD34+ progenitors. In our study (part 2 of the chapter “Results and discussions”), we compared circulating HSCs from MS patients after natalizumab treatment and HSCs mobilized by G-CSF in healthy volunteers, with regard to their migratory potential, clonogenicity and gene expression. CD34+ cells in the blood and marrow of natalizumab-treated patients expressed less of the stem cell marker CD133, were enriched for erythroid progenitors (CFU-E) and expressed lower levels of adhesion molecules. The level of surface CXCR-4 expression on CD34+ cells from patients treated with natalizumab was higher compared to that of CD34+ cells mobilized by granulocyte-colony stimulating factor (G-CSF) (median 43.9% vs. 15.1%). This was associated with a more than doubled migration capacity towards a chemokine stimulus. Furthermore, CD34+ cells mobilized by natalizumab contained more m-RNA for p21 and less MMP9 compared to G-CSF mobilised HSC. Our data indicate that G-CSF and CD49d blockade mobilize different HSC subsets and suggest that both strategies may be differentially applied in specific cell therapy approaches.
In order to further improve the clinical outcome of HSC transplantation, many groups are focusing on ex vivo maintain or expand HSC. Unfortunately, the maintenance of HSC in vitro is difficult to achieve because of their differentiation. This is presumably caused by a lack of appropriate cues that are provided in vivo by the microenvironment. Indeed, HSCs located in the bone marrow are interacting with a specific microenvironment referred to as the stem cell niche, which regulates their fate in terms of quiescence, self-renewal and differentiation. An orchestra of signals mediated by soluble factors and/or cell-to-cell contact keeps the balance and homeostasis of self-renewal, proliferation and differentiation in vivo. To investigate the communication between HSCs and the niche, coculture assays with mesenchymal stromal cells (MSCs) were performed in vitro. Here, we can demonstrate that cell-to-cell contact has a significant impact on hematopoietic stem cells expansion, migratory potential and stemness. In this study (part 3 of the chapter “Results and discussions”), we investigated in more detail the spatial relationship between hematopoietic stem cells and mesenchymal stromal cells during ex-vivo expansion. And we defined three distinct localizations of HSCs relative to MSC layer: (i) those in supernatant (non-adherent cells); (ii) cells adhering on the surface of mesenchymal stromal cells (phase-bright cells) and (iii) cells beneath the mesenchymal stromal cells (phase-dim cells). Our data suggest that the mesenchymal stromal cell surface is the dominant location where hematopoietic stem cells proliferate, whereas the compartment beneath the mesenchymal stromal cell layer seems to be mimicking the stem cell niche for more immature cells. Our data provide novel insight into the construction and function of three-dimensional HSC–MSC microenvironments.
In summary, we provided a new method to isolate fetal stem cells from extra-embryonic tissue (i.e. placenta) in the first part, then we discussed an innovative strategy with CD49d blockade to improve clinical modality for adult stem cell mobilization in the second part, and finally we investigated HSC maintenance and expansion in vitro and provided feasible way to mimic HSC niche in vitro in the last part.
This thesis contributes to HSC-based stem cell therapy in two aspects, i.e. 1) fetal and adult stem cell isolation holding great therapeutic potential for blood diseases; 2) ex vivo stem cell manipulation providing a valuable platform to model HSC niche regulation.
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Selection procedures relating to Australian vocal repertoire for mid-adolescent HSC performers.Dixon, Wendy P January 2007 (has links)
Master of Music (Music Education) / This thesis documents an investigation of the selection procedures relating to Australian vocal repertoire for mid-adolescent and Higher School Certificate (New South Wales) performers, as used by private singing teachers, school music teachers and singing students. It explores the similarities and differences in the criteria employed in these selections. Semi-structured interviews were the source of data and were conducted with participants from these three categories as well as two composers. The participants evinced highly disparate views. The private singing teachers believed that repertoire should be dictated by the technical ability and physiological constraints of mid-adolescent students and that their role in selecting repertoire was related to the long term vocal growth of each individual. They felt that the school music teachers vetted their repertoire choices with no useful explanation of their reasoning, while the school music teachers noted that students frequently presented repertoire that was too difficult or that was not readily communicated with the audience. The ability of mid-adolescent singers to communicate with and engage an audience was the prime concern of the school music teachers. The students wanted to impress their examiners and believed that infrequently heard repertoire was the best choice, though this was not endorsed by the teachers. There was a perception that the students would perform at their best when they chose repertoire to which they could relate emotionally. Many private singing teachers and school music teachers are not aware of the very broad range of contemporary Australian music and its divergent characteristics. However, there is a shortage of appropriate Australian repertoire that addresses the physiological and emotional needs of mid-adolescent singers.
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The role of hsc-70 in very low density lipoprotein tranport vesicle golgi fusion complex formationNafi Valencia, Erika 01 December 2012 (has links)
Excess production and secretion of very low-density lipoprotein (VLDL) by the liver into the circulatory system is directly related to atherosclerosis, a chronic cardiovascular disease that threatens the lives of many worldwide and continues to be a leading cause of death in the United States. The rate-limiting step in VLDL secretion is its transport from the site of biogenesis, the hepatic endoplasmic reticulum to the cis-Golgi. This step is mediated by a specialized ER- derived vesicle, the VLDL transport vesicle (VTV). Upon exit of the ER the VTV targets, fuses and delivers VLDL into the lumen of the Golgi. The targeting and fusion of the VTV with the Golgi is facilitated by specific set of soluable N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins that form a SNARE complex, which is required for the VTV-Golgi fusion and thus delivery to the Golgi. Data from our laboratory indicates that the formation of the SNARE complex requires cytosolic factors. Through the purification of liver cytosol, chromatographic steps, detailed mass spectrometry, immunodepletion and western blotting data it was identified that the protein necessary for SNARE complex formation is Hsc-70. Although Hsc-70's identification is significant, the role it plays in SNARE complex formation for VTV -Golgi fusion is a predicament and yet to be unraveled. In this study we performed a series of co-immunoprecipitation reactions to identify its role in SNARE-complex assembly. Using western blot data we confirmed binding of Hsc-70 with Sec22b, the v-SNARE on the VTV. Moreover, we confirmed the interaction of Hsc-70 with t-SNAREs, (syn5, rBet1 and GOS28) on the Golgi membrane. Removal of Hsc-70 from the liver cytosol resulted in significant reduction of SNARE-complex formation. Ultimately, the identification proteins involved in the process of VLDL delivery to the Golgi would offer therapeutic targets to control VLDL secretion into the blood by the liver.
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Rôle des gènes HOX du paralogue 4 dans l'autorenouvellement des cellules souches et progéniteurs hématopoïétiquesFournier, Marilaine 08 1900 (has links)
La transplantation de cellules souches hématopoïétiques (CSH) est un traitement couramment utilisé pour traiter plusieurs types de maladies hématologiques telles que les leucémies. Par contre, une limite importante de ce type de traitement est la quantité restreinte de CSH disponibles pour la transplantation. Il importe donc de trouver des moyens pour expandre efficacement ces cellules ex vivo tout en préservant leurs propriétés. Le gène HOXB4 est présentement un candidat très prometteur pour atteindre cet objectif. Il a en effet été montré que HOXB4 est capable d’expandre les CSH in vivo et in vitro sans mener au développement de leucémie. Le gène HOXC4, qui appartient au même paralogue est aussi en mesure d’expandre les cellules hématopoïétiques primitives suggérant un rôle commun pour les gènes HOX du paralogue 4 dans l’autorenouvellement des CSH.
Le gène HOXA4 est dix fois plus exprimé que le gène HOXB4 dans des CSH du foie fœtal au moment de leur principale expansion. De plus, les CSH mutantes pour Hoxa4, contrairement aux CSH mutantes pour Hoxb4, sont incapables de reconstituer un receveur irradié lorsqu’elles sont transplantées en condition de compétition. HOXA4 pourrait donc jouer un rôle plus important que les autres gènes du paralogue 4 pour l’expansion des CSH au niveau physiologique. Nous avons donc posé l’hypothèse que HOXA4 est capable d’expandre des CSH de façon plus importante que HOXB4.
Les résultats obtenues dans le cadre de ce projet de recherche ont montré que la surexpression de HOXA4 était capable d’expandre les CSH et les progéniteurs hématopoïétiques primitifs dans le même ordre que ce qui est connu pour HOXB4. Des cultures et des essais de transplantation en situation de compétition ont confirmé la capacité égale des CSH surexprimant HOXA4 et HOXB4 de proliférer et de reconstituer les receveurs irradiés à long terme. Par contre, nous avons observé une meilleure reconstitution périphérique à court terme par les CSH HOXA4+ par rapport aux CSH HOXB4+, associée à une meilleure reconstitution lymphoïde. Nous avons aussi comparé les niveaux d’expression de gènes cibles potentiels dans des CSH surexprimant HOXA4 ou HOXB4 et observer que plusieurs gènes importants pour la fonction des CSH était régulé positivement suite à leur surexpression, notamment plusieurs gènes impliqués dans les voies de signalisation Notch et Wnt, tels que des récepteurs et ligands. Les gènes HOX du paralogue 4 pourraient donc réguler la communication entre les CSH et leur microenvironnement via ces voies de signalisation majeures et ainsi réguler leur autorenouvellement. La modulation de différents gènes codant pour des facteurs de transcription et des molécules impliquées dans la pluripotence suggère également que HOXA4 et HOXB4 utilisent des mécanismes intrinsèques et extrinsèques pour réguler leur potentiel d’autorenouvellement.
Ces connaissances pourront ainsi être utilisées pour optimiser les protocoles d’expansion ex vivo des CSH dans un but thérapeutique. / Transplantation of hematopoietic stem cells (HSC) is a treatment commonly used to treat several types of hematological diseases such as leukemia. However, a major limitation of this type of treatment is the limited number of HSC available for transplantation. It is therefore important to develop ways to expand these cells ex vivo. The HOXB4 gene is a promising candidate for achieving this goal. It has indeed been shown that HOXB4 is able to expand HSC in vivo and in vitro without inducing leukemia. HOXC4, which belongs to the same paralog group, is also able to expand primitive hematopoietic cell suggesting a common role for paralog 4 HOX genes in the self-renewal of HSC.
HOXA4 is ten times more expressed in fetal liver HSC during their primary expansion. Furthermore, Hoxa4 mutant HSC, unlike Hoxb4 mutant HSC, are unable to reconstitute an irradiated recipient when transplanted in competition. Therefore, HOXA4 could play a more important role than other paralog 4 genes for HSC expansion at the physiological level and we hypothesized that HOXA4 can expand HSC more efficiently than HOXB4.
The results obtained during this research project showed that the overexpression of HOXA4 expand HSC and primitive hematopoietic progenitors in the same order as HOXB4. Direct competitive culture and transplantation assays confirmed the equal capacity of HSC overexpressing HOXA4 and HOXB4 to proliferate and engraft at long-term. However, we observed a better short-term peripheral reconstitution by HOXA4+ HSC compared to HOXB4+ HSC, which was associated with a better lymphoid reconstitution. We also compared the expression levels of potential target genes in HSC overexpressing HOXA4 or HOXB4 and observed that many genes important for HSC function were upregulated following their overexpression, including several genes involved in the Notch and Wnt signaling pathway. These included both receptors as well as ligands, indicating that HOX4 genes might regulate the communication of primitive HSCs with their environment through these major signaling pathways and promote self-renewal. In addition, modulation of genes coding for transcription factors and molecules known for their function in pluripotency suggest that HOXA4 and HOXB4 have both intrinsic and extrinsic potential to control self renewal potential.
This knowledge can then be further explored and used to optimize ex vivo HSC expansion protocols for clinical purposes.
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Semi-supervised anomaly detection in mask writer servo logs : An investigation of semi-supervised deep learning approaches for anomaly detection in servo logs of photomask writers / Semiövervakad anomalidetektion i maskritares servologgar : En undersökning av semi-övervakade djupinlärningsmetoder för anomalidetektion i servologgar av fotomaskritareLiiv, Toomas January 2023 (has links)
Semi-supervised anomaly detection is the setting, where in addition to a set of nominal samples, predominantly normal, a small set of labeled anomalies is available at training. In contrast to supervised defect classification, these methods do not learn the anomaly class directly and should have better generalization capability as new kinds of anomalies are introduced at test time. This is applied in an industrial defect detection context in the logs of photomask writers. Four methods are compared: two semi-supervised one-class anomaly detection methods: Deep Semi-Supervised Anomaly Detection (DeepSAD), hypersphere classifier (HSC) and two baselines, a reconstructive GAN method based on the Dual Autoencoder GAN (DAGAN) and a non-learned distance method based on the Kullback-Leibler divergence. Results show that semi-supervision increases performance, as measured by ROC AUC and PRO AUC, of DeepSAD and HSC, but at the tested supervision levels, do not surpass the performance of DAGAN. Furthermore, it is found that autoencoder pretraining increases performance of HSC similarly to as it does for DeepSAD, even though only the latter is recommended in literature. Lastly, soft labels are utilized for HSC, but results show that this has no or negative effect on the performance. / Inom semiövervakad anomalidetektion finns det förutom en mängd nominella datapunkter (huvudsakligen normala), även en liten mängd märkta anomalier tillgängliga vid träning. I motsats till övervakad defektklassifikation lär sig dessa metoder inte att känna igen anomaliklassen direkt och bör ha större generaliseringsförmåga när nya sorters anomalier introduceras vid testning. Detta appliceras inom industriell defektdetektion i loggarna för fotomaskritare. Fyra metoder jämförs: Djup Semiövervakad Anomalidetektion (DeepSAD), hypersfärklassificerare (HSC) och två basnivåer, en rekonstruktiv GAN-metod baserad på Dual Autoencoder GAN (DAGAN) och en ickke-lärd avståndsmetod baserad på Kullback-Leibler-divergens. Resultaten visar att semiöervakning förbättrar prestationen, mätt med hjälp av ROC AUC och PRO AUC, för DeepSAD och HSC. Däremot överträffar det inte, för de testade övervakningsnivåerna, prestationen för DAGAN. Vidare kan det ses att autokodningsförträning förbättrar prestationen för HSC på ett liknande sätt som det gör för DeepSAD, trots att bara det senare rekommenderas i litteraturen. Slutligen används mjuka märkningar för HSC, men resultaten visar att detta har liten eller till och med negativ påverkan på resultatet.
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Обоснование выбора способа плавки медных концентратов : магистерская диссертация / Rationale for the choice of method of melting copper concentratesЗвонцов, Н. О., Zvontsov, N. O. January 2018 (has links)
Выпускная квалификационная работа, 67 с., 9 рис., 9 табл., 25 источников.
Объект исследования – способы плавки медных концентратов.
Предмет исследования – поиск наиболее эффективного способа плавки медных концентратов.
Цель работы – провести ряд технологических и экономических расчетов, на основании которых определить наиболее эффективный способ плавки медных концентратов.
В процессе работы были проведены расчеты материальных балансов и тепловых балансов для выбранных способов плавки медных концентратов. Также проведен сравнительный анализ выбранных способов плавки по полученным результатам расчетов и проведена предварительная экономическая оценка инвестиционного проекта, предполагающего замену отражательных печей предприятия ОАО «Святогор» на печи, являющимися основным оборудованием для того способа плавки, который будет выбран наилучшим по результатам анализа.
В результате работы был сделан вывод об эффективности выбранных способов плавки медных концентратов относительно друг друга, среди которых был выбран наиболее эффективный способ плавки. А также проведена предварительная экономическая оценка инвестиционного проекта, предполагающего замену отражательных печей предприятия ОАО «Святогор» на печи, являющимися основным оборудованием для того способа плавки, который будет выбран наилучшим по результатам анализа. / Final qualifying work, 67 pp., 9 pics., 9 tables, 25 references
Object – methods of melting copper concentrates.
Subject – search for the most effective method of melting copper concentrates.
The purpose of the work is to carry out a number of technological and economic calculations on the basis of which the most effective method of melting copper concentrates is determined.
In the course of work, material balances and heat balances were calculated for the selected methods for melting copper concentrates. The analysis of the selected melting methods was also carried out based on the results of calculations, and a preliminary economic evaluation of the investment project was carried out, involving the replacement of the reflector furnaces of the Svyatogor enterprise with the furnace, which is the main equipment for the melting method to be chosen as best according to the analysis.
As a result of the work, a conclusion was made about the effectiveness of the chosen methods of melting copper concentrates relative to each other, among which the most effective method of melting was chosen. Also, a preliminary economic evaluation of the investment project was carried out, which involves replacing the reflective furnaces of the Svyatogor enterprise with the furnace, which is the main equipment for the melting process, which will be chosen the best according to the results of the analysis.
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Habitat Suitability Criteria for Zuni Bluehead Sucker Catostomus discobolus yarrowi and Navajo Nation Genetic Subunit Bluehead Sucker Catostomus discobolus and Comparing Efficiency of AFS Standard Snorkeling Techniques to eDNA Sampling TechniquesUlibarri, Roy M. January 2016 (has links)
I quantified habitat selection for the endangered Zuni Bluehead Sucker Catostomus discobolus yarrowi and the Navajo Nation Genetic Subunit (NNGS) Bluehead Sucker Catostomus discobolus - a recent taxon described from genetic information. Both taxa are found in northern Arizona and New Mexico border regions. I examined fish [≥50 millimeters (mm) total length (TL)] selection of microhabitat conditions (i.e., water velocity, substrate size, overhead cover, water depth, instream cover, and mesohabitat conditions [i.e., pool, run riffle], during summer base flow conditions for NNGS Bluehead Suckers, and during both summer base flow and high spring flow conditions for Zuni Bluehead Suckers in six streams). Electrofishing, seining, and snorkeling were used to evaluate fish occupancy. From this information, I developed stream specific habitat suitability criteria (HSC) and then generalized HSC for each taxon, and tested transferability of the generalized HSC to individual streams. Zuni Bluehead Suckers and NNGS Bluehead Suckers occupied similar habitats: low velocity pools; sand, silt, and pebble substrate; high percent of instream cover; and water temperatures ranging from 2-21°C. However, Zuni Bluehead Suckers selected for low (0-25%) overhead cover where as NNGS Bluehead Sucker selected for high (0-75%) overhead cover. This was likely due to the source of instream cover–aquatic macrophytes that required sunlight in the Zuni Bluehead Sucker streams, and large woody debris falling from overhead branches in the NNGS Bluehead Sucker streams. Suggestions for managers includes maintaining existing cover or artificially construct additional instream cover; promote overhead cover (e.g., maintaining large trees along streams) and pool mesohabitats. In addition to this work I also tested the new method of environmental DNA (eDNA) to further help conservation efforts for these taxa. Environmental DNA has typically been used to detect invasive species in aquatic environments through water samples. I compared the efficacy of eDNA methodology to American Fisheries Society standard snorkeling surveys to detect presence of a rare fish species. My study site included three streams on the Navajo Nation in northern Arizona and northern New Mexico containing Navajo Nation Genetic Subunit Bluehead Sucker Catostomus discobolus and the Zuni Bluehead Sucker Catostomus discobolus yarrowi. To determine sample sites, I first divided entire wetted area of streams into 100-m consecutive reaches. I systematically selected 10 of those reaches for snorkel and eDNA surveys. Water samples were taken in 10-m sections within each 100-m reach, and fish presence via snorkeling was noted in each 10-m section as well. Water samples were collected at the downstream starting point of each reach, and continued upstream in each section 5 to 8 m ahead of the snorkeler. A qPCR was run on each individual water sample in quadruplicate to test for sucker presence or absence. I was able to positively detect both species with eDNA sampling techniques in two out of three streams. Snorkeling resulted in positive detections of both species in all three streams. In streams where fish were detected with eDNA sampling, snorkeling detected fishes at 11-29 sites per stream, where as eDNA detected fish at 3-12 sites per streams. My results suggested that AFS standard snorkeling was more effective at detecting target fish species than eDNA. To improve eDNA sampling, the amount of water collected and tested should be increased. Additionally, filtering water on site may improve eDNA techniques for detecting fish. Future research should focus on standardizing eDNA sampling to provide a widely operational sampling tool similar to electrofishing, netting, and hydroacoustics.
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T cells development in vitro : a minimalist approachLapenna, Antonio January 2012 (has links)
T lymphocytes are considered an essential and advanced component of the immune system, since these cells are able to discriminate self from non-self, start up an immune reaction and further develop into memory cells. However, therapies based on the use of patient derived newly generated T cells reinoculated into humans do not exist. This is due to difficulties in replicating the peculiar conditions required for T cell development in vitro. The systems developed so far are based on the use of animal or unrelated human thymic tissue and therefore they would not be adequate to be used in any clinical application. Having conjectured that human skin cells, rearranged in a threedimensional fashion, would be able to support the development of human T lymphocytes from hematopoietic stem cells, we developed a model consisting of human skin keratinocytes and fibroblasts arrayed on a synthetic matrix so to create a prototype suitable to be translated into the clinic. In this way we were able to induce few hundred cord blood CD34⁺ haematopoietic stem cells to entirely develop into mature CD4⁺ or CD8⁺ T lymphocytes in vitro. However, circulating adult peripheral CD34⁺ precursors failed to survive in the same conditions. Finally we were able to explain our success as consequence of strong induction of the Notch delta ligand Dll-4 by the keratinocytes cultured in the construct. In synthesis, we report here for the first time that skin keratinocytes, in the presence of fibroblasts and reconfigured in a three-dimensional arrangement, are able to induce the differentiation of a minimal amount of cord but not adult blood stem cells into fully differentiated T cells by acting through the Dll-4 Notch signaling pathway in vitro.
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Les rôles des gènes Hoxa9 et Meis1 dans l'hématopoïèse normale et leucémiqueMamo, Aline January 2005 (has links)
Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.
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Spatio-Temporal Characterization of Ligand-Receptor Interactions in Haematopoietic Stem Cell Rolling during HomingAl Alwan, Bader 11 1900 (has links)
Researches on Hematopoietic Stem Cell (HSC) have been expanding that leads to an increase in our understanding of HSC normal behaviors and abnormal alterations. One of the most important issues in the research on HSCs is to understand the mechanism of the homing process of these cells to settle in their niche in the bone marrow and establish the production of various blood cell types after bone marrow transplantation. The cells first must come in contact with the endothelial cells. This contact is known as adhesion and occurs through a multi-step paradigm ending with transmigration to the bone marrow niche. The initial step of the homing, tethering and rolling of HSC, is mediated by P- and E-Selectins present on endothelial cell surface through their interactions with the ligands expressed on the surface of HSC. Thus, understanding the adhesion process and its contribution for efficient HSCs homing will have great impact on HSC therapy. The selectin – ligands interaction has been intensively studied using in vivo and in vitro approaches. However, the molecular mechanism involved by HSCs at single molecule level is poorly understood. Here in this study, a novel experimental method to unravel the
molecular mechanisms of the Selectin-ligands interactions in vitro at the single molecule level is developed by combining microfluidics, epi-fluorescence microscopy and live cells. In this work, the new single-molecule imaging technique enabled us to directly visualize the nanoscale spatiotemporal dynamics of the membrane protein-ligand interactions under conditions of shear stress acting on the cells at the molecular level in real time. Using this method, we revealed that selectin ligands on membrane-tethers and slings show unique spatiotemporal dynamics that is distinct from those on the cell body. We demonstrated that the membrane tethers are formed from single microvilli on the cells, which provides a mechanism to spatially localize selectin ligands, PSGL-1 and CD44 on the tethers and slings. We also demonstrated that the selectin ligands show fast diffusional motion along the tethers and slings compared with that on the cell body due to the detachment of cell membranes from actin cytoskeleton during the formation of the tethers. Our results suggest that the spatial confinement of the selectin ligands together with the fast scanning of a large area by the selectin ligands increase the efficiency of selectin-ligands interaction during the rolling, resulting in slow and stable rolling of the cell on selectin. Our findings contribute significantly to molecular level understanding of the initial step of HSCs. This single-molecule imaging technique that we developed in this study will find wide applications in the molecular-level studies on cell-cell interactions including cancer cell metastasis.
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