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Santi, Juliana de 1982. "Gerenciamento ativo de filas para o protocolo "High Speed Transmission Control Protocol" em redes com produto banda-atraso elevado." [s.n.], 2008. http://repositorio.unicamp.br/jspui/handle/REPOSIP/276151.
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Orientador: Nelson Luis Saldanha da FonsecaDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Computação
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Resumo: A utilização eficiente da banda passante em redes de alta velocidade e grandes atrasos, denominadas redes com produto banda-atraso elevado (PBA), tornou-se um grande desafio. Isto ocorre devido aos ajustes do protocolo Transmission Control Protocol (TCP). O High Speed TCP (HSTCP), uma variante do TCP para redes com PBA elevado, emprega ajustes mais agressivos permitindo, assim, que a utilização da banda seja escalável. As políticas de Gerenciamento Ativo de Filas ou Active Queue Management (AQM), monitoram o nível de ocupação das filas nos roteadores e notificam o congestionamento incipiente aos emissores TCP através do descarte/marcação de pacotes. O sistema de controle de congestionamento apresenta natureza de retroalimentação, na qual a taxa de transmissão dos nós fontes é ajustada em função do nível de ocupação da fila. Os controladores AQM determinam a probabilidade de descarte/marcação para maximizar a vazão e minimizar perdas, garantindo, assim, a estabilidade do tamanho da fila independentemente das variações das condições da rede. Neste trabalho, define-se a política de gerenciamento ativo de filas HSTCP-H2 para redes com PBA elevado que utilizam o protocolo HSTCP. Para a derivação de HSTCPH2: são utilizadas técnicas de Teoria de Controle Ótimo. A principal característica desta política é considerar o atraso do sistema o que permite melhor utilização dos recursos disponíveis. A estabilidade e os objetivos de desempenho do sistema são expressos e solucionados através de Desigualdades Matriciais Lineares, permitindo que os parâmetros do controlador possam ser calculados através da solução de um problema convexo simples. Diferentes controladores foram derivados considerando-se diferentes objetivos de desempenho, os quais consideram as características de redes com produto banda-atraso elevado. Através de simulações, os desempenhos dos controladores derivados são avaliados e a eficácia do controlador que apresentou o melhor desempenho foi comparado com o desempenho da política de AQM RED. São considerados cenários com enlace gargalo único e com múltiplos gargalos.
Abstract: The efficient utilization of bandwidth in high speed and large delay networks, called high bandwidth-delay product networks (BDP), has become a major challenge. This is due to adjustments of the Transmission Control Protocol (TCP). The High Speed TCP HSTCP): a TCP variant to high BDP networks, employs more aggressive adjustments, allowing scalable bandwidth utilization. The Active Queue Management (AQM) policies monitor the queue length in the routers and notify incipient congestion to TCP source by marking or dropping packets. The congestion control system presents intrinsic feedback nature, where the transmission rates of the sources are adjusted according to the level of congestion inferred by the queue occupancy. The AQM controllers determine the dropping marking probability values to maximize throughput and minimize losses, giving guarantees to stabilize the queue length independent of network conditions. In this work, it is defined HSTCP-H2, an active queue management policy to high BDP networks, which adopt the HSTCP as their transport protocol. Optimal control theory is used to conceive HSTCP-H2. The novelty of the proposed approach lies in consider the delay of the system which allows better use of available resources. Furthermore, in the proposed approach, stability and performance objectives are completely expressed as Linear Matrix Inequalities (LMIs), thus requiring the solution of a single convex problem for the computation of the controller parameters. Different controllers are derived considering different design goals, which take into account the characteristics of the high bandwidth-delay product networks. The performance produced by different optimal controllers was investigated. The efficacy of the controller with the best performance was then compared to the performance of RED policy. The simulation experiments were carried out using topologies with single and multiple bottleneck.
Mestrado
Redes de Computadores
Mestre em Ciência da Computação
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Haeberle, Raphael. "Search for new heavy stable charged particles with the CMS experiment." Electronic Thesis or Diss., Strasbourg, 2024. http://www.theses.fr/2024STRAE008.
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Cette thèse présente les résultats obtenus en utilisant deux stratégies différentes de recherche de particules lourdes, chargées et à long temps de vie dans l'expérience CMS auprès du LHC, utilisant des données issues de collisions proton-proton à une énergie de √s= 13 TeV, collectées entre 2017 et 2018 et qui correspondent à une luminosité intégrée de 101 fb-1. Cette recherche est divisée en deux stratégies, la première se concentre sur l'utilisation de signature s exotiques de dépôts de haute énergie dans le trajectographe en silicium, et la seconde utilise le temps de vie comme signature additionelle. Les bruits de fond associés sont estimés dans les données par deux méthodes différentes, et des procédures de validation sont effectuées dans des regions de contrôle. Les résultats de la première stratégie ont été soumis à une interpétation statistique puis rendus publics, tandis que les résultats de la seconde stratégie ne le sont pas encoreThis thesis presents the results obtained using two different strategies in the search for heavy stable charged particles in the CMS experiment at the LHC, using proton-proton collision data at an energy of √s= 13 TeV corresponding to an integrated luminosity of 101 fb-1 collected between 2017 and 2018. This research is divided into two strategies: the first focuses on using exotic signatures of high-energy deposits in the silicon tracker, and the second uses lifetime as an additional signature. Backgrounds are estimated in the data using two different methods, and validation procedures are performed in control regions. The results of the first strategy have been subjected to statistical interpretation and made public, while the results of the second strategy are not yet public
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Jayavaradhan, Rajeswari. "Optimization of Gene Editing Approaches for Human Hematopoietic Stem Cells." University of Cincinnati / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1543919940219677.
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Husain, I. "Studies on the mitochondrial Hsup(+)-ATPase complex and its interaction with the Hsup(+)-ATPase inhibitor protein." Thesis, University of Leeds, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.355705.
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Paula, Ana Carolina Barbosa de [UNESP]. "Imunomarcação de proteínas de estresse (HSP 27, HSP 72, HSP 90) e proteína P53 em neoplasias mamárias de cadelas." Universidade Estadual Paulista (UNESP), 2010. http://hdl.handle.net/11449/95943.
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Os tumores de mama são a principal causa de morte em cães e isso vem despertando maior interesse no desenvolvimento de estudos relacionados a este distúrbio. A proximidade com os seres humanos, tanto na convivência quanto aos hábitos, podem influenciar o aparecimento das neoplasias. A semelhança dos tumores mamários caninos com os tumores de mama da mulher leva a um interesse no estudo da patologia comparada, estimulando o uso de modelos animais. Apesar dos muitos estudos, pouco se conhece sobre o prognóstico e as causas dos tumores mamários caninos, observando-se um esforço crescente na tentativa de acrescentar aos fatores prognósticos clássicos novos parâmetros, de natureza molecular, que auxiliem a decisão clínica, à semelhança do verificado em Medicina Humana, estando entre eles os marcadores moleculares, como a proteína P53 e as proteínas de estresse. A expressão de proteína P53 e das proteínas de estresse tem sido observada em muitas neoplasias, incluindo o câncer de mama. Nesse sentido, o objetivo do presente estudo foi investigar a imunomarcação de HSP 27, HSP 72, HSP 90 e proteína P53 em tecido mamário normal e neoplásico de cadelas e estabelecer uma relação entre a expressão destas proteínas e o grau histológico das neoplasias. Foi realizada análise estatística e o nível de significância (α) adotado foi de 5%. Dentre os tumores malignos, os carcinomas simples foram o tipo histológico predominante. Para a proteína P53, não houve diferença significativa em sua expressão entre os grupos de tumores malignos avaliados, ocorrendo o mesmo para as HSPs 27, 72 e 90. A sensibilidade do teste de imuno-histoquímica para a proteína P53, nesta amostra, foi de 67,5%, a especificidade foi de 100%, o valor preditivo positivo foi de 100%, o valor preditivo negativo foi de 25% e acurácia do teste foi de 92%. Ainda para a proteína P53, comparando-se o grupo...
Breast tumors are the leading cause of death in dogs and this has aroused great interest in developing studies related to this disease. The proximity with humans, much as in living habits, may influence the onset of tumors. The similarity of canine mammary tumors with breast tumors of women take an interest in the study of comparative pathology, stimulating the use of animal models. Despite many studies, little is known about the prognosis and causes of canine mammary tumors, observing a growing effort in trying to add to the classic prognostic factors new parameters of molecular nature, that help the clinical decision, like that seen in Human Medicine, and among them the molecular markers such as P53 and stress proteins. The expression of P53 protein and the stress proteins has been observed in many cancers, including breast cancer. Accordingly, the purpose of this study was to investigate the immunostaining of HSP 27, HSP 72, HSP 90 and P53 protein in normal and neoplastic breast tissue of female dogs and establish a relationship between the expression of these proteins and the histological grade of tumors. Statistical analysis was performed and significance level (α) was 5%. Among the malignant tumors, simple carcinomas were the predominant histologic type. Protein P53, presented no significant difference in expression between the evaluated groups of malignant tumors, the same occurring for HSPs 27, 72 and 90. The test sensitivity of immunohistochemistry for protein P53 in this sample was 67,5%, specificity was 100%, positive predictive value was 100%, negative predictive value was 25% and accuracy of test was 92%. Although protein P53, compared to the control group with other groups in relation to staining intensity and proportion score, there was a significant difference only between the group of simple carcinomas. With regard to staining intensity, we tested the correlation between... (Complete abstract click electronic access below)
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Paula, Ana Carolina Barbosa de. "Imunomarcação de proteínas de estresse (HSP 27, HSP 72, HSP 90) e proteína P53 em neoplasias mamárias de cadelas /." Jaboticabal : [s.n.], 2010. http://hdl.handle.net/11449/95943.
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Orientador: Antônio Carlos AlessiBanca: Karin Werther
Banca: Felipe Augusto Ruiz Sueiro
Resumo: Os tumores de mama são a principal causa de morte em cães e isso vem despertando maior interesse no desenvolvimento de estudos relacionados a este distúrbio. A proximidade com os seres humanos, tanto na convivência quanto aos hábitos, podem influenciar o aparecimento das neoplasias. A semelhança dos tumores mamários caninos com os tumores de mama da mulher leva a um interesse no estudo da patologia comparada, estimulando o uso de modelos animais. Apesar dos muitos estudos, pouco se conhece sobre o prognóstico e as causas dos tumores mamários caninos, observando-se um esforço crescente na tentativa de acrescentar aos fatores prognósticos clássicos novos parâmetros, de natureza molecular, que auxiliem a decisão clínica, à semelhança do verificado em Medicina Humana, estando entre eles os marcadores moleculares, como a proteína P53 e as proteínas de estresse. A expressão de proteína P53 e das proteínas de estresse tem sido observada em muitas neoplasias, incluindo o câncer de mama. Nesse sentido, o objetivo do presente estudo foi investigar a imunomarcação de HSP 27, HSP 72, HSP 90 e proteína P53 em tecido mamário normal e neoplásico de cadelas e estabelecer uma relação entre a expressão destas proteínas e o grau histológico das neoplasias. Foi realizada análise estatística e o nível de significância (α) adotado foi de 5%. Dentre os tumores malignos, os carcinomas simples foram o tipo histológico predominante. Para a proteína P53, não houve diferença significativa em sua expressão entre os grupos de tumores malignos avaliados, ocorrendo o mesmo para as HSPs 27, 72 e 90. A sensibilidade do teste de imuno-histoquímica para a proteína P53, nesta amostra, foi de 67,5%, a especificidade foi de 100%, o valor preditivo positivo foi de 100%, o valor preditivo negativo foi de 25% e acurácia do teste foi de 92%. Ainda para a proteína P53, comparando-se o grupo... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Breast tumors are the leading cause of death in dogs and this has aroused great interest in developing studies related to this disease. The proximity with humans, much as in living habits, may influence the onset of tumors. The similarity of canine mammary tumors with breast tumors of women take an interest in the study of comparative pathology, stimulating the use of animal models. Despite many studies, little is known about the prognosis and causes of canine mammary tumors, observing a growing effort in trying to add to the classic prognostic factors new parameters of molecular nature, that help the clinical decision, like that seen in Human Medicine, and among them the molecular markers such as P53 and stress proteins. The expression of P53 protein and the stress proteins has been observed in many cancers, including breast cancer. Accordingly, the purpose of this study was to investigate the immunostaining of HSP 27, HSP 72, HSP 90 and P53 protein in normal and neoplastic breast tissue of female dogs and establish a relationship between the expression of these proteins and the histological grade of tumors. Statistical analysis was performed and significance level (α) was 5%. Among the malignant tumors, simple carcinomas were the predominant histologic type. Protein P53, presented no significant difference in expression between the evaluated groups of malignant tumors, the same occurring for HSPs 27, 72 and 90. The test sensitivity of immunohistochemistry for protein P53 in this sample was 67,5%, specificity was 100%, positive predictive value was 100%, negative predictive value was 25% and accuracy of test was 92%. Although protein P53, compared to the control group with other groups in relation to staining intensity and proportion score, there was a significant difference only between the group of simple carcinomas. With regard to staining intensity, we tested the correlation between... (Complete abstract click electronic access below)
Mestre
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David, Dylan Naitraj. "Inflammation-Induced HSPC Dysfunction: Towards a Better Understanding of the Role of MAVS, ASC, and Caspase-1 in HSPC Dysfunction and Bone Marrow Failure." University of Cincinnati / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1626356668978688.
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Möhrle, Bettina Maria [Verfasser]. "Stem cell specific mechanisms ensure genomic fidelity within hematopoietic stem cells (HSCs) and upon aging of HSCs / Bettina Maria Möhrle." Ulm : Universität Ulm, 2016. http://d-nb.info/1104840006/34.
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Holuša, Jan. "Modelování protokolů HSRP a GLBP pro redundanci brány." Master's thesis, Vysoké učení technické v Brně. Fakulta informačních technologií, 2016. http://www.nusl.cz/ntk/nusl-255366.
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This thesis deals with theoretical analysis of First Hop Redundancy Protocols. It describes Hot Standby Router Protocol, Virtual Router Redundancy Protocol and Gateway Load Balancing Protocol. It also shows examples of configuration of each protocol on Cisco devices with supported version of the Cisco IOS. Furthermore, this thesis includes design of two of these protocols, Hot Standby Router Protocol and Gateway Load Balancing Protocol, and their implementation in discrete event simulator OMNeT++ and Automated Network Simulation and Analysis library. Finally, the thesis presents results of testing of the implementations in comparison with actual Cisco devices.
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Galeas, Pena Trilce Michelle. "Thermoregulation of capsule production of Streptococcus pyogenes strain HSC5 /." Available to subscribers only, 2009. http://proquest.umi.com/pqdweb?did=1967978671&sid=7&Fmt=2&clientId=1509&RQT=309&VName=PQD.
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Golovidov, Oleg. "Variable-Complexity Approximations for Aerodynamic Parameters in Hsct Optimization." Thesis, Virginia Tech, 1997. http://hdl.handle.net/10919/36789.
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A procedure for generating and using polynomial approximations to the range or to the cruise drag components in terms of 29 design variables for the High Speed Civil Transport (HSCT) configuration and performance design is presented.
Response surface model methodology is used to fit quadratic polynomials to data gathered from a series of numerical analyses of different HSCT designs. Several techniques are employed to minimize the number of required analyses and to maintain accuracy. Approximate analysis techniques
are used to find regions of the design space where reasonable HSCT designs could occur and response surface models are built using higher fidelity analysis results of the designs in this "reasonable" region. Regression analysis and analysis of variance are then used to reduce the number of polynomial terms in the response surface model functions.
Optimizations of the HSCT are then carried out both with and without the response surface models, and the effect of the use of the response surface models is discussed. Results of the work showed that considerable reduction of the amount of numerical noise in optimization is achieved with response surface models and the convergence rate was slightly improved. Careful attention was required to keep the accuracy of the models at an acceptable level.
NOTE: (07/2012) An updated copy of this ETD was added after there were patron reports of problems with the file.Master of Science
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Zhang, Jianbing. "Characterization of HSCs in zebrafish using label-retaining strategy /." View abstract or full-text, 2009. http://library.ust.hk/cgi/db/thesis.pl?BICH%202009%20ZHANG.
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Galeas, Trilce Michelle. "Thermoregulation of Capsule Production of Streptococcus pyogenes Strain HSC5." OpenSIUC, 2009. https://opensiuc.lib.siu.edu/theses/122.
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Group A Streptococcus (GAS) is responsible for mild and common infections like tonsillitis and pharyngitis, and more serious invasive disorders like necrotizing fasciitis and glomerulonephritis. The ability to invade tissues is closely linked to the virulence factors expressed by the bacterium. Hyaluronic acid capsule expression is variable among all the strains in S. pyogenes and confers the capacity to evade the immune response. In a previous study, it was found that capsule production in CovR mutants was temperature-regulated, showing no capsule production at 37℃ but increased production was observed at 25℃. In this study, the objective is to find the elements involved in the thermoregulation using a genetic approach. First, mutants were created by knocking-out CovR, the response regulator of the CovRS two-component system that controls about 15% of GAS genome. Transposon mutants were screened to find changes in capsular phenotype. Colonies expressing capsule at 37℃ were selected for sequencing. The sequencing revealed three different events in different mutants. Two of them pointed at hypothetical proteins, one of them, SpyM3_1255, was phage associated protein with a DnaD domain and the other one, SpyM3_1377, encoded cvfA. A third over-producer mutant showed an insertion in the promoter area of the has operon, the operon that encodes for hyaluronan synthase production, upstream from other disruptions in the promoter area that generated non-producing mutants. This suggest that there is more than one factor involved in thermoregulation of capsule production.
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Stein, Sebastian [Verfasser]. "HSC-Kantenbearbeitung von Blech / Sebastian Stein." Aachen : Shaker, 2010. http://d-nb.info/1081884592/34.
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Faulds, Gary Bryan. "Hsp 90 in lupus-prone mice." Thesis, University College London (University of London), 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.281722.
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Larsson, Hilmersson Annika. "Att leva med HSP : Högkänsligas berättelser." Thesis, Mälardalens högskola, Akademin för hälsa, vård och välfärd, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:mdh:diva-44811.
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Högkänslighet är än så länge ett relativt okänt karaktärsdrag i personligheten. Trots att 15-20 procent av befolkningen tros ha en högkänslighet, enligt tidigare studier, så är forskningen begränsad om fenomenet och dess konsekvenser på individnivå. Syftet med denna undersökning var att ta reda på upplevelsen av att leva med högkänslighet med hjälp av en kvalitativ metod. Intervjuer med nio stycken intervjupersoner med en högkänslig personlighet utfördes, spelades in, transkriberades och analyserades. Resultatet visade att överväldigande, strategier, uppvaknande och visioner var gemensamma teman för samtliga respondenter. Konsekvenserna var både negativa och positiva, såsom uttröttning, depression, lyckorus och en god empatisk förmåga. Det som kunde hjälpa en högkänslig individ var strategier för återhämtning och en förståelse från omgivningen. Undersökningen visade både gemensamma faktorer och skillnader i upplevelsen av högkänslighet. Om högkänslighet kan ses som en resurs istället för en nackdel, skulle kombinationen av högkänsliga och icke högkänsliga vara bra och till nytta för samhället.
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Knill, Duane L. "Implementing Aerodynamic Predictions from Computational Fluid Dynamics in Multidisciplinary Design Optimization of a High-Speed Civil Transport." Diss., Virginia Tech, 1997. http://hdl.handle.net/10919/29530.
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A method to efficiently introduce supersonic drag predictions from computational fluid dynamics (CFD) calculations in a combined aerodynamic-structural optimization of a High-Speed Civil Transport (HSCT) is presented. To achieve this goal, the method must alleviate the large computational burden associated with performing CFD analyses and reduce the numerical noise present in the analyses. This is accomplished through the use of response surface (RS) methodologies, a variation of the variable-complexity modeling (VCM) technique, and coarse grained parallel computing. Variable-complexity modeling allows one to take advantage of the information gained from inexpensive lower fidelity models while maintaining the accuracy of the more expensive high fidelity methods. The utility of the method is demonstrated on HSCT design problems of five, ten, fifteen, and twenty design variables.
Motivation for including CFD predictions into the HSCT optimization comes from studies detailing the differences in supersonic aerodynamic predictions from linear theory, Euler, and parabolized Navier-Stokes (PNS) calculations for HSCT configurations. The effects of these differences in integrated forces and distributed loads on the aircraft performance and structural weight are investigated. These studies indicate that CFD drag solutions are required for accurate HSCT performance and weight estimates.
Response surface models are also used to provide useful information to the designer with minimal computational effort. Investigations into design trade-offs and sensitivities to certain design variables, available at the cost of evaluating a simple quadratic polynomial, are presented. In addition, a novel and effective approach to visualizing high dimensional, highly constrained design spaces is enabled through the use of RS models.
NOTE: An updated copy of this ETD was added in July 2012 after there were patron reports of problems with the original file.Ph. D.
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Burgee, Susan L. "A coarse-grained variable-complexity MDO paradigm for HSCT design." Thesis, This resource online, 1995. http://scholar.lib.vt.edu/theses/available/etd-08142009-040544/.
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QUARANTA, PAMELA. "Unveiling the biological role of human circulating Hematopoietic Stem and Progenitor cells." Doctoral thesis, Università Vita-Salute San Raffaele, 2022. http://hdl.handle.net/20.500.11768/128257.
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Although mostly resident in the bone marrow (BM), few circulating HSPC (cHSPC) regularly traffic in the peripheral blood (PB) of un-mobilized subjects. Mainly descriptive studies have been published so far about this rare population in humans, and a complete evaluation of their composition, functional features and hierarchical relationship with respect to BM HSPC is still missing.
In the present study, we phenotypically characterized cHSPC composition during aging, by applying multi-parametric flow cytometry on 114 PB and, as control, 48 BM samples of healthy donors (HD) of diverse age. These analyses were integrated with single-cell transcriptome profiling (scRNAseq), and ad hoc designed in vitro and in vivo assays to investigate the transcriptional and functional properties of steady-state cHSPC with respect to BM counterpart. Moreover, to study circulating vs. resident HSPC relationships and differentiation potential in vivo in humans, we exploited integration site (IS) clonal tracking of cHSPC, BM HSPC and mature PB lineages isolated from patients treated with HSPC-gene therapy (GT).
We observed that cHSPC show a progressive reduction in number during aging and a different composition than BM counterpart, with Multi Lymphoid Progenitors (MLP) displaying the highest PB circulation capability. cHSPC are endowed with multilineage differentiation potential both in vitro and in vivo, with comparable BM homing capability but reduced long-term survival after transplantation in immune-deficient mice than BM HSPC. This latter finding can be explained by the low primitive HSC content and the transcriptional pre-activated state observed in steady-state PB HSC.
Indeed, applying scRNA-seq, we identified a unique transcriptional profile of both primitive and lineage-committed cHSPC subpopulations, characterized by lower replicative, metabolic and transcriptional activity, but increased differentiation-, adhesion- and immune response-priming than BM counterpart. The enrichment of lymphoid phenotypic and transcriptional signatures found in PB HSPC, together with their higher IS sharing with PB lymphoid than myeloid mature lineages suggest that cHSPC could have a role in seeding lymphoid organs. Moreover, the higher expression of erythroid marker genes detected in trafficking than resident HSPC was consistent with cHSPC erythroid differentiation bias observed after transplantation and in single-cell in vitro differentiation assay. These findings suggest cHSPC as a source of erythroid-committed progenitors, able to sustain stress-responsive extramedullary erythropoiesis.
Finally, our preliminary data on a cohort of HSPC-GT patients suggest that cHSPC may sustain clonal redistribution to distant BM sites, both during active hematopoietic reconstitution and, at a lower extent, during steady-state conditions.
Altogether, our findings indicate PB trafficking HSPC as a peculiar steady-state reservoir of low-cycling, pre-activated hematopoietic progenitors, which continuously recirculate among multiple BM sites and are poised for promptly sustaining activation and in situ local hematopoietic differentiation in case of demand.Nonostante risiedano principalmente nel midollo osseo (BM), poche HSPC circolanti (cHSPC) ricircolano regolarmente nel sangue periferico (PB) di donatori sani in assenza di mobilizzazione. Soprattutto studi di tipo descrittivo sono stati pubblicati sino ad ora su questa rara popolazione nell’uomo e al momento manca una valutazione completa della loro composizione, caratteristiche funzionali e relazione gerarchica rispetto alle HSPC di BM. In questo studio abbiamo caratterizzato fenotipicamente la composizione delle cHSPC durante l'invecchiamento fisiologico, applicando la citometria a flusso multiparametrica su 114 campioni di PB e, come controllo, 48 campioni di BM di donatori sani (HD) di età diversa. Queste analisi sono state integrate con l’analisi del profilo trascrizionale a livello di singola cellula (scRNAseq) e studi in vitro e in vivo, al fine di studiare rispettivamente le proprietà trascrizionali e funzionali delle cHSPC in condizioni fisiologiche rispetto alla controparte di BM. Inoltre, per studiare la relazione tra HSPC circolanti e residenti nel midollo osseo e il loro potenziale di differenziamento in vivo nell'uomo, abbiamo effettuato il monitoraggio clonale dei siti d’integrazione (IS) di cHSPC, BM HSPC e cellule mature di PB isolate da pazienti trattati con terapia genica (GT) basata su HSPC. Abbiamo osservato che le cHSPC si riducono progressivamente durante l'invecchiamento e sono caratterizzate da una composizione diversa rispetto alla controparte di BM, mostrando una più alta capacità di ricircolo dei progenitori linfoidi. Le cHSPC sono in grado di differenziare nelle varie popolazioni ematopoietiche sia in vitro che in vivo, mostrando una simile capacità di migrazione nel BM ma una ridotta sopravvivenza a lungo termine dopo trapianto in topi immunodeficienti rispetto a HSPC di BM. Quest'ultima scoperta può essere spiegata dal basso contenuto di HSC primitive e da uno stato trascrizionale pre-attivato presente nelle HSC di sangue periferico in condizioni fisiologiche. Applicando scRNA-seq, abbiamo identificato un profilo trascrizionale unico nelle sottopopolazioni di cHSPC, sia primitive che più differenziate, caratterizzato da una minore attività replicativa, metabolica e trascrizionale, ma una maggiore capacità di differenziamento, adesione e risposta immunitaria rispetto alla controparte di BM. L'arricchimento dei profili fenotipici e trascrizionali di tipo linfoide osservato nelle HSPC di PB, in associazione con la loro più elevata condivisione di IS con cellule di PB di lignaggio linfoide rispetto a quello mieloide, suggeriscono che le cHSPC potrebbero seminare organi linfoidi. Inoltre, la maggiore espressione di geni di differenziamento eritroide rilevati nelle HSPC circolanti rispetto a quelle residenti è coerente con il bias di differenziamento eritroide osservato nelle cHSPC post-trapianto e nel test di differenziamento in vitro. Questi risultati suggeriscono che le cHSPC costituiscono una fonte di progenitori eritroidi, in grado di sostenere l'eritropoiesi extramidollare in risposta a stress. Infine, i nostri dati preliminari su una coorte di pazienti trattati con HSPC-GT suggeriscono che il ricircolo di HSPC può sostenere la ridistribuzione clonale di cellule staminali in siti midollari distanti, sia durante la ricostituzione ematopoietica attiva che, in misura minore, in condizioni fisiologiche. Complessivamente, i nostri risultati indicano le HSPC circolanti come una peculiare popolazione di progenitori ematopoietici in stato pre-attivato, che ricircolano continuamente tra diversi siti di midollo osseo e sono pronti per sostenere rapidamente un'attivazione e un differenziamento ematopoietico locale in situ in caso di necessità.
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Seemampillai, Borggia. "Role of hsp-27 in cardiac transplant rejection." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/23925.
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Prevention of allograft rejection following cardiac transplantation is the major obstacle to long-term graft survival. Previous studies have suggested that overexpression of hsp-27 protects against non-transplant atherosclerosis and cardiac allograft vasculopathy in humans and ex vivo induced I/R injury in mice. The purpose of this study was to investigate whether overexpression of hsp-27 protects the heart from acute and chronic rejection using mice overexpressing HA-tagged human hsp-27 as donors. Overexpression of HA-tagged hsp-27 was confirmed by western blot and immunocytochemistry. ELISA showed presence of hsp-27 in the serum of transgenic animals. In the acute rejection model, B10.A hearts from transgenic or littermate controls were transplanted into C57BL/6 wild-type recipients. Survival of transgenic allografts was significantly prolonged compared to littermate control allografts. Furthermore, RT-PCR and immunohistochemistry results demonstrated decreased cellular infiltration of CD3+ and CD8+ T-cells and decreased inflammatory cytokines in transgenic allografts compared to controls. A chronic rejection model was established by grafting B10.A donor hearts into CD4+ T-cell depleted CBA recipients. Cardiac allografts harvested at 4, 6 and 8 weeks post-transplant showed reduced intimal thickening and less vessels affected in transgenic allografts compared to littermate controls. This was associated with significant diminution of infiltrating T-cells but augmentation of IL-4 production in transgenic grafts. Flow cytometry analyses showed that hsp-27 may not influence alloantibody production. Ex vivo studies suggest that overexpression of hsp-27 significantly decreased the activity of caspase-3, -9 and -1 following ischaemia. In addition, the increase in caspase-3 activity was significantly reduced in transgenic hearts following I/R injury in vivo. However, this study failed to demonstrate the immunomodulatory effect of hsp-27 in vitro. Our data suggest that hsp-27 protects against acute and chronic rejection. Protective mechanisms include a delay in inflammatory responses and protection against apoptosis of cardiomyocytes.
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Meiklejohn, Stuart J. "The role of BMP signalling in HSC ontogeny." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:305597a8-b8cb-42ff-88fd-34b3dd5bf39b.
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The haematopoietic stem cell (HSC) is found in the adult human bone marrow, where it gives rise to all the circulating blood cells throughout adulthood. Understanding the signalling events that programme these cells during development will improve HSC in vitro culture, their generation from embryonic stem cells or induced pluripotent stem cells, and their potential therapeutic application. HSCs bud from the floor of the dorsal aorta and seed the bone marrow via circulation. The precursors to the dorsal aorta and HSCs are called haemangioblasts, which are found in the dorsal lateral plate mesoderm in Xenopus. The knowledge of the location of these precursors allows their programming to be studied in detail during embryonic development. A key pathway implicated in the programming of HSCs is the BMP signalling pathway. Here, using both a small molecule inhibitor and a transgenic Xenopus line, BMP signalling has been inhibited post-gastrulation without perturbing the gross morphology of the embryo. This has shown that BMP signalling is required for HSC programming in the dorsal lateral plate mesoderm via the expression of a critical haematopoietic transcription factor, gata2. Morpholino knockdown of evi3has revealed it to be essential for HSC programming in the dorsal lateral plate mesoderm, where it is required for the expression of gata2. Furthermore, as evi3 is known to bind to the active BMP signalling complex, and as evi3 knockdown phenocopies post-gastrulation BMP inhibition, evi3 appears to be required for BMP signalling to initiate gata2 expression in the DLP. Taken together, the findings presented here demonstrate an essential post-gastrulation role of BMP signalling and Evi3 for programming HSCs in Xenopus.
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SCARFÒ, REBECCA. "Precise characterization of hemogenic endothelial cells during human hematopoietic development." Doctoral thesis, Università Vita-Salute San Raffaele, 2022. http://hdl.handle.net/20.500.11768/128259.
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During embryonic development, blood cells emerge from a subset of specialized endothelial cells, named hemogenic endothelium (HE), via a process known as endothelial-to-hematopoietic transition (EHT). HE represents a heterogeneous population found in various anatomical sites, including the yolk sac (YS) and the aorta-gonad-mesonephros (AGM). It comprises cells that differ in developmental potential, thus defining distinct hematopoietic programs. Despite the endothelial descendancy of blood cells is well established, the identity of HE is still debated. A more thorough characterization of HE is therefore essential to guide the efforts to derive this population from human pluripotent stem cells (hPSCs), a critical step to generate therapeutic blood products in vitro. However, current known markers used to isolate HE are insufficient as they also enrich associated arterial cells. To identify specific human HE markers, we performed transcriptomic analysis of 4-5-week-old human embryos, a developmental stage characterized by active EHT. We identified FCGR2B encoding for the Fc receptor CD32, previously associated with other specialized endothelia, as enriched gene in the ACE+CD34+ population that contains HE. Functional ex vivo analyses confirmed that multilineage hematopoietic potential is highly enriched in CD32+ endothelial cells isolated from the AGM and YS of human embryos. In addition, CD32 emerged as selective marker for hPSC-derived HE across different hematopoietic programs. Remarkably, our analyses showed that CD32 specificity for cells with hemogenic potential is superior to other known HE markers in hPSC-derived hematopoietic cultures. These findings provide a simple method for isolating HE from human embryos and hPSCs, allowing its molecular characterization as well as the efficient generation of hematopoietic cells in vitro.Durante lo sviluppo embrionale, le cellule del sangue emergono da un sottoinsieme di cellule endoteliali specializzate, denominate endotelio emogeno (HE), attraverso un processo noto come transizione endoteliale-ematopoietica (EHT). HE è una popolazione eterogenea che si trova in vari siti anatomici, tra cui il sacco vitellino (YS) e l'aorta-gonade-mesonefro (AGM). Comprende cellule che differiscono per il potenziale di sviluppo, definendo così programmi ematopoietici distinti. Nonostante la discendenza endoteliale delle cellule del sangue sia ben consolidata, l'identità di HE è ancora dibattuta. Una caratterizzazione più approfondita di HE è quindi essenziale per guidare gli sforzi nel derivare questa popolazione dalle cellule staminali pluripotenti umane (hPSC), un passaggio fondamentale per generare emoderivati terapeutici in vitro. Tuttavia, gli attuali marcatori noti utilizzati per isolare l'HE non sono sufficienti poiché arricchiscono anche le cellule arteriose associate. Per identificare specifici marcatori HE umani, abbiamo eseguito l'analisi trascrittomica di embrioni umani di 4-5 settimane, una fase di sviluppo caratterizzata da EHT attivo. Abbiamo identificato la codifica FCGR2B per il recettore Fc CD32, precedentemente associato ad altri endoteli specializzati, come gene arricchito nella popolazione ACE+CD34+ che contiene HE. Analisi funzionali ex vivo hanno confermato che il potenziale ematopoietico multilineare è altamente arricchito nelle cellule endoteliali CD32+ isolate dall'AGM e dall'YS di embrioni umani. Inoltre, CD32 è emerso come marcatore selettivo per HE derivato da hPSC in diversi programmi ematopoietici. Sorprendentemente, le nostre analisi hanno mostrato che la specificità del CD32 per le cellule con potenziale emogeno è superiore ad altri marcatori HE noti nelle colture ematopoietiche derivate da hPSC. Questi risultati forniscono un metodo semplice per isolare HE da embrioni umani e hPSC, consentendo la sua caratterizzazione molecolare e la generazione efficiente di cellule ematopoietiche in vitro.
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Garcia, Luisa Almeida Deragon. "HSP-1 e HSP-2 no plasma seminal equino: efeitos da sazonalidade na concentração e relação com a fertilidade de garanhões." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2014. http://hdl.handle.net/10183/108170.
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Proteínas presentes no plasma seminal (PS) vêm sendo estudadas em relação a níveis reprodutivos de fertilidade ou infertilidade, em várias espécies de mamíferos, particularmente em animais domésticos. As proteínas do plasma seminal equino 1 (HSP-1) e 2 (HSP-2) são as proteínas mais abundantes nesta espécie. O objetivo deste estudo foi investigar a concentração das proteínas HSP-1/2 presentes no plasma seminal bem como o conteúdo de proteína total, em garanhões adultos durante a estação reprodutiva e fora dela, para determinar se essas concentrações estão relacionadas com a fertilidade. O PS foi obtido a partir de 42 ejaculados de 11 garanhões adultos (3-25 anos). Os animais foram alocados em dois grupos (alta e baixa fertilidade) de acordo com as taxas de prenhez de éguas e dos dados de viabilidade do sêmen avaliados no primeiro dia de coleta. As concentrações das HSP-1/2 (mg/mL) foram medidas e analisadas por um sistema de Cromatografia Líquida de Ultra Eficiência utilizando uma coluna UHPLC. Houve diferença significativa (P<0,05) na concentração de proteínas totais e das proteínas HSP-1/2 (mg/mL, média ± DP) entre os ejaculados de animais de alta e baixa fertilidade. Não houve diferença na concentração das HSP-1/2 no primeiro e segundo ejaculados de garanhões de alta fertilidade, tanto dentro ou fora da estação reprodutiva. O PS de animais classificados no grupo de baixa fertilidade apresentou diferença significativa (P<0,05) na concentração das HSP-1/2 entre o primeiro e segundo ejaculado, tanto no período da estação reprodutiva quanto fora dele. Em conclusão, a concentração das principais proteínas do plasma seminal em garanhões, as HSP-1/2, foi maior em ejaculados de garanhões de baixa fertilidade, o que não parece ser influenciado pelo período de coleta, podendo assim, ser indicada como biomarcador da baixa fertilidade em garanhões.Seminal plasma (SP) proteins have been assessed in relation to reproductive fertility levels or infertility, in several species of mammals, particularly domestic animals. Horse seminal plasma proteins 1 (HSP-1) and 2 (HSP-2) are the most abundant proteins in equine seminal plasma. The aim of this study was to investigate in adult stallions the concentrations of seminal plasma HSP-1/2 and total protein in the breeding season and non-breeding season and to determine if these concentrations were related with fertility. SP was obtained from 42 ejaculates of 11 adult stallions (3-25 yrs). Stallions were allocated into two groups (good and poor fertility) according to pregnancy rates of mares, and to their semen viability data in the first collection day. Seminal plasma HSP- 1/2 concentrations (mg/mL) were measured and analyzed by an Ultra High Performance Liquid Chromatography using a UHPLC column. There were significant differences (P<0.05) in total protein and HSP-1/2 concentration (mg/mL, mean ± SD) in the ejaculates from good and poor fertility stallions. The HSP-1/2 concentration did not show differences in the first and second ejaculates of good fertility stallions in both the non-breeding and breeding season. SP of stallions classified as poor fertility showed significant difference (P<0.05) in HSP-1/2 concentration between the first and second ejaculate in both the non-breeding and breeding season. In conclusion, the concentration of the major proteins of stallion seminal plasma HSP-1/2 was higher in ejaculates from stallions with poor fertility, is not influenced by the season and could serve as biomarker for poor fertility in stallions.
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Monteiro, Janaína Munuera. "Imunolocalização das Heat Shock Proteins (HSPs) 60 e 70 na placenta bovina." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/10/10132/tde-27062006-105146/.
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As Heat Shock Proteins (HSPs) ou proteínas do choque térmico são encontradas em todas as células e são classificadas de acordo com seu peso molecular. Dentre elas encontram-se as de 27, 60, 70, 90 e 110 kDa, sendo as mais estudadas no contexto da reprodução as da família 60 e 70. Essas proteínas são ditas como chaperoninas, em razão do seu importante papel no dobramento e desdobramento de outras proteínas celulares sem alterar sua conformação final, e são expressas frente a qualquer tipo de estresse como calor, vírus, bactéria, hormônios, diferenciação celular, etc, e influenciam nas respostas imune inata e adquirida. A placenta também expressa essas proteínas, uma vez que é um órgão de intenso estresse e diferenciação celular durante toda a gestação. Nesse estudo, busca-se avaliar a expressão ou não dessas proteínas na placenta bovina e para isso foram utilizadas 30 amostras de diferentes animais em estágios distintos de gestação, fixadas em formol tamponado a 10% e processadas pela técnica de imuno-istoquímica. O mesmo numero de amostras foi também processado para a análise de imuno-microscopia eletrônica de transmissão pelas técnicas de \"freeze-substitution\" e marcação por pós-embebição. Na imuno-istoquímica, as HSPs 60 e 70 foram localizadas nos trofoblastos, epitélio materno e células binucleadas. A expressão da HSP 60 foi maior no início declinando no segundo e terceiro terço. Já a expressão da HSP 70 manteve-se praticamente constante, evidenciando a forte expressão dessa proteína durante todo o período. Na análise de imuno-microscopia eletrônica de transmissão, ambas as famílias foram localizadas nas células binucleadas (núcleo, citoplasma e vesículas) e epitélio materno (núcleo e citoplasma) em todos os terços gestacionais. O perfil das proteínas estudadas na placenta bovina foi diferente quando comparada à placenta humana, pois nesta última, a intensidade da expressão para a HSP 70 diminuiu com o decorrer da gestação enquanto para a HSP 60 foi constante durante todo a gestação. Provavelmente essas diferenças podem estar relacionadas ao fato dessas amostras terem sido coletadas de mulheres com gravidez interrompidas e também pelo tipo de placentação distinta. A bovinocultura de corte é de extrema importância para a econômica para o Brasil e se faz necessário o conhecimento de fatores que possam melhorar suas características reprodutivas. Dessa forma os resultados obtidos nesse estudo contribuirão certamente de subsídio para experimentos futuros sobre o papel das Heat Shock Proteins na placenta bovina.Heat Shock Proteins (HSP) can be found in any kind of cell. These proteins are classified according to their molecular weight and their known families include the HSP 27, 60, 70, 90 and 110 kDa. Among these, HSP 60 and 70 are the ones of interest in reproduction. They were known as chaperonines because of their capacity to fold and unfold other proteins into the cell, without changing their own conformation. They are expressed during several stress conditions likes virus and bacteria infections, hormones, heat, cellular differentiation, etc, and also take part signalizing for innate and acquired immune responses. Heat shock proteins are expressed in several tissues and organs, including the placenta. In this study we have evaluated the expression of these proteins in the bovine placenta, using thirty samples from different animais with distinct gestational periods, fixed in 10% formalin and processed for immunohistochemistry. The same numbers of samples were processed for immunoelectron microscopy using freeze-substitution and post embedding labeling techniques. The immunohistochemistry results show the expression of HSP 60 and 70 in trophoblasts, maternal epithelia and binucleated cells. The HSP 60 expression was higher in the beginning of gestation, becoming lower during the second and third trimester. Heat shock protein 70 expression were practically constant throughout the gestation. The immunoelectron microscopy analysis revealed that both HSP 60 and 70 were located in the cytoplasm and nucleio binucleated cells and maternal epithelia from the beginning to the end of pregnancy. The immunolocalization of HSP 60 and 70 in the bovine placenta were distinct from the ones found in studies on women, probably due to the differences of the placentation type and to the fact that those samples were collected from abnormal or discontinuous pregnancy. Beef production in Brazil is an important economical activity and studies to improve the bovine reproductive characteristics are necessary and must be expended, therefore our results certainly contributes for further studies on HSP function during pregnancy in this species.
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ZARPELON, LIA M. C. "Contribuicao ao estudo da separacao zirconio/hafnio no sistema MIBK-HSCN-HCL." reponame:Repositório Institucional do IPEN, 1995. http://repositorio.ipen.br:8080/xmlui/handle/123456789/10423.
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Made available in DSpace on 2014-10-09T12:38:32Z (GMT). No. of bitstreams: 0Made available in DSpace on 2014-10-09T14:04:40Z (GMT). No. of bitstreams: 1 06010.pdf: 10090735 bytes, checksum: 27ba3c575e2c6be05736a4e1e736812a (MD5)
Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP
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Kaufman, Matthew Douglas. "Variable-Complexity Response Surface Approximations For Wing Structural Weight in HSCT Design." Thesis, Virginia Tech, 1998. http://hdl.handle.net/10919/36566.
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A procedure for generating and using a polynomial approximation to wing bending material weight of a High Speed Civil Transport (HSCT) is presented. Response surface methodology is used to fit a quadratic polynomial to data gathered from a series of structural optimizations. Several techniques are employed in order to minimize the number of required structural optimizations and to maintain accuracy. First, another weight function based on statistical data is used to identify a suitable model function for the response surface. In a similar manner, geometric and loading parameters that are likely to appear in the response surface model are also identified. Next, rudimentary analysis techniques are used to find regions of the design space where reasonable HSCT designs could occur. The use of intervening variables along with analysis of variance reduce the number of polynomial terms in the response surface model function. Structural optimization is then performed by the program GENESIS on a 28-node Intel Paragon. Finally, optimizations of the HSCT are completed both with and without the response surface.Master of Science
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Leite, Jaqueline Santos Moreira. "Efeito da suplementação oral crônica com L-glutamina e L-alanina livres ou como dipeptídeo sobre o estresse oxidativo e HSP27 em ratos submetidos a exercícios resistido." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/9/9132/tde-10062015-170852/.
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Introdução: O exercício resistido em nível atlético pode promover estresse oxidativo crônico, fato que implica em uma resposta imuno-inflamatória exacerbada com consequente redução de desempenho e efeitos à saúde. Ao mesmo tempo, exercícios de caráter intenso elevam o consumo de glutamina por células e tecidos, reduzindo assim a disponibilidade deste aminoácido ao organismo, Todavia, estudos avaliando o metabolismo da glutamina em exercício do tipo resistido ainda são escassos. A síntese de antioxidantes, tais como a glutationa (GSH) e proteínas citoprotetoras como proteínas de choque térmico (HSPs) podem ser influenciadas pela disponibilidade de glutamina. Objetivo: Avaliar o efeito da suplementação oral crônica com L-glutamina e L-alanina, ambas na forma livre ou como Dipeptídeo sobre o estresse oxidativo e citoproteção mediado pela HSP 27 em ratos submetidos a exercício resistido. Métodos: Cinquenta ratos Wistar machos adultos (n = 10 por grupo) foram distribuídos em 5 grupos experimentais: Sedentário (SED), Treinado (CTRL) e suplementados com DIP, solução com L-glutamina e L-alanina livres (GLN+ALA) e somente L-Alanina (ALA), e foram submetidos ao protocolo de subida em escada durante 6 semanas, suplementados na água de beber em uma solução à 4%, nos últimos 21 dias do experimento. Foram analisados: teste de carga máxima, lactato sanguíneo, glutamina e glutamato (plasma, fígado e músculo -Tibial e EDL), creatina kinase e mioglobina (plasma) transaminases (plasma), glutationa oxidada (GSSG) e reduzida (GSH) (papa de hemácias, fígado e músculo - Tibial e EDL), TBARS (fígado e músculo- Tibial e EDL) e, expressão de HSP-27 e Glutamina Sintetase (músculo Tibial). Resultados: Os resultados demonstraram que o protocolo de exercício resistido reduziu a concentração de glutamina no músculo (p<0,05), aumentou a razão [GSSG/GSH] no fígado, papa de hemácia e músculo (p<0,05), e consequentemente houve aumento de TBARS nos tecidos. Já as suplementações com L-glutamina e L-alanina livres e como dipeptídeo aumentaram as concentrações de glutamina no plasma e tecidos (p<005), melhoraram a razão de [GSSG/GSH] no fígado, papa de hemácias e músculo (p<0,05). Também foi encontrado aumento da expressão de HSP 27 no Tibial, redução de TBARS nos tecidos, e creatina kinase no plasma (p<0,05). Conclusão: As suplementações com L- glutamina e L- alanina livres ou como dipeptídeo aumentam a síntese de GSH e a expressão de HSP 27, atenuando assim o estresse oxidativo causado pelo exercício resistido.Introduction: Athletic Resistance exercise way promotes chronic oxidative stress, which implies in exacerbated immune inflammatory response with consequent reduction in performance and health effects. At the same time, intense exercise improves consumption of glutamine for cells and tissues, thereby reducing the availability of this amino acid to the body. However, studies evaluating the glutamine metabolism in resistance exercise are still scarce. The synthesis of antioxidants such as glutathione (GSH) and cytoprotective proteins such as heat shock proteins (HSPs) can be influenced by the availability of glutamine. Objective: To evaluate the effect of chronic oral supplementation with L-glutamine and L-alanine, both in its free form or as dipeptide on oxidative stress and the cytoprotection mediated by HSP 27 in rats subjected to resistance exercise. Methods: Fifty (n = 10 per group) Wistar adult rats were divided into 5 groups: Sedentary (SED), Trained (CTRL) and supplemented with DIP, solution with L-glutamine and free L-alanine (GLN + ALA) and L-alanine (ALA). The trained groups were underwent to climb stairs protocol for six weeks. Supplementations were offered in 4% solution in drinking water in the last 21 days of the experiment. Were analyzed: maximum load test, blood lactate, glutamine and glutamate (in plasma, liver and muscle Tibialis and EDL), creatine kinase and myoglobin (plasma), transaminase (plasma), oxidized (GSSG) and reduced (GSH) glutathione (erythrocytes, liver and muscle- Tibialis and EDL), TBARS (liver and muscle Tibialis- and EDL), HSP-27 and Glutamine Synthetase expression (Tibialis muscle). Results: The results showed that resistance exercise protocol reduced glutamine concentration in muscle (p <0.05) increased the ratio [GSSG / GSH] in the liver, erythrocytes and muscle (p <0.05), and TBARS increase the tissue. The Supplementation with L-glutamine and L-alanine and free dipeptide and increased glutamine concentrations in the plasma and tissues (p <0.05), improved the ratio of [GSSG / GSH] in liver, erythrocytes and muscle (p <0.05). HSP 27 expression was also increased in Tibialis Muscle. There was reduction of TBARS in tissues and creatine kinase in plasma (p <0.05). Conclusion: Supplementations with L-glutamine and L-alanine in its free form or as dipeptide increase the synthesis of GSH and Expression HSP 27, thus reducing oxidative stress caused by resistance exercise.
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Sahm, Alexander [Verfasser]. "Prognose der Schnittkräfte bei der HSC-Bearbeitung / Alexander Sahm." Aachen : Shaker, 2003. http://d-nb.info/1174513950/34.
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Löfling, Andreas, and Lukas Juza. "Utformning av ett materialflödessystem anpassat för HSP-Gripens produktion." Thesis, Högskolan i Gävle, Avdelningen för Industriell utveckling, IT och Samhällsbyggnad, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:hig:diva-19798.
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Like many small-time companies there is a need to improve the flow of materials at HSP-Gripen AB. The company develops and manufactures hydraulic-powered grapples for the machine- and forest industry. By mapping the material flow in the current production and gathering necessary data, suggestions on how different parts of the production could be controlled is presented in this dissertation. This has been achieved by the study of relevant scientific literature. The suggested ways of controlling the production consist of 2-bin systems, hybrid material flow system as well as a cyclic product-planning. In the later part of this dissertation it is discussed how reliable the calculated bin-size is, the need for the suggested production system to have a headstart and the fact that 90 percent of the grapple models are handled in this dissertation.
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Bonkhofer, Florian. "Identification of novel Runx1 targets involved in HSC development." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:4badf9f4-f796-4063-8176-dd57644fd811.
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Haematopoietic stem and progenitor cells (HSPCs) are de novo generated within in the ventral aspects of the embryonic dorsal aorta (DA). Cells of this haemogenic endothelium (HE) will eventually undergo an endothelial to haematopoietic transition (EHT) that involves cell budding out of the aortic wall. Despite the detailed description of the cellular events, the exact haemogenic lineage path and the underlying molecular mechanism that establish full haematopoietic competence are still not entirely understood. The transcription factor Runx1 is critical for the emergence of HSPCs and shows expression in the zebrafish HE as early as 24 hpf. To facilitate a detailed analysis of the transient HE population I generated a TgBAC(runx1P2:Citrine) reporter line under the control of the endogenous runx1 promoter on a bacterial artificial chromosome (BAC). Double-transgenic reporter lines for runx1 and the endothelial marker kdrl allowed us to isolate specifically cells of the DA away from the whole endothelial population, which could be further sub-divided into HE and non-haemogenic cells. Genomewide expression analysis within the respective tissues and upon Runx1 loss of function enabled the identification of HE-specific Runx1-regulated genes. Hereby, the gfi1ab gene appeared as the functional homologue of the murine Gfi1. I show that in zebrafish, EHT is orchestrated through a conserved Runx1-Gfi1-Lsd1 axis. The cellular functions of the remaining Runx1 targets imply that maturation into fully functional HSCs depends on epigenetic regulation due to the up-regulation of de novo DNAmethyltransferases, as well as on factors that allow the developing HSCs to respond to extrinsic cues from haematopoietic niches. Lastly, it became evident that the early HE expresses dll4 at similar levels to the rest of the aortic endothelium, indicating a common lineage path. In the absence of RUNX1 the HE remains essentially arterial and persists as an integrated part of the DA.
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Hotakainen, L. (Lari). "HSC-3 -solujen eksosomit ja CAV-1 -proteiinin ilmeneminen." University of Oulu, 2017. http://urn.fi/URN:NBN:fi:oulu-201705312281.
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Kielisyövän tuumorimikroympäristö ja sen eri komponentit ovat olleet viime aikoina kiivaan tutkimuksen kohteena. Näillä tekijöillä näyttäisi olevan merkitystä syövän kasvussa, invaasiossa ja leviämisessä. Mikroympäristön ominaisuuksiin vaikuttaa todennäköisesti syöpäsolujen ja tuumorin mikroympäristön molekyylivälitteinen keskustelu, ja tämän vuoropuhelun viestinvälittäjiä ovat esimerkiksi eksosomit. Eksosomit ovat kehon normaalien solujen ja syöpäsolujen erittämiä lipidikaksoiskalvollisia mikrovesikkeleitä, jotka voivat spesifisesti sitoutua kohdesoluunsa, ja muokata sen toimintaa esimerkiksi sen sisältämien proteiinien ja RNA:n välityksellä. Syöpäsolujen eksosomien kohdesoluina voivat olla esimerkiksi mesenkymaaliset kantasolut, tulehdussolut tai ns. syöpään liittyvät fibroblastit (CAFs, cancer associated fibroblasts). Kielisyövän on havaittu ilmentävän normaalia enemmän kaveoliini-1 -proteiinia (CAV-1), jonka määrä näyttäisi nousevan syövän kehitysasteen mukaan. Tässä tutkimuksessa selvitimme in vitro -laboratoriokokeilla, sisältävätkö aggressiivisen liikkuvan kielen syöpäsolulinjan (HSC-3) erittämät eksosomit CAV-1 proteiinia.
Tutkimuksiemme alkuvaiheessa loimme pohjaa eksosomieristyksen menetelmille yhteistyössä israelilaisen tutkimusryhmän kanssa. Varsinaisessa tutkimuksissamme eristimme eksosomeja HSC-3 solulinjalta käyttäen kaupallista ExoQuick-TC™ -valmistetta. Solujen eksosomieritystä indusoitiin käyttäen 4-aminofenyylielohopea-asetaattia (APMA). Kerättyjä eksosomeja ja sen sisältämiä proteiineja (CD63 ja CAV-1) tarkasteltiin immunoelektronimikroskopialla sekä Western blot -analyysillä.
Sekä immunoelektronimikroskopiassa että Western blot -analyysissä havaittiin eristettyjen eksosomien sisältävän niille tyypillistä CD63-proteiinia sekä CAV-1 -proteiinia. Saamamme tulokset viittaavat siihen, että kielisyöpäsolut saattavat olla osana CAV-1-proteiinin kertymissä tuumorin mikroympäristöön. Tällä saattaa olla merkitystä mikroympäristön muuttumisessa edullisemmaksi syövän jakaantumiselle, invaasiolle ja leviämiselle. Vaikka tutkimuksemme osoittavat eksosomien ja CAV-1-proteiinin yhteyden, tarvitaan lisätutkimuksia myös näiden kahden yhteydestä sekä in vitro -kokeissa muilla kielisyöpäsolulinjoilla että in vivo -kielisyöpätutkimuksissa. CAV-1 proteiinin rooli syövän kehityksessä on monimutkainen ja osin epäselvä, ja tämänhetkiset tutkimustulokset ovatkin osin ristiriidassa keskenään. Myös eksosomien ja CAV-1 proteiinin erityksen säätely solutasolla vaatii lisätutkimuksia. Tulokset kuuluvat BMC Cancer -lehdessä julkaistuun alkuperäisartikkeliin, jossa allekirjoittaneen työpanos on käsitelty tiivistelmäosuudessa.
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Enk, Dirk. "Untersuchungen zum dynamischen Stabilitätsverhalten von Fräswerkzeugen zur HSC-Bearbeitung." Essen Vulkan-Verl, 2009. http://d-nb.info/995794677/04.
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Silveira, Camila Oliveira. "Formas de CRISP-3, HSP-1 e HSP-2 como candidatas a marcadores de congelabilidade do sêmen de garanhões da raça Mangalarga Marchador." Universidade Federal de Viçosa, 2017. http://www.locus.ufv.br/handle/123456789/10918.
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Com este estudo objetivou-se avaliar o perfil proteômico total e diferencial do plasma seminal de garanhões da raça Mangalarga Marchador, que apresentam variações na motilidade espermática do sêmen após descongelamento, visando identificar proteínas candidatas a marcadores de congelabilidade. Foram utilizados quatro garanhões de fertilidade comprovada, divididos em dois grupos, com dois animais em cada grupo: T1: alta congelabilidade (n= 4 ejaculados) e T2: baixa congelabilidade (n= 4 ejaculados), sendo cada tratamento composto por dois ejaculados de cada garanhão. As análises físicas do sêmen foram realizadas antes e após a criopreservação espermática. Os grupos estudados foram divididos pós-descongelamento pela avaliação da motilidade espermática total. O perfil protéico do plasma seminal foi obtido por eletroforese bidimensional, as análises de imagem dos géis foram realizadas pelo programa ImageMaster 2D Platinum 6.0 (GE Healtcare ® ) e a identificação das proteínas foi por espectrometria de massas usando MALDI-TOF/TOF, com o software Mascot Daemon para a busca em banco de dados. A validação das proteínas identificadas entre os tratamentos foi realizada pelo programa SCAFFOLD (95%). Os dados foram analisados pela ANOVA com 5 % de probabilidade de erro, correlações simples de Pearson e análise de regressão linear. Observou-se diferença (p<0,05) em relação à motilidade espermática total do sêmen fresco e pós-descongelamento. No perfil protéico identificou-se 5 famílias de proteínas (HSP-1, HSP-2, CRISP-3, Calecreína e Albumina sérica). Destas famílias, algumas proteínas se apresentaram com diferenças em abundância (p<0,05) entre os grupos estudados, em que quatro destas podem ser consideradas possíveis candidatas a marcadores de congelabilidade (HSP-1: spot 175; HSP-2: spot 39; CRISP-3: spot 66 e 166). Conclui-se então que os garanhões da raça Mangalarga Marchador apresentam variação individual ao processo de criopreservação do sêmen e possuem possíveis proteínas candidatas a marcador de congelabilidade para raça.
This study aimed to evaluate the total and differential proteomic profile of the seminal plasma of stallions of Mangalarga Marchador breed that presented variations in sperm motility after thawing, intending to identify candidate proteins as freezability markers. Four proven fertility stallions were used and splited in two groups with two animals in each group: T1: high freezability (n= 4 ejaculates) and T2: low freezability (n= 4 ejaculates) with two ejaculates of each stallion in each treatment. Semen analysis were made before and after criopreservation. The studied groups were divided after thawing by the evaluation of total sperm motility. The protein profile of the seminal plasma was obtained by bidimensional electrophoresis. The analyses of the images of the gels were performed by the ImageMaster 2D Platinum 6.0 (GE Healtcare ® ) program and the proteins identification were made by the mass spectrometry using MALDI-TOF/TOF with the software Mascot Daemon for database search. The validation of identified proteins between treatments was performed by SCAFFOLD (95%) program. The data were analyzed by ANOVA, Pearson's simple correlations and linear regression analysis with 5 % of error probability. Differences were observed (p<0.05) in relation to total sperm motility of fresh and after thawing semen. At protein profile, 5 protein families (HSP-1, HSP-2, CRISP-3, Kallikrein and Serum albumin) were identified. Of these families some proteins presented differences in abundance (p<0.05) between studied groups in which four of them can be considered as possible markers candidates of semen freezability (HSP-1: spot 175; HSP-2: spot 39; CRISP-3: spot 66 and 166). It can be concluded that stallions of Mangalarga Marchador breed present individual variation to semen cryopreservation process and have possible proteins as markers candidates of semen freezability.
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Monteiro, Janaína Munuera. "O papel da HSP 60 nas células leucocitárias da placenta bovina." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/10/10132/tde-18012010-140946/.
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A tolerância materno-fetal envolve, além do sistema imune, muitas peculiaridades quanto ao tipo de placenta e placentação. A placenta em bovinos é considerada não invasiva, e como conseqüência de uma implantação superficial, sem invasão endometrial, se estabelece uma barreira placentária complexa que se interpõe entre a circulação materna e fetal. Aliado a isso, a placenta um órgão que se encontra em constante diferenciação e proliferação celular além da alta atividade metabólica, fontes constantes de estresse e desafio para o sistema imune. Atuando nesse contexto, destacamos as proteínas de choque térmico (heat shock proteins HSP), proteínas que são expressadas em condições fisiológicas mas, em situações de estresse, sua expressão aumenta. Na placenta bovina já se constatou a expressão das HSP 60 e 70; contudo notou-se uma maior peculiaridade na expressão da HSP 60, que é maior no primeiro trimestre da gestação. Observando-se o perfil da presença e expressão dessa proteína na placenta bovina e, tendo em vista a característica desse órgão em apresentar populações leucocitárias funcionalmente distintas das do sangue periférico, levantou-se a hipótese da influência desta proteína sobre as células placentárias, particularmente sobre os leucócitos no processo de tolerância materno fetal. Desta forma, esse trabalho analisou a participação da HSP 60 em mecanismos imunes junto à alguns leucócitos placentários por meio de ensaio de proliferação pela técnica de CFSE, ensaio de fagocitose e ensaios bioquímicos como peroxidação lipídica; ciclo celular e potencial de membrana mitocondrial de células placentárias em cultivo. Nossos resultados mostraram que a HSP 60 não influencia a proliferação de linfócitos e outras células placentárias nas diversas condições testadas. Em contrapartida a HSP 60 aumenta a fagocitose e apoptose no terceiro terço gestacional, bem como a produção de radicais oxidados poliinsaturados e o potencial de membrana mitocondrial. Tendo em vista esses resultados, podemos observar que a HSP 60 nas células da placenta bovina possivelmente esteja modulando a cinética da ativação de mecanismos de sinalização de morte celular programada entre as células da placenta e o sistema imunológico. Esta hipótese assegura que, no concepto a termo, as variações hormonais e o sistema imune possam estar regulando o processo para o nascimento do feto.The maternal-fetal tolerance involves, besides the immune system, many peculiarities regarding the type of placenta and placentation. The bovine placenta is considered not invasive and, as consequence of a superficial implantation without endometrial invasion, it establishes itself as complex barrier that interposes between the maternal and fetal circulation. In addition to that, placenta is an organ that undergoes continuous differentiation and cellular proliferation besides the high metabolic activity that represents a constant source of stress and challenge for the immune system. As an important component in this context, we highlight the heat shock proteins - HSP, that are usually expressed in physiological conditions but, in situations of stress, their expression increases. In the bovine placenta the expression of HSP 60 and 70 has already been verified; however HSP 60 showed a peculiar expression behavior, with higher staining in the first trimester of pregnancy. Considering the presence and expression profiles of HSP 60 in the bovine placenta allied to the particularity of presenting functional distinct leucocytes populations, a hypothesis was raised regarding the influence of this protein on the placental cells, particularly on the leucocytes in the process of fetal-maternal tolerance. Therefore, this work analyzed the role of HSP 60 in immune mechanisms related to some placental leucocytes by several approaches like proliferation assay by CFSE technique, phagocytosis assay and biochemical assays as lipoperoxidation; cell cycle phases and mitochondrial membrane potential of placental cells in culture. Our results showed that HSP 60 does not influence the proliferation of lymphocytes or any other placental cells in various conditions tested. On the other hand, HSP 60 increases phagocytosis and apoptosis in the third trimester, as well as the production of polyunsaturated oxide radicals and the mitochondrial membrane potential. In view of these results, we may infer that HSP 60 may be modulating the kinetics of activation mechanisms for signaling programmed cellular death between placental and immune cells. This hypothesis assures that, on the termed conceptus, the hormonal variation and the immune tolerance may be responsible for regulating the parturition process.
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SIGHINOLFI, SILVIA. "INTRACELLULAR IRON OVERLOAD AFFECTS HSC METABOLISM BY IMPAIRING MITOCHONDRIAL FITNESS IN β-THALASSEMIA." Doctoral thesis, Università Vita-Salute San Raffaele, 2023. https://hdl.handle.net/20.500.11768/137019.
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Mitochondrial activity and metabolism significantly control hematopoietic stem cell (HSC) function and fate. HSCs change the metabolic state in response to stress signals, such as reactive oxygen species (ROS), which drive HSC entry into cell cycle accompanied by increased mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis. However, excessive accumulation of ROS results in oxidative damage of cellular organelles, including mitochondria. Iron is one of the sources of ROS and HSCs can uptake iron but little is known about the effects of iron on HSC metabolism.
Recently, we demonstrated an impaired function of HSCs in β-Thalassemia (BThal), a condition of systemic iron overload (IO). We also observed that IO reduces the hematopoietic supportive capacity of BThal BM mesenchymal stromal cells. However, there is no evidence of the direct effect of IO on HSCs in BThal. We hypothesized that IO and the resulting oxidative stress could alter HSC metabolism and function.
We found a positive enrichment of iron homeostasis genes in HSCs from thalassemic th3 mice, suggesting increased iron uptake and storage. Consistently, we detected high levels of free reactive iron in the cytoplasm and in mitochondria of th3 HSCs, correlating with high ROS levels. As a result, mitochondria are impaired, with low mass and activity. Interestingly, th3 multipotent progenitors inherited dysfunctional mitochondria since the rescue of mitochondrial activity occurred in the transition to more committed progenitors. In line with mitochondrial dysfunction, th3 HSCs had reduced OXPHOS-derived ATP and relied on glycolysis. In vivo reduction of mitochondrial ROS rescued mitochondrial activity and metabolism, and increased th3 HSC frequency and quiescence, thus indicating that oxidative stress is the cause of mitochondrial dysfunction and potentially HSC defects. Importantly, in vivo administration of iron dextran to wt mice generated intracellular IO and mitochondrial oxidative stress and decreased mitochondrial activity in HSCs, indicating that IO alone is sufficient to impair mitochondria.
Our study unveils that IO directly impacts on HSC metabolism by inducing oxidative stress and mitochondrial dysfunction. Alterations in mitochondrial activity and metabolic profile, in response to IO, are expected to alter HSC function. This research will add novel insight about the role of iron in regulating HSC metabolism and provide clues for improving clinical conditions associated to IO, such as BThal.L'attività e il metabolismo mitocondriali controllano in modo significativo la funzione e il destino delle cellule staminali ematopoietiche (HSC). Le HSC modificano lo stato metabolico in risposta a segnali di stress, come le specie reattive dell'ossigeno (ROS), che guidano l'ingresso delle HSC nel ciclo cellulare accompagnato da un aumento della fosforilazione ossidativa mitocondriale (OXPHOS) e della glicolisi. Tuttavia, l'eccessivo accumulo di ROS provoca il danno ossidativo degli organelli cellulari, compresi i mitocondri. Il ferro è una delle fonti di ROS e le HSC possono assorbire il ferro, ma si sa poco sugli effetti del ferro sul metabolismo delle HSC. Recentemente, abbiamo dimostrato una funzione alterata delle HSC nella β-talassemia (BThal), una condizione di sovraccarico sistemico di ferro (IO). Abbiamo anche osservato che l'eccesso di ferro riduce la capacità di supporto ematopoietica delle cellule stromali mesenchimali talassemiche. Tuttavia, non ci sono prove dell'effetto diretto del sovraccarico di ferro sulle HSC in BThal. Abbiamo ipotizzato che il sovraccarico di ferro e il conseguente stress ossidativo alterino il metabolismo e la funzione delle HSC. Abbiamo trovato un arricchimento positivo dei geni dell'omeostasi del ferro nelle HSC dei topi talassemici th3, suggerendo un aumento dell'assorbimento e dell'immagazzinamento del ferro. Coerentemente, abbiamo rilevato alti livelli di ferro reattivo libero nel citoplasma e nei mitocondri di th3 HSC, che correlano con alti livelli di ROS. Di conseguenza, i mitocondri sono alterati, con ridotta massa e attività. I progenitori multipotenti th3 hanno ereditato mitocondri disfunzionali poiché la correzione dell'attività mitocondriale si è verificata nella transizione verso progenitori più differenziati. In linea con la disfunzione mitocondriale, le HSC th3 hanno una ridotta produzione di ATP mediante OXPHOS e dipendono dalla glicolisi. La riduzione in vivo dei ROS mitocondriali ha ripristinato l'attività e il metabolismo mitocondriali e ha aumentato la frequenza e la quiescenza delle HSC th3, dimostrando così che lo stress ossidativo è la causa della disfunzione mitocondriale e dei potenziali difetti delle HSC. È importante sottolineare che la somministrazione in vivo di ferro destrano a topi wt ha generato eccesso di ferro intracellulare e stress ossidativo mitocondriale e una ridotta attività mitocondriale nelle HSC, indicando che il sovraccarico di ferro da solo è sufficiente per compromettere i mitocondri. Il nostro studio rivela che il sovraccarico di ferro ha un impatto diretto sul metabolismo delle HSC inducendo stress ossidativo e disfunzione mitocondriale. Le alterazioni dell'attività mitocondriale e del profilo metabolico, in risposta al sovraccarico di ferro, potrebbero alterare la funzione delle HSC. Questa ricerca aggiungerà nuove informazioni sul ruolo del ferro nella regolazione del metabolismo delle HSC e fornirà nuove conoscenze utili per migliorare le condizioni cliniche caratterizzate da sovraccarico di ferro, come BThal.
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Balabanov, Vladimir Olegovich. "Development of Approximations for HSCT Wing Bending Material Weight using Response Surface Methodology." Diss., Virginia Tech, 1997. http://hdl.handle.net/10919/30730.
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A procedure for generating a customized weight
function for wing bending material weight of a High Speed Civil Transport
(HSCT) is described. The weight function
is based on HSCT configuration parameters. A response
surface methodology is used to fit a quadratic polynomial to data gathered
from a large number of structural optimizations.
To reduce the time of performing a large number of structural optimizations,
coarse-grained parallelization with a master-slave processor
assignment on an Intel Paragon computer is used.
The results of the structural optimization are noisy.
Noise reduction in the structural optimization results is discussed.
It is shown that the response surface filters out this noise.
A statistical design of experiments technique is used
to minimize the number of required structural optimizations and to maintain
accuracy. Simple analysis techniques
are used to find regions of the design space where reasonable HSCT designs
could occur, thus customizing the weight function to the design requirements of
the HSCT,
while the response surface itself is created employing detailed analysis
methods.
Analysis of variance is used to
reduce the number of polynomial terms in the response surface model function.
Linear and constant corrections based on a small number of high fidelity
results
are employed to improve the accuracy of the
response surface model.
Configuration optimization of the HSCT employing a customized weight function
is compared to the configuration optimization of the HSCT with
a general weight function.Ph. D.
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Ghiaur, Gabriel. "The role of Rho GTPases in hematopoietic stem cell biology RhoA GTPase regulates adult HSC engraftment and Rac1 GTPases is important for embryonic HSC /." Cincinnati, Ohio : University of Cincinnati, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1204374567.
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Bour, Pierre Gilbert Louis. "Investigating the role of Runx1 in the specification of haematopoietic stem cells from early precursors in the embryo using a Runx1 reactivatable knockout mouse model." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/7835.
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Runx1 is a central transcription factor in the development of the murine haematopoietic system and in the emergence and specification of its main key component, the haematopoietic stem cell. Previous studies suggested a requirement for Runx1 in a window of time stretching from mesoderm specification (E6.5) to mid-gestation (E11), but these studies did not investigate each primary haematopoietic site separately. During this PhD project, a Runx1 reactivatable knockout mouse model was used to study the impact of the absence of Runx1 from E9.5 to E11 in primary haematopoietic sites on early precursor populations, especially PreHSC Type I and II. At E9.5, the KO conceptus was already developmentally retarded, lacking progenitors and PreHSC Type II but was not devoid of PreHSC Type I, as demonstrated by flow cytometry, thus suggesting a requirement for Runx1 in the transition from PreHSC Type I to PreHSC Type II stage. Using a novel culture system that enables the potent in vitro maturation of precursors of HSCs into fully mature adult-repopulating HSCs, it was found that maturation of PreHSC Type I into HSCs was hindered in KO tissues, despite the expression of Runx1 in OP9 niche compartment, thus pointing towards a cell autonomous requirement for Runx1. In this model, the Runx1 allele was subsequently reactivated to a functional state by tamoxifen-induced Cre-mediated recombination (CreERT2 system). Tamoxifen / Cre toxicity on HSC maturation was evaluated during AGM reaggregate culture to achieve the best balance between the highest recombination levels and the lowest toxicity when Cre was induced in cell suspension prior to reaggregation. It was found that reactivation of Runx1 at E9.5 in primary haematopoietic sites was not sufficient to rescue haematopoietic development, thus suggesting a requirement for Runx1 before E9.5.
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Stern, Lauren Michelle. "Immune reconstitution patterns in allogeneic haematopoietic stem cell transplant recipients with human cytomegalovirus reactivation." Thesis, The University of Sydney, 2023. https://hdl.handle.net/2123/29958.
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Allogeneic haematopoietic stem cell transplantation (HSCT) is a treatment with curative potential for a range of haematological malignancies and non‐malignant haematological disorders. Opportunistic infections are a major cause of transplant‐related morbidity and mortality in HSCT patients due to the profound immunodeficiency and immunosuppression associated with the procedure. Human cytomegalovirus (HCMV) reactivation is one of the most significant infectious complications following HSCT and frequently occurs in the first 100 days post‐transplant.
This thesis aimed to investigate patterns of immune reconstitution associated with HCMV reactivation the first 100 days post‐HSCT. High parameter immune profiling technologies (mass cytometry and multiplex cytokine assays) were employed to characterise peripheral blood immune cell subset recovery and plasma cytokine profiles at key phases of HCMV reactivation (prior to detection, at the initial detection, at the peak and near resolution) in a cohort of allogeneic HSCT recipients. The data illustrate the emergence of distinct patterns of immune cell reconstitution and circulating cytokine signatures in patients who experienced HCMV reactivation, including elevated frequencies of activated T cell subsets and an inflammatory cytokine profile at the peak of reactivation. Mucosal associated invariant T (MAIT) cell levels at the initial detection of HCMV reactivation were significantly lower in patients who subsequently developed high‐level HCMV reactivation compared to those with low‐level HCMV reactivation. Faster effector memory CD4+ T cell recovery was also found to distinguish low‐level and high‐level reactivators. Together, this work provides comprehensive insight into immune reconstitution signatures linked with the evolution and magnitude of HCMV reactivation after allogeneic HSCT.
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Zayas, Jennifer. "Regulation of HSC Self-Renewal and Differentiation by Pumilio Proteins." Scholarly Repository, 2008. http://scholarlyrepository.miami.edu/oa_dissertations/300.
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Evolutionarily conserved Pumilio (Pum) RNA-binding proteins act as translational repressors during embryo development and cell fate specification. Previous work in the lab has shown that over-expression of Pum2 (Pum2-EML) supports maintenance and suppresses mutilineage differentiation of murine multipotent HSC/MPP-like cell line EML. The subsequent analysis of HSC markers and functional analysis has revealed that wt EML cells share the LKS CD34 positive phenotype, whereas the majority Pum2-EML cells are similar to LKS CD34 negative. The CD34 positive wt EML cells can be divided into CD34low, CD34med and CD34high subpopulations, whereas Pum2-EML CD34 positive cells correspond to CD34low subpopulation. Colony forming assays have revealed that the overall multilineage differentiation of wt EML and Pum2-EML cells strongly correlates with the CD34 expression levels. Multiple experiments have revealed that purified CD34 negative and CD34 positive wt EML cells can generate each other and among CD34 positive wt EML cells the CD34low cells have the highest capacity to give rise to CD34 negative EML cells. We have proposed a model in which CD34 negative EML cells are more primitive cells in an "inactive" (differentiation inhibited) state, that give rise to CD3low "active" (differentiation ready) EML cells. The CD34low EML cells can revert back to the CD34 negative state or give rise to CD34med/high cells that can readily differentiate into multiple lineages. Based on that model, the over-expression of Pum2 leads to increased maintenance of cells in inactive CD34 negative state, and blocks development of CD34 positive cells past the CD34low stage. Cumulatively, these results support the notions that Pum2 could be involved in maintaining the balance between inactive and active state of multipotent hematopoietic cells. The c-kit receptor plays a vital role in self-renewal and differentiation of hematopoietic stem cells (HSC) and multipotent progenitors (MPPs). We have discovered that besides c-kit, the murine multipotent HSC/MPP-like cell line EML expresses the transcript and protein for a truncated form of c-kit, called tr-kit. Notably, the tr-kit transcript and protein levels were down-regulated during cytokine induced differentiation of HSC/MPP-like cell line EML into myelo-erythroid lineages. RT-PCR results show tr-kit is transcribed solely in cell populations enriched for LTR-HSC, STR-HSC and MPPs. The observation that tr-kit is co-expressed with c-kit only in more primitive, HSC and MPP-enriched cell populations raises an exciting possibility that tr-kit functions either as a new component of SCF/c-kit pathway, or is involved in a novel signaling pathway, present exclusively in HSC and MPPs. These findings necessitate functional characterization of tr-kit, and analysis of its potential role in the self-renewal, proliferation and/or differentiation of HSC and multipotent progenitors.
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Marrs, Kevin L. "The cystic fibrosis transmembrane conductance regulator regulation by HSP-90 /." View the abstract Download the full-text PDF version, 2007. http://etd.utmem.edu/ABSTRACTS/2007-031-Marrs-index.html.
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Thesis (Ph.D.)--University of Tennessee Health Science Center, 2007.Title from title page screen (viewed on July, 18, 2008). Research advisor: Anjaparavanda Naren, Ph.D. Document formatted into pages (xv, 72 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 66-72).
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Піддубний, Артем Михайлович, Артем Михайлович Поддубный, Artem Mykhailovych Piddubnyi, Роман Андрійович Москаленко, Роман Андреевич Москаленко, Roman Andriiovych Moskalenko, Анатолій Миколайович Романюк, et al. "Hsp 90 overexpression in chronic bacterial prostatitis with corpora amylacea." Thesis, Springer, 2017. http://essuir.sumdu.edu.ua/handle/123456789/64954.
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Objective: To study the expression of heat shock protein Hsp90 in patients with chronic bacterial prostatitis (CBP) and corpora amylacea formation. Method: Hsp90 expression was investigated in tissue of prostate of 22 CBPs with corpora amylacea by immunohistochemistry. Samples were fixed, embedded in paraffin and analized for Hsp90 accumulation using
the anti-Hsp90 antibody, followed by DAB detection substrate and counterstained with Mayer’s hematoxylin. Microbiological examinations were carried out in intraoperative collection o f material. The identification of
accumulated bacterial cultures was carried out using conventional
methods based, on morphological, tinctirial, cultural, biochemical and
antigenic properties.
Results: In prostates with CBP E.coli was defined in 63.6 % of cases, S.aureus and P. vulgaris in 9.1 % of patients, Klebsiella spp. in 18.2 % of samples. CBP was characterized by significant inflammatory infiltration around glands and in the stroma. Immunohistochemical examination revealed significant expression of Hsp90 in the prostate gland epithelium. The reaction in the stroma was observed around foci of inflammation. Corpora amylacea had a rounded shape and lamellar structure, between layers of deposits of Hsp 90 was revealed. Hsp90 overexpression was found in points corpora amylacea and glandular epithelium contact.
Conclusion: Overexpression of Hsp 90 in prostate tissue with CBP and corpora amylacea indicates a participation of the heat shock proteins in the development and formation of corpora amylacea.
Hsp90 overexpression may be regarded as a prospective (potential) role in the corpora amylacea development.
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Піддубний, Артем Михайлович, Артем Михайлович Поддубный, Artem Mykhailovych Piddubnyi, Роман Андрійович Москаленко, Роман Андреевич Москаленко, Roman Andriiovych Moskalenko, Анатолій Миколайович Романюк, et al. "Hsp 90 overexpression in chronic bacterial prostatitis with corpora amylacea." Thesis, Springer, 2017. http://essuir.sumdu.edu.ua/handle/123456789/75157.
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Munirathnam, Madhu. "Einfluss masseoptimierter Kragarmstrukturen auf die dynamische Bahngenauigkeit von HSC-Fräsmaschinen /." Aachen : Shaker, 2008. http://d-nb.info/990312801/04.
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Geraldo, Ana Carina Alves Pereira de Mira. "Termotolerância em fêmeas bovinas: abordagens celular e fisiológica." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/74/74131/tde-07102013-091616/.
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Este estudo teve como objetivo a compreensão dos efeitos desencadeados pelas temperaturas elevadas na expressão gênica de proteínas de choque térmico, nomeadamente da HSPA1A e HSP90AA1, em linfócitos de fêmeas bovinas de diferentes raças e origens. Parte do projeto foi desenvolvida em Portugal, onde foram utilizadas 20 novilhas da raça Limousine e 22 da raça Mertolenga. A parte desenvolvida no Brasil decorreu com 11 novilhas da raça Holstein Frísia e 20 da raça Brahman. Os animais da raça Holstein participaram do Experimento I no qual foram coletados 4 tubos de sangue para posterior choque térmico in vitro, onde cada tudo de sangue foi submetido a diferentes temperaturas: 40 °C e 42 °C (ambos através de banhos-maria), 23 °C (mantido a temperatura ambiente) e 10 °C (na geladeira), durante duas horas. Todos os animais de todas as raças participaram no Experimento II, onde foram sujeitos ao teste de tolerância ao calor, tendo sido aferidas a temperatura retal e a frequência respiratória, e coletado sangue (após os tratamentos sombra e sol). A todas as amostras de ambos os experimentos foi realizada a lise das hemácias de modo a obter o buffy-coat. O RNA foi isolado através do método do TRIzol e a RT-PCR realizada com SuperScript III após digestão com DNAse I. A PCR em tempo real decorreu no aparelho 7500 Fast Real Time, utilizando TaqMan Gene Expression Assays para os genes alvo HSPA1A e HSP90AA1, e ACTB e PPIA como genes endógenos. Foram calculados os ΔCt (Ctalvo - Ctendógeno) assim como a expressão gênica através do método 2-ΔΔCt. A análise estatística foi realizada através de modelos lineares mistos, recorrendo ao programa R Software Project (versão 3.0.1). No Experimento I foi atestada a qualidade da relação para ambos os endógenos nas relações que estabelecem com o mRNA-HSPA1A e o mRNA-HSP90AA1, através de uma análise de regressão pairwise. Tal como era esperado os valores de expressão gênica a uma temperatura de 23 °C foram os menores, seguidos 10 °C (estresse por frio), 40 °C e 42 °C para o gene HSPA1A. No caso da HSP90AA1, foi a 40 °C que se verificou uma maior expressão gênica. No Experimento II verificou-se uma variação intra-raça algo acentuada para valores de frequência respiratória, permitindo supor que o esforço termorregulatório dos animais de uma mesma raça possa ter sido diferente. Em relação à temperatura retal a raça Brahman apresentou valores significativamente diferentes das restantes raças para a situação sol. As diferenças observadas foram provavelmente consequência dos diferentes níveis de estresse aos quais os animais estiveram sujeitos. As diferenças observadas nos ΔCt não foram muito expressivas e apenas se observaram diferenças significativas na expressão gênica relativa na raça Mertolenga entre as situações sombra e sol. Podemos concluir que o aumento planificado da temperatura de linfócitos bovinos leva a diferentes expressões gênicas relativas de HSPA1A e HSP90AA1. A expressão de mRNA-HSPA1A é tanto maior quanto maior o estresse térmico, quer por frio quer por calor. As expressões gênicas relativas de HSPA1A e HSP90AA1 exibidas por animais com diferentes capacidades termolíticas são também elas diferentes, existindo uma variabilidade individual na expressão gênica relativa de proteínas de choque térmico.The present study aimed to understand the effects triggered by high temperatures in heat shock proteins gene expression, HSPA1A and HSP90AA1, in cow lymphocytes from different breeds and origins. Part of the project was developed in Portugal, where 20 Limousin and 22 Mertolenga breed heifers were used and the second part was developed in Brazil with 11 Holstein Friesian and 20 Brahman heifers. The Holstein animals participated in Experiment I in which four blood samples were collected for subsequent in vitro heat shock, where each one of the blood samples was submitted to different temperatures: 40 ° C and 42 ° C (through water baths), 23 ° C (kept at room temperature) and 10 ° C (in refrigerator) for two hours. All animals from all breeds participated in Experiment II, where they were subjected to the heat tolerance test, where rectal temperature and respiratory rate were measured, and blood samples were collected (after shadow and after sun treatments). In all samples of both experiments was carried out the erythrocytes lysis so as to obtain the buffycoat. The RNA was isolated by the TRIzol method and RT-PCR performed with SuperScript III after digestion with DNase I. The real-time PCR apparatus took place in 7500 Real Fast Time, using TaqMan Gene Expression Assays for HSPA1A and HSP90AA1 target genes, ACTB and PPIA as endogenous genes. The ΔCt (Cttarget - Ctendogenous) were calculated as well as gene expression through the 2-ΔΔCt method. Statistical analysis was performed using linear mixed models, using the program R Project Software (version 3.0.1). In Experiment I it was attested the quality of the relationship for both endogenous with the mRNA-HSPA1A and mRNAHSP90AA1 through a pairwise regression analysis. As expected values, gene expression at a temperature of 23 °C were lower, followed by 10 °C (cold stress), 40 °C and 42 °C for the HSPA1A gene. In the case of HSP90AA1 the higher gene expression was found at 40 °C. In Experiment II, there was a slightly pronounced intra-race variation for respiratory rate values, allowing the assumption that the thermoregulatory effort of animals of the same breed may have been different. Regarding the rectal temperature, in Brahman breeds it was significantly different from the other breeds in the sun treatment. The differences observed were probably a result of different levels of stress to which the animals were subjected. The differences observed in ΔCt were not very expressive and significant differences were only observed in relative gene expression of Mertolenga breed between shade and sun treatments. We conclude that planned increasing temperature in bovine lymphocytes leads to different relative gene expression of HSPA1A and HSP90AA1. The mRNA-HSPA1A expression is greater in higher thermal stress, either by cold or by heat. The HSPA1A and HSP90AA1 relative gene expressions exhibited by animals with different thermolytic capabilities, are also different, existing an individual variability in relative gene expression of heat shock proteins.
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Cruzat, Vinicius Fernandes. "Efeito da suplementação com L-glutamina livre e na forma de dipeptídeo sobre eixo glutamina-glutationa, sistema imune, sistema inflamatório e vias de sinalização proteica em camundongos submetidos à endotoxemia." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/9/9132/tde-23052013-152405/.
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A sepse é a principal causa de morte em unidades de terapia intensiva (UTIs) no mundo. A reduzida disponibilidade do aminoácido mais abundante do organismo, a glutamina contribui para o complicado estado catabólico da sepse. No presente estudo investigamos os efeitos da suplementação oral com L-glutamina e L-alanina (GLN+ALA), ambos na norma livre e como dipeptídeo, L-alanil-L-glutamina (DIP), sobre o eixo glutamina-glutationa (GSH), sistema imune, inflamação, proteínas de choque térmico (HSPs) e expressão de genes envolvidos com vias de sinalização proteica em animais endotoxêmicos. Camundongos C57/B6 foram submetidos à endotoxemia (Escherichia coli LPS, 5 mg.kg-1, grupo LPS) e suplementados por 48 horas com L-glutamina (1 g.kg-1) e L-alanina (0,61 g.kg-1, grupo GLN+ALA-LPS) ou 1,49 g.kg-1 de DIP (grupo DIP-LPS). A endotoxemia promoveu depleção da concentração de glutamina no plasma (71%), músculo esquelético (50%) e fígado (49%), quando comparado ao grupo CTRL, sendo restauradas nos grupos DIP-LPS e GLN+ALA-LPS (P<0,05), fato que atenuou a redução da GSH e o estado redox (taxa GSSG/GSH) em eritrócitos circulantes, musculo e fígado (P<0,05). A suplementação em animais endotoxêmicos resultou em uma upregulation dos genes GSR, GPX1 e GCLC no músculo e fígado. A concentração das citocinas plasmáticasTNF-α, IL-6, IL-1β e IL-10 foi atenuada pelas suplementações, bem como a expressão de mRNAs envolvidos com a resposta inflamatória, ativadas pela via do NF-κB(P<0,05). Concomitantemente, verificou-se aumento da capacidade proliferativa de linfócitos T e B circulantes nos grupos GLN+ALA-LPS e DIP-LPS. A expressão de mRNAs e a concentração de HSPs no tecido muscular foi restabelecida pelas suplementações, contudo, a expressão mRNAs relacionados às vias de síntese e degradação proteica foi somente estimulada no tecido hepático(P<0,05). Os resultados do presente estudo demonstram que a suplementação por via oral com GLN+ALA ou DIP podem ser utilizados clinicamente como métodos nutricionais em reverter o quadro de depressão da disponibilidade de glutamina corporal da sepse induzida por LPS, tendo impacto no eixo glutamina-glutationa, sistema imune e inflamatório.Sepsis is the leading cause of death inintensive care units (ICUs) in the world.The availability ofthe most abundant amino acid in the body, glutamine, is reduced in this situation, fact that contribute to the complicated catabolic state of sepsis. In the present study, we investigated the effects of oral supplementation with L-glutamine and L-alanine (GLN+ALA), both in their free form and as a dipeptide, L-alanyl-L-glutamine (DIP) on glutamine-glutathione axis (GSH), immune and inflammatory system, heat shock proteins (HSPs) expression and gene expressions involved in protein signaling pathways during endotoxemia. C57/B6 mice were subjected to endotoxemia (Escherichia coli LPS, 5 mg.kg-1, LPS group) and supplemented for 48 hours with L-glutamine (1 g.kg-1) plus L-alanine(0.61 g.kg-1, GLN+ALA-LPS group) or 1.49 g.kg-1of DIP (DIP-LPS group). Endotoxemia promoted depletion glutamine concentration in plasma (71%), skeletal muscle (50%) and liver (49%), when compared to the CTRL group, and was restored in the DIP-LPS e GLN+ALA-LPS (P<0.05), fact that attenuate the reduction of GSH and the redox state (GSSG/GSH rate) in circulating erythrocytes, liver and muscle (P<0.05). Supplementations in endotoxemic mice resulted in upregulation of GSR, GCLC and GPX1 genes in muscle and liver. Plasma concentration of TNF-α, IL-6, IL-1β and IL-10 were attenuated by supplementation as well as the expression of mRNAs involved in the inflammatory response, activated by NFκ-B pathway (P <0.05). At the same time, high proliferative capacity of circulating T and B lymphocytes GLN+ALA-LPS e DIP-LPS were observed. HSPs (protein and mRNAs) and in muscle were restored by the supplements, however, the mRNAs expression related to the synthesis and degradation of protein pathways was only stimulated in the liver (P <0.05). Our results demonstrate that oral supplementation with GLN+ALA or DIP can be used as clinically nutritional methods to reverse the depression of body glutamine availability during sepsis induced by LPS, impacting on the glutamine-glutathione axis, immune and inflammatory system.
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Sakamaki, Taro. "Hoxb5 defines the heterogeneity of self-renewal capacity in the hematopoietic stem cell compartment." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263564.
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Duarte, Marta Maria Medeiros Frescura. "Hidrólise de nucleotídeos de adenina em plaquetas de pacientes com diferentes níveis de colesterol e sua relação com o processo inflamatório." Universidade Federal de Santa Maria, 2006. http://repositorio.ufsm.br/handle/1/11120.
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The activity of NTPDase (EC 3.6.1.5, apyrase, CD39) was verified in platelets from patients with increasing cholesterol levels. A possible association between cholesterol levels and inflammatory markers, such as oxidized low density lipoprotein (oxLDL), highly sensitive C-reactive protein (hsCRP) and oxLDL autoantibodies was also investigated. The following groups were studied: group I (< 150 mg/dl), group II (151 to 200 mg/dl); group III: (201 to 250 mg/dl); group IV (> 251 mg/dl) of cholesterol. The results demonstrated that both ATP and ADP hydrolysis were enhanced as a function of cholesterol levels. The LDL levels increased concomitantly with total cholesterol levels. The triglyceride levels were increased in the group with total cholesterol above 251 mg/dl. oxLDL levels were elevated in groups II, III and IV. hsCRP was elevated in the group with cholesterol higher than 251 mg/dl. oxLDL autoantibodies were elevated in groups III and IV. TBARS content was enhanced as a function of cholesterol levels. In summary, hypercholesterolemia is associated with an enhanced of inflammatory response and ATP and ADP hydrolysis. The increase in NTPDase activity is possibly related to a compensatory response to the
inflammatory and pro-oxidative state associated with hypercholesterolemia.A atividade da NTPDase (EC 3.6.1.5, apyrase, CD39) foi verificada em plaquetas de pacientes com diferentes níveis de colesterol. Uma possível associação entre os níveis de colesterol e os marcadores inflamatórios como LDL oxidado (oxLDL), proteína C reativa ultrasensível (hsCRP) e anticopos anti-LDL oxidado (Anti-oxLDL) foi investigado. Os seguintes grupos foram estudados: grupo I (< 150 mg/dl), grupo II (151 a 200 mg/dl); grupo III: (201 a 250 mg/dl); grupo IV (> 251 mg/dl) de colesterol. Os resultados demonstraram que a hidrólise dos nucleotídeos (ATP e ADP) aumentou em função dos níveis de colesterol. Os níveis de LDL aumentaram concomitantemente com os níveis de colesterol total. Os níveis de triglicerídeos foram elevados no grupo com colesterol total acima de 251 mg/dl. Os níveis de oxLDL foram elevados nos grupos II, III and IV. A hsCRP foi elevada no grupo com cholesterol maior que 251 mg/dl. Os Anti-oxLDL foram elevados nos grupos III e IV. O conteúdo de TBARS foi aumentando em função dos níveis de colesterol. Em resumo, a hipercolesterolemia está associada com o aumento da resposta inflamatória e hidrólise de ATP e ADP. O aumento da atividade da NTPDase está possivelmente relacionado com uma resposta compensatória ao estado inflamatório e pró-oxidativo associado com a hipercolesterolemia.
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AZARIO, ISABELLA MARIA REBECCA. "Neonatal transplantation of umbilical cord blood as a new therapeutic option for Mucopolysaccharidosis type I." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2018. http://hdl.handle.net/10281/199019.
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Lo scopo di questo progetto è stato lo sviluppo preclinico di una nuova terapia per la Mucopolisaccaridosi di tipo I (MPS-I): il trapianto di cellule staminali da sangue del cordone ombelicale, eseguito in epoca neonatale.
L’MPS-I è una rara malattia lisosomiale dovuta a mutazioni nel gene IDUA, che codifica per l’enzima lisosomiale α-L-iduronidasi (IDUA). L’assenza di attività iduronidasica provoca l’accumulo di glicosaminoglicani nei tessuti, causando una disfunzione multi-organo progressiva e, in particolare, gravi anomalie scheletriche. La terapia d’elezione per la MPS-I è il trapianto di cellule staminali ematopoietiche (HSCT) da donatore sano, che riduce l’accumulo dei substrati e attenua molte manifestazioni cliniche, ma non è del tutto efficace sulle anomalie ossee.
L’obiettivo di questa tesi è stato quello di testare nel modello murino di MPS-I l’efficacia di una nuova strategia trapiantologica, che combinava l’intervento precoce (neonatale) e l’uso del sangue del cordone ombelicale come fonte. Infatti, è stato deciso di trattare gli animali nel periodo peri-natale, per prevenirne le manifestazioni fenotipiche, e di impiegare il sangue cordonale come fonte di cellule da trapiantare, perché il trapianto cordonale in clinica dà diversi vantaggi rispetto al trapianto di midollo osseo, in particolare in questi pazienti.
Il primo risultato ottenuto in questo lavoro è stata la caratterizzazione delle proprietà fenotipiche e funzionali delle cellule cordonali murine. E’ stato poi eseguito il trapianto di tali cellule in topi adulti wild type (WT) C57BL/6: si è ottenuto un buon attecchimento a lungo termine ed è stata verificata la presenza di cellule di origine del donatore in tutti i lineages ematopoietici. Infine, è stato valutato l’esito del trapianto neonatale di cellule cordonali nei topi MPS-I: si è ottenuto un aumento di attività IDUA negli organi periferici dei topi MPS-I con attecchimento elevato (>50%), e una conseguente riduzione dei livelli di glicosaminoglicani. Il fenotipo scheletrico dei topi a 20 settimane d’età è stato dettagliatamente descritto tramite indagini radiografiche, microCT e istologiche. Le radiografie hanno rivelato che, mentre nei topi MPS-I non trattati si aveva un generale ispessimento osseo rispetto ai WT, nei topi affetti con elevato attecchimento questa anomalia non si riscontrava. Le analisi microCT e istologiche hanno dimostrato che la porzione corticale del femore dei topi MPS-I presentava irregolarità che erano invece ridotte nei topi trattati con alto attecchimento. Questi dati confermano che il trapianto neonatale di cellule del cordone ombelicale risulta una terapia efficace nel modello murino di MPS-I.
In collaborazione con il Prof. Aiuti presso l’Ospedale San Raffaele-Tiget, è stato intrapreso un nuovo progetto di ricerca con lo scopo di correggere geneticamente le cellule di cordone ombelicale per ottenere livelli sovra-fisiologici di espressione dell’enzima IDUA. L’obiettivo è quello di trasdurre cellule di cordone murino MPS-I con un vettore lentivirale PGK-IDUA e di trapiantare le cellule così corrette in topi MPS-I neonati. Verificheremo se sarà possibile ottenere nei riceventi livelli di attività enzimatica più alti che nel trapianto di cellule WT, e se l’esito della terapia genica sarà quindi ancora migliore.
Finora, abbiamo sviluppato una procedura per isolare dal sangue cordonale murino le cellule ematopoietiche staminali e progenitrici, che sono state in grado di ripopolare topi C57BL/6 adulti e neonati. A questo punto verranno effettuate le prime prove di infezione con vettori lentivirali GFP e IDUA su tali cellule, per trovare le migliori condizioni per il loro trapianto nei neonati MPS-I.
Questi risultati potrebbero aprire la strada verso lo sviluppo di un approccio di terapia genica neonatale con cellule di cordone ombelicale geneticamente corrette nei pazienti MPS-I.The aim of this PhD project was the preclinical testing of a new possible therapeutic option for Mucopolysaccharidosis type I (MPS-I): the transplantation of umbilical cord blood (UCB) in neonatal age. MPS-I is a rare lysosomal disease due to mutations in the IDUA gene, which encodes for the lysosomal enzyme α-L-iduronidase (IDUA). The absence of IDUA activity leads to the accumulation of glycosaminoglycans (GAGs) in patients’ tissues, which causes a progressive multi-organ dysfunction, with a wide spectrum of skeletal anomalies. The first-choice therapy for MPS-I is hematopoietic stem cell transplantation (HSCT) from healthy donor, because it reduces the accumulation of substrates and it solves many clinical symptoms, but this treatment is not very effective on the skeletal defects. The aim of this thesis was to test in the murine model a novel transplantation strategy for MPS-I, combining early (neonatal) intervention and the use of murine UCB as a source. Indeed, we decided to treat our mouse model at early age, in order to prevent the anomalies, and to employ UCB cells (UCBCs) as a source for transplantation, because UCB transplantation (UCBT) has shown advantages over bone marrow transplantation in patients suffering from inherited metabolic disorders. The first result of this work was the characterization of the phenotypical and functional properties of murine hematopoietic UCBCs compared to adult bone marrow cells. Then, adult wild-type (WT) C57BL/6 mice were transplanted with UCBCs, and they reached good long-term engraftment levels, with the presence of cells of donor origin among all the hematopoietic lineages. Finally, the outcome of UCBT on neonatally-transplanted MPS-I mice was evaluated: an increase of IDUA activity was evident in the peripheral organs of high-engrafted MPS-I mice (engraftment >50%), and, consequently, GAG levels reduced. An extensive characterization of the skeletal phenotype was performed by radiographs, microCT, and histology. Radiographic images showed that MPS-I untreated mice had increased radio-opacity and diameter of the bones at 20 weeks of age, compared to WT, and these parameters were reduced in high-engrafted mice. MicroCT scans and histomorphometry revealed that the cortical region of MPS-I femurs appeared irregular, and returned to normal in treated mice with high-engraftment, confirming the benefit of neonatal UCBT on MPS-I mice. In collaboration with Alessandro Aiuti’s group at Ospedale San Raffaele-Tiget (Telethon Institute for gene therapy), a new project started with the aim of gene-correcting murine UCBCs to obtain a supra-physiological expression of IDUA enzyme. The goal is to transduce MPS-I murine UCBCs with a PGK-IDUA lentiviral vector, and to transplant gene-corrected cells into MPS-I newborns. We will verify if we can obtain in the recipients higher levels of IDUA activity than transplanting WT UCBCs, and if the outcome of gene therapy could be better than the one obtained with normal UCBCs. A method was developed to isolate hematopoietic stem and progenitor cells (HSPCs) from murine UCB and to culture them for lentiviral infection. WT untransduced UCB-HSPCs engrafted WT adult and newborn mice at high levels. We are now in the process of testing the GFP and IDUA vector on MPS-I UCBCs, to set the best conditions for their transplantation in MPS-I neonates. These results will hopefully pave the way for developing a neonatal gene therapy approach with lentivirally-corrected UCB cells in MPS-I babies.
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Atance, Joel William. "Does sodium salicylate treatment enhance HSP 72 expression and myocardial protection?" Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0005/MQ40704.pdf.
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