Multiple Defekte der Hämatopoese und T-Zellentwicklung in Gfi-1-defizienten Mäusen

Das Gen gfi-1 (engl. ?growth factor independence-1?) kodiert für ein Zinkfingerprotein, welches wahrscheinlich als transkriptioneller Repressor wirkt. Zudem stellt gfi-1 ein potentielles Onkogen dar und spielt besonders während der Lymphomagenese von T-Zellen ein Rolle. Um nähere Einblicke in die Funktion des Zinkfingerproteins Gfi-1 in vivo zu erlangen, wurde im Rahmen dieser Arbeit, über die Methodik der homologen Rekombination und dem Einsatz von embryonalen Stammzellen, ein Gfi-1-defizientes Mausmodell generiert. Anhand von Protein und RNA Analysen konnte bestätigt werden, daß ein vollständiger Verlust an Gfi-1 Protein in den Mäusen erzielt wurde. Dieser Verlust hatte dramatische Konsequenzen und führte zu einer 100 %igen Letalität innerhalb der ersten Lebenswochen. Die durchgeführten Untersuchungen der Gfi-1-defizienten Mäuse zeigten Veränderungen im gesamten hämatopoetischen System auf. Mit zunehmendem Alter der Mäuse konnte eine Akkumulation von atypischen, monozytären Gr-1+Mac-1+ Zellen im Knochenmark beobachtet werden. Mit dem Auftreten dieser Zellen ging eine Reduktion der Erythropoiese im Knochenmark einher. Eine weitere Veränderung in den Gfi-1-/- Tieren betraf die Entwicklung von T-Lymphozyten im Thymus. Es konnte gezeigt werden, daß bereits früheste T-Zellpopulationen von dem Gfi-1 Verlust betroffen waren. Insbesondere die c-Kit positiven Thymozyten zeigten eine verminderte Proliferation und gleichzeitig eine erhöhte Apoptoserate. Eine weitere Störung der Thymozytenentwicklung konnte bei dem Prozeß der positiven/negativen Selektion und der Linienentscheidung CD4 versus CD8 gezeigt werden. Neben einer Verschiebung des Verhältnisses von CD4 zu CD8 SP T-Zellen im Thymus konnte indirekt über die Expression von CD69 und CD62L nachgewiesen werden, daß Störungen der positiven Selektion vorliegen. Eine Bestätigung hierfür ergaben Experimente mit foetalen Thymi, die eine reduzierte Entwicklung von CD4 T-Zellen aufzeigten. Trotz massiv gestörter T-Zelldifferenzierung und verminderter Zahl an Thymozyten, wurde in der Peripherie eine Lymphadenopathie der zervikalen Lymphknoten beobachtet, mit einer Akkumulation aller Lymphozytenpopulationen. Auffällig war ein gehäuftes Auftreten von Gedächtniszellen in den Lymphknoten, das vor allem die CD4+ T-Zellen betraf. Experimente in vitro an isolierten T-Zellen zeigten, daß der Verlust von Gfi-1 zu einer verminderten Aktivierbarkeit der T-Zellen führte und letztlich zu einer reduzierten Proliferationsrate. Hierin dürfte u.a. die beobachtete Immunsuppression der Gfi-1-/-Tiere begründet liegen

The Transcriptional Repressor Gfi1 Affects Development of Early, Uncommitted c-Kit+ T Cell Progenitors and CD4/CD8 Lineage Decision in the Thymus

In the thymus, several steps of proliferative expansion and selection coordinate the maturation of precursors into antigen-specific T cells. Here we identify the transcriptional repressor Gfi1 as an important regulator of this maturation process. Mice lacking Gfi1 show reduced thymic cellularity due to an increased cell death rate, lack of proliferation, and a differentiation block in the very early uncommitted CD4−/CD8−/c-Kit+ cytokine-dependent T cell progenitors that have not yet initiated VDJ recombination. In addition, Gfi1-deficient mice show increased major histocompatibility complex class I–restricted positive selection and develop significantly more CD8+ cells suggesting a requirement of Gfi1 for a correct CD4/CD8 lineage decision. Absence of Gfi1 correlates with high level expression of the genes for lung Krüppel-like factor (LKLF), inhibitor of DNA binding (Id)1 and Id2, suggesting the existence of new regulatory pathways in pre-T cell development and thymic selection in which Gfi1 acts upstream of LKLF as well as the E-proteins, which are negatively regulated by Id1 and Id2

Zum Umsetzungsstand von Gender Mainstreaming in Einrichtungen der Kinder- und Jugendhilfe auf kommunaler Ebene:Von Bejing über Brüssel, Berlin nach Bottrop : Was von Gender Mainstreaming im Mainstream der Kinder- und Jugendhilfe übrig bleibt

Gender Mainstreaming ist eine gleichstellungspolitische Strategie, die aus internationalen frauen- und entwicklungspolitischen Kontexten stammend seit Anfang des 21. Jahrhunderts Einzug hält in die nationale Gleichstellungspolitik. Über diesen Weg hat sie Eingang gefunden in das Handlungsfeld der Kinder- und Jugendhilfe. Eine Vielzahl an Publikationen, Praxishilfen, Best-Practice-Projekten etc. ist das öffentliche Aushängeschild der Auseinandersetzungen mit dieser Strategie innerhalb der Kinder- und Jugendhilfe. Dagegen zeigt sich der empirisch gesicherte Erkenntnisstand über den Bekanntheitsgrad von Gender Mainstreaming, das diesbezügliche Fachwissen von Fachkräften sowie über die von ihnen unternommenen Aktivitäten zur Umsetzung äußerst fragmentarisch. An diese Forschungslücke knüpft die vorliegende Arbeit an. Auf der Grundlage einer in nordrhein-westfälischen Großstädten durchgeführten Online-Befragung wird eine auf Repräsentativität abzielende Untersuchung des Wissens- und Umsetzungsstandes von Gender Mainstreaming in Einrichtungen der Kinder- und Jugendhilfe vorgelegt, die alle Arbeitsfelder und Trägergruppen einbezieht und somit differenzierte Aussagen über den Status quo zulässt

Expression of TCR-Vβ peptides by murine bone marrow cells does not identify T-cell progenitors

Germline transcription has been described for both immunoglobulin and T-cell receptor (TCR) genes, raising questions of their functional significance during haematopoiesis. Previously, an immature murine T-cell line was shown to bind antibody to TCR-Vβ8.2 in absence of anti-Cβ antibody binding, and an equivalent cell subset was also identified in the mesenteric lymph node. Here, we investigate whether germline transcription and cell surface Vβ8.2 expression could therefore represent a potential marker of T-cell progenitors. Cells with the TCR phenotype of Vβ8.2(+) Cβ(-) are found in several lymphoid sites, and among the lineage-negative (Lin(-) ) fraction of hematopoietic progenitors in bone marrow (BM). Cell surface marker analysis of these cells identified subsets reflecting common lymphoid progenitors, common myeloid progenitors and multipotential progenitors. To assess whether the Lin(-) Vβ8.2(+) Cβ(-) BM subset contains hematopoietic progenitors, cells were sorted and adoptively transferred into sub-lethally irradiated recipients. No T-cell or myeloid progeny were detected following introduction of cells via the intrathymic or intravenous routes. However, B-cell development was detected in spleen. This pattern of restricted in vivo reconstitution disputes Lin(-) Vβ8.2(+) Cβ(-) BM cells as committed T-cell progenitors, but raises the possibility of progenitors with potential for B-cell development.This study was supported by grants to H.C.O. from the National Health and Medical Research Council of Australia. J.A. was supported by a fellowship from the Australian Academy of Science

Flt3 Ligand Regulates Dendritic Cell Development from Flt3+ Lymphoid and Myeloid-committed Progenitors to Flt3+ Dendritic Cells In Vivo

Stimulation of Flt3 receptor tyrosine kinase through its cognate ligand expands early hematopoietic progenitor and dendritic cells (DCs) in humans and mice. The exact developmental stages at which hematopoietic progenitors express Flt3, are responsive to its ligand, and subsequently develop to DCs, are not known. Here we show that common lymphoid and common myeloid progenitors, as well as steady state DCs in thymus, spleen, and epidermis, express Flt3. The receptor is down-regulated once definitive B cell, T cell, and megakaryocyte/erythrocyte commitment occurs, and Flt3 is not detectable on other steady state hematopoietic cell populations. Upon in vivo Flt3 ligand (Flt3L) administration, Flt3+ progenitor cells and their progeny DCs are expanded, whereas Flt3− downstream progenitors are not, or are only slightly increased. Transplantation of common lymphoid and common myeloid progenitors and subsequent Flt3L injection increases progeny DCs of both precursor populations. These findings provide a definitive map of Flt3 expression in the hematopoietic hierarchy and directly demonstrate that Flt3L can drive DC development along both the lymphoid and myeloid developmental pathways from Flt3+ progenitors to Flt3+ DCs

Comprehensive methylome map of lineage commitment from haematopoietic progenitors.

Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Haematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. Although DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs, a comprehensive DNA methylation map of haematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Marked epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, indicating a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome seems to be important in haematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions

Self-renewal of the long-term reconstituting subset of hematopoietic stem cells is regulated by Ikaros

Hematopoietic stem cells (HSCs) are rare, ancestral cells that underlie the development, homeostasis, aging, and regeneration of the blood. Here we show that the chromatin-associated protein Ikaros is a crucial self-renewal regulator of the long-term (LT) reconstituting subset of HSCs. Ikaros, and associated family member proteins, are highly expressed in self-renewing populations of stem cells. Ikaros point mutant mice initially develop LT-HSCs with the surface phenotype cKit+Thy1.1(lo)Lin(-/lo)Sca1+Flk2-CD150+ during fetal ontogeny but are unable to maintain this pool, rapidly losing it within two days of embryonic development. A synchronous loss of megakaryocyte/erythrocyte progenitors results, along with a fatal, fetal anemia. At this time, mutation of Ikaros exerts a differentiation defect upon common lymphoid progenitors that cannot be rescued with an ectopic Notch signal in vitro, with hematopoietic cells preferentially committing to the NK lineage. Althoughdispensable for the initial embryonic development of blood, Ikaros is clearly needed for maintenance of this tissue. Achieving successful clinical tissue regeneration necessitates understanding degeneration, and these data provide a striking example by a discrete genetic lesion in the cells underpinning tissue integrity during a pivotal timeframe of organogenesis

Проектирование системы электроснабжения базы по обслуживанию нефтегазодобывающего месторождения

Выпускная квалификационная работа 136 с., 19 рис., 60 табл., 24 источника, 4 приложения. Объектом исследования является ремонтно-механический цех базы по обслуживанию нефтегазодобывающего месторождения. Цель работы: Проектирование системы электроснабжения базы по облуживанию нефтегазодобывающего месторождения. Экономическое обоснование принятых решений. В процессе исследования произведен расчет нагрузок ремонтно-механического цеха и базы в целом с применением различных методов, выбор метода расчета производился на основе исходных данных, а также осуществлен выборThis graduate qualification work includes 136 p., 19 fig., 60 tables, 24 ones in list of references and 4 transcripts. The subject of research is mechanical repair shop of upstream and gas production field operation base. Work objective is energy supply system designing of upstream and gas production field operation base and its business case. In the course of investigations there was estimated a load analysis of upstream and gas production field operation base and its mechanical repair shop by different methods, based on input data. Also there were steps of the equipment selection and its testing of several operations. As a result of investigation the model of such base was engineered, and there was shown its commercial importance and ecological security. Range of application: petro

A hitchhiker's guide to myeloid cell subsets: practical implementation of a novel mononuclear phagocyte classification system

The classification of mononuclear phagocytes as either dendritic cells or macrophages has been mainly based on morphology, the expression of surface markers, and assumed functional specialization. We have recently proposed a novel classification system of mononuclear phagocytes based on their ontogeny. Here, we discuss the practical application of such a classification system through a number of prototypical examples we have encountered while hitchhiking from one subset to another, across species and between steady-state and inflammatory settings. Finally, we discuss the advantages and drawbacks of such a classification system and propose a number of improvements to move from theoretical concepts to concrete guidelines

Increased S-nitrosylation and proteasomal degradation of caspase-3 during infection contribute to the persistence of adherent invasive escherichia coli (AIEC) in immune cells

Adherent invasive Escherichia coli (AIEC) have been implicated as a causative agent of Crohn's disease (CD) due to their isolation from the intestines of CD sufferers and their ability to persist in macrophages inducing granulomas. The rapid intracellular multiplication of AIEC sets it apart from other enteric pathogens such as Salmonella Typhimurium which after limited replication induce programmed cell death (PCD). Understanding the response of infected cells to the increased AIEC bacterial load and associated metabolic stress may offer insights into AIEC pathogenesis and its association with CD. Here we show that AIEC persistence within macrophages and dendritic cells is facilitated by increased proteasomal degradation of caspase-3. In addition S-nitrosylation of pro- and active forms of caspase-3, which can inhibit the enzymes activity, is increased in AIEC infected macrophages. This S-nitrosylated caspase-3 was seen to accumulate upon inhibition of the proteasome indicating an additional role for S-nitrosylation in inducing caspase-3 degradation in a manner independent of ubiquitination. In addition to the autophagic genetic defects that are linked to CD, this delay in apoptosis mediated in AIEC infected cells through increased degradation of caspase-3, may be an essential factor in its prolonged persistence in CD patients