Les cellules sanguines proviennent d’une cellule souche hématopoïétique (CSE), présente dans la moelle osseuse chez l’homme adulte, qui nécessite d’être en contact étroit avec une zone particulière du microenvironnement médullaire (appelée niche hématopoïétique) pour sa différenciation et son autorenouvellement. La nature exacte des cellules qui composent cette niche (appelée cellules stromales) n’est pas encore bien connue, en particulier concernant sa relation avec les cellules souches mésenchymateuses (CSM). Le but de cette thèse a été d’étudier le rôle des CSM dans la régulation de l’hématopoïèse en fonction de leur type et leur stade de différenciation mésenchymateuse et d’évaluer leur rôle dans la migration des progéniteurs hématopoïétique (PH). Nous montrons que les CSM non différenciées possèdent la capacité de soutien de l’hématopoïèse primitive la plus importante (par culture à long terme pendant 5 semaines) et que cette capacité est rapidement perdue dès 3 jours de différenciation adipogénique, otéogénique et vasculaire musculaire lisse. Par ailleurs, nous montrons que le G-CSP agit directement sur les CSM pour augmenter la migration des CSH/PH hors de la niche (par un test de migration trans-stromale) via un mécanisme MMP-2 dépendant. / Blood cells arise from a hematopoietic stem cell (HSC), present into bone marrow (3M) in adult humans, which needs close contacts with a special zone of 3M micro environment (named hematopoietic niche) for its differentiation and self’-renewal. The precise nature of the niche-forming cells (named stromale cells) are not yet well known, particularly in their relationship with mesenchymal stem cells (MSCs). The aim of the study was to investigate further the role of the MSCs in the hematopoiesis control according to their differentiation pathway and state and to evaluate their role in the migration process of hematopoietic progenitor cells (HPÇ,[ We show that non-differentiated MSCs display the best hematopoietic supporting activity (using 5-week long term cultures) that is completely lost after in vitro differentiation into adipocytes, osteoblasts and vascular smooth muscle cells. In addition, we show that G-CSP stimulation of 3M MSCs promotes HSC/HPC migration (using trans-stromal migration assay) via a MMP-2-dependent mechanism.

BIM (Building Information Modelling) är idag ett exponentiellt ökande område inom byggindustrin, som inte visar några tecken att sakta ner. I takt med att BIM som koncept och process används i större omfattning både internationellt och nationellt, dyker problem dock oundvikligen upp vid dess implementering. De faktorer som orsakar dessa problem har i detta examensarbete givits termen CSF, ett begrepp som hittills ej använts inom svensk byggindustri. CSF står för Critical Success Factors, vilket innefattar det som i svenska arbeten annars till exempel kallats ”kritiska faktorer”, ”hindrande faktorer”, ”bromsande faktorer” etc. Syftet med detta arbete är därmed att utforska både begreppet CSF och dessa faktorer, samt deras betydelse och relevans. Utifrån tidigare studier, främst utländska, identifierades och introducerades termen CSF samt 14 sådana punkter, vilka gavs en utförligare beskrivning. För att ge dessa 14 punkter en lokal anknytning, intervjuades fem personer i svensk byggindustri med olika erfarenheter av BIM. Det framträdde då klara likheter mellan tidigare diskussion kring de identifierade punkterna och intervjuresultaten. De centrala tankar som kontinuerligt återkom under arbetets gång var värdet av gemensam och delad förståelse, samt vikten av att ta vara på redan etablerad kunskap inom byggindustrin. Det finns i branschen en ofta påtalad klyfta mellan olika generationer, vilket enligt detta arbete ses som det tydligaste exemplet både på en bristande förståelse och ett dåligt utnyttjande av tidigare kunskaper. Vid ett ineffektivt möte mellan olika erfarenheter och kunskaper så leder detta till friktion istället för utveckling, med bortslösade resurser och kunskaper som resultat. Ett stort fokus med detta arbete blev därför att betona den mänskliga aspekten av BIM-implementering snarare än de tekniska delarna. Slutligen konstaterar detta examensarbete att de 14 utvalda punkterna samt användandet av deras övergripande term CSF har ett mervärde både för framtida studier och framtidens implementeringar av BIM, vilket därmed även uppfyller rapporten syfte. / BIM (Building Information Modeling) is an exponentially growing field within the construction industry, with no signs of slowing down. However, as the process continues to spread on both an international and national scale, issues with implementation inevitably arise due to many different factors, in this work identified as CSF (Critical Success Factors). The purpose of this work is therefore to examine these factors, as well as the term CSF itself, with regards to meaning and relevance. Based on previous studies the term CSF, as well as 14 factors, were established. To correlate these internationally described factors to the Swedish market, five interviews featuring people with experience of BIM in Sweden were conducted, after which similarities and patterns could be recognized. The core concepts continually established throughout the work was the value of mutual and shared understanding, as well as the importance of utilizing previously established knowledge within the construction industry. The report concludes that the identified factors as well as the term CSF are both valid and beneficial for future discussions and implementations of BIM

Os neutrófilos são glóbulos brancos do sangue e primeira linha de defesa contra as bactérias. A proliferação destas células é principalmente controlada pelo Fator Estimulador de Colônia de Granulócito (G-CSF). O G-CSF humano recombinante (rhG-CSF) é um medicamento amplamente utilizado para tratar a neutropenia, condição caracterizada pela contagem de neutrófilos abaixo de 1.500/mm3. O rhG-CSF já foi obtido no Centro de Biotecnologia do Instituto Butantan em E. coli como corpos de inclusão (CI), reportando-se instabilidade do plasmídeo e baixo rendimento. Neste trabalho, o plasmídeo pARKAN-I foi avaliado para produzir rhG-CSF em E. coli e viabilizar sua obtenção em biorreator. Esse vetor carrega a sequência pAR, inserida para aumentar sua estabilidade. E. coli BL21 (DE3) Star pLysS transformada com pARKAN-I/rhG-CSF foi cultivada em frascos agitados para avaliação da estabilidade do plasmídeo em meios complexos (2YT e de autoindução) e quimicamente definido (HDF). Avaliou-se a influência de indutores (IPTG e lactose), glicose e antibióticos sobre a estabilidade plasmidial. A fim de se obter material para purificação, foram realizados cultivos em biorreator com meio de autoindução sem antibióticos em modo batelada e HDF com antibióticos em modo batelada alimentada, a 30ºC, 30% de oxigênio e pH 6,8. As etapas de purificação incluíram: lise em homogeneizador contínuo de alta pressão, lavagens, solubilização e renaturação dos CI, e cromatografia de troca catiônica em SP-Sepharose. Avaliaram-se três métodos de renaturação: diafiltração, diálise e diluição. A estabilidade do plasmídeo foi avaliada pela percentagem de colônias obtidas em placas de LB-ágar com antibiótico em relação às colônias que cresceram nas placas de LB-ágar sem antibiótico. A identidade proteica foi determinada por SDS-PAGE e Western Blotting. A pureza relativa e a produção específica do rhG-CSF foram determinadas por densitometria das bandas da eletroforese. A quantificação proteica foi feita pelo método do ácido bicinconínico. Nos cultivos em frascos com meio 2YT sem glicose, o crescimento foi mais lento, porém a fase exponencial mais prolongada, alcançando concentração celular (Cx) mais elevada (2,56 g/L) do que as culturas com glicose (Cx1,35 g/L), que cresceram mais rápido e chegaram primeiro à fase estacionária. O cultivo com meio de autoindução proporcionou crescimento similar ao do meio 2YT sem glicose. Em meio HDF, os cultivos tiveram o crescimento mais lento e menor Cx (0,94 g/L). Os meios 2YT e de autoindução mostraram 100% de colônias resistentes à canamicina antes e após indução, exceto o 2YT sem antibiótico e sem glicose, com 97,5% e 94,1% após 6 e 8 h de indução, respectivamente, coincidindo com a maior produção de rhG-CSF. Em frascos, o meio HDF com antibiótico teve 93% de colônias resistentes antes da indução dos cultivos em frascos, diminuindo até 85% após 4 h de indução, com baixa produção do rhG-CSF, verificada apenas por Western Blotting. Os cultivos em biorreator mostraram baixa velocidade específica de crescimento (0,30 h-1), porém elevada Cx e produção de rhG-CSF, sendo superior no meio de autoindução, que também resultou mais barato do que o HDF. O método de diluição em tampão tris 20 mM pH 8,0 com EDTA 2 mM, Triton X-100 0,1% e glicerol 10% apresentou maior percentagem de renaturação. Foi estabelecido um processo de purificação que permitiu obter rhG-CSF com 87% de pureza, integridade estrutural e atividade biológica. O plasmídeo pARKAN-I, vetor sem restrições de propriedade intelectual e proteção de patente, mostrou alta estabilidade nos meios complexos avaliados, tanto em frasco como em biorreator, permitindo produzir rhG-CSF em larga escala sem adição de antibióticos ao meio de cultura, o que reduziu o custo do processo de obtenção. / Neutrophils are white blood cells and part of the first line of defense against bacteria. The proliferation of these cells is mainly controlled by the Granulocyte Colony Stimulating Factor (GCSF). Recombinant human G-CSF (rhG-CSF) is widely used to treat neutropenia, condition characterized by a neutrophil count below 1,500/ mm3. The rhG-CSF was already obtained at the Biotechnology Center of the Butantan Institute in E. coli as inclusion bodies (IB), but plasmid instability and low yield were reported. In this work, the plasmid pARKAN-I was evaluated to produce rhG-CSF in E. coli and enable the bioreactor production. This vector carries a Par sequence, inserted to increase its stability. E. coli BL21 (DE3) Star pLysS transformed with pARKAN-I / rhG-CSF was grown in shaken flasks to evaluate the plasmid stability in complex (2YT and autoinduction) and chemically defined (HDF) media. Also, the influence of inducers (IPTG and lactose), the addition of glucose and the presence of antibiotics on the plasmid stability were evaluated. In order to obtain material for rhG-CSF purification, a batch culture with autoinduction medium without antibiotic and fed-batch culture HDF medium with antibiotic were performance in bioreactor at 30º C, 30% oxygen and pH 6.8. The purification steps included: lysis in high pressure continuous homogenizer, wash, solubiliztion and refolding of IB and cation exchange chromatography in SP-Sepharose. Three refolding methods were evaluated: diafiltration, dialysis and dilution. Plasmid stability was evaluated by the percentage of colonies on LB-agar plates with antibiotic in relation to the colonies that grew on LB-agar plates without antibiotics. Protein identity was determined by SDS-PAGE and Western Blotting. Relative purity and specific production of rhG-CSF were determined by densitometry of SDS-PAGE bands. Protein quantification was performed by the bicinchoninic acid method. In shaken flask cultures with 2YT medium without glucose, the growth was slower, but the exponential phase was longer, reaching higher cell concentration (Cx=2.56 g/L) than the cultures in 2YT medium with glucose (Cx≈1.35 g/L), which grew faster and reached the stationary phase earlier. Cultures with autoinduction medium provided similar growth to the 2YT medium without glucose. Cultures with HDF medium displayed slower growth and lower Cx (0.94 g/L). Shaken flask cultures with 2YT and autoinduction media showed 100% of kanamycin resistant colonies before and after induction, except for 2YT without antibiotic and without glucose, which presented 97.5% and 94.1% of kanamycin resistant colonies after 6 and 8 h of induction, respectively, despite being the medium with higher production of rhG-CSF. In flasks, HDF medium with antibiotic presented 93% of kanamycin resistant colonies before induction, decreasing to 85% after 4 h of induction, the rhGCSF production was very low, and could be verified only by Western Blotting. Bioreactor cultivations showed low specific growth rate (0.30 h-1), but high cell density and recombinant protein production. The rhG-CSF production was higher in autoinduction medium, which was cheaper than HDF. The dilution method using 20 mM tris buffer pH 8.0, 2 mM EDTA, 0.1% Triton X-100 and 10% glycerol showed the highest percentage of refolding. A purification process was established and allowed to obtain rhG-CSF with 87% purity, structural integrity and biological activity. The expression vector pARKAN-I, which is free of intellectual property and patent protection, showed high plasmid stability in the complex media evaluated in flask and bioreactor and allowed to produce rhG-CSF in large scale without addition of antibiotics to the culture medium, reducing the cost of the production process.

On May 25, 2018, the Directive, 95/46/EC, is superseded by the General Data Protection Regulation (GDPR), (EU) 2016/679. Companies and organizations will have to revise routines, restructure organizations' processes and rebuild IT systems. The purpose of this study is to identify the critical success factors for implementing GDPR in the Swedish banking and finance sector. The study carried out a literature study as a foundation for the qualitative interviews with which empirical was gathered. Subjects for interviews was data protection officers (DPO) at four banks, as well as the Swedish Data Protection Authority and Forum för dataskydd, a national forum for DPO:s. The study's results showed a number of significant success factors for implementation processes. Based on these success factors, three were identified as critical to the implementation of GDPR from a computer science perspective: data governance, privacy-by-design, and documentation. / Den 25 maj 2018 ersattes dataskyddsdirektivet, 95/46/EG, med EU-förordningen 2016/679, General Data Protection Regulation (GDPR). För företag och organisationer kommer detta bland annat innebära nya rutiner, omstrukturering av organisationers processer och ombyggnation av IT-system. Syftet med denna studie är att identifiera de kritiska framgångsfaktorerna för implementering av GDPR inom den svenska bank- och finanssektorn. I studien genomfördes litteraturstudie som låg till grund för insamlande av empiri genom kvalitativa intervjuer med dataskyddsombud vid fyra banker. Intervjuer genomfördes även med Datainspektionen och Forum för dataskydd. Studiens resultat visade på ett flertal betydande framgångsfaktorer för implementeringsprocesser. Utifrån dessa framgångsfaktorer identifierades tre som kritiska för implementation av GDPR ur ett datavetenskapligt perspektiv: data governance, privacy-by-design samt dokumentation.

Submitted by Fabiana Gonçalves Pinto (benf@ndc.uff.br) on 2017-08-28T20:29:28Z No. of bitstreams: 1 Elizabeth Maria Oliveira da Silva.pdf: 2307471 bytes, checksum: 8d4691afbac0bcd5899bff0e397c4b96 (MD5) / Made available in DSpace on 2017-08-28T20:29:28Z (GMT). No. of bitstreams: 1 Elizabeth Maria Oliveira da Silva.pdf: 2307471 bytes, checksum: 8d4691afbac0bcd5899bff0e397c4b96 (MD5) Previous issue date: 2016 / Mestrado Profissional em Enfermagem Assistencial / Problema: Atualmente o câncer infantil representa a segunda causa de morte na faixa etária de 5 a 19 anos, ultrapassado apenas pelos óbitos provocados por causas externas. E, a partir do diagnóstico, traça-se uma das propostas de tratamento, a terapêutica com os agentes antineoplásicos, conhecidos como quimioterápicos, os quais são responsáveis pelo longo período de internação, tendo em vista que um dos efeitos adversos desta terapêutica com maior incidência é a neutropenia pós-quimioterapia. Tal condição representada pela contagem de neutrófilos inferior a 1500/m3 torna necessário o uso de moduladores de resposta muitas vezes em domicílio. Objetivo: Este estudo tem como objetivo construir uma ação educativa durante a utilização em domicílio do fator estimulador de colônias granulocíticas (G-CSF), pelo paciente e/ou familiar, buscando conhecer o significado desta ação educativa a ser construída em conjunto com os familiares, considerando e respeitando o conhecimento e saber emanado por eles, em todo o processo holístico de saúde onde o indivíduo apresenta necessidades interligadas ao meio que o cerca. Método: É um estudo de natureza qualitativa, tipo descritivo com apropriação do método fenomenológico na percepção de Maurice Merleau Ponty, e a teoria transpessoal de Jean Watson e teve como cenário o centro de quimioterapia infantil do Instituto Nacional de Câncer, que é uma Instituição Pública Federal de referência na área oncológica, e obteve aprovação no CEP em 22/12/2015n° do CAAE-51715515.500005243 e no CEP da instituição coparticipante em 07/01/2016 n0 do CAAE-51715515.5.3001.5274. O estudo foi desenvolvido em conformidade com os princípios éticos estabelecidos na Resolução do Conselho Nacional de Saúde (CNS) 466/12. Resultado: A coleta de dados ocorreu no serviço de quimioterapia infantil por meio de entrevista com participação de dezoito familiares. As entrevistas foram transcritas na integra com apropriação do método de Giorgi e após analise a luz da fenomenologia da Percepção de Maurice Merleau-Ponty originou-se a formação de quatro categorias: Categoria 1-O impacto do diagnóstico de câncer na perspectiva do familiar; Categoria 2-O significado do câncer e tratamento na percepção do familiar; Categoria 3-A importância da espiritualidade na percepção do familiar da criança com câncer; 4-O significado do cuidado domiciliar na perspectiva do familiar. Considerações finais: Durante o decorrer deste estudo percorreu-se um caminho direcionado ao cuidado, tanto ao cuidador quanto do ser a ser cuidado com o objetivo de compreender e auxiliar os familiares no processo do cuidar a partir da percepção da experiência vivenciada por estes em um contexto familiar. Tais propósitos incluíram o respeito e valorização do conhecimento e das experiências desses familiares, incluindo suas crenças, cultura, e hábitos sociais. E, neste contexto é de extrema importância que o enfermeiro vivencie a experiência a partir da percepção do sujeito deste estudo, considerando os conceitos humanísticos, visando almejar uma assistência cada vez mais qualificada e de excelência, o que propiciou a construção de uma tecnologia educacional (cartilha educativa), no que se refere ao uso em domicilio do fator estimulador de crescimento de colônias de granulócitos / Problem: Currently, childhood cancer represents the second cause of death in the age group of 5 to 19 years, surpassed only by deaths caused by external causes. From the diagnosis, one of the treatment proposals is the treatment with antineoplastic agents, known as chemotherapeutic agents, which are responsible for the long period of hospitalization, considering that neutropenia is one of the adverse effects of this therapy with a higher incidence after chemotherapy. That condition, represented by the neutrophil count below 1500 / m3 makes it necessary to use response modulators often at home. Objective: This study aimed to construct an educational action during the use of the granulocytic colony-stimulating factor (G-CSF), by the patient and / or family member at home. It seeks to know the meaning of this educational action built together with the family, considering and respecting the knowledge and knowledge emanated by them, throughout the holistic process of health where the individual present needs interconnected to the environment that surrounds him. Method: It is a descriptive qualitative study, based on the phenomenological method from the perspective of Maurice Merleau Ponty, and the transpersonal theory of Jean Watson. It had as setting the child chemotherapy center of the National Cancer Institute, which is a public federal hospital, specialized in oncology area, and obtained approval in the CEP on 12/22/2015 under protocol number CAAE-51715515.500005243 and in the CEP of the participating Institution on 07/01/2016 under protocol number CAAE-51715515.5.3001.5274. The study was developed in conformity with the ethical principles set out in National Health Council (CNS) Resolution No.466/12. Results: Data collection took place in the infant chemotherapy service through an interview with the participation of eighteen relatives. We transcribed the interviews in full, with appropriation of the Giorgi method, followed by analysis under the perspective of the phenomenology of Perception by Maurice Merleau Ponty. Then, four categories originated category 1. The impact of the cancer diagnosis from the family perspective; category; 2. The meaning of the cancer and treatment under the perception of the family member; category 3. The importance of the spirituality under the perception of the family member of the child with cancer; -the meaning of home care from the perspective of the family member. Final Considerations. During the development of this study, we went through a path directed to care that reached both the caregiver and the being to be cared for with the objective of understanding and helping the family in the care process from the perception of experiences lived by them in a family context. Such purposes included the respect and valorization of the knowledge and experiences of these relatives, including their beliefs, culture, and social habits. And in this context, it is extremely important that nurses live the experience from the perception of the subject of this study, considering the humanistic concepts, aiming for an increasingly qualified and excellent assistance, which has enabled the construction process of an educational technology (educational leaflet) as regards the use at home of granulocytes colony stimulating factor

For many years, it has been known that Rheumatoid arthritis (RA) is a negative risk factor for the development of Alzheimer’s disease (AD). It has been commonly assumed that RA patients’ usage of non-steroidal anti-inflammatory drugs (NSAIDs) have helped prevent the onset and progression of AD pathogenesis. Furthermore, experiments in animal models of Alzheimer’s disease have looked to inhibit inflammation, and have demonstrated some efficacy against AD-like pathology in these models. Thus many NSAID clinical trials have been performed over the years, but all have proven unsuccessful in AD patients. This suggests that intrinsic factors within RA pathogenesis itself may underlie RA’s protective effect. My dissertation research goal was to investigate this inverse relationship between RA and AD, in order to more precisely pinpoint critical events in AD pathogenesis toward developing therapeutic strategies against AD. It seemed improbable that any secreted factors, produced in RA pathogenesis, could maintain high enough concentrations in the circulatory system to cross the blood brain barrier and inhibit AD pathogenesis, without affecting all other organ systems. It did seem possible that the leukocyte populations induced in RA, could traverse the circulatory system, extravasate into the brain parenchyma, and impede or reverse AD pathogenesis. We thus investigated the colony-stimulating factors, which are up-regulated in RA and which induce most of RA’s leukocytosis, on the pathology and behavior of transgenic AD mice. We found that G-CSF and more significantly, GM-CSF, reduced amyloidosis throughout the treated brain hemisphere one week following bolus intrahippocampal administration into AD mice. We then found that 20 days of subcutaneous injections of GM-CSF (the most amyloid-reducing CSF in the bolus experiment) significantly reduced brain amyloidosis and completely reversed cognitive impairment in aged cognitively-impaired AD mice, while increasing hippocampal synaptic area and microglial density. These findings, along with two decades of accrued safety data using Leukine, the recombinant human GM-CSF analogue, in elderly leukopenic patients, suggested that Leukine should be tested as a treatment to reverse cerebral amyloid pathology and cognitive impairment in AD patients. It was also implied that age-related depressed hematopoiesis may contribute to AD pathogenesis.

Background The monoclonal antibody natalizumab (NAT) inhibits the migration of lymphocytes throughout the blood–brain barrier by blocking very late antigen (VLA)-4 interactions, thereby reducing inflammatory central nervous system (CNS) activity in patients with multiple sclerosis (MS). We evaluated the effects of different NAT treatment regimens. Methods We developed and optimised a NAT assay to measure free NAT, cell-bound NAT and VLA-4 expression levels in blood and cerebrospinal fluid (CSF) of patients using standard and prolonged treatment intervals and after the cessation of therapy. Results In paired CSF and blood samples of NAT-treated MS patients, NAT concentrations in CSF were approximately 100-fold lower than those in serum. Cell-bound NAT and mean VLA-4 expression levels in CSF were comparable with those in blood. After the cessation of therapy, the kinetics of free NAT, cell-bound NAT and VLA-4 expression levels differed. Prolonged intervals greater than 4 weeks between infusions caused a gradual reduction of free and cell-bound NAT concentrations. Sera from patients with and without NAT-neutralising antibodies could be identified in a blinded assessment. The NAT-neutralising antibodies removed NAT from the cell surface in vivo and in vitro. Intercellular NAT exchange was detected in vitro. Conclusions Incorporating assays to measure free and cell-bound NAT into clinical practice can help to determine the optimal individual NAT dosing regimen for patients with MS.

Background The monoclonal antibody natalizumab (NAT) inhibits the migration of lymphocytes throughout the blood–brain barrier by blocking very late antigen (VLA)-4 interactions, thereby reducing inflammatory central nervous system (CNS) activity in patients with multiple sclerosis (MS). We evaluated the effects of different NAT treatment regimens. Methods We developed and optimised a NAT assay to measure free NAT, cell-bound NAT and VLA-4 expression levels in blood and cerebrospinal fluid (CSF) of patients using standard and prolonged treatment intervals and after the cessation of therapy. Results In paired CSF and blood samples of NAT-treated MS patients, NAT concentrations in CSF were approximately 100-fold lower than those in serum. Cell-bound NAT and mean VLA-4 expression levels in CSF were comparable with those in blood. After the cessation of therapy, the kinetics of free NAT, cell-bound NAT and VLA-4 expression levels differed. Prolonged intervals greater than 4 weeks between infusions caused a gradual reduction of free and cell-bound NAT concentrations. Sera from patients with and without NAT-neutralising antibodies could be identified in a blinded assessment. The NAT-neutralising antibodies removed NAT from the cell surface in vivo and in vitro. Intercellular NAT exchange was detected in vitro. Conclusions Incorporating assays to measure free and cell-bound NAT into clinical practice can help to determine the optimal individual NAT dosing regimen for patients with MS.

Mobilization of peripheral blood stem cells (PBSC) in healthy volunteers with granulocyte colony-stimulating factor (G-CSF) is currently carried out at many institutions worldwide. This report presents the experience of the Dresden center regarding donor evaluation and mobilization schedule. Data regarding efficacy, short- and long-term safety of G-CSF treatment gained from 8290 PBSC collections in healthy donors are outlined. These results are discussed against the background of the available evidence from the literature. Although established as a standard procedure, G-CSF application to allogeneic donors will always be a very delicate procedure and requires the utmost commitment of all staff involved to ensure maximum donor safety. (PBSC) donation does not require hospitalization and is generally assumed to be less physically demanding for the donor. However, application of mobilizing agents is stringently required for successful HSC mobilization. The standard substance, which is almost exclusively used in healthy donors worldwide, is recombinant human granulocyte colony-stimulating factor (rhG-CSF). Two preparations – filgrastim and lenograstim – are available and have been approved for PBSC mobilization for about 15 years in Germany. Currently, more than 20,000 healthy donors worldwide receive rhG-CSF for PBSC mobilization every year [7]. At the Dresden University Hospital, PBSC collections have been performed since 1996. In the two collection facilities associated with the university hospital, 8,290 allogeneic PBSC collections from 8,005 donors (i.e. 285 second collections) have been documented in a database up until May 2012. This paper presents the data of our own group, and summarizes the current knowledge regarding the short- and long-term effects of G-CSF treatment in healthy stem cell donors.

CSF-1R (colony-stimulating factor 1 receptor) est un récepteur transmembranaire à activité tyrosine kinase exprimé à la surface des monocytes, des macrophages et de leurs progéniteurs. Son ligand, CSF-1, oriente les cellules souches hématopoïétiques vers le lignage myéloïde et permet la différenciation des monocytes en macrophages. Une localisation nucléaire de CSF-1R a été décrite dans certaines lignées tumorales, dans des tumeurs mammaires primitives et dans les macrophages murins. Dans le noyau de ces cellules, CSF-1R régulerait la phosphorylation de protéines nucléaires et l'expression de gènes de la prolifération. Nous avons identifié une localisation nucléaire de CSF-1R dans les monocytes primaires humains par différentes approches et différents anticorps. La forme nucléaire de CSF-1R correspond à la protéine entière monomérique qui est transportée depuis la membrane plasmique vers le noyau, de manière rétrograde, après activation par son ligand et avec celui-ci. L'utilisation d'inhibiteurs de l'activité kinase de CSF-1R diminue la quantité de récepteur dans le noyau. En revanche le blocage des mécanismes d'export nucléaire dépendant de CRM1 par la leptomycine B conduit à l'accumulation de la protéine dans ce compartiment. Dans les monocytes, CSF-1R est localisé sur la chromatine, dans les régions intergéniques et introniques et colocalise avec la marque H3K4me1 présente au niveau des enhancers activés. CSF-1R est situé à proximité de gènes régulant la morphogénèse, le développement du système nerveux, l'ossification et la différenciation cellulaire. Le récepteur est présent sur le promoteur du gène PU.1, facteur de transcription clé dans la différenciation myéloïde et la génération des monocytes, ainsi que sur des gènes impliqués dans la différenciation, la polarisation, la survie et les fonctions des macrophages. Au niveau de la chromatine, CSF-1R interagit avec des facteurs de transcription comme EGR1 sur lequel il exerce un effet co-répresseur. Cette localisation nucléaire de CSF-1R est conservée lorsque les monocytes se différencient en macrophages en réponse à CSF-1. CSF-1R nucléaire est alors relocalisé vers les régions promotrices et exoniques où il colocalise avec la marque H3K4me3. Il est présent à proximité de gènes régulant la vascularisation, la phagocytose, le métabolisme, la réponse au stress et à l'hypoxie. Il interagit avec les facteurs de transcription ELK1 et YY1, et joue un rôle de co-activateur. Lorsque les monocytes sont différenciés en macrophages par une autre cytokine, le GM-CSF, CSF-1R reste dans le noyau des cellules mais sa localisation sur la chromatine et ses interacteurs diffèrent de ceux des monocytes et des macrophages générés par CSF-1, démontrant un régulation différentielle de CSF-1R nucléaire selon le stade de différenciation et les signaux environnementaux. Dans des monocytes de patients atteints de leucémie myélomonocytaire chronique, l’expression, la localisation sur l’ADN et les interacteurs de CSF-1R sont modifiés, indiquant une dérégulation des fonctions nucléaires du récepteur en condition pathologique. CSF-1R est donc localisé dans le noyau des monocytes et des macrophages où il exerce un rôle de régulation de l'expression des gènes dont PU.1. Des résultats préliminaires suggèrent une localisation nucléaire du récepteur dans certaines populations de progéniteurs myéloïdes où il pourrait participer à la regulation de la différenciation. De nombreux inhibiteurs de CSF-1R sont en développement afin de cibler les macrophages infiltrant les tumeurs. Nos résultats démontrent que certains inhibiteurs ont la capacité de cibler la forme membranaire et la forme nucléaire du récepteur et donc d'inhiber l'ensemble des activités de CSF-1R dans les cellules, renforçant l'activité potentielle de ces traitements. / CSF-1R (colony-stimulating factor 1 receptor) is a transmembrane receptor with a tyrosine kinase activity. It is expressed at the cell surface of monocytes, macrophages and their progenitors. Its ligand, CSF-1, has an instructive role on hematopoietic stem cells to direct their differentiation into the myeloid lineage. CSF-1R is also able to differentiate monocytes into macrophages. A nuclear location was described for CSF-1R in cancer cell lines, primary breast tumors and murine macrophages. In the cell nucleus, CSF-1R was suggested to regulate nuclear protein phosphorylation and gene expression. We demonstrate that a small part of CSF-1R is in the nucleus of primary human monocytes, using different antibodies and technical approaches. Nuclear CSF-1R corresponds to full length monomeric receptor. After activation by its ligand, CSF-1R is translocated form cell surface to the nucleus through a retrograde transport, together with CSF-1. Kinase activity inhibitors impaired this process while inhibitors of CRM1-dependant nuclear export (leptomycin B) can revert this effect. In monocytes, CSF-1R is localized on chromatin, mainly on intergenic and intronic regions. It colocalizes with H3K4me1 mark which signs active enhancers. The receptor is present around genes involved in morphogenesis, nervous system development, ossification and cell differentiation. CSF-1R is also located on PU.1 promoter, which is a master transcription factor involved in myeloid and monocyte differentiation. CSF- 1R is also present on genes implicated in macrophage functions, differentiation, polarization and survival. At the chromatin level, CSF-1R interacts with different transcription factors like EGR1 and exerts a co-repressive role to decrease or limit gene expression. CSF-1R nuclear localization persists in macrophages generated by exposure of monocytes to CSF-1. It entails CSF-1R relocalization on promoter-TSS and exonic regions where it colocalizes with H3K4me3 mark. The receptor is close to genes regulating vascularization, phagocytosis, metabolism, stress and hypoxia responses. CSF-1R interacts with ELK1 and YY1 to promote macrophage functions. When monocytes are differentiated into macrophages with GM-CSF, CSF-1R also remains in the nucleus. However, its chromatin localization and interactions change compared to monocytes and CSF-1 differentiated macrophages. This indicates that nuclear CSF-1R is differentially regulated, depending on the cytokine that triggers cell differentiation. In monocytes from chronic myelomonocytic leukemia, CSF-1R expression, chromatin localization and interactors are modified, indicating a deregulated CSF-1R nuclear function under pathological state. Altogether, we showed that CSF-1R is localized in the nucleus of human monocytes and macrophages where it regulates gene expression including PU.1. Preliminary results suggest CSF-1R nuclear location in myeloid progenitor subsets where the receptor could directly regulate the expression of myeloid differentiation genes. Targeting CSF-1R is currently tested as a therapeutic strategy to impair tumor infiltrating macrophages. Our results show that CSF-1R inhibitors are able to target both membrane and nuclear forms and thus to inhibit all CSF-1R activities in the cells, enhancing the potential therapeutic effects of these molecules.