Adenine nucleotides hydrolysis is an important step of the neuromodulation of CNS and some of its breakdown products are able to protect the nervous tissue against brain injuries. The activity of NTPDase (EC 3.6.1.5, apyrase, CD39) was verified in cerebrospinal fluid (CSF) from patients without neural inflammatory process under different conditions and in the presence of several inhibitors. The samples were chose considering the low protein levels, normal glucose levels, low leukocyte count and CSF differential count. We chose use the supernatant 1 (S1) for enzyme assay due to the best enzymatic activities in this fraction. The best hydrolyze temperature was 37°C to ATP and ADP. This enzyme was cation-dependent, with a maximal rate for ATP and ADP hydrolysis in pH 8.0 in the presence of 5mM Ca+2. Sodum azide inhibited both nucleotide hydrolysis at concentrations higher than 10 mM. Sodium fluoride inhibited the ATP and ADP hydrolysis at concentrations of 15 mM and 20 mM. The Na+ K+ ATPase inhibitor ouabain, did not affect ATP or ADP hydrolysis. The inhibitor P-type ATPase lanthanum 5mM was ineffective on ATPDase hydrolysis. Suramin (30-300 μM) inhibited ATP and ADP hydrolysis and presented a maximal inhibitory effect of 50% at 300 μM. The results of the present study demonstrated that ATP and ADP hydrolysis from human CSF presented a similar response those obtained from rat synaptosomes. / A análise do líquor é de grande importância para a detecção de desordens neurológicas de diversas etiologias. O ATP junto a seus produtos de hidrólise (ADP, AMP e adenosina) desempenha importantes funções junto ao SNC, as quais envolvem ações neuroprotetoras em doenças de etiologias variadas. O presente estudo tem como objetivos verificar a ocorrência de hidrólise de nucleotídeos da adenina em líquor de pacientes sem doença inflamatória do sistema nervoso. A determinação da atividade enzimática da NTPDase foi feita em líquor humano em diferentes condições experimentais e na presença de inibidores. As amostras foram escolhidas de acordo com os baixos níveis protéicos, valores normais de glicose e contagem celular diminuída. Foi escolhido o sobrenadante 1 (S1) por apresentar melhores atividades enzimáticas. A melhor temperatura de hidrólise do ATP e ADP foi 37°C. Esta enzima é cátion dependente, sendo a atividade de hidrólise ótima do ATP em presença de 5 mM Ca+2 e o ADP 5-7 mM de Ca+2, ambos em pH 8.0. A azida sódica alterou a atividade enzimática do ATP e do ADP somente nas concentrações mais altas deste inibidor da ATPase mitocondrial. A ouabaína, um inibidor da Na+/K+ ATPase não afetou a hidrólise do ATP/ADP. O inibidor de ATPase tipo-P lantânio (5 mM) foi ineficaz na hidrólise dos nucleotídeos. O suramin (30-300 XM), inibidor específico de NTPDase, inibiu a hidrólise do ATP/ADP e apresentou máximo efeito inibitório na concentração de 300 XM. Os resultados deste estudo mostraram que a hidrólise de ATP/ADP em líquor humano apresentou uma resposta semelhante àquelas obtidas em sinaptossomas de ratos.

No description available.

Os projetos de desenvolvimento realizados em colaboração são fundamentais para que as empresas de máquinas agrícolas consigam aumentar o grau de inovação em produtos, permitindo-as acompanhar os novos desafios de um mercado mais competitivo. Há na literatura vários trabalhos que identificam fatores críticos de sucesso (FCS) para projetos colaborativos, porém, não há muitos estudos sobre a verificação desses fatores no Brasil, em especial na indústria de máquinas agrícolas (IMA). Este trabalho tem como objetivo verificar se os FCS identificados na literatura poderiam ser empregados nesse contexto. Empreendeu-se uma revisão bibliográfica para identificar os fatores críticos de sucesso em projetos colaborativos de desenvolvimento de produtos e uma revisão sobre indústria de máquinas agrícolas (IMA), de forma a entender as especificidades desse setor no Brasil. Em seguida, realizou-se um levantamento em uma empresa do setor, com nível de maturidade elevado em desenvolvimento de produto, medido conforme Simões (2007), para verificar se os FCS identificados poderiam ser utilizados em levantamentos no setor e verificar se não havia outros fatores a serem considerados, específicos para o segmento e região. Utilizou-se o método do estudo de caso único, do tipo incorporado. A unidade de análise é constituída por projetos do tipo colaborativo, realizados com sucesso pela empresa. Foram analisados dois projetos de produtos inovadores, com diferentes tipos de parceiro: um com um cliente e outro com um fornecedor. Os dados foram coletados por meio de entrevistas e o instrumento de pesquisa é um roteiro. As instalações da empresa foram visitadas com o objetivo de conhecer os projetos analisados. Como resultado afirma-se que há fatores descritos na literatura que podem não ser críticos; indica-se a existência de novos FCS e reforça-se a importância dos fatores ligados à garantia de igualdade e fatores universais de sucesso. A contribuição do trabalho é uma lista de fatores críticos de sucesso que podem servir como passo inicial para levantamentos gerais de campo no setor industrial estudado. / The collaborative projects of development are one of the main tools in order to agricultural machines companies get to increase the innovation degree in products, allowing them to follow the new challenges of a more competitive market. There is in the literature several works that identify critical success factors (CSF) for collaborative projects; however, there are not many studies about the verification of those factors in Brazil, especially in the agricultural machinery industry. The aim of this work is to verify if identified CSF in the literature could be used in that context. A bibliographical revision was undertaken to identify the \"critical success factors\" in collaborative projects of development of products and a revision on agricultural machines industry in order to understand the specificities of that section in Brazil. After that it has taken place a survey in a company of that sector, with high level of maturity in development of measured product according to Simões (2007), to verify if identified CSF could be used in surveys in that sector and to verify if there were any other factors to be considered, specific for that segment and field. A case study was used and the units of analysis are collaborative projects accomplished successfully by the company. Two projects development of products were analyzed, with different kinds of partner: one with a customer and the other with a supplying company. Besides the interviews it has taken visits to the companies in order to know the analyzed projects. There are factors described that could not be critical; the results indicate the existence of new critical factors and they confirm the importance of equality and universal factors as critical for collaborative projects. The main contribution of these projects is a list of critical factors of success that one can serve as initial step for general survey in this field.

Au cours d’une infection, les cellules de l’immunité innée sont capables de reconnaître via les Toll-like receptors (TLR) des motifs appelés pathogen-associated molecular patterns. Les cellules natural killer (NK) contribuent au processus inflammatoire en produisant de nombreuses cytokines. Chez la souris, nous avons montré que l’expression du TLR2 et du TLR4 dans les cellules NK spléniques est intracellulaire, comme pour le TLR9. La réponse des NK aux agonistes des TLR2, 4 et 9 nécessite la présence de cytokines accessoires (IL-15 et IL-18), afin d’obtenir une production significative des cytokines pro-inflammatoires IFN- et GM-CSF. En revanche, dans un modèle de sepsis polymicrobial, les NK spléniques de souris présentent une diminution dramatique de leur production d’IFN- et de GM-CSF en réponse aux agonistes des TLR. Cette diminution est sous le contrôle des cellules T régulatrices (Treg) et due au TGF-1. L’analyse des voies de signalisation nous a permis de montrer que la production de GM-CSF est abolie chez les cellules NK de souris déficientes pour STING en réponse au CpG-DNA. Ces résultats mettent en lumière une voie alternative et cytoplasmique pour la détection de l’ADN bactérien dans les cellules NK, différente de la voie classique TLR9-MyD88 dépendante. De plus, nous avons montré un trafic du récepteur TLR2 depuis l’intérieur vers la surface des cellules NK. La migration du TLR2 à la surface des NK nécessite la molécule UNC93B1, précédemment décrite comme transporteur endosomal de TLR.Chez les cellules NK humaines circulantes (sous-populations CD3-CD56bright et CD3-CD56dim), nous avons montré que l’expression des TLR2 et 4 est majoritairement intracellulaire, comme pour le TLR9 et comme chez la souris. La production d’IFN- par les NK de sujets sains en réponse aux agonistes des TLR nécessite également la présence de cytokines accessoires. Nous montrons que cette production est fortement altérée pour les NK des patients admis en soins intensifs et ayant un sepsis ou un syndrome de réponse inflammatoire systémique (SIRS). De même nous avons trouvé des différences entre les patients et les sujets sains dans l’expression du CD69 (marqueur d’activation précoce) et des TLR eux-mêmes. Cette étude indique que les NK des patients sepsis et SIRS deviennent tolérants aux agonistes des TLR en terme de production d’IFN-, de manière similaire à ce qui a été décrit pour d’autres cellules comme les monocytes / As sensors of infection, innate immune cells are able to recognize pathogen-associated molecular patterns by receptors such as Toll-like receptors (TLR). NK cells contribute to inflammatory processes by the production of numerous cytokines. In mice, we have shown that the protein expression of TLR2 and TLR4 in naive NK cells from spleen is predominantly intracellular, similarly to TLR9. The responsiveness of purified NK cells to TLR2, 4 or 9 agonists in vitro requires the presence of accessory cytokines (IL-15 and 18) to trigger a significant production of IFN- and GM-CSF. In contrast, NK cells purified from a model of in vivo polymicrobial sepsis, showed a dramatic reduction in their capacity to respond to TLR agonists in terms of IFN- and GM-CSF release due an inhibitory cross talk with Treg cells mediated by TGF-1. Analyzing the signaling pathways involved in cytokine production in response to CpG-DNA, we found that GM-CSF production was abolished in NK cells from STING-deficient mice, revealing that this intracytoplasmic receptor acts as a TLR9/MyD88-independent alternative sensor to bacterial DNA in NK cells. Additionally we show that intracellularly expressed TLR2 traffics to the cell surface of NK cells, by a mechanism involving UNC93B1, a protein previous described as an endosomal TLR carrier.In human peripheral blood NK cells (CD3-CD56bright and CD3-CD56dim subsets), we show that TLR2 and 4 protein expression is primarily intracellular, similar to TLR9, and similar to our findings in murine NK cells. The ex vivo responsiveness of human blood NK cells to TLR2, 4 or 9 agonists also requires accessory cytokines, to promote secretion of IFN-. In intensive care patients diagnosed with systemic inflammatory response syndrome (SIRS) and sepsis, IFN- production was significantly decreased. We also discovered modulations in the expression of CD69 (early activation marker) and in that of TLR themselves. This study indicates that NK cells undergo tolerance in response to TLR agonists during SIRS or sepsis, similarly to other cells, such as monocytes.

The meningitis is a serious inflammatory illness of meninges, the membranes that recover and protect encephalo and the spinal cord. The main causing agents of the meningitis are the viruses and the bacteria. Some works affirm that the adenosine, the final product of ATP hydrolysis (when degraded by ectonucleotidases), have an important protective role in brain disorders. Breakdown of extracellular ATP to adenosine is catalyzes by ecto-nucleotidases family, the NTPDase hydrolyze extracellular nucleotide tri and diphosphates (ATP and ADP) to nucleotide monophosphate (AMP) and the 5'-nucleotidase catalyzes the hydrolysis of AMP to adenosine. In this work we verified adenine nucleotides hydrolysis in the CSF of aseptic and bacterial meningitis patients, in order to compare with adenine nucleotides hydrolysis in controls patient (absence of neural inflammatory process). Were used patients with age between 18 and 59 years of both the sex in a sampling of 15 patient controls, 15 patients with aseptic meningitis and 15 patients with bacterial meningitis. For the criteria of inclusion and exclusion had been analyzed parameters like, aspect of the CSF, total counting of leukocytes, distinguishing counting of leukocytes, values of protein, glucose and lactate. We got as resulted a significant reduction of hydrolysis of the ATP in the patients with aseptic and bacterial meningitis when compared with patient controls. In contrast we got a significant increase of hydrolysis of the ADP and the AMP in the two groups of meningitis when compared with the controls. The increase in ADP and AMP hydrolysis maybe means the attempt of increase the adenosine production, that have a recognized neuroprotective actions in brain insults. / A meningite é uma grave doença inflamatória das meninges, as membranas que recobrem e protegem o encéfalo e a medula espinhal. Os principais agentes causadores da meningite são os vírus e as bactérias. Vários trabalhos afirmam que a adenosina, produto final da degradação do ATP (quando degradado pelas ectonucleotidases), possui um importante papel protetor nas desordens cerebrais. A degradação do ATP até adenosina é catalisada pela família das ecto-nucleotidases, a NTPDase hidrolisa nucleotídeos extracelulares tri e di-fosfatados (ATP e ADP) até nucleotídeos mono-fosfatados (AMP), e a 5'-nucleotidase catalisa a hidrolise do AMP até adenosina. Neste trabalho verificou-se a hidrólise dos nucleotídeos de adenina no líquor de pacientes com meningite asséptica e bacteriana, a fim de comparar a hidrólise destes nucleotídeos com pacientes controles (ausência de processo inflamatório neural). Foram utilizados pacientes com idade entre 18 e 59 anos de ambos os sexos em uma amostragem de 15 pacientes controles, 15 pacientes com meningite asséptica e 15 paciente com meningite bacteriana. Para os critérios de inclusão e exclusão foram analisados parâmetros tais como: aspecto do líquor, contagem total de leucócitos, contagem diferencial de leucócitos, valores de proteína, glicose e lactato. Obtivemos como resultado uma diminuição significativa da hidrólise do ATP nos pacientes com meningite asséptica e bacteriana quando comparados com pacientes controles. Ao contrário obtivemos um aumento significativo da hidrólise do ADP e do AMP nos dois grupos de meningites quando comparados com os controles. A hidrólise aumentada do ADP e do AMP refere-se talvez a tentativa de aumentar a produção de adenosina, que por sua vez possui um conhecido papel neuroprotetor frente a insultos cerebrais.

La sclérose en plaques (SEP) est une maladie inflammatoire auto-immune du SNC qui affecte 90 000 personnes en France et génère un coût important pour la société. La forme la plus fréquente (85% des cas) est la SEP récurrente-rémittente (RRMS) caractérisée par des poussées démyélinisantes entrecoupées de périodes de rémission après un syndrome cliniquement isolé (CIS). La conversion en RRMS après un CIS, caractérisée par une nouvelle poussée ou de nouvelles lésions sur les IRM de suivi, survient après un délai variant de quelques mois à plus de 10 ans (20% des cas, « SEP bénigne »). Les traitements disponibles (immunomodulateurs et immunosuppresseurs) ont une efficacité indéniable pour la prévention des poussées et doivent être initiés dès le diagnostic pour une efficacité maximale, surtout chez les patients montrant une conversion rapide en SEP après un CIS. Leur intérêt est en revanche négligeable pour les formes bénignes. Ainsi, il existe un besoin d’identifier des biomarqueurs pronostiques précoces de la SEP pour une prise en charge optimale des patients. Le but de ma thèse est d’identifier des biomarqueurs pronostiques de SEP en utilisant différentes approches de protéomique quantitative,. Dans un premier temps, nous avons utilisé des échantillons cliniques de LCR de patients. Ces travaux m’ont permis d’identifier, en protéomique quantitative utilisant un marquage chimique des peptides (TMT), plusieurs candidats biomarqueurs de SEP. Ceux-ci incluent deux chitinase-3-like protéines, CHI3L1 et CHI3L2 dont le taux mesuré en ELISA, dans le LCR, augmente avec l’évolution de la maladie. Ces travaux ont également démontré que la concentration sanguine de CHI3L1 constituait un biomarqueur de la progression de la maladie. Dans un second temps, nous avons combiné l’analyse du LCR de différents patients SEP et contrôles en protéomique Label-Free avec un modèle préclinique analysant les modifications du sécrétome d’oligodendrocytes murins en culture primaire par approche SILAC. Les biomarqueurs potentiels, 87 protéines correspondant à 226 peptides cibles, ont été ensuite vérifiés en protéomique quantitative ciblée ou parallel reaction monitoring (PRM), à l’aide de peptides marqués servant de référence. Les 11 candidats les plus discriminants issus de cette étape ont ensuite été vérifiés en PRM sur une cohorte plus large d’échantillons de patients. Finalement, 7 candidats ont montré un profil significatif au cours de cette première validation. Ces candidats biomarqueurs permettent notamment de discriminer les différentes formes évolutives de la SEP et de distinguer cette pathologie d’autres maladies inflammatoires et neurologiques. De plus, ces derniers pourraient avoir un intérêt prognostique permettant d’identifier les patients qui vont développer une SEP après la découverte fortuite de lésions typiques de la maladie à l’IRM. Ce travail de thèse a donc caractérisé de nouveaux biomarqueurs de SEP devant être validés sur de larges cohortes multicentriques de patients et ouvre de nouvelles perspectives sur la compréhension des mécanismes physiopathologiques de la SEP. / Discovery, confirmation and verification of candidate biomarkers for multiple sclerosis diagnosis and prognosis in cerebrospinal fluid.Multiple sclerosis (MS) is an inflammatory disease of the central nervous system. Most often the disease initiates by a first demyelinating event called clinically isolated syndrome (CIS), followed by remission periods and relapses occurring at irregular intervals. Clinical symptoms and MRI are used for diagnosis, but clinicians lack tools to predict the rate of disease progression. This study aims at identifying biomarkers that predict the delay of conversion to MS after a CIS. We compared the cerebrospinal fluid (CSF) proteome from MS patients and symptomatic controls and the CSF from CIS patients with rapid conversion to MS (<1 year) and CIS patients with slow conversion to MS (>2 years). For the discovery step, human CSF samples (n=40) depleted of the 20 major plasma proteins were digested using a modified filter-assisted sample preparation (FASP) and analysed by high-resolution mass spectrometry using isobaric mass tag labelling or label-free quantification procedures. Proteins upregulated in CSF from MS patients included two proteins involved in tissue remodeling, namely chitinase-3-like protein-1 (CHI3L1) and chitinase-3-like protein-2 (CHI3L2). Their increased level in CSF of MS patients was confirmed by ELISA in a new cohort comprising CIS and MS patients (n=123) at different disease stages. Moreover, CHI3L1 levels in CSF and serum from CIS patients discriminated patients with rapid conversion to MS (< one year) from those with slower conversion.We also implemented a PRM method (peptide selection, dilution optimization of heavy isotope labeled non-purified peptides, reproducibility evaluation and method validation) to qualify a larger set of candidate biomarkers (226 peptides corresponding to 87 proteins) in a cohort different from the one used for the discovery step (n=60), including CSF from controls and MS patients at different disease stages. Finally, to further verify the 11 candidate biomarkers that passed this qualification step, we monitored 16 peptides in a new PRM assay, using shorter gradient and high-purity heavy isotope labeled peptides. This new PRM analysis was performed on a larger cohort (n=189) that included CSF of patients with other inflammatory and non-inflammatory neurological disorders in addition to control and MS patients. These analyses identified seven robust candidate biomarkers, which might help to discriminate patients suffering from MS or other neurological disorders.

The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2015-11-16T22:08:40Z No. of bitstreams: 1 Disserta??o_PAMELA_SANTANA.pdf: 760607 bytes, checksum: c2d79c30297aa8834b72575b1f4dd8d1 (MD5) / Made available in DSpace on 2015-11-16T22:08:40Z (GMT). No. of bitstreams: 1 Disserta??o_PAMELA_SANTANA.pdf: 760607 bytes, checksum: c2d79c30297aa8834b72575b1f4dd8d1 (MD5) Previous issue date: 2013-04-30 / Dietary habits composed of high levels of fat are a trigger for obesity. This pandemic is involved with the development of cardiovascular diseases, due to structural and hemodynamic changes in the heart. The G-CSF has emerged as a potential treatment for many degenerative diseases, emphasizing its beneficial effect on the structural and functional regeneration of the heart in ischemic cardiomyopathy and Chagas disease, with diminished inflammation and fibrosis, and improvement of cardiac impulse generation and conduction. We used C57BL / 6 mice fed a high-fat (HF) diet that developed obesity, hyperglycemia and glucose intolerance. The changes in cardiac function were assessed by electrocardiogram, echocardiogram and treadmill. The animals were divided into two groups: standard diet (n = 8) and HF diet (n = 16) to induce obesity. 37 weeks after HF protocol, the animals were divided into three groups: treated with G-CSF (n = 8) + standard diet, not treated with G-CSF (n = 8) + standard diet, and the third group with standard diet only (n = 8). Results showed that replacing the standard diet following HF diet was responsible for the return to baseline of anthropometric and biochemical values, which were significantly elevated. Diet replacement alone did not reverse exercise intolerance or prevent cardiac abnormalities, such as bradycardia and increases in QRS amplitude, septal wall thickness and left ventricular posterior thickness. Diet replacement and treatment with G-CSF reversed structural changes in the heart and recovery of physical capacity. / H?bitos alimentares de altos teores de gordura s?o fatores desencadeantes da obesidade. Essa pandemia est? envolvida com o desenvolvimento de doen?as cardiovasculares, devido a altera??es estruturais e hemodin?micas no cora??o. O G-CSF surge como um tratamento em potencial para a terapia de diversas doen?as degenerativas, destacando-se seu efeito ben?fico na regenera??o estrutural e funcional do cora??o nas cardiomiopatias isqu?mica e chag?sica cr?nica com diminui??o de inflama??o e fibrose, e melhora na gera??o e condu??o do impulso card?aco. Utilizou-se camundongos C57Bl/6 machos alimentados com dieta hipergordurosa (high-fat HF) que desenvolveram obesidade, hiperglicemia e intoler?ncia ? glicose. As altera??es funcionais card?acas foram avaliadas por eletrocardiograma, ecocardiograma e ergometria. Os animais foram separados em 2 grupos experimentais: dieta padr?o (n=8) e dieta HF (n = 16) para indu??o da obesidade. 37 semanas ap?s a introdu??o da HF, os animais foram separados em 3 grupos: tratado com G-CSF (n=8) + dieta padr?o; n?o tratado com G-CSF (n=8) + dieta padr?o, e o terceiro grupo somente com dieta padr?o (n=8). Resultados mostraram que a substitui??o da dieta HF pela dieta padr?o foi respons?vel pelo retorno ao n?vel basal dos valores bioqu?micos e antropom?tricos, que apresentavam-se elevados. Apenas a substitui??o da dieta n?o reverteu ? intoler?ncia ao exerc?cio nem preveniu as altera??es card?acas, como bradicardia, aumento da amplitude de QRS e aumento da espessura das paredes septal e posterior do ventr?culo esquerdo. A substitui??o da dieta e o tratamento com G-CSF reverteram as altera??es estruturais do cora??o e recuperarem a capacidade f?sica.

Die erfolgreiche Übertragung der vielversprechenden Ergebnisse aus zahlreichen Kleintierstudien zu den neuroprotektiven und -regenerativen Eigenschaften des Wachstumsfaktors G-CSF auf eine multizentrische randomisierte Phase-IIa-Studie misslang. Bei näherer Betrachtung fiel eine deutlich höhere Dosierung des Wachstumsfaktors in der humanen Studie im Vergleich zu den erfolgreichen präklinischen Studien auf, sodass Fragen nach einem schädigenden Einfluss möglicher immunologischer Veränderungen aufkamen.Um dies zu überprüfen, wurde in einem murinen Schlaganfallmodell eine quantitative FACS-Analyse des frühen entzündlichen Hirninfiltrats und der Immunzellpopulationen im peripheren Blut mit besonderem Augenmerk auf neutrophile Granulozyten 24 Stunden nach (Schein-)Schlaganfall durchgeführt. Ergänzend erfolgte eine immun-histochemische Beurteilung des Infiltrationsverhaltens neutrophiler Granulozyten, wobei neben der räumlichen Verteilung die Zellmorphologie untersucht wurde.:Abbildungsverzeichnis IV Tabellenverzeichnis VI Abkürzungsverzeichnis VII 1 Einleitung 1 1.1 Der Schlaganfall 1 1.1.1 Epidemiologie und sozioökonomische Relevanz 1 1.1.2 Ätiologie und Pathogenese 1 1.1.3 Klinische Symptomatik 2 1.1.4 Rekanalisierende Therapie 2 1.2 Immunologie des Schlaganfalls 3 1.2.1 Das Gehirn als immunprivilegiertes Organ 3 1.2.2 Immunantwort nach Schlaganfall 3 1.2.3 Bedeutung neutrophiler Granulozyten für die Immunantwort 6 1.2.4 Rolle des neutrophilen Granulozyten im Schlaganfall 7 1.3 Der Granulozyten Kolonie-stimulierende Faktor 10 1.3.1 G-CSF im Schlaganfall 10 1.3.2 Einführung 12 1.3.3 Präklinische Schlaganfallstudien mit G-CSF 14 1.3.4 klinische Schlaganfallstudien mit G-CSF 14 1.4 Dosistranslation von experimentellen zu klinischen Studien 16 2 Zielstellung der Arbeit 17 3 Material und Methoden 18 3.1 Versuchstiere 18 3.2 Schlaganfallmodell 18 3.3 Ein- und Ausschlusskriterien 19 3.4 Versuchsplan 20 3.5 Behandlung mit G-CSF 21 3.6 Euthanasie und transkardiale Perfusion 22 3.7 Quantifizierung der Leukozytenzahl im Vollblut 23 3.8 Durchflusszytometrische Aufarbeitung 23 3.8.1 Lyse- und Färbeprotokoll Blut 23 3.8.2 Isolation von Immunzellen aus dem Maushirn und Färbeprotokoll 24 3.9 Durchflusszytometrische Analyse 25 3.10 Histopathologische Aufarbeitung 26 3.10.1 Anfertigung histologischer Schnitte im Kryostat 26 3.10.2 Hämatoxylin-Eosin-Färbung 27 3.10.3 Immunfluoreszenz 27 3.10.4 Differenzierung von Granulozyten und Monozyten 29 3.10.5 Immunhistochemische Auszählung 29 3.10.6 Darstellung der räumlichen Verteilung 31 3.11 Statistische Auswertung 33 4 Ergebnisse 34 4.1 Durchflusszytometrie 34 4.1.1 Veränderungen des Körper- und Milzgewichts 34 4.1.2 Immunzellpopulationen im peripheren Blut 35 4.1.3 Immunzellpopulationen im Hirnisolat 38 4.2 Immunhistochemie 42 4.2.1 Differenzierung von Monozyten und Granulozyten 42 4.2.2 Quantitative Analyse 43 4.2.2.1 Ipsilateral / kontralateral 44 4.2.2.2 Meningeal / intrazerebral 45 4.2.2.3 Rund / stäbchenförmig 46 4.2.3 Räumliche Verteilung der neutrophilen Granulozyten 47 5 Diskussion 51 5.1 Zusammenfassung der Ergebnisse 51 5.2 G-CSF und die Einwanderung neutrophiler Granulozyten 53 5.3 G-CSF und die Zusammensetzung des frühen entzündlichen Infiltrats im Gehirn 54 5.4 G-CSF und die räumliche Verteilung einwandernder neutrophiler Granulozyten 56 5.5 G-CSF und die Zellmorphologie einwandernder neutrophiler Granulozyten 58 5.6 G-CSF und die periphere bzw. systemische Immunantwort 60 5.7 Methodische Einflussfaktoren und Limitationen der Arbeit 63 5.7.1 Berechnung der murinen Äquivalenzdosis 63 5.7.2 Wahl des Schlaganfallmodells 64 5.7.3 Immunologische Unterschiede zwischen Maus und Mensch 65 5.7.4 Methoden und Versuchstieranzahl 66 5.7.4.1 Durchflusszytometrie 66 5.7.4.2 Immunhistochemie 67 5.8 Ausblick 68 6 Zusammenfassung 70 Literaturverzeichnis XI Erklärung über die eigenständige Abfassung der Arbeit XXVII Tabellarischer Lebenslauf XXVIII Publikationen XXIX Danksagung XXX

Kuwait is planning for huge infrastructure and development projects through long term strategies and visions from 2007 until 2035 to overcome the needs for the welfare housing and to move the country to be the region’s top commercial and financial hub. These goals wouldn’t be achieved without having a strong partner (private sector) to drive the economy by participating in construction mega projects. Public-private partnership (PPP) is the framework which engages both parties to successfully achieve long term plans to deliver what the country and citizens need. To attract the private sector to participate and provide the services needed with the expected high qualities and techniques, the public sector needs to improve the environment to facilitate PPP implementation. This research investigates the existence of critical success factors of PPP project in construction sector of Kuwait: Effective procurement, Project implementability, Available financial market, Government guarantee and Favorable economic conditions and give recommendations to focus on improving them to achieve successful PPP projects.