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Background and Aims Gliomas account for over half of all primary brain tumours and have a very poor prognosis, with a median survival of less than two years. There is an urgent and unmet clinical need to develop new therapies against glioma. Recent reports have indicated the overexpression of FPR1 in gliomas particularly in high grade gliomas. The aim of this project was to identify and synthesise small molecule FPR1 antagonists, and to demonstrate a proof of principle in preclinical in vitro and in vivo models that small molecule FPR1 antagonism can retard expansion of glioma. Methods A number of small molecule FPR1 antagonists were identified by in silico design, or from the literature and then were prepared using chemical synthesis. FPR1 antagonists were evaluated in vitro for their ability to abrogate FPR1-induced cellular responses in a range of models including calcium mobilisation, cell migration, and invasion. The efficacy of FPR1 antagonist ICT12035 in vivo was assessed in a U-87 MG subcutaneous xenograft model. Results Virtual high throughput screening using a homology model of FPR1 led to the identification of two small molecule FPR1 antagonists. At the same time chemical synthesis of two other antagonists, ICT5100 and ICT12035 as well as their analogues were carried out. The FPR1 antagonists were assessed in calcium flux assay which gave an insight into their structure-activity relationship. Further investigation of both ICT5100 and ICT12035 demonstrated that both small molecule FPR1 antagonists were effective at abrogating FPR1-induced calcium mobilisation, migration, and invasion in U- 87 MG in vitro models in a dose-dependent manner. ICT12035 is a particularly selective and potent inhibitor of FPR1 with an IC50 of 37.7 nM in calcium flux assay. Additionally, it was shown that the FPR1 antagonist ICT12035 was able to arrest the growth rate of U-87 MG xenografted tumours in mice. Conclusion The results demonstrate that targeting FPR1 by a small molecule antagonist such as ICT12035, could provide a potential new therapy for the treatment of glioblastoma. / Yorkshire Cancer Research

Sustainable energy sources are of great importance, and will become even more important in the future. Gasification of biomass is an important process for utilization of biomass, as a renewable energy carrier, to produce fuels and chemicals. Density functional theory (DFT) calculations were used to investigate i) the effect of co-adsorption of water and CO on the Ni(111) catalysed water splitting reaction, ii) water adsorption and dissociation on Ni(111), Ni(100) and Ni(110) surfaces, as well as iii) formyl oxidation and dissociation, iv) hydrocarbon combustion and synthesis, and v) the water gas shift (WGS) reaction on these surfaces. The results show that the structures of an adsorbed water molecule and its splitting transition state are significantly changed by co-adsorption of a CO molecule on the Ni(111) surface. This leads to less exothermic reaction energy and larger activation barrier in the presence of CO which means that far fewer water molecules will dissociate in the presence of CO. For the adsorption and dissociation of water on different Ni surfaces, the binding energies for H2O and OH decrease in the order Ni(110) > Ni(100) > Ni(111), and the binding energies for O and H atoms decrease in the order Ni(100) > Ni(111) > Ni(110). In total, the complete water dissociation reaction rate decreases in the order Ni(110) > Ni(100) > Ni(111). The reaction rates for both formyl dissociation to CH + O and to CO + H decrease in the order Ni(110) > Ni(111) > Ni(100). However, the dissociation to CO + H is kinetically favoured. The oxidation of formyl has the lowest activation energy on the Ni(111) surface. For combustion and synthesis of hydrocarbons, the Ni(110) surface shows a better catalytic activity for hydrocarbon combustion compared to the other surfaces. Calculations show that Ni is a better catalyst for the combustion reaction compared to the hydrocarbon synthesis, where the reaction rate constants are small. It was found that the WGS reaction occurs mainly via the direct pathway with the CO + O → CO2 reaction as the rate limiting step on all three surfaces. The activation barrier obtained for this rate limiting step decreases in the order Ni(110) > Ni(111) > Ni(100). Thus, the WGS reaction is fastest on the Ni(100) surface if O species are present on the surfaces. However, the barrier for desorption of water (as the source of the O species) is lower than its dissociation reaction on the Ni(111) and Ni(100) surfaces, but not on the Ni(110) surface. Therefore the direct pathway on the Ni(110) surface will dominate and will be the rate limiting step at low H2O(g) pressures. The calculations also reveal that the WGS reaction does not primarily occur via the formate pathway, since this species is a stable intermediate on all surfaces. All reactions studied in this work support the Brønsted-Evans-Polanyi (BEP) principles.

In Acute Myocardial Infarction (AMI), inflammation is a prerequisite for healing but it can paradoxically extend tissue injury; hence it needs to be modulated. Here, we investigated the role of the pro resolving GPCR FPR2/ALX and its agonist Annexin A1 (AnxA1) in AMI using mice lacking of the Fpr2/3 genes and with an in-frame GFP gene ‘knocked-in’. We developed protocols aimed to determine GFP expression as an indication of Fpr2 gene activity. Also, the Left Anterior Descending Coronary Artery of male Fpr2/3 KO and littermate controls (WT) was occluded for 30min and re-opened for 90min. At the end tissue injury and inflammatory response were studied. A significant proportion of Fpr2/3 KO perished during the procedure. The rest survived up to 90 min and exhibited a larger infarct size, with higher troponin I and inflammation markers (KC, TNFα) than WT animals. At the end of reperfusion, Fpr2/3 KO displayed an unbalanced production of pro and anti-inflammatory lipids (higher PGE2, PGI2, LTB4 and attenuated PGA1, RvD2, LXA4) and a deregulated activation of the cardioprotective IL-6/JAK/STAT3 signalling. Administration of AnxA1 afforded cardioprotection (reduction of infarct size; Troponin I, Caspase3 activity and TNFα) in WT but not in Fpr2/3 KO. A parallel in vitro investigation on the functional FPR2/ALX domains required by AnxA1 and other agonists was also conducted. HEK-293 cells transfected with FPR1, FPR2/ALX and FPR1/FPR2 chimeric receptor were used and calcium flux, 4 pERK and gene modulation analysed. AnxA1 required the N-terminus and the II and III extracellular loops of FPR2/ALX to evoke canonical responses. SAA interacted/activated the I and the II extracellular loops of FPR2/ALX, whereas the compound 43 suffices the I extracellular loop. In summary, the FPR2/AnxA1 pathway exerts a protective role in AMI. AnxA1 mimetic that activated selective FPR2/ALX domains can be synthetize to prevent tissue damage caused by AMI.

A tese encontra-se em formato de artigos conforme norma do Programa de Pós-graduação em Genética e Bioquímica da Universidade Federal de Uberlândia. Esta tese é composta por 4 capítulos, sendo o primeiro capítulo a revisão bibliografica a qual foi escrita em português de acordo com as normas da ABNT e os demais capítulos refere-se a artigos científicos os quais estão em inglês e obdecem as normas das revistas científicas as quais serão submetidos. / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / A sepse é uma síndrome clínica grave responsável por grande parte dos óbitos nas Unidades de Terapia Intensiva. Há uma diversidade de causas primárias que podem evoluir para um quadro clínico de sepse o que dificulta o diagnóstico precoce e a descoberta de drogas eficientes para tratamento. Os altos custos dos tratamentos e as taxas de letalidade mundial demonstram a necessidade de uma melhor compreensão desta síndrome. Este estudo buscou avaliar a expressão gênica dos Receptores de Peptídeos Formilados (FPR) em leucócitos totais, análise de marcadores sistêmicos e o perfil proteínas globais dos monócitos para averiguar as alterações presentes no organismo do indivíduo com a síndrome da sepse. Foi realizado a caracterização das alterações sistêmicas responsáveis pelo óbito de pacientes com sepse através da quantificação simultânea de 27 biomarcadores; verificou-se o silenciamento da expressão gênica do FPR1 e o FPR2 em pacientes com sepse e choque, relatando as possíveis disfunções imunológicas provocadas pela ausência desses FPRs; e a análise proteômica dos monócitos de pacientes com choque séptico permitiu averiguar a contribuição dessa célula no colapso desta síndrome, sua influência na lesão vascular, endotoxemia, inflamação e trombose. Esta investigação identificou pela primeira vez alvos biológicos relevantes os quais podem desempenhar papeis importantes no diagnóstico, prognóstico e terapêutica. / Sepsis is a serious clinical syndrome responsible for most of the deaths in the Intensive Care Units. There are a number of primary causes that may develop for a clinical picture of sepsis, making it difficult to diagnose early and find effective treatment medications. The high costs of treatments and global lethality rates demonstrate the need for a better understanding of this syndrome. This study aimed to evaluate the gene expression of Formulated Peptide Receptors (FPR) in total leukocytes, analysis of systemic markers and the profile of monocyte global proteins to ascertain the changes present in the body of the individual with sepsis syndrome. The characterization of the systemic changes responsible for the death of patients with sepsis was carried out through the simultaneous quantification of 27 biomarkers. The silencing of the gene expression of FPR1 and FPR2 in patients with sepsis and septic shock, reporting as possible immunological dysfunctions caused by the absence of these FPRs. The proteomic analysis of the monocytes of patients with septic shock allowed us to investigate the contribution of this cell in the collapse of this syndrome, its influence on vascular injury, endotoxemia, inflammation and thrombosis. This research identified for the first time, what is more important for the diagnosis, prognosis and therapeutics. / Tese (Doutorado)

Tous les êtres vivants présentent un arsenal de défenses contre les pathogènes, et la réponse inflammatoire constitue le processus initial de cette défense, qui s’achève par la réparation des tissus lésés. Paradoxalement, un processus inflammatoire prolongé est également associé à de nombreuses pathologies comme l’athérosclérose, l’asthme, les maladies auto-immunes mais aussi certains cancers. Le recrutement excessif de leucocytes au site de l’inflammation est un processus commun à ces pathologies. Dès lors, la compréhension et la maîtrise du phénomène complexe et finement orchestré de la migration sélective des populations leucocytaires, appelée chimiotactisme, sont des enjeux majeurs de la recherche médicale contemporaine. <p>Les récepteurs aux peptides formylés bactériens et mitochondriaux (FPRs) forment la première famille de récepteurs chimiotactiques identifiée. Elle comprend trois membres, FPR1, 2 et 3, présentant un haut niveau de similitude et partageant certains de leurs multiples ligands. Le troisième membre de ce groupe, FPR3, reste actuellement le moins bien connu. Récemment, un agoniste de FPR3, affin et spécifique, a été identifié dans le laboratoire. Il s’agit du peptide F2L, qui correspond aux 21 premiers acides aminés de la protéine intracellulaire HEBP1.<p><p>Dans le cadre de ce travail de thèse, nous nous sommes attelé à la caractérisation approfondie du récepteur FPR3 et son ligand peptidique F2L. <p>Dans un premier temps, et à l’aide d’anticorps validés dans le cadre de ce travail, nous avons montré que le peptide F2L induit le chimiotactisme d’un ensemble de populations leucocytaires qui expriment FPR3, dont les sous-populations de macrophages des poumons, du colon et de la peau, les éosinophiles et les cellules dendritiques plasmacytoïdes. Cette distribution suggère, pour FPR3, une fonction dans la réponse inflammatoire. <p>Nous avons pu montrer ensuite que F2L peut être généré par la protéolyse de son précurseur, HEBP1, sous l’action de la cathepsine D des macrophages. La cathepsine D est une aspartique protéase lysosomiale impliquée dans l’homéostasie cellulaire, les processus apoptotiques et inflammatoires physiologiques et pathologiques, et dans le développement tumoral. Il s’agit désormais d’identifier dans quel compartiment et sous quelles conditions F2L est produit et sécrété. <p>Enfin, parallèlement à ces travaux, nous avons démontré que la cathepsine G, une sérine protéase contenue dans les granules azurophiles des neutrophiles, active également le récepteur FPR3. Des résultats préliminaires suggèrent un mode d’activation alternatif du récepteur, impliquant la protéolyse d’un troisième partenaire et la génération d’un agoniste actuellement non identifié. <p><p>Le couple FPR3-F2L semble dès lors impliqué dans l’induction ou la résolution de la réponse inflammatoire en recrutant les éosinophiles, monocytes, macrophages et cellules dendritiques au site de la lésion. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Neutrophil activation is the most important initial immune defense against invading microbes in newborn infants. The reduced neutrophil migration and uncontrolled regulation of reactive oxygen species (ROS) production observed in neonates, could result in a diminished infectious response or in tissue damage. The aims were to study neutrophil chemotactic response towards IL-8 and fMLP in term neonates; to examine neutrophil receptor expression involved in adhesion, migration, phagocytosis and complement after stimulation with IL-8 and fMLP in term neonates; and to investigate neutrophil production of ROS, induced by PMA and E.coli, after preincubation with IL-8 and fMLP in term and preterm newborn infants. Comparisons were made to neutrophils from healthy adults. Chemotaxis was distinguished from randomly migrating neutrophils, and the neutrophil migration distance and the number of migrating neutrophils per distance was evaluated. Neutrophils were labeled with antibodies to cell surface antigens (CD11b, CD18, CD65, CD15S, CD162, CD44, CD35, CD88, CD181, CD182 and CD64) after stimulation with IL-8 and fMLP. After preincubation of neutrophils with fMLP or IL-8 and stimulation with PMA or E.coli, respiratory burst was detected. The same analyses were also made in preterm infants (median 25+3weeks GA; range 23+0–29+2) within 3 days postnatal age. Neutrophils from neonates exhibited different migratory and receptor responses to IL-8 and fMLP, with a diminished response towards IL-8 in term newborn infants in terms of reduced chemotaxis and modulation of receptors involved in adhesion, chemotaxis, complement and phagocytosis as compared to adults. fMLP reduced PMA- and E.coli-induced respiratory burst in neutrophils from term neonates and adults. The reduced respiratory burst by fMLP may be a mechanism for reducing the detrimental effects of uncontrolled inflammation. Although a similar burst reduction was observed in preterm infants born &gt;25 weeks GA with fMLP, a diminished neutrophil respiratory burst modulation in very preterm infants cannot be excluded and requires further studies at different gestational and postnatal ages.

Submitted by SARA DE SOUZA COSTA null (sarah_sc_0705@hotmail.com) on 2017-03-30T13:36:06Z No. of bitstreams: 1 Dissertação Sara de Souza Costa.pdf: 2766240 bytes, checksum: 20b447fe5bf3c49e40aa0b5fdf4dff1f (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-04-06T13:29:26Z (GMT) No. of bitstreams: 1 costa_ss_me_sjrp.pdf: 2766240 bytes, checksum: 20b447fe5bf3c49e40aa0b5fdf4dff1f (MD5) / Made available in DSpace on 2017-04-06T13:29:26Z (GMT). No. of bitstreams: 1 costa_ss_me_sjrp.pdf: 2766240 bytes, checksum: 20b447fe5bf3c49e40aa0b5fdf4dff1f (MD5) Previous issue date: 2017-03-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As lesões uterinas são causas importantes de desconforto, infertilidade e óbito entre as mulheres no Brasil e no mundo. O câncer de endométrio é um tumor maligno frequente e sua incidência vem aumentando nas últimas décadas. Enquanto, o tumor uterino benigno mais comum, o leiomioma, acomete cerca de 40% das mulheres na idade reprodutiva, sendo relacionado à menorragia, dismenorreia e infertilidade. Investigações indicam que o microambiente tumoral é crucial para o avanço do câncer, sendo caracterizado, principalmente, pela composição alterada da matriz extracelular, alta densidade de microvasos e abundância de células inflamatórias, como mastócitos (MCs). MCs desgranulados liberam fatores quimiotáticos e proteases, como triptase e quimase, para o meio extracelular, contribuindo na degradação da matriz extracelular, promoção da angiogênese, propiciando ambiente favorável para invasão tumoral e remodelação tecidual por meio de proteólises seletivas na matriz e ativação de metaloproteinases. Outro aspecto importante no crescimento tumoral é a proteína anti-inflamatória Anexina A1 (ANXA1), relacionada à regulação dos processos de crescimento e migração/invasão celular, sendo seus efeitos mediados por receptores para peptídeos formilados (FPRs), especialmente FPR1 e FPR2. Diante da importância dos MCs e da ANXA1/FPR no desenvolvimento tumoral, o objetivo desta investigação foi analisar a heterogeneidade dos MCs e a expressão das proteínas ANXA1, FPR1 e FPR2 em biópsias humanas das variáveis clínico-patológicas de útero normal: hiperplasia endometrial simples (HES), adenomiose, leiomiomas e adenocarcinoma (ADC) endometrial de graus I e II. Os MCs foram quantificados de acordo com seu estado de ativação e expressão das proteases triptase e quimase. A expressão da ANXA1 e seus receptores FPR1 e FPR2 nos MCs e tecidos uterinos foram analisadas nas diferentes biópsias estudadas. Nossos resultados mostraram MCs intactos e desgranulados, no endométrio e miométrio normais e aumentados na HES, margens tumorais nos leiomiomas, adenomiose e ADC endometrial de graus I e II, e diminuídos significantemente na região tumoral do leiomioma. Com relação à heterogeneidade, os MCs triptase-positivos foram observados em maior quantidade. As expressões endógenas da ANXA1 e do FPR1 foram observadas nos tecidos uterinos e MCs, com ausência para o FPR2. As modulações dos MCs, da proteína ANXA1 e de modo específico do receptor FPR1, nas variáveis clínico-patológicas das lesões uterinas investigadas indicam o envolvimento dessas células e a interação ANXA1/FPR1 no desenvolvimento de inflamação e neoplasia uterina. / Uterine lesions are important causes of discomfort, infertility and death among women in Brazil and in the world. Endometrial cancer is a frequent malignant tumor and its incidence has been increasing in the last decades. Besides, the most common benign uterine tumor, leiomyoma, affects about 40% of women at reproductive age, being related to menorrhagia, dysmenorrhea and infertility. Investigations indicate that the tumor microenvironment is crucial for the advancement of cancer, being characterized mainly by the altered composition of the extracellular matrix, high microvessel density and abundance of inflammatory cells, such as mast cells (MCs). Degranulated MCs release chemotactic and protease factors, such as tryptase and chymase, to the extracellular medium, contributing to the degradation of the extracellular matrix, promoting angiogenesis, providing a favorable environment for tumor invasion and tissue remodeling through selective proteolysis in the matrix and activation of metalloproteinases. Another important aspect of tumor growth is the anti-inflammatory protein Annexin A1 (ANXA1), related to the regulation of growth and migration / invasion processes, and its effects mediated by receptors for formylated peptides (FPRs), especially FPR1 and FPR2. The objective of this investigation was to analyze the heterogeneity of the MCs and the expression of the ANXA1, FPR1 and FPR2 proteins in human biopsies of clinical-pathological variables of normal uterus: simple endometrial hyperplasia (HES), adenomyosis, leiomyomas and endometrial adenocarcinoma (ADC) of grades I and II. MCs were quantified according to their state of activation and expression of tryptase and chymase proteases. Expression of ANXA1 and its FPR1 and FPR2 receptors in the MCs and uterine tissues were analyzed in the different biopsies studied. Our results showed intact and degranulated MCs in the normal endometrium and myometrium and increased MCs in HES, tumor margins in leiomyomas, adenomyosis and endometrial ADC of grades I and II, but significantly decreased in the leiomyoma tumor region. In relation to the heterogeneity, it was observed that the tryptase-positive MCs were observed in greater quantity. Endogenous expressions of ANXA1 and FPR1 were observed in uterine tissues and MCs, but absent for FPR2. Modulations of MCs and ANXA1 protein expression and the specificity of FPR1 receptor immunolabeling in the clinical-pathological variables of the investigated uterine lesions indicate the involvement of these cells and the interaction ANXA1/FPR1 in the development of inflammation and uterine neoplasia.

After cooking, kraft pulps always contain not only residual lignin but also significant amounts of hexenuronic acid and other non-lignin structures oxidizable by permanganate under the standard kappa number determination conditions. These here referred to as false lignin. Like ordinary lignin, the false lignin also consumes bleaching chemicals, thus increasing both the production costs and the environmental impact of bleach plant effluents. The false lignin also has an effect on pulp properties such as brightness stability. This necessitates the development of efficient experimental routines for the determination of false lignin in different types of unbleached and bleached kraft pulps, together with studies of its formation, chemical behaviour, and ultimate fate. The main aim of this work has been to establish a method for the quantification of various types of oxidizable structures in bleached kraft pulps and to study their impact on pulp quality, particularly, on the brightness stability of pulps bleached in elemental-chlorine-free (ECF) and a totally-chlorine-free (TCF) processes. Part of this research deals with the relationship between the kappa number and the lignin content in the case of partly oxidized lignins. Spruce and birch kraft pulps processed according to the ODEQP and OQ(OP)Q(PO) bleaching sequences, respectively, have been analyzed. It has been found that the oxidation equivalent of the residual lignin decreases with increasing degree of oxidation along each bleaching sequence. This finding has been further supported by experiments with a number of model compounds. The Ox-Dem kappa number method has been shown to be an accurate means of determining the residual lignin content and of monitoring the efficiency of lignin removal along different bleaching sequences. It has been demonstrated that the kappa number can always be fractioned into partial contributions, the first of which comes from the residual lignin and is measured by the Ox-Dem kappa number, and the second from the false lignin and is given by the difference between the standard kappa number and the Ox-Dem kappa number. The effect of false lignin on the pulp kappa number is most pronounced in unbleached and oxygen-delignified kraft pulps. The extractability of residual and false lignin in different solvents has been investigated. The changes that occurred in the kappa number following different extraction steps have been compared with corresponding changes in the chemical composition and the conclusion has been drawn that the hemicellulose component of a kraft pulp is a major sourse of non-lignin structures contributing to the kappa number. The influence on the brightness stability of various oxidizable structures, viz.: residual lignin, hexenuronic acid and other non-lignin structures, in spruce, birch and eucalyptus kraft pulps bleached in ECF and TCF type processes was studied. It was demonstrated that the selective removal of all false lignin structures significantly improves the brightness stability. The degree of yellowing was found to be proportional to the content of HexA groups in pulps. It has been shown that 2-furancarboxylic acid, 5-formyl-2furancarboxylic acid and reductic acid are formed during the course of thermal yellowing. The influence of two bleaching sequences, D0(EP)D1 (ECF-type) and Q1(OP)Q2(PO) (TCF)-type, on the content of different oxidizable structures in eucalyptus kraft pulp was studied in relation to the brightness stability of the pulp. It was shown by kappa number fractionation that pulp bleached to full brightness with ECF- and TCF-type sequences contains different amounts of HexA. The most significant discoloration was observed in the case of TCF-bleached pulp having an especially high content of HexA. The mechanism of the moist (8 % moisture) thermal yellowing of fully bleached kraft pulps was further studied using dissolving pulp impregnated with a set of model compounds representing the most likely HexA degradation products, viz. as 2-furancarboxylic acid (FA), 5-formyl-2-furancarboxylic acid (FFA) and reductic acid (RA), either alone or in combination with Fe(II) or Fe(III) ions. It was found that the latter two acids take part in reactions leading to colour formation whereas 2-furancarboxylic acid does not. The effect of iron ions on the colour formation appears to vary with their oxidation state. The brightness loss caused by either FFA or RA, present in an amounts similar to the content of HexA in industrial pulps, was of the same order of magnitude as that observed in industrial pulps aged under the same conditions. Based on these findings, it is suggested that the overall mechanism of moist thermal yellowing involves several stages, including the degradation of hexenuronic acid and the formation of reactive precursors, such as 5-formyl-2-furancarboxylic acid and reductic acid. The presence of ferrous ions further enhances the discoloration. / QC 20101005

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23760号 / 医博第4806号 / 新制||医||1056(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 湊谷 謙司, 教授 森信 暁雄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM