Dissertations / Theses on the topic 'NOS inhibition'

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Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde
O presente estudo é uma aplicação prática do conceito dos Três Rs (3Rs) de Russell e Burch (1959) nos ensaios de controle da qualidade de imunobiológicos para Raiva preconizados pela Farmacopéia Brasileira através de: uma análise retrospectiva de dados para a Redução do nº de animais no Ensaio de Potencia NIH para vacina contra raiva de uso humano (vaccinum rabiei ad usum humanum); mudanças no método de avaliação da inativação viral destas vacinas utilizando animais e células e validação de um ensaio in vitro para substituir o ensaio in vivo no teste de potência de imunoglobulinas anti-rábicas (Immunosera rabicum ex animali ad usum humanum e immunoglobulinum humanum rabicum). Todos os três protocolos de ensaio testados no estudo demonstraram viabilidade de utilização.
The present study is a practical application of the Russell and Burch 3Rs concept (1959) in the in vivo tests described in the Farmacopéia Brasileira for quality control of rabies biologicals by testing: A reduction in the number of mice in the NIH potency test for rabies vaccine for human use (vaccinum rabiei ad usum humanum); changes in the evaluation of virus inactivation method using suckling mice and cells as an alternative to the current Brazilian Pharmacopoeia official test (Reduction, Refinement and Replacement) and the validation of an in vitro assay to replace the in vivo assay for the potency test of Rabies Immunoglobulins (Immunosera rabicum ex animali ad usum humanum e immunoglobulinum humanum rabicum) (Replacement). All the three assay protocols have shown viability of use.

Plasma membrane calcium ATPase 4 (PMCA4) is a calcium extrusion pump which may also modulate Ca2+-triggered signal transduction pathways. Previous studies postulate that PMCA4 modulates signalling via an interaction with neuronal nitric oxide synthase (nNOS) in localised plasmalemmal microdomains. The effect of PMCA4 on vascular contractility is unclear. This project has utilised PMCA4 ablated mice (PMCA4 KO (-/-)) and a novel specific PMCA4 inhibitor (termed AP2) to study the role of PMCA4 in mouse resistance artery contractility.Immunohistochemistry, Western blotting and polymerase chain reaction (PCR) confirmed the absence of PMCA4 in the brain, vasculature and ear snips obtained from PMCA4 KO (-/-) mice whereas it was present in those from wild type (WT (+/+)) mice. Pressure myography was employed to assesss contractile function of isolated, pressurised (to 60 mmHg) mesenteric resistance arteries from 3 months old male PMCA4 KO (-/-) and WT (+/+) mice, in response to high K+ physiological salt solution (KPSS) (40mM & 100mM) and noradrenaline (NA) (Log[NA] -9.0 to -5.0M). Passive lumen diameter and left and right wall thicknesses of arteries from PMAC4 KO (-/-) and WT (+/+) mice were taken at transmural pressures of 5-140 mmHg. Effects of acute PMCA4 inhibition with AP2 (10µM and 1µM), nitric oxide synthase (NOS) inhibition with LNNA (100µM) and specific nNOS inhibition with Vinyl-L-Nio (10µM) were also investigated. Effects of PMCA4 ablation and AP2 (10µM) on global intracellular Ca2+ changes ([Ca2+]i) in pressurised mesenteric arteries were assessed after loading arteries with the Ca2+-sensitive indicator indo-1. PMCA4 ablation had no effect on the magnitude of arterial constrictions or on the changes of [Ca2+]i in response to KPSS (40mM & 100mM) or to noradrenaline. The passive intra-lumen diameter, wall thickness, wall to lumen diameter and cross sectional area of mesenteric arteries across the intravascular pressure range studied were also not modulated by PMCA4 ablation. A leftwards shift in the stress to strain relationship and significant increase in beta elastic modulus (β) were revealed in arteries from PMCA4 KO (-/-) mice compared to those from WT (+/+) mice, suggesting that PMCA4 ablation reduces mesenteric arterial distensibility. Acute PMCA4 inhibition with AP2, significantly reduced arterial constrictions and the increase in [Ca2+]i in response to noradrenaline in arteries from WT (+/+) mice, but had no effect on arterial constrictions elicited by arteries from PMCA4 KO (-/-) mice. Inhibitory effects of AP2 were not present in arteries after NOS inhibition by LNNA and also after nNOS inhibition with Vinly-L-Nio. Hence, PMCA4 inhibition with AP2 reduces vascular constriction by a nNOS-dependent mechanism.In conclusion, the main findings of the study were that ablation and acute inhibition of PMCA4 with AP2 have different effects on mouse mesenteric resistance arterial contractility. This study provides more insight into PMCA4 as a significant modulator of signalling within the vasculature via effects on nNOS.

Des petits ARN non codants dérivant d'ARN de transfert (tRF) ont été identifiés dans tous les domaines de la vie, et de plus en plus de fonctions importantes leur sont attribuées chez de nombreux organismes. Dans ce travail mené sur la plante modèle Arabidopsis, nous avons d’abord montré que la population en tRF varie en fonction des tissus et des conditions de stress. Concernant leur biogenèse, les endoribonucléases responsables du clivage des ARNt ont été identifiées, il s'agit des RNases T2, RNS1, 2 et 3. Afin de réaliser une étude structure/fonction, une approche d’expression en système de levure a été initiée pour permettre l’obtention de quantité suffisante de RNS1 purifiée. L’étude des fonctions des tRF montre que certains d’entre eux sont associés à AGO1, qu'ils semblent cibler entre-autres des éléments transposables et qu’ils pourraient avoir une localisation nucléaire. Enfin, deux tRF, le tRF-5D (Ala) et le tRF-5D (Asn) inhibent efficacement la traduction in vitro. Une association de tRF-5D (Ala) aux polyribosomes de plantules d'Arabidopsis a pu être visualisée, suggérant que certains tRF puissent agir en tant que régulateur global de la traduction
Small non-coding RNAs derived from transfer RNAs (tRFs) have been identified in all domains of life, and more and more important functions are attributed to them in many organisms. In this work on the model plant Arabidopsis, we first showed that the tRF population varies according to tissues and stress conditions. With regard to their biogenesis, the endoribonucleases responsible for tRNA cleavage were identified, it is the RNases T2, RNS1, 2 and 3. In order to carry out a structure / function analysis, heterologous expression in yeast has been developed with the hope to get sufficient amount of purified RNS1. The question of tRF functions has also been studied. It has been shown that some tRFs are associated with AGO1, that they often seem to target transposable elements and could have a nuclear localization. Finally, the study of the involvement of the tRFs in the regulation of translation was tackled. Two tRFs, tRF-5D (Ala) and tRF-5D (Asn) efficiently inhibit translation in vitro. An association of tRF-5D (Ala) with polyribosomes of Arabidopsis seedlings could be visualized suggesting that some plant tRFs could as global regulator of the translation process

A partial nitritation-anammox continuous flow reactor (CFR) was operated for eight months demonstrating that a mixture of large anammox-supported aerobic granules (ASAGs) and small conventional aerobic granules (CAGs) can be maintained stably for extended periods of time. The influent NH4+ was kept at 50 - 60 mg N L-1 to verify that the upper range of total ammonia nitrogen (TAN) for domestic wastewater can supply an inhibitory level of free ammonia (FA) for nitrite oxidizing bacteria (NOB) suppression in CFRs at pH around 7.8. The ammonia oxidizing bacteria (AOB):NOB activity ratio was determined for a series of granule sizes to understand the impact of mass diffusion limitation on the FA inhibition of NOB. When dissolved oxygen (DO) limitation is the only mechanism for NOB suppression, the AOB:NOB ratio was usually found in previous studies to increase with the granule size. However, the trend is reversed when FA has an inhibitory effect on NOB, as was observed in this study. The decrease in AOB:NOB ratio indicates that the resistance to the diffusion of FA along the granule radius limited its ability to inhibit NOB. This means smaller granules, e.g. diameter < 150 microns, are preferred for nitrite accumulation when high FA is present, e.g. in the partial nitritation-anammox process. The trend was further verified by observing the increase in the apparent inhibition coefficient, KI,FAapp, as granule size increased. This study for the first time quantified the effect of diffusion limitation on the KI,FAapp of NOB in granules and biofilms. A mathematical model was then utilized to interpret the observed suppression of NOB. The model predicted that NOB suppression was only complete at the granule surface. The NOB that did survive in larger granules was forced to dwell within the granule interior, where the FA concentration was lower than that in the bulk solution. This means FA inhibition can be taken advantage of as an effective means for NOB suppression in small granules and thin biofilms. Further, FA and DO were found to be both required for the stratification of AOB and NOB in partial nitritation-anammox CFRs. The structural stratification commonly observed in granules is then concluded to be a consequence but not a cause of the NOB suppression.
MS
A partial nitritation-anammox continuous flow reactor (CFR) was operated for eight months demonstrating that granular sludge can be maintained stably for extended periods of time. In this approach, NH3 is only partially converted to NO2 - (partial nitritation), and the conversion to NO3 - is prevented by the suppression of nitrite oxidizing bacteria (NOB). NH3 and NO2 - are then utilized by anammox bacteria to create N2 gas. The influent NH4 + fed to the reactor was kept at 50 to 60 mg N L-1 to verify that the upper range of total ammonia nitrogen (TAN) for domestic wastewater can supply a sufficiently high level of free ammonia (FA) to inhibit NOB growth in CFRs at a pH around 7.8. It is expected that the penetration of a substrate into granule sludge will experience diffusional resistance as it moves from water to denser solid material and is consumed by bacteria. The ammonia oxidizing bacteria (AOB):NOB activity ratio was determined for a series of granule sizes to understand the impact of mass diffusion limitation on the FA inhibition of NOB. When dissolved oxygen (DO) limitation is the only mechanism for NOB suppression, the AOB:NOB ratio was usually found in previous studies to increase with the granule size. However, the trend is reversed when FA has an inhibitory effect on NOB, as was observed in this study. The decrease in AOB:NOB ratio indicates that the resistance to the diffusion of FA, which increases with increasing granule size, along the granule radius limited its ability to inhibit NOB. This means smaller granules, e.g. diameter < 150 µm, are preferred for NO2 - accumulation when high FA is present. The trend was further verified by observing the increase in the apparent inhibition coefficient, KI,FAapp, as granule size increased. This coefficient quantifies the effectiveness of an inhibitor, with larger values indicating weaker inhibition. This study for the first time quantified the effect of diffusion limitation on the KI,FAapp of NOB in granules and biofilms. A mathematical model was then utilized to interpret the observed suppression of NOB. The model predicted that NOB suppression was only complete at the granule surface. The NOB that did survive in larger granules was forced to dwell within the granule interior, where the FA concentration was lower than that in the bulk solution. This means FA inhibition can be taken advantage of as an effective means for NOB suppression in small granules and thin biofilms. Further, FA and DO were found to be both required for the stratification of a layer of AOB at the surface over a layer of NOB in partial nitritation-anammox CFRs. The structural stratification commonly observed in granules is then concluded to be a consequence but not a cause of the NOB suppression.

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CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
A presente dissertação tem como proposta investigar no campo de interlocução entre Psicanálise e Educação a problemática das dificuldades de aprendizagem. Partimos da pergunta: “nos quadros de dificuldades de aprendizagem trata-se de um Outro que não sabe, ou um Outro que sabe demais”? O nosso caminho para responder a essa questão partirá da verificação lógica da sugestão de Lacan (1967): a segregação do real pelo Ideal. Com esse percurso, pretendemos avançar nesse ponto da segregação que a dimensão do Ideal coloca em jogo. Subsequentemente, visaremos a uma interlocução entre a abordagem psicanalítica e a teoria piagetiana das aprendizagens, discutindo a noção do egocentrismo infantil reivindicado por Piaget. Também, visaremos posteriormente uma interlocução com uma abordagem crítica das políticas públicas educacionais que denuncia uma forte dimensão idealizante no fundamento dessas políticas. Tal interlocução se dará no sentido de verificar se essa visada é a mesma da visada psicanalítica, avançando um pouquinho mais nesse tema. O passo seguinte será a investigação detida das dificuldades de aprendizagem na psicanálise a partir da abordagem da inibição neurótica da qual a inibição na aprendizagem faz parte, à luz da tragédia “Hamlet”, de Shakespeare, onde podemos encontrar a inibição de Hamlet intimamente articulada com um Outro que sabe demais. Trabalharemos com a hipótese de uma dimensão excessivamente idealizante no cerne de quadros de séria inibição na aprendizagem. Dimensão de um saber idealizado demais que obstrui o ato de aprender. É justamente essa hipótese que será investigada.
This dissertation aims at investigating learning difficulties at the intersection of Psychoanalysis and Education. We begin with the following question: “Learning difficulties pictures: the Other does not knowledge or knowledge too much”? To deal with this question we will begin with the logical verification for Lacan’s suggestion (1967) about the segregation of the real order comprised by Ideal dimension. With this approach, we aim at advancing in this segregation notion introduced by the Ideal. Subsequently, we will be concerned with the conversation between psychoanalysis and Piaget’s theory about learning, discussing child’s egocentrism claimed by this researcher. After, we will also be concerned with the conversation with a critical approach of educational public policies denounces a strong Ideal dimension in the core of these policies. Such a conversation will take place in the sense of verifying whether this reading and psychoanalytical one are the same, advancing a bit more, in this theme. The following step will be the learning difficulties detailed investigation from the neurotic inhibition approach from which the learning inhibition takes part, on the lights from Shakespeare’s Hamlet, where we can find the Hamlet’s inhibition closely related to an Other that kowledges too much. We will be concerned with the excessively idealized dimension hypothesis in the core of the learning difficulties pictures. Dimension which comprises an idealized knowledge that prevents the act of learning. It is justly this hypothesis which will be investigated.

This master thesis considers an investigation of protective formulations (ointment, cream) containing enzyme inhibitors. Model experiments have been made on the enzyme trypsin. It is well accepted that feces and urine are an important causing factor for skin irritation (dermatitis) while using diaper. A protective formulation is a physical barrier that separates the harmful substances from the skin. It can also be an active barrier containing active substances, which can be active both towards the skin, and the substances from feces and urine. By preventing contact from these substances the skin will not be harmed, at least for a period of time. A number of different inhibitors were tested towards trypsin and they all showed good inhibition, two of the inhibitors were selected to be immobilized with the help of NHS-­activated Sepharose. Immobilization of these two inhibitors leads to a lesser extent of the risk of developing allergy and also that the possible toxic effect can be minimized.

The type 1 insulin-like growth factor receptor (IGF-1R) is a receptor tyrosine kinase that mediates diverse cellular functions including growth, differentiation, migration and apoptosis protection. IGF-1R signalling has been implicated in tumorigenesis in a variety of cancers, and IGF-1R inhibitory drugs are currently undergoing clinical evaluation. Previous work in our laboratory has shown IGF-1R over-expression in urological cancers at both the mRNA and protein level, thus making it a potential therapeutic target. The first aim of this project was to develop a protocol for IGF-1R immunohistochemistry, investigate the expression and cellular distribution of the IGF-1R receptor in clear cell renal cell carcinomas (ccRCC), and assess correlation with clinical parameters. In tissue microarray analysis, IGF-1R was detected in ~90% of 195 ccRCCs, with signal in the plasma membrane, cytoplasm and also in the nucleus. The presence of nuclear IGF-1R in up to 50% of ccRCCs and its association with adverse prognosis was a novel finding, and suggests that nuclear IGF-1R may influence ccRCC biology. Further investigations will clarify its role in the nucleus and its potential as a prognostic biomarker. The second aim was to investigate effects of IGF-1R inhibition on radiosensitivity and DNA repair, following previous work in our laboratory showing that IGF-1R depletion enhances chemo- and radio-sensitivity, delays double strand break (DSB) resolution, and may play a role in the homologous recombination (HR) pathway of DNA DSB repair. However, the repair defect seen in these early experiments was larger than could be entirely explained by a defect in HR. The current project used a small molecule IGF-1R tyrosine kinase inhibitor AZ12253801 (AstraZeneca), which blocked IGF-1 induced IGF-1R activation and inhibited cell survival. AZ12253801 enhanced the radiosensitivity of prostate cancer cells, which appeared to be independent of effects of IGF-1R inhibition on cell cycle distribution and apoptosis induction. IGF-1R inhibition delayed the resolution of γH2AX foci, supporting a potential role for the IGF-1R in DSB repair. This delay in focus resolution was apparent at early time-points (less than 4 hr), and was epistatic with DNA dependent protein kinase (DNAPK) inhibition in prostate cancer cells and DNAPK deficiency in glioblastoma cells. These results suggest a role for the IGF-1R in the non-homologous end-joining (NHEJ) pathway of DNA DSB repair. A cell-based reporter assay in HEK-293 cells confirmed that IGF-1R inhibition suppressed DSB repair by NHEJ, helping to explain the radiosensitization demonstrated upon IGF-1R inhibition. There was lack of support for a transcriptional effect, with no significant change observed in gene expression on microarray analysis. Although the mechanism of this effect remains unclear, the observed inhibition of NHEJ has implications for the use of IGF-1R inhibitors in combination with DNA damaging agents in cancer treatment.

Connor O’Hara July 29, 2019 Inhibition of Cancer Stem Cells by Glycosaminoglycan Mimetics In the United States cancer is the second leading cause of death, with colorectal cancer (CRC) being the third deadliest cancer and expected to cause over 51,000 fatalities in 2019 alone.1 The current standard of care for CRC depends largely on the staging, location, and presence of metastasis.2 As the tumor grows and invades nearby lymph tissue and blood vessels, CRC has the opportunity to invade not only nearby tissue but also metastasize into the liver and lung (most commonly).3 The 5-year survival rate for metastasized CRC is <15%, and standard of care chemotherapy regimens utilizing combination treatments only marginally improve survival.3-5 Additionally, patients who have gone into remission from late-stage CRC have a high risk of recurrence despite advances in treatment.6-7 The Cancer Stem-like Cell (CSC) paradigm has grown over the last 20 years to become a unifying hypothesis to support the growth and relapse of tumors previously regressed from chemotherapy (Figure 1).8 The paradigm emphasizes the heterogeneity of a tumor and its microenvironment, proposing that a small subset of cells in the tumor are the source of tumorigenesis with features akin to normal stem cells.9 The CSCs normally in a quiescent state survive this chemotherapy and “seed” tumor redevelopment.10 First observed in acute myeloid lymphoma models, CSCs have since been identified in various other cancers (to include CRC) by their cell surface antigens and unique properties characterizing them from normal cancer cells.11-12 These include tumor initiation, limitless self-renewal capacity to generate clonal daughter cells, as well as phenotypically diverse, mature, and highly differentiated progeny.13-14 Previously our lab has identified a novel molecule called G2.2 (Figure 2) from a unique library of sulfated compounds showing selective and potent inhibition of colorectal CSCs in-vitro.15 G2.2 is a mimetic of glycosaminoglycans (GAGs) and belongs to a class of molecules called non-saccharide GAG mimetics (NSGMs). Using a novel dual-screening platform, comparisons were made on the potency of G2.2 in bulk monolayer cells, primary 3D tumor spheroids of the same cell line, and subsequent generations of tumor spheroids. This work has shown in-vitro the fold-enhancement of CSCs when culturing as 3D tumor spheroids. Spheroid culture serves as a more accurate model for the physiological conditions of a tumor, as well as the functional importance of upregulating CSCs. Evaluation of G2.2 and other NSGMs was performed in only a few cell lines, developing a need to better understand the ability of G2.2 to inhibit spheroids from a more diverse panel of cancer cells to better understand G2.2’s mechanism. The last few decades have seen the advancement in fundamental biological and biochemical knowledge of tumor cell biology and genetics.16 CRC, in particular, has served as a useful preclinical model in recapitulating patient tumor heterogeneity in-vitro.17 Recent work has characterized the molecular phenotypes of CRC cell lines in a multi-omics analysis, stratifying them into 4 clinically robust and relevant consensus molecular subtypes (CMS).18-19 Our work was directed to screen a panel of cells from each of the molecular subtypes and characterize the action of G2.2 and 2nd generation lipid-modified analogs, synthesized to improve the pharmacokinetic properties of the parent compound. Four NSGMs, namely G2.2, G2C, G5C, and G8C (Figure 2) were studied for their ability to inhibit the growth of primary spheroids across a phenotypically diverse panel. Compound HT-29 IC50 (μM) Panel Average IC50 (μM) G2.2 28 ± 1 185 ± 55 G2C 5 ± 2 16 ± 15 G5C 8 ± 2 63 ± 19 G8C 0.7 ± 0.2 6 ± 3 Primary spheroid inhibition assays were performed comparing the potency of new NSGMs to G2.2. Fifteen cell lines were evaluated in a panel of colorectal adenocarcinoma cell lines with several cell lines representing each CMS. Primary spheroid inhibition assays revealed 3 distinct response with regard to G2.2’s ability to inhibit spheroid growth. Cells from CMS 3 and 4, which display poor clinical prognosis, metabolic dysregulation, and enhanced activation of CSC pathways, showed the most sensitivity to G2.2 (mean IC50 = 89 ± 55 μM). Mesenchymal CMS 4 cell lines were over 3-fold more sensitive to treatment with G2.2 when compared to CMS 1 cell lines. Resistant cell lines were composed entirely of CMS 1 and 2 (mean IC50 = 267 ± 105 μM). In contrast, all lipid-modified analogs showed greater potency than the parent NSGM in almost every CRC cell line. Of the three analogs, G8C showed the greatest potency with a mean IC50 of less than 15 μM. Of the CRC spheroids studied, HT-29 (CMS 3) was most sensitive to G8C (IC50 = 0.73 μM). To evaluate the selectivity of NSGMs for CSC spheroid inhibition, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) cytotoxicity assays were performed on monolayer cell culture, and the fold-selectivity of NSGM for spheroids was analyzed. Data shows that NSGMs preferentially target CSC-rich spheroids compared with monolayer cellular growth, with G2.2 having over 7-fold selectivity for spheroid conditions. This fold selectivity was enhanced in CMS 3/4, supporting the idea that G2.2 targets a mesenchymal and stem-like phenotype. To further validate this selectivity, limiting dilution assays were performed across the panel to determine the tumor-initiating capacity of each cell line. Cell lines which showed a sensitive response to G2.2 were over 2-fold more likely to develop into spheroids, validating the previous hypothesis. Further characterization was performed analyzing the changes G2.2 induced on CSC markers, as well as the basal expression of a unique pair of cancer cells. Western blots showed a reduction in self-renewal marker across all CMS after treatment with G2.2, and that cell lines sensitive to G2.2-treatment overexpress mesenchymal and stem-like markers. G2.2-resistant cell lines show an epithelial phenotype, lacking this expression. The positive results observed in these studies enhance the understanding of G2.2 and analogs, and further evaluation with additional cell lines of various tissues would improve the knowledge thus far gained. However, all experiments described take valuable time to perform and analyze. Thus, there became a need to develop a high-throughput screening (HTS) platform for our assays that standardized analysis and enhanced productivity. Initial development of the method for this assay are underway, and recent evidence from these evaluations of breast cancer spheroids suggests that G2.2 and analogs may be tissue-specific compounds for the treatment of cancer. Future work entails refining the application of this method for evaluation of the NCI-60 (National Cancer Institute) tumor cell panel. Overall, these results make several suggestions concerning the NSGMs evaluated against the panel. First, G2.2 selectively targets CSCs with limited toxicity to monolayer cells of the same cell line. Further, G2.2 has the greatest potency with CMS 3/4, whose mesenchymal phenotypes are associated with poor clinical prognosis and enrichment of CSCs. Supporting evidence include that sensitive cell lines are highly tumorigenic and show enhanced expression of mesenchymal/CSC markers compared to resistant cell lines. Lipid-modification of G2.2 enhances in-vitro potency against spheroid growth, with nM potency reached in the most sensitive cell lines. Evidence in the development of a HTS platform also suggests these NSGMs show tissue specificity to cancers of the intestine. Further work characterizing the mechanism of NSGMs in a broader multi-tissue panel will enhance our understanding of the compounds as a potential therapy to dramatically improve patient survival through specific targeting of tumorigenesis. References 1. Colorectal Cancer Facts & Figures 2017-2019. American Cancer Society 2017. 2. Compton, C. C.; Byrd, D. R.; Garcia-Aguilar, J.; Kurtzman, S. H.; Olawaiye, A.; Washington, M. K. Colon and rectum. In AJCC Cancer Staging Atlas, 2nd ed.; Ed. Springer Science: New York, 2012; pp 185–201. 3. Van Cutsem, E.; Cervantes, A.; Adam, R.; Sobrero, A.; Van Krieken, J. H.; Aderka, D.; Aranda Aguilar, E.; Bardelli, A.; Benson, A.; Bodoky, G.; et al. ESMO consensus guidelines for the management of patients with metastatic colorectal cancer. Ann. Oncol. 2016, 27, 1386–422. 4. Siegel, R. L.; Miller, K. D.; Fedewa, S. A.; Ahnen, D. J.; Meester, R. G. S.; Barzi, A.; Jemal, A. Colorectal cancer statistics, 2017. CA Cancer J. Clin. 2017, 67, 177–193. 5. Moriarity, A.; O'Sullivan, J.; Kennedy, J.; Mehigan, B.; McCormick, P. Current targeted therapies in the treatment of advanced colorectal cancer: a review. Ther. Adv. Med. Oncol. 2016, 8, 276–293. 6. Seidel, J.; Farber, E.; Baumbach, R.; Cordruwisch, W.; Bohmler, U.; Feyerabend, B.; Faiss, S. Complication and local recurrence rate after endoscopic resection of large high-risk colorectal adenomas of >/=3 cm in size. Int. J. Colorectal Dis. 2016, 31, 603–611. 7. Pugh, S. A.; Shinkins, B.; Fuller, A.; Mellor, J.; Mant, D.; Primrose, J. N. Site and stage of colorectal cancer influence the likelihood and distribution of disease recurrence and postrecurrence survival: data from the FACS randomized controlled trial. Ann. Surg. 2016, 263, 1143–1147. 8. Batlle, E.; Clevers, H. Cancer stem cells revisited. Nat. Med. 2017, 23, 1124–1134. 9. Hanahan, D.; Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 2011, 144, 646–674. 10. Tirino, V.; Desiderio, V.; Paino, F.; De Rosa, A.; Papaccio, F.; La Noce, M.; Laino, L.; De Francesco, F.; Papaccio, G. Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization. FASEB J. 2013, 27, 13–24. 11. Bonnet, D.; Dick, J. E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 1997, 3, 730–737. 12. Desai, A.; Yan, Y.; Gerson, S. L. Concise reviews: cancer stem cell targeted therapies: toward clinical success. Stem Cells Transl. Med. 2019, 8, 75–81. 13. Munro, M. J.; Wickremesekera, S. K.; Peng, L.; Tan, S. T.; Itinteang, T. Cancer stem cells in colorectal cancer: a review. J. Clin. Pathol. 2018, 71, 110–116. 14. Zhou, Y.; Xia, L.; Wang, H.; Oyang, L.; Su, M.; Liu, Q.; Lin, J.; Tan, S.; Tian, Y.; Liao, Q.; Cao, D. Cancer stem cells in progression of colorectal cancer. Oncotarget 2018, 9, 33403–33415. 15. Patel, N. J.; Karuturi, R.; Al-Horani, R. A.; Baranwal, S.; Patel, J.; Desai, U. R.; Patel, B. B. Synthetic, non-saccharide, glycosaminoglycan mimetics selectively target colon cancer stem cells. ACS Chem. Biol. 2014, 9, 1826–1833. 16. Punt, C. J.; Koopman, M.; Vermeulen, L. From tumour heterogeneity to advances in precision treatment of colorectal cancer. Nat. Rev. Clin. Oncol. 2017, 14, 235–246. 17. Mouradov, D.; Sloggett, C.; Jorissen, R. N.; Love, C. G.; Li, S.; Burgess, A. W.; Arango, D.; Strausberg, R. L.; Buchanan, D.; Wormald, S.; et al. Colorectal cancer cell lines are representative models of the main molecular subtypes of primary cancer. Cancer Res. 2014, 74, 3238–3247. 18. Guinney, J.; Dienstmann, R.; Wang, X.; de Reynies, A.; Schlicker, A.; Soneson, C.; Marisa, L.; Roepman, P.; Nyamundanda, G.; Angelino, P.; et al. The consensus molecular subtypes of colorectal cancer. Nat. Med. 2015, 21, 1350–1356. 19. Berg, K. C. G.; Eide, P. W.; Eilertsen, I. A.; Johannessen, B.; Bruun, J.; Danielsen, S. A.; Bjornslett, M.; Meza-Zepeda, L. A.; Eknaes, M.; Lind, G. E.; et al. Multi-omics of 34 colorectal cancer cell lines - a resource for biomedical studies. Mol. Cancer 2017, 16, 116–132.

Através de Testes de Diluição em Sistema de Tubos Múltiplos determinou-se a Intensidade de Atividade de Inibição Bacteriana (IINIB/bacteriostasia) e a Intensidade de Atividade de Inativação Bacteriana (IINAB/bactericidia) de soluções contendo extratos hidroetanólicos e hídricos (decocto e infuso) de três acessos de Bixa orellana L. (urucum) a saber: Arroio do Meio/RS, Eldorado do Sul/RS e Maringá/PR, sobre inóculos padronizados de Salmonella Enteritidis (ATCC 11076), Escherichia coli (ATCC 11229), Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 19433) e Listeria monocytogenes (ATCC 19114). Determinou-se, paralelamente, o teor de bixina presente nas sementes. Os extratos hídricos apresentaram baixa atividade de inibição e/ou inativação sobre os inóculos bacterianos, enquanto que a forma de extração hidroetanólica apresentou atividade antibacteriana seletiva e significativamente mais intensa (inibição/inativação) entre as cinco bactérias testadas. Independente da forma de extração, as bactérias Enterococcus faecalis e Listeria monocytogenes foram as mais sensíveis à atividade antibacteriana, enquanto que Escherichia coli apresentou a menor sensibilidade. Houve diferença significativa entre os teores de bixina dos três acessos, e, consequentemente, a atividade antibacteriana determinada mostrou-se diretamente proporcional a estes teores.
Through of Dilution Tests in Multiple Tubes System it was determined the intensity of bacterial inhibition activity (IINIB/bacteriostasy) and the intensity of bacterial inactivation activity (IINAB/bactericidie) from solutions containing hidroetanolic and hydric (decoction and infusion) extracts of tree accesses from Bixa orellana L. (annatto) at know: Arroio do Meio/RS, Eldorado do Sul/RS and Maringá/PR, on standardized inocula of Salmonella Enteritidis (ATCC 11076), Escherichia coli (ATCC 11229), Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 19433) and Listeria monocytogenes (ATCC 19114). It was determined, in parallel, the content of bixin present in the three different accesses of the seeds. The forms of hydric extraction showed low inhibition and/or inactivation activity of the bacterial inocula, and the hidroetanolic extract form showed selective antibacterial activity and significantly pronounced inhibition/inactivation against the five bacteria tested. Independent of the extraction forms, Enterococcus faecalis and Listeria monocytogenes were the more sensitive agents to the antibacterial activity. Escherichia coli had the lowest sensitivity to all forms of extraction. The bixin contents were significantly different between the accesses and, consequently, the antibacterial activity was directly proportional to this contents.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第17830号
農博第2015号
新制||農||1016(附属図書館)
学位論文||H25||N4787(農学部図書室)
30645
京都大学大学院農学研究科農学専攻
(主査)教授 稲村 達也, 教授 冨永 達, 教授 奥本 裕
学位規則第4条第1項該当

Cardiovascular disease is the leading cause of death worldwide and ischemic heart disease is the most common manifestation. Despite improved outcomes during the last decades, patients with acute coronary syndromes (ACS) are still at substantial risk of recurrent ischemic events and mortality. The aims of this thesis were to investigate the effect of the novel antiplatelet agent ticagrelor versus clopidogrel in patients with non-ST-elevation ACS (NSTE-ACS), overall and in relation to initial revascularization, and to explore this effect in relation to cardiac biomarkers. The impact of timing of revascularization in non-ST-elevation myocardial infarction (NSTEMI) was also studied, by assessing risk of mortality and recurrent myocardial infarction in relation to delay of percutaneous coronary intervention (PCI) in a nation-wide cohort. Finally, a novel clinical prediction model based on angiographic findings, biomarkers, and clinical characteristics was developed to estimate risk of ischemic events after performed revascularization. Ticagrelor treatment compared with clopidogrel was associated with a reduction in the composite endpoint of cardiovascular death/myocardial infarction/stroke and mortality alone, without any increase in overall major bleeding, but increased non-CABG-related major bleeding. The effect of ticagrelor over clopidogrel was consistent independent of initial revascularization. Elevated high-sensitivity cardiac troponin-T predicted benefit of ticagrelor over clopidogrel, while no difference between treatments was detected at normal levels. In patients with NSTEMI, PCI treatment within two days after hospital admission was associated with lower risk of all-cause death and recurrent myocardial infarction compared with delayed PCI. The new clinical prediction model included the following variables: prior vascular disease, extent of coronary artery disease, level of N-terminal pro-B-type natriuretic peptide and estimated glomerular filtration rate; and showed good discriminatory ability for the risk prediction of cardiovascular death/myocardial infarction/stroke and cardiovascular death alone. In conclusion, these results show that ticagrelor reduces the risk of recurrent ischemic events and mortality in patients with NSTE-ACS when compared with clopidogrel, and this effect seems independent of performed revascularization. The results also indicate that biomarkers could be used to select patients who would benefit most from more intense platelet inhibition. Furthermore, early PCI in NSTEMI seems to be associated with improved outcome. Finally, the novel clinical prediction model based only on four variables showed good discriminatory ability, which makes it a potentially effective and simple tool for tailored treatment based on individual risk of recurrent events.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
A glicoproteína-p (gp-P) foi descrita pela primeira vez em 1976 como uma glicoproteína de superfície presente na membrana citoplasmática. Esta é uma bomba de efluxo que pertence à família de transportadores ABC e que participa no fenómeno de resistência a múltiplos fármacos. Encontra-se amplamente distribuída nos tecidos e participa em variadas funções fisiológicas importantes. É expressa nos pontos de entrada dos xenobióticos (trato gastrointestinal, fígado, rins, cérebro, placenta) e consegue efetuar o transporte destes para o exterior das células. É também expressa em barreiras hemato-teciduais e em células tumorais, impedindo a entrada de substâncias nestes tecidos graças ao seu transporte para fora das células. Desempenha um papel crucial na absorção, distribuição, metabolização e excreção de muitos fármacos no organismo. Atua na proteção dos tecidos contra xenobióticos tóxicos e metabolitos endógenos, através da excreção destes compostos para o lúmen intestinal, bílis, urina e promovendo também a sua expulsão do sistema nervoso central. As interações farmacológicas ocorrem quando os efeitos de um fármaco são alterados pela presença de outro fármaco, alimento, ou outro agente químico. Neste trabalho são discutidas algumas das interações fármaco-fármaco mais importantes envolvendo a gp- P. A co-adminstração de fármacos que são indutores ou inibidores da gp-P pode originar uma interação farmacológica. Quando os fármacos atuam como inibidores de gp-P originam um aumento dos seus substratos a nível intracelular, graças ao decréscimo do transporte de efluxo de fármacos. Para além dos fármacos que têm a capacidade de inibir a gp-P, outras moléculas como os surfactantes (tween-20) usados na indústria farmacêutica podem também inibi-la. A gp-P é ainda considerada de fácil indução quer in vivo quer in vitro, sendo conhecidos vários tipos de indutores. Estes atuam aumentando o transporte de efluxo, diminuindo assim a biodisponibilidade dos fármacos que são transportados por esta proteína. Quanto maior o número de fármacos co-administrados maior é a probabilidade de ocorrer interação farmacológica. The p-glycoprotein (P-gp) was first described in 1976 as a surface glycoprotein present in the cytoplasmic membrane. It is an efflux pump that belongs to the ABC transporter family and is involved in multidrug resistance phenomenon. It is widely distributed in tissues and participate in a variety of important physiological functions. Since it is expressed in the entry points of xenobiotics (gastrointestinal tract, liver, kidneys, brain and placenta) it can make the transportation of these substances outside of the cells. It is also expressed in the blood-tissue barriers and tumour cells, preventing the entry of xenobiotics in these tissues. P-gp plays a crucial role in the absorption, distribution, metabolism and excretion of many drugs in the body. This protein protects tissues against toxic xenobiotics and endogenous metabolites through the excretion of these compounds into the intestinal lumen, bile, urine, and also promoting the expulsion of the central nervous system. A drug interaction occurs when the effect of a drug is changed by the presence of another drug, food, or other chemical. A discussion of some of the most important drugdrug interactions involving P-gp is presented. The co-adminstration of drugs that are P-gp inducers or inhibitors may lead to a pharmacological interaction. P-gp drug inhibitors may increase substrates intracellularly thanks to the decrease in drug efflux transport. In addition, other molecules such as surfactants (Tween-20) used in the pharmaceutical industry may also inhibit it. The Pgp is also easily induced both in vivo and in vitro and various types of inductors are known. These inductors act by enhancing the transport efflux and thereby decreasing the bioavailability of drugs that are transported by this protein. The likelihood of interactions rises as the number of drugs co-administered increases.

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Plantas medicinais s?o comumente usadas por comunidades tradicionais, principalmente em regi?es com menor desenvolvimento humano. Algumas esp?cies vegetais possuem entre seus componentes subst?ncias denominadas inibidores de proteases. Os inibidores de proteases se destacam na prote??o de fluidos e tecidos contra sua degrada??o por prote?lise e poss?veis falhas na degrada??o de prote?nas de meia-vida que podem interferir de forma dr?stica nas fun??es celulares. Diante do exposto, esse estudo objetivou identificar e caracterizar inibidores de proteases em cinco esp?cies vegetais nativas do Cerrado e da Mata Atl?ntica. As esp?cies de Punica granatum L. (Rom?), Plantago major L. (Tansagem), Ocimum gratissimum L. (Alfavaca), Anadenanthera colubrina Vellozo (Angico) e Stryphnodendron adstringens Mart. Coville (Barbatim?o) foram selecionados nas cidades de Pot?, Ladainha, Atal?ia, Te?filo Otoni e Ara?ua?, devido ao seu uso tradicional como anti-inflamat?rio. A sequencia gen?mica de inibidores de proteases foi pesquisada para essas esp?cies vegetais no GenBank, mas nenhuma sequencia foi descrita para as esp?cies selecionadas. As amostras provenientes dos procedimentos de extra??o foram submetidas ?s quantifica??o de prote?nas e a presen?a de inibidores de proteases foi detectada por eletroforese em gel de poliacrilamida 12% SDS-PAGE. Somente os extratos das sementes de Punica granatum e das folhas do Anadenanthera colubrina tiveram detec??o satisfat?ria de inibidores de proteases e foram submetidos ? an?lise por cromatografia l?quida de alta efici?ncia em sistema de HPLC. Este trabalho demonstra pela primeira vez a detec??o e extra??o de inibidores de proteases em folhas de Anadenanthera colubrina e sementes de Punica granatum.
Disserta??o (Mestrado Profissional) ? Programa de P?s-Gradua??o em Tecnologia, Sa?de e Sociedade, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016.
Traditional communities, especially in regions with lower human development, commonly use medicinal plants. Some plant species have among their components substances called protease inhibitors. Protease inhibitors act protecting fluids and tissues from degradation by proteolysis and possible failures in the degradation of half-life proteins that can drastically interfere with cellular functions. This study aimed to identify and characterize protease inhibitors in five native plant species of Cerrado and Atlantic Forest. The species of Punica granatum L. (Rom?), Plantago major L. (Tansagem), Ocimum gratissimum L. (Alfavaca), Anadenanthera colubrina Vellozo (Angico) and Stryphnodendron adstringens Mart. Coville (Barbatim?o) were selected in the cities of Pot?, Ladainha, Atal?ia, Te?filo Otoni and Ara?ua? due to their traditional use. The genomic sequence of protease inhibitors was screened for these plant species in GenBank, but no sequence was described for the selected species. Samples from the extraction procedures were subjected to protein quantification and the presence of protease inhibitors was detected by 12% SDS-PAGE polyacrylamide gel electrophoresis. Only the extracts of the seeds of Punica granatum and of the leaves of Anadenanthera colubrina had satisfactory detection of proteases inhibitors and were submitted to the analysis by high performance liquid chromatography system. This work demonstrates for the first time the detection and extraction of protease inhibitors in leaves of Anadenanthera colubrina and seeds of Punica granatum.

La corrélation étroite entre l'activation de la saccharase intestinale de lapin (sucrose α-D-glucohydrolase, EC. 3. 2. 1. 48) par le Na⁺ et l'activation par déprotonation de l'enzyme dans la zone de pH 4. 8 à 7. 2 a conduit à la formulation d'un modèle cinétique, le Modèle II ( Vasseur, 1982). Ce modèle fait le lien entre le modèle allostérique, indépendant du pH, le Modèle I (Alvarado, 1974), et les modèles classiques, dépendants du pH, de Michaelis, Alberty, Dixon ou Cleland, excepté que l'activation de la saccharase, au-dessous de pH 7, est liée à la perte simultanée de deux protons clés (pK₁ = 5. 7). Le modèle II, édifié essentiellement sur la base des modèles classiques, s'est révélé insuffisant pour expliquer l'effet antagoniste du Na⁺ (ou du substrat) avec lin seul des deux protons clés, un proton, Hₓ, se comportant comme un inhibiteur compétitif. L'autre proton, Hy, est responsable de l'activité catalytique. Nous proposons que les deux protons impliqués dans pK₁ jouent un rôle fonctionnel distinct et appartiennent à deux familles différentes: la famille K et la famille V, responsables respectivement des effets sur l’affinité et sur la capacité. Si Hₓ se fixe ou non, l'enzyme oscille entre deux formes ayant des propriétés de liaison différentes vis à vis des deux coactivateurs allostériques. L'effet des métaux alcalins, Li⁺ et Na⁺, consiste à déplacer cet équilibre. A faibles concentrations, ils facilitent la déprotonation de l'enzyme, donnant une activation de type K. Au contraire, à fortes concentrations, ils favorisent la reprolonation de l'enzyme causant une inhibition compétitive pure. Aussi, pour expliquer les mécanismes moléculaires d'activation et/ou d'inhibition de la saccharase par les métaux alcalins dans la zone de pH 4. 8 à 8. 8, nous proposons un modèle cinétique complet, le Modèle TETARD, qui considère la fonction et le nombre (3) des protons clés de l'enzyme. Contrairement aux concepts classiques d'activation des enzymes en fonction du pH, le modèle' têtard inclut la participation simultanée des deux familles de protons, V et K, dans la même réaction d'ionisation. Afin de généraliser, nous proposons que chaque famille de protons puisse contenir un ou plusieurs protons liés fonctionnellement qui sont perdus (ou gagnés) comme un seul bloc. Puisque, en principe, une enzyme possède deux pK caractéristiques et que chacun de ces pK peut contenir un maximum de deux familles de protons, nous obtenons un modèle à quatre familles de protons, le Modèle PAPILLON, qui peut servir de base à la construction de la plupart des modèles enzymatiques. Ce modèle peut générer des modèles-fils, le plus simple étant celui décrit par Michaelis et Davidsohn où seulement 2 familles de protons sont visibles au 11eu des 4 théoriquement possibles. Nous décrivons une méthode mathématique générale capable de donner directement l'équation globale décrivant pratiquement tous les modèles enzymatiques; cette équation est, de plus, directement adaptable pour le traitement global des résultats par ordinateur
The close relationship between sodium-dependent activation of rabbit intestinal sucrase (sucrose α-D-glucohydrolase, EC. 3. 2. 1. 48 ) and activation by enzyme deprotonation in the pH range 4. 8 to 7. 2 led us to build a kinetic model, Model II (Vasseur, 1982), derived in turn from an earlier pH-independent allosteric model, Model (Alvarado, 1974). Ln principle, Model II resembles the one first proposed by Alberty for anion and H⁺-dependent enzyme activation, except that formation of the active species involves loss of two protons rather than one ( pK₁ = 5. 7). Model Il, essentially based on classical models of Michaelis, Dixon or Cleland, does not explain fully the peculiar antagonistic relationship between Na⁺ (or the substrate) and one single key proton, Hₓ, which behaves as a fully competitive inhibitor. The second proton, Hy, is responsible for changes in enzyme catalytic activity. We postulate, therefore, that the two protons involved in pK₁ are functionally distinguishable and belong to two different families: the K family and the V family, responsible for affinity and capacity-type effects, respectively. Depending on whether Hₓ is bound or not, the enzyme alternates between two distinct forms exhibiting quite different binding properties. The alkali-metal ions Na⁺ and Li⁺ have a concentration-dependent dual effect on this equilibrium. At low concentrations, they facilitate the release of Hₓ, resulting in K-type activation. On the contrary, at higher concentrations they favor enzyme reprotonation, causing K-type (fully competitive) inhibition. To explain the molecular mechanisms underlying the complex activatory and inhibitory effects of the alkali-metal ions in the pH range 4. 8 to 8. 8, we propose a complete sucrase model, the Tetard (Tadpole) , which considers both the function and the number (three) of key protons participating. In contrast to classical concepts on pH-dependent enzyme activation, the Tetard model considers the participation of two proton families, V and K, in the same ionization reaction. Generalizing, we propose that a proton family consists of one or several protons that perform the same kinetic function and are either gained or lost as a bloc. Because all enzymes exhibit in principle two characteristic pK values, and since each of these pK values may involve a maximum of two proton families, we obtain, as a result, a four-proton-families general model, the Papillon (Butterfly), which should be able to explain any enzyme mechanism. By introducing appropriate simplifications, this model can generate any of a series of better known models, the simplest one of which is the classical Michaelis- Davidsohn one, where only two proton families are experimentally demonstrable instead of the theoretical four. We also describe a general mathematical treatment capable of yielding directly the global equation describing practically any enzyme, an equation which, furthermore, is readily adaptable for computer analysis

Acetylcholinesterase (AChE) is an essential enzyme with an evolutionary conserved function: to terminate nerve signaling by rapid hydrolysis of the neurotransmitter acetylcholine. AChE is an important target for insecticides. Vector control by the use of insecticide-based interventions is today the main strategy for controlling mosquito-borne diseases that affect millions of people each year. However, the efficiency of many insecticides is challenged by resistant mosquito populations, lack of selectivity and off-target toxicity of currently used compounds. New selective and resistance-breaking insecticides are needed for an efficient vector control also in the future. In the work presented in this thesis, we have combined structural biology, biochemistry and medicinal chemistry to characterize mosquito AChEs and to develop selective and resistance-breaking inhibitors of this essential enzyme from two disease-transmitting mosquitoes.We have identified small but important structural and functional differences between AChE from mosquitoes and AChE from vertebrates. The significance of these differences was emphasized by a high throughput screening campaign, which made it evident that the evolutionary distant AChEs display significant differences in their molecular recognition. These findings were exploited in the design of new inhibitors. Rationally designed and developed thiourea- and phenoxyacetamide-based non-covalent inhibitors displayed high potency on both wild type and insecticide insensitive AChE from mosquitoes. The best inhibitors showed over 100-fold stronger inhibition of mosquito than human AChE, and proved insecticide potential as they killed both adult and larvae mosquitoes.We show that mosquito and human AChE have different molecular recognition and that non-covalent selective inhibition of AChE from mosquitoes is possible. We also demonstrate that inhibitors can combine selectivity with sub-micromolar potency for insecticide resistant AChE.

In addition to its canonical role in aminoacylation, threonyl-tRNA synthetase (TARS) possesses pro-angiogenic activity that is susceptible to the TARS-specific antibiotic borrelidin. However, the therapeutic benefit of borrelidin is offset by its strong toxicity to living cells. The removal of a single methylene group from the parent borrelidin generates BC194, a modified compound with significantly reduced toxicity but comparable anti-angiogenic potential. Biochemical analyses revealed that the difference in toxicities was due to borrelidin's stimulation of amino acid starvation at ten-fold lower concentrations than BC194. However, both compounds were found to inhibit in vitro and in vivo models of angiogenesis at sub-toxic concentrations, suggesting a similar mechanism that is distinct from the toxic responses. Crystal structures of TARS in complex with each compound indicated that the decreased contacts in the BC194 structure may render it more susceptible to competition with the canonical substrates and permit sufficient aminoacylation activity over a wider concentration of inhibitor. Conversely, both borrelidin and BC194 induce identical conformational changes in TARS, providing a rationale for their comparable effects on angiogenesis. The mechanisms of TARS and borrelidin-based compounds on angiogenesis were subsequently tested using zebrafish and cell-based models. These data revealed ectopic branching, non-functional vessels, and increased cell-cell contracts following BC194-treatment or knockdown of TARS expression, suggesting a role for the enzyme in the maturation and guidance of nascent vasculature. Using various TARS constructs this function was found to be dependent on two interactions or activities associated with the TARS enzyme that are distinct from its canonical aminoacylation activity. Furthermore, observations that TARS may influence VEGF expression and purinergic signaling suggest the possibility for a receptor-mediated response. Taken together, the results presented here demonstrate a clear role for TARS in angiogenesis, independent of its primary function in translation. Although the exact molecular mechanisms through which TARS and borrelidin regulate this activity remain to be determined, these data provide a foundation for future investigations of TARS's function in vascular biology and its use as a target for angiogenesis-based therapy.

Peacebuilding NGOs are increasingly aware that religion is a steadfast and sometimes growing influence in the contexts in which they work. Despite this, many fail to meaningfully integrate religious perspectives into their initiatives. This thesis examines and consolidates criticisms of NGO responses to religion in peacebuilding programmes, identifies factors inhibiting responses from advancing, and explores NGO staff attitudes regarding religion, including perspectives on whether a conducive environment exists for developing alternative responses. It then formulates recommendations for advancing practice and suggests future research directions.  The research approach consists of an examination of literature regarding NGO responses to religion, accompanied by a survey using semi-structured interviews of nine people who have worked on NGO peacebuilding programmes across the world. The main conclusions drawn from this study are that whilst NGOs consider religious actors as potential allies, they rarely utilise the role of religion in society as an analytical lens. This impinges their ability to understand contexts holistically. The contemporary funding environment is also found to discourage alternative approaches from emerging. This thesis recommends that further research is conducted in order to produce examples of improved NGO responses to religion. This will provide practical evidence of how to enhance practice.

Orientador: Brenda Paula Figueiredo de Almeida Gomes
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Introdução: O objetivo do presente trabalho foi avaliar a interação entre as diferentes substâncias químicas auxiliares, utilizadas em endodontia, e seus efeitos nas etapas do tratamento endodôntico. Métodos: Soluções de hipoclorito de sódio (NaOCl) a 0,16%, 1%, 2,5% e 5,25%, clorexidina 2% solução e gel, EDTA 17%, ácido cítrico 10% e ácido fosfórico 37% foram utilizadas isoladamente ou associadas entre si na proporção 1:1. As mesmas foram analisadas quimicamente por espectrometria de massa; e microbiologicamente pelo método de difusão em ágar, contra diferentes patógenos. Adicionalmente, foram avaliados onze protocolos de irrigação em relação à formação de smear layer química por microscopia eletrônica de varredura. Por fim, foram avaliados 9 protocolos de irrigação associados a dois diferentes sistemas de obturação, guta-percha/ AH Plus e Resilon/ Real Seal SE, em relação à microinfiltração coronária e à resistência de união da dentina aos materiais obturadores. Resultados: O NaOCl, em todas as concentrações, associado à clorexidina em ambas as formulações, levou à formação de precipitado, assim como a associação entre a clorexidina e o EDTA e entre clorexidina e solução salina. Todas as associações avaliadas apresentaram algum grau de inibição contra os patógenos testados. Irrigação intermediária, com 10 mL de água destilada, entre as soluções de NaOCl e clorexidina, não foram capazes de inibir a formação de smear layer química, assim como irrigações com EDTA e ácido cítrico. Dos diferentes protocolos de irrigação avaliados, aqueles que apresentaram uma irrigação final com solução de clorexidina 2% tiveram níveis reduzidos de microinfiltração coronária. Em relação à resistência de união à dentina, no sistema obturador guta-percha/AH Plus, os grupos em que se utilizou as associações NaOCl/ácido fosfórico e clorexidina/EDTA apresentaram maiores valores de resistência de união, ao passo que no sistema Resilon/Real Seal SE, os maiores valores foram encontrados nos grupos clorexidina/ácido fosfórico. A utilização da clorexidina como irrigante final não afetou negativamente os sistemas obturadores avaliados. Conclusões: A interação entre as substâncias químicas auxiliares pode levar à formação de precipitados. As diferentes substâncias químicas auxiliares, quando associadas, possuem atividade antimicrobiana. Irrigações intermediárias entre as diferentes substâncias químicas auxiliares são necessárias para reduzir ou até mesmo impedir a formação de precipitados, visualizados na superfície dentinária como uma smear layer química. Durante o preparo químicomecânico, as diferentes substâncias químicas auxiliares geram modificações na superfície dentinária que influenciam na microinfiltração coronária e na resistência de união dos sistemas obturadores guta-percha/AH Plus e Resilon/Real Seal SE
Abstract: Introduction: The aim of the present study was to evaluate the interaction among different chemical auxiliary substances used in endodontics and their effects on different steps of endodontic treatment. Methods: 0.16%, 1%, 2.5% and 5.25% sodium hypochlorite solutions (NaOCl), 2% chlorhexidine solution and gel, 17% EDTA, 10% citric acid, 37% phosphoric acid, distilled water, and saline solution were used both associated with each other (1:1 ratio) and not associated. The solutions were chemically examined with mass spectrometry. They were microbiologically examined using the Agar Diffusion Method, against different pathogens. In addition, eleven protocols were analyzed in regards to chemical smear layer with scanning electron microscopy. Finally, nine irrigation protocols (associated with two filling systems, i.e. gutta-percha/AH Plus and Resilon / Real Seal SE) were examinated regarding coronal microleakage and were examined as well regarding their bond strength with dentin. Results: NaOCl, at all concentrations, associated with both chlorhexidine formulations, led to precipitate formation, similar to the association between chlorhexidine/EDTA, and the chlorhexidine/saline solution association. All associations had some degree of inhibition against the evaluated pathogens. Intermediate flush, with 10 mL of distilled water, between NaOCl and chlorhexidine did not inhibit chemical smear layer formation, similar to intermediate flushes with EDTA and citric acid. Among the different irrigation protocols that were evaluated, when final flush with 2% chlorhexidine was present, the lowest levels of coronal microleakage were found. Regarding the bond strength to dentin, in the gutta-percha/AH Plus system, the groups with NaOCl/phosphoric acid and chlorhexidine/EDTA associations showed higher bond strength values. In the Resilon/Real Seal SE system, the highest values were found in the chlorhexidine/phosphoric acid groups. The use of chlorhexidine as a final flush did not negatively affect the filling systems evaluated. Conclusions: The interaction among the auxiliary chemical substances may lead to precipitate formation. The association between the different substances has antimicrobial activity. Intermediate flushes are necessary to reduce or even avoid the formation of chemical smear layer on the dentin surface. During the chemo-mechanical preparation, the various auxiliary chemical substances used do modify the dentine surface. These modifications have influence in the coronal microleakage and in the bond strength of the filling systems gutta-percha/AH Plus and Resilon / Real Seal SE
Doutorado
Endodontia
Doutora em Clínica Odontológica

L’objet de ce travail est de comprendre les mécanismes de corrosion par piqûres d’un acier faiblement allié (S235) dans une solution aqueuse aérée de NaHCO3/NaCl et d’étudier l’effet de deux inhibiteurs de corrosion (HPO42- et NO2-) sur la résistance à la piqûration de l’acier. La Piqûration de l’acier a été étudiée en couplant des mesures électrochimiques et des analyses spectroscopiques. Un suivi in situ de l’activité électrochimique des piqûres, a été réalisé par imagerie électrochimique à électrode vibrante (SVET). La microspectroscopie Raman a permis d’identifier puis de suivre, in situ, l’évolution des produits de corrosion au cours du temps. En parallèle, une étude chimique de synthèse et d’oxydation des produits de corrosion susceptibles de se former dans les piqûres a été effectuée dans des conditions expérimentales similaires à celles régnant dans les piqûres. La caractérisation, in situ, des produits de corrosion formés dans les piqûres a montré que le premier produit de corrosion formé est un carbonate de fer FeCO3 appelé sidérite. L’action oxydante du dioxygène dissous présent dans la solution conduit à une transformation de la sidérite en rouille verte carbonatée, qui est ensuite elle-même oxydée en goethite. Ces résultats semblent montrer qu’il existe une relation significative entre la nature des produits de corrosion formés et le processus de développement des piqûres. En effet, la formation de sidérite et de rouille verte signe un développement rapide des piqûres, alors que la formation de composées ferriques est associée à un ralentissement du développement des piqûres et à une accélération de leur repassivation. La même démarche a été réutilisée pour l’étude de l’effet des ions HPO42- et NO2- sur le processus de piqûration de l’acier étudié. L’inhibition de la piqûration par l’ion HPO42- se traduit par une diminution de la proportion de la sidérite formée au profit d’un phosphate de fer (la vivianite), produit connu être plutôt protecteur. Pour des concentrations élevées en HPO42-, la formation exclusive de la vivianite conduit à une meilleure protection contre la piqûration. Pour l’ion NO2-, l’effet inhibiteur semble provenir de la formation d’un complexe de Fe(III) [Fe(H2O)6]3+. En effet, la caractérisation des produits de corrosion formés dans les piqûres en présence de l’ion nitrite a donné le complexe [Fe(H2O)6]3+ avec une faible proportion de la sidérite. L’ion NO2-, connu pour son action oxydante, réduit énormément la quantité de sidérite formée dans les piqûres et oxyde rapidement le Fe(II) dissous, conduisant à des complexes de Fe(III) probablement précurseurs des oxydes de Fe(III) constitutifs du film passif
The aim of this work is to understand the pitting mechanisms of S235 carbon steel in aerated aqueous NaHCO3/NaCl solutions and to study the effect of two inhibitors (HPO42- and NO2-) on pitting of steel in the same media. Electrochemical measurements coupled to spectroscopic analysis were used to characterize pitting corrosion. The activity of a given pit has been studied by scanning Vibrating Electrode Technique (SVET). The micro Raman spectroscopy is used to identify and to follow, in situ, the evolution of the corrosion products formed in the pits. In parallel, a chemical study of synthesis and oxidation of the products likely to form in the pit has been done in experimental conditions similar to those found in the pits. The characterization, in situ, of the products of corrosion formed in the pits showed that the first corrosion product formed is an iron carbonate FeCO3 named siderite. The oxidizing action of the dissolved oxygen present in solution, transforms siderite into carbonated green rust, that is then oxidized into goethite. These results seem to show that a meaningful relation exists between the nature of the corrosion products and the process of development of the pits. Indeed, the formation of siderite and green rust signs a fast development of the pit, whereas the formation of ferric compounds is associated to a slowing of the development of the pit and an acceleration of their repassivation. The same methodology has been used for the study of the effect of two corrosion inhibitors (HPO42- and NO2-) on the process of pitting of the studied steel. The inhibition by the HPO42- ions is explained by a reduction of the proportion of siderite formed to the profit of an iron phosphate (vivianite), product known to be rather protective. For elevated concentrations of HPO42-, the exclusive formation of the vivianite gives a better protection against pitting. For the NO2- ion, the inhibiting effect seems to come from the formation of Fe(III) complexes [Fe(H2O)6]3+. Indeed, the characterization of the corrosion products formed in the pits in presence of the nitrite ions gave the complex [Fe(H2O)6]3+ with a weak proportion of siderite. NO2- ions oxidize quickly the dissolved Fe(II), giving a Fe(III) complex probably precursors of the Fe(III) spinel oxides of the passive film

The aim of this research is to investigate the extent to which Open Source Software (OSS) adoption behaviour can empirically be shown to be governed by a set of self-reported (driving and inhibiting) salient beliefs of key informants in a sample of organisations. Traditional IS adoption/usage theory, methodology and practice are drawn on. These are then augmented with theoretical constructs derived from IT governance and organisational diagnostics to propose an artefact that aids the understanding of organisational OSS adoption behaviour, stimulates debate and aids operational management interventions. For this research, a combination of quantitative methods (via Fisher's Exact Test) and complimentary qualitative method (via Content Analysis) were used using self-selection sampling techniques. In addition, a combination of data and methods were used to establish a set of mixed-methods results (or meta-inferences). From a dataset of 32 completed questionnaires in the pilot study, and 45 in the main study, a relatively parsimonious set of statistically significant driving and inhibiting factors were successfully established (ranging from 95% to 99.5% confidence levels) for a variety for organisational OSS adoption behaviours (i.e. by year, by software category and by stage of adoption). In addition, in terms of mixed-methods, combined quantitative and qualitative data yielded a number of factors limited to a relatively small number of organisational OSS adoption behaviour. The findings of this research are that a relatively small set of driving and inhibiting salient beliefs (e.g. Security, Perpetuity, Unsustainable Business Model, Second Best Perception, Colleagues in IT Dept., Ease of Implementation and Organisation is an Active User) have proven very accurate in predicting certain organisational OSS adoption behaviour (e.g. self-reported Intention to Adopt OSS in 2014) via Binomial Logistic Regression Analysis.

Since the outbreak of the HIV/AIDS pandemic in the 1980s, the disease has cost the lives of over 30 million people, and a further 33 million are currently living with the HIV infection. With the appropriate treatment, HIV/AIDS can today be regarded as a chronic but manageable disease. However, treatment is not available globally and UNAIDS still estimates that there are currently 5000 AIDS-related deaths worldwide per day. HIV protease inhibitors (PIs) constitute one of the fundaments of HIV treatment, and are commonly used in so-called highly active antiretroviral therapy (HAART), together with reverse transcriptase inhibitors. Although there are ten PIs on the market, there is still a need for novel structures. The rapid development of resistant strains, due to the high frequency of mutations, together with the commonly observed adverse effects of the drugs available, illustrate the need to develop new potent structures. Two novel scaffolds were investigated in this work. A tertiary alcohol-containing scaffold comprising a three-carbon tether, and a β-hydroxy γ-lactam-based scaffold were designed, synthesized and evaluated using enzyme- and cell-based assays. X-ray analyses of inhibitors from each class provided information on inhibitor–protease interactions. The inhibitors containing the tertiary alcohol provided at best an enzymatic inhibition (Ki) of 2.3 nM, and an inhibition in the cell-based assay (EC50) of 0.17 µM. The γ-lactam-based inhibitors exhibited better inhibition than the first series; the best values being Ki = 0.7 nM and EC50 = 0.04 µM. The second part of these studies involved the evaluation of a novel non-resonance continuous-flow microwave instrument. The instrument was validated regarding heating capacity, temperature stability and temperature homogeneity. A number of model reactions were performed with low- and high-microwave-absorbing solvents. It was found that the microwave heating source allowed rapid temperature adjustment, together with easily regulated, flow-dependent reaction times, providing an efficient tool for reaction optimisation.

A substantial number of works have aimed at modeling the receptive field properties of the primary visual cortex (V1). Their evaluation criterion is usually the similarity of the model response properties to the recorded responses from biological organisms. However, as several algorithms were able to demonstrate some degree of similarity to biological data based on the existing criteria, we focus on the robustness against loss of information in the form of occlusions as an additional constraint for better understanding the algorithmic level of early vision in the brain. We try to investigate the influence of competition mechanisms on the robustness. Therefore, we compared four methods employing different competition mechanisms, namely, independent component analysis, non-negative matrix factorization with sparseness constraint, predictive coding/biased competition, and a Hebbian neural network with lateral inhibitory connections. Each of those methods is known to be capable of developing receptive fields comparable to those of V1 simple-cells. Since measuring the robustness of methods having simple-cell like receptive fields against occlusion is difficult, we measure the robustness using the classification accuracy on the MNIST hand written digit dataset. For this we trained all methods on the training set of the MNIST hand written digits dataset and tested them on a MNIST test set with different levels of occlusions. We observe that methods which employ competitive mechanisms have higher robustness against loss of information. Also the kind of the competition mechanisms plays an important role in robustness. Global feedback inhibition as employed in predictive coding/biased competition has an advantage compared to local lateral inhibition learned by an anti-Hebb rule.

Cell migration is a behaviour critical to many key biological effects, including wound healing, cancerous cell invasion and morphogenesis, the development of an organism from an embryo. However, given that each of these situations is distinctly different and cells are extremely complicated biological objects, interest lies in more basic experiments which seek to remove conflating factors and present a less complex environment within which cell migration can be experimentally examined. These include in vitro studies like the scratch assay or circle migration assay, and ex vivo studies like the colonisation of the hindgut by neural crest cells. The reduced complexity of these experiments also makes them much more enticing as problems to mathematically model, like done here. The primary goal of the mathematical models used in this thesis is to shed light on which cellular behaviours work to generate the travelling waves of invasion observed in these experiments, and to explore how variations in these behaviours can potentially predict differences in this invasive pattern which are experimentally observed when cell types or chemical environment are changed. Relevant literature has already identified the difficulty of distinguishing between these behaviours when using traditional mathematical biology techniques operating on a macroscopic scale, and so here a sophisticated individual-cell-level model, an extension of the Cellular Potts Model (CPM), is been constructed and used to model a scratch assay experiment. This model includes a novel mechanism for dealing with cell proliferations that allowed for the differing properties of quiescent and proliferative cells to be implemented into their behaviour. This model is considered both for its predictive power and used to make comparisons with the travelling waves which result in more traditional macroscopic simulations. These comparisons demonstrate a surprising amount of agreement between the two modelling frameworks, and suggest further novel modifications to the CPM that would allow it to better model cell migration. Considerations of the model’s behaviour are used to argue that the dominant effect governing cell migration (random motility or signal-driven taxis) likely depends on the sort of invasion demonstrated by cells, as easily seen by microscopic photography. Additionally, a scratch assay simulated on a non-homogeneous domain consisting of a ’fast’ and ’slow’ region is also used to further differentiate between these different potential cell motility behaviours. A heterogeneous domain is a novel situation which has not been considered mathematically in this context, nor has it been constructed experimentally to the best of the candidate’s knowledge. Thus this problem serves as a thought experiment used to test the conclusions arising from the simulations on homogeneous domains, and to suggest what might be observed should this non-homogeneous assay situation be experimentally realised. Non-intuitive cell invasion patterns are predicted for diffusely-invading cells which respond to a cell-consumed signal or nutrient, contrasted with rather expected behaviour in the case of random-motility-driven invasion. The potential experimental observation of these behaviours is demonstrated by the individual-cell-level model used in this thesis, which does agree with the PDE model in predicting these unexpected invasion patterns. In the interest of examining such a case of a non-homogeneous domain experimentally, some brief suggestion is made as to how this could be achieved.

La pandémie de l'obésité entraine une augmentation de la prévalence des maladies chroniques du foie ou stéatopathies métaboliques (NAFLD). Le spectre des NAFLD va de la stéatose caractérisée par une accumulation de lipides dans le foie à la stéatohépatite (NASH) associant une inflammation, de la mort hépatocytaire et de la fibrose. Lors de l'obésité, l'élévation de signaux de dangers métaboliques perturbe les fonctions du réticulum endoplasmique (RE) essentielles pour l’homéostasie cellulaire. Les perturbations sont transmises par 3 senseurs : IRE1α, ATF6 et PERK pour activer une réponse adaptative. Si ce stress est sévère ou devient chronique, la cellule enclenchera une réponse terminale apoptotique. La protéine Bax Inhibitor-1 (BI-1) pourrait jouer un rôle hépatoprotecteur en inhibant l’hyperactivation de la voie de signalisation IRE1α.En combinant des études chez l’homme et dans des modèles animaux, l’objectif de cette étude était de mieux caractériser l'activation chronique du stress du RE dans les NAFLD. Ce travail a émis l’hypothèse qu’une déficience en BI-1 entrainerait l’activation soutenue de la voie IRE1α qui serait responsable de la transition de la stéatose à la NASH. Cette étude s'intéresse au dialogue potentiel entre le stress du RE et l’activation de l'inflammasome NLRP3, qui induit la sécrétion des cytokines pro-inflammatoires (IL-1β, IL-18) grâce aux caspases pro-inflammatoires (caspase-1, caspase-4/11). L’utilisation d’un inhibiteur global du stress du RE ou des inhibiteurs pharmacologiques spécifiques à la voie IRE1α améliorerait les caractéristiques pathophysiologiques de la NASH et pourrait ouvrir de nouvelles perspectives thérapeutiques
Due to the obesity pandemic, the last decades have been marked by a constantly increasing prevalence of Non-Alcoholic Fatty Liver Disease (NAFLD). NAFLD covers a spectrum of hepatic disorders ranging from steatosis, characterized by the ectopic accumulation of lipids in the liver, to steatohepatitis (NASH), featuring inflammation, hepatocellular death and fibrosis. During obesity, an increase in metabolic danger signals leads to disrupted endoplasmic reticulum (ER) function, essential for cellular homeostasis. The resulting ER stress activates a signaling network involving three sensors: IRE1α, ATF6 and PERK to enforce adaptive programs. If this stress is severe or becomes chronic, the cell will trigger a terminal apoptotic response. The protein Bax Inhibitor-1 (BI-1), as a negative endogenous regulator of the IRE1α signaling pathway in the liver, may play a hepatoprotective role.By combining data from obese patients with liver complications and experimental approaches in mice, this thesis aimed to better characterize the chronic activation of ER stress in NAFLD pathogenesis. This work also emitted the hypothesis that a deficiency in BI-1 leads to unrestrained IRE1α signaling that may be responsible for the steatosis to NASH transition. This study further investigated the potential dialogue between ER stress and the activation the NLRP3 inflammasome, which induces the secretion of pro-inflammatory cytokines (IL-1β, IL-18) by activating pro-inflammatory caspases (caspase-1, caspase-4/11). The administration of a broad spectrum ER stress inhibitor or specific inhibitors of IRE1α improved the pathophysiological features of NASH and may open novel therapeutic perspectives

Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma (NHL). The current standard of care is the combination of rituximab with cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP), but this only results in a 60% over-all 5-year survival rate, thus highlighting a need for new therapeutic approaches. Histone deacetylase inhibitors (HDACi) are novel therapeutics that is being clinically evaluated for combination therapy. Rational selection of companion therapeutics for HDACi is difficult due to their poorly understood, cell-type specific mechanisms of action. To understand these mechanisms better, we developed a pre-clinical model system of response to the HDACi belinostat. Using this model system, we identified two major responses. Resistance, consisting of a reversible G1 cell cycle arrest with little induction of apoptosis; or sensitivity, consisting of mitotic arrest and high levels of apoptosis. In this dissertation, we determine that the induction of G1 cell cycle arrest is due to the increased expression of cyclin dependent kinase inhibitors (CDKi) that bind to and inhibit the cyclin E/CDK2 complex thereby blocking the final repressive phosphorylation steps of Rb protein. Repression of transcriptional elongation blocked CDKi upregulation and prevented G1 cell cycle arrest in belinostat-resistant cells. Additionally, we identified that belinostat arrests sensitive cells prior to metaphase and belinostat-resistant cells slow-down in mitosis but complete the process prior to arresting in G1. The combination of belinostat with the microtubule-targeting agent, vincristine resulted in strong synergistic induction of apoptosis by targeting mitotic progression. Furthermore, this combination prevents polyploidy, a key mechanism of resistance to microtubule targeting agents. Finally, we utilized selective class one HDAC inhibitors to identify the individual contributions of HDACs in the eliciting the responses observed with belinostat treatment. HDAC1&2 inhibition recapitulated the belinostat-resistant phenotype of G1 cell cycle arrest with little apoptosis, in both belinostat-resistant and sensitive cell lines. HDAC3 inhibition resulted in the induction of DNA damage, increased S phase and the induction of apoptosis in belinostat-resistant cells. Belinostat-resistant cells did not have observable effects to HDAC3 inhibitor alone but when combined with vincristine had significantly increased G2/M population at early time points. This suggests that HDAC3 maintains roles in DNA replication and also in mitotic progression. HDAC3 inhibition combined with vincristine resulted in a significant increase in polyploidy, suggesting that HDAC3 might not regulate the expression of apoptotic regulating factors as belinostat does.

Les résultats insuffisants de la radiochimiothérapie conventionnelle dans les cancers bronchiques non à petites cellules (CBNPC) ont motivé l’évaluation d’une nouvelle stratégie de modulation pharmacologique de la radiosensibilité tumorale basée sur les modifications épigénétiques. Pour cela nous avons évalué la combinaison de la radiothérapie et d’un nouvel pan-inhibiteur des histones déacétylases (HDACi), l’abexinostat en préclinique in vitro et in vivo sur deux modèles de CBNPC puis en phase clinique précoce chez l’homme dans le cadre d’un essai de phase I. Nous avons d’abord montré que l’abexinostat augmente la radiosensibilité des cellules de CBNPC de manière dépendante de la séquence thérapeutique en normoxie et en hypoxie en augmentant l’apoptose caspase dépendante ainsi que les cassures doubles brins radio-induites et en réduisant la signalisation et la réparation de ces dommages de l’ADN. L’abexinostat potentialise également le retard de croissance tumorale induit par la radiothérapie in vivo dans des xénogreffes sous-cutanées de CBNPC avec un profil de toxicité acceptable. Nous avons également montré pour la première fois que la triple combinaison de radiothérapie, abexinostat et cisplatine potentialise le retard de croissance tumorale in vivo.Les premiers résultats in vitro et in vivo confortant le rationnel pour la combinaison abexinostat-radiothérapie nous avons réalisé étude de phase I exploratoire d’escalade de dose combinant l’abexinostat à la radiothérapie palliative hypofractionnée standard délivrant 30y en 10 fractions pour des tumeurs solides métastatiques. Parmi les 58 patients traités, d’âge médian 61 ans (20-82), on note 71% de stade M1 et 88% de patients ayant déjà reçu des traitements préalables par chimiothérapie et/ou radiothérapie et/ou chirurgie. La dose recommandée pour un essai de phase 2 que nous avons établie est de 90mg/m². Sur les 51 patients évaluables, on observe un taux de réponse globale de 7,8% (1 réponse complète (RC) et 3 réponses partielles (RP)) et un taux de réponse locorégionale de 11,8% (1 RC et 5 RP) avec un suivi médian de 16 semaines. Les patients présentant des lésions (cibles ou non) cérébrales ont présenté des taux de réponse encourageant avec notamment un patient en RC. Nous avons retrouvé peu d’effets secondaires de grade ≥3, les plus fréquents étant la thrombopénie (17,2%), la lymphopénie (12,1%) et l’hypokaliémie (6,9%). Au total, 6 patients (10%) ont interrompu leur traitement du fait des effets secondaires. Nous n’avons pas observé de prolongation de l’intervalle QTc de grade ≥3 et il n’y a pas eu d’interruption de traitement en rapport avec cet effet secondaire. Dans l’ensemble nos données in vitro et in vivo montrent une potentialisation de l’effet antitumoral par la combinaison d’abexinostat et radiothérapie. Chez les patients présentant des tumeurs solides avancées l’abexinostat oral en combinaison à la radiothérapie est bien toléré. Cette combinaison pourrait avoir un potentiel particulièrement intéressant dans le traitement des métastases cérébrales.De plus nos travaux précliniques suggèrent pour la première fois un effet prometteur d’une triple combinaison avec HDACi, cisplatine et radiothérapie qui justifie de plus amples investigations et pourrait guider de nouvelles stratégies thérapeutiques dans les CBNPC.Nos travaux s’inscrivent dans une stratégie de recherche translationnelle et montrent l’importance de la recherche préclinique pour les études d’association aux rayonnements ionisants. Seuls un développement préclinique rationnel et un développement clinique méthodique permettront l’émergence de combinaisons modulant la radiosensibilité tumorale de manière suffisamment efficace pour modifier nos standards de traitement et améliorer le pronostic de nos patients
Insufficient results of conventional chemoradiotherapy in non-small cell lung carcinomas (NSCLC) have encouraged assessment of new pharmacological strategies for modulation of radiosensitization based on epigenetic changes. We have investigated the combination of radiotherapy and a novel pan-histone deacetylase inhibitor (HDACi), abexinostat in vitro and in vivo in two NSCLC models and in an early phase clinical trial. Our findings demonstrate that abexinostat enhances radiosensitivity of NSCLC cells in a time dependent way in vitro in normoxia and hypoxia increasing radio-induced caspase dependent apoptosis and persistent DNA double strand breaks associated with decreased DNA damage signaling and repair. Interestingly, abexinostat potentiates tumor growth delay in vivo in combined modality treatments associating not only abexinostat and irradiation but also in the triplet combination of abexinostat, irradiation and cisplatin.We conducted an exploratory phase 1, dose-escalation study of abexinostat in combination with standard hypofractionated radiotherapy (30Gy in 10 fractions) in patients with advanced solid tumors treated in a palliative setting. Among 58 treated patients, the median age was 61.5 years (range, 20-82); 47% of the patients had M1 stage disease, and 95% had received previous chemotherapy alone or chemotherapy in combination with surgery and/or radiotherapy. The recommended phase 2 dose was determined to be 90 mg/m2. Of the 51 patients evaluable for response, best overall response was 8% (1 complete response [CR], 3 partial responses [PRs]), and best loco-regional response was 12% (1 CR and 5 PRs) at a median follow-up of 16 weeks. Of note, patients with target or non-target brain lesions showed encouraging responses, with 1 patient achieving a best loco-regional response of CR. Treatment-emergent grade ≥3 adverse events (AEs) were few, with most common being thrombocytopenia (17%), lymphopenia (12%), and hypokalemia (7%). Six patients (10%) discontinued treatment due to AEs. No grade ≥3 prolongation of the QTc interval was observed, with no treatment discontinuations due to this AE.Altogether, our data demonstrate in vitro and in vivo a potentiation of anti-tumor effect by abexinostat in combination with irradiation in NSCLC. Oral abexinostat combined with radiotherapy was well tolerated in patients with advanced solid tumors. The combination may have potential for treatment of patients with brain lesions.Moreover, our work suggest for the first time to our knowledge promising triple combination opportunities with HDACi, irradiation and cisplatin which deserves further investigations and could be of major interest in the treatment of NSCLC.Our studies which are part of a translational research strategy highlight the importance of preclinical investigations in the area of radiotherapy and drug combination research. Only rationale preclinical development and methodologically well conducted clinical development will allow new standards of treatment to emerge and improve patient’s prognostic

Spike timing-dependent plasticity (STDP) has been suggested as one of the key mechanism underlying learning and memory. Due to its importance, timing-dependent plasticity studies have been approached in the living human brain by means of non-invasive brain stimulation (NIBS) protocols such as paired associative stimulation (PAS). However, contrary to STDP studies at a cellular level, functional plasticity induction in the human brain implies the interaction among target cortical networks and investigates plasticity mechanisms at a systems level. This thesis comprises of two independent studies that aim at understanding the importance of considering broad cortical networks when predicting the outcome of timing-dependent associative plasticity induction in the human brain. In the first study we developed a new protocol (ipsilateral PAS (ipsiPAS)) that required timing- and regional-specific information transfer across hemispheres for the induction of timing-dependent plasticity within M1 (see chapter 3). In the second study, we tested the influence of individual brain structure, as measured with voxel-based cortical thickness, on a standard PAS protocol (see chapter 4). In summary, we observed that the near-synchronous associativity taking place within M1 is not the only determinant influencing the outcome of PAS protocols. Rather, the online interaction of the cortical networks integrating information during a PAS intervention determines the outcome of the pairing of inputs in M1.

Protein Methyltransferasen sind oftmals fehlreguliert in Tumorzellen und stellen potenzielle Ziele in der Krebstherapie dar. Das SET und MYND Domain enthaltene Protein 2 (SMYD2) wurde als potenzielles Onkogen beschrieben und eine Überexpression korreliert mit einer schlechten Prognose. Für SMYD2 wurden verschiedene Substrate beschrieben u.a. Histon H3 und der Tumorsuppressor p53, allerdings ist die Biologie dieses Enzymes kaum verstanden. Durch die Entwicklung einer Testsubstanz zur spezifischen Hemmung von SMYD2 könnte ein möglicher therapeutischer Nutzen besser untersucht werden. Hierfür wurde ein zellulärer mechanistischer Test zur Messung der SMYD2 Aktivität mittels eines methylierungs-spezifischen Antikörpers etabliert. Mit Hilfe dieses Tests wurde BAY-598 als selektiver und potenter zellulärer Hemmer für SMYD2 identifiziert. Im weiteren Verlauf dieser Arbeit wurden mittels eines Proteomansatzes nach SMYD2 Überexpression hunderte neue zelluläre Lysinmethylierungen identifiziert. Hierbei wurde das AHNAK Protein als neues SMYD2-Substrat identifiziert und validiert. Die AHNAK Methylierung konnte in verschiedenen Zelllinien und im Muskelgewebe von Mäusen nachgewiesen werden. Im letzten Teil der Arbeit wurde die spezifische Testsubstanz BAY-598 genutzt, um verschiedene in der Literatur aufgekommene Hypothesen zur SMYD2 Funktion zu testen. Die vorliegende Arbeit hat dazu beigetragen die potente und selektive SMYD2 Testsubstanz BAY-598 zu entwickeln. Außerdem wurde mit AHNAK ein neues SMYD2 Substrat identifiziert und validiert. Die Relevanz des SMYD2 Enzymes und der AHNAK Methylierung erfordert weitere Forschungsarbeit, die durch die Bereitstellung der spezifischen Testsubstanz BAY-598 deutlich verbessert werden sollte.
Protein methyltransferases are often misregulated in tumor cells and display a potential target for cancer therapy. The SET and MYND domain containing protein 2 (SMYD2) was described as a potential oncogene and overexpression correlated with a worse prognosis. Several substrates for SMYD2 had been described among them histone H3 and p53. However, the biology of SMYD2 is poorly understood. By developing a small molecule inhibitor of SMYD2 its therapeutic role could be better evaluated. Therefore, a cellular mechanistic assay was developed using a methylation specific antibody. With that assay BAY-598 was identified as a potent and selective cellular inhibitor of SMYD2. In the following a proteomic approach revealed hundreds of novel cellular lysine methylation sites in SMYD2 overexpression cells. Among these AHNAK protein was validated as a novel SMYD2 substrate, which was present in several cell lines as well as in muscle of mice. Finally, BAY-598 was used to test several hypothesized functions of SMYD2 in different cell line models. Taken together, the current work strongly supported the development of the probe inhibitor BAY-598 and the discovery of AHNAK as a novel SMYD2 methylation substrate. The relevance of SMYD2 and AHNAK methylation needs further investigation, which should be supported by BAY-598.

This project contributed to a greater understanding of chemotherapy resistance in non-small cell lung cancer (NSCLC), a prevalent issue to chemotherapy. The potential of the inflammatory transcription factor NF-ĸB as a therapeutic target was investigated to combat NSCLC chemo-resistance using in vitro and in vivo approaches. The activity of NF-ĸB was elucidated in models of chemo-resistant NSCLC and where specific inhibitors of NF-ĸB were used to block activity. In addition, a novel 3D model of chemo-resistance in NSCLC was generated which will serve as an important model in future studies.

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Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico
Ameloblastomas and keratocystic odontogenic tumors (KOT) represent odontogenic lesions that, despite their benign nature, are distinguished by a distinct biological behavior, characterized by locally aggressive growth and recurrent episodes. The gnathic bone resorption caused by the growth of these lesions is a key to the expansion of the same, both being mediated by osteoclastic cells like enzymatic activity of various matrix metalloproteinases (MMPs) factor. The expression of stimulatory factors and inhibitors of bone resorption has been correlated with the development of these lesions, with emphasis to some MMPs such as collagenases and gelatinases and tissue inhibitors of metalloproteinases (TIMPs), among others. Based on the premise that stimulatory and inhibitory factors of osteolytic processes can be decisive for the growth rate of intraosseous odontogenic lesions, this experiment evaluated the immunoreactivity of MMP-9, -13 and TIMP-1 protein in the epithelium and mesenchyme of ameloblastoma and the KOT specimens, by a quantitative analysis of the immunoreactivity cells. Statistical analysis was performed using the Mann-Whitney and Wilcoxon tests with a significance level set at 5 %. Immunohistochemical expression of MMP-9, -13 and TIMP-1 was observed in 100% of cases both in the epithelium and in mesenchyme. The immunoreactivity in the epithelium of KOT and ameloblastomas revealed a predominance of score 3 for MMP-9 (p=0.382) and MMP-13 (p=0.069) and no statistically significance for TIMP-1, the latter being significantly higher immunoreactivity in ameloblastomas. In the mesenchyme, there was a higher score immunoreactivity of MMP-13 (p=0.031) in ameloblastomas in relation to KOT, whereas for MMP-9 and TIMP-1 no statistically significant difference (p=0.403 was observed, p=1.000). The calculation of the ratio of scores revealed expression of proteins in general, similarity of the lesions, a significant predominance of equal expression of TIMP-1 and MMP-9 was observed only in the epithelium of ameloblastoma. The marked immunostaining of MMP-9 , MMP-13 and TIMP-1 in epithelium and mesenchyme of the lesion indicate that these proteins involved in ECM remodeling required for tumor progression, however, specific differences in the expression of some of these proteins, are not sufficient to suggest differences in the biological behavior of ameloblastomas and KOTs
Os ameloblastomas e tumores odontog?nicos ceratoc?sticos (TOC) representam les?es odontog?nicas que, apesar de sua natureza benigna, se destacam por um comportamento biol?gico distinto, caracterizado pelo crescimento localmente agressivo e epis?dios recidivantes. A reabsor??o dos ossos gn?ticos provocada pelo crescimento dessas les?es constitui um fator determinante ? expans?o das mesmas, sendo mediada tanto por c?lulas osteocl?sticas como pela a??o enzim?tica de diversas metaloproteinases de matriz (MMPs). A express?o de fatores estimuladores e inibidores da reabsor??o ?ssea vem sendo correlacionada com o desenvolvimento destas les?es, merecendo destaque algumas MMPs como as colagenases e as gelatinases e os inibidores teciduais de metaloproteinases (TIMPs), dentre outros. Baseados na premissa de que fatores estimuladores e inibidores de processos osteol?ticos podem ser determinantes para o ritmo de crescimento de les?es odontog?nicas intra?sseas, o objetivo de estudo foi avaliar a imunoexpress?o das prote?nas MMP-9, -13 e TIMP-1 no epit?lio e mes?nquima de esp?cimes de ameloblastomas e TOC. A an?lise estat?stica foi realizada atrav?s dos testes de Mann-Whitney e Wilcoxon com n?vel de signific?ncia estabelecido em 5%. Atrav?s de uma an?lise quantitativa das c?lulas imunomarcadas, foi observada a express?o imuno-histoqu?mica das MMP-9, -13 e TIMP-1 em 100% dos casos, tanto no epit?lio quanto no mes?nquima tumoral. Mais de 76% das c?lulas epiteliais (escore 3) dos TOC e ameloblastomas apresentaram imunomarca??o para MMP-9 (p=0,382) e MMP-13 (p=0,069), sendo estatisticamente significativa para o TIMP-1 (p=0,003) nos ameloblastomas. No mes?nquima, observou-se maior escore de imunomarca??o da MMP-13 (p=0,031) nos ameloblastomas em rela??o aos TOC, enquanto para a MMP-9 e TIMP-1 n?o se observou diferen?a estatisticamente significativa (p=0,403; p=1,000). O c?lculo da raz?o entre os escores de express?o das prote?nas revelou, de uma maneira geral, similaridade entre as les?es, sendo observado predom?nio significante de igualdade de express?o do TIMP-1 e da MMP-9 apenas no epit?lio dos ameloblastomas. A imunoexpress?o marcante das MMP-9, MMP-13 e TIMP-1 no epit?lio e mes?nquima das les?es estudadas indica que estas prote?nas participam na remodela??o da MEC necess?ria ? progress?o tumoral, no entanto, as diferen?as pontuais observadas na express?o de algumas destas prote?nas, n?o s?o suficientes para sugerir diferen?as no comportamento biol?gico dos ameloblastomas e dos TOCs

Introdução: Micose fungoide poiquilodérmica (MFp) é uma variante clínica de micose fungoide (MF). É mais indolente e caracterizada pela presença da poiquilodermia. As metaloproteinases (MMP) e seus inibidores específicos TIMP (Tissue Inhibitors of Metaloproteinases) estão envolvidos na oncogênese. Especificamente as MMP2 e MMP9 e seus inibidores, TIMP-2 e TIMP-1, respectivamente, foram relacionados ao prognóstico em tumores. Poucos trabalhos estudaram MMP e nenhum estudou a ação dos TIMP na MF. Objetivos: avaliar a relação entre MMP2 e MMP9 e seus inibidores TIMP2 e TIMP1 e a agressividade da MF e descrever a casuística de micose fungoide poiquilodérmica no ambulatório de linfomas cutâneos da Divisão de Clínica Dermatológica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Métodos: análise retrospectiva de 54 casos de MFp, sendo 25 de MFp localizada 14 de MFp generalizada e 15 de MFp mista. Para análise das MMP e TIMP, os grupos de MFp foram comparados com 7 amostras de pele normal (PN), 10 casos de MF clássica inicial (MFi), 9 casos de MF tumoral não-transformada (MFT nt) e 10 de MF tumoral transformada (MFT t). Resultados: A proporção de mulheres: homens foi 2,44. MFp apresentou maior tempo entre os primeiros sintomas e o diagnóstico. MFpG apresentou maior prevalência de lesões do tipo pitiríase liquenoide crônica (PLC) (79%). Houve alta prevalência de MF hipocromiante (62%) no grupo MFp mista. A histologia da MFp apresentou características típicas de MF e, adicionalmente, atrofia, telangectasias e derrame pigmentar, específicos da forma poiquilodérmica. Na imuno-histoquímica predominou o fenótipo CD3+, CD4+, CD7-, CD8- em todos os grupos, e MFp apresentou significantemente menor predomínio do fenótipo CD8+ que o grupo MFi. O grupo MFpG apresentou baixa positividade para pesquisa de clonalidade T da pele (12,5%). A MMP2 esteve mais presente na epiderme em MFi e MFp relativamente a MFT. Na derme superficial, os grupos MFi e MFp marcaram mais MMP2 que a pele normal, mas sem diferença estatística entre eles. Não houve diferença estatística em MMP2 na derme profunda entre os grupos. À zimografia, houve maior atividade de MMP2 ativa no grupo MFTt. Não houve expressão de TIMP-2 pela epiderme da pele normal. Os grupos MFi e MFp marcaram TIMP-2 na epiderme de forma semelhante, porém menos que os grupos MFT. Na derme superficial, não houve diferença estatística entre os grupos MFi e MFp. TIMP-2 foi mais expresso na derme profunda dos dois grupos de MFT comparativamente a todos os outros grupos. Na epiderme e na derme superficial, MMP9 foi mais expressa no grupo MFi comparativamente a MFp. Na derme profunda, a expressão de MMP9 foi maior nos grupos MFT, seguido por MFi e, por último, MFp. A atividade de MMP9 foi maior no grupo MFT não transformada comparativamente aos outros grupos. TIMP-1, ne epiderme e na derme superficial e na derme profunda foi mais expresso no grupo MFi, comparativamente aos outros grupos. Discussão: MFpG apresentou mais lesões tipo PLC e a forma mista, lesões hipocrômicas. A histologia da MFp foi semelhante à descrita previamente na literatura, mas a baixa positividade de CD8 difere de relatos prévios. A MMP2 pareceu ser um marcador de atividade para MF, principalmente quando a sua presença por imunohistoquímica foi associada a dados de zimografia. A expressão de MMP9 nas amostras foi compatível com os dados prévios de literatura, tendo sido mais expressa nas formas mais agressivas de MF e, histologicamente, mais localizada nos locais de maior atividade do tumor. TIMP-1 foi expresso de forma análoga à MMP9, conforme descrito previamente na literatura. TIMP-2, por sua vez, seguiu o padrão de distribuição de MMP2. No entanto, não foi expresso pela pele normal e foi mais expresso pelos grupos de MFT, o que não ocorreu com a MMP2 na imuno-histoquímica. Conclusões: A expressão de MMP e TIMP correlacionou-se com o local de maior atividade linfocitária e com a agressividade da MF. A atividade da MMP2 e MMP9 foi maior nos grupos MFT comparativamente aos grupos mais indolentes. Separar os casos de MFp de acordo com suas apresentações localizadas, generalizada e mista foi relevante do ponto de vista clínico, laboratorial e evolutivo
Introduction: poikilodermatous mycosis fungoides (pMF) is a clinical variant of mycosis fungoides (MF). It is more indolent than classic MF and is characterized by the presence of poikiloderma. The matrix metalloproteinases (MMPs) and their specific inhibitors TIMP (Tissue Inhibitors of Metalloproteinases) are involved in oncogenesis. Specifically, MMP2 and MMP9 and their inhibitors, TIMP-2 and TIMP-1, respectively, have been related to prognosis in tumors. There are few studies on MMP and none on the role of TIMPs in MF. Objectives: To evaluate if there is a relationship between the presence and activity of MMP2 and MMP9 and their inhibitors TIMP2 and TIMP1, and the aggressiveness of MF. To describe a casuistic of poikilodermatous mycosis fungoides in an outpatient clinic in the Dermatological Division of Hospital das Clinicas of University of Sao Paulo Medical School. Methods: Retrospective analysis of 54 cases of pMF, this included 25 localized pMF (LpMF), 14 generalized pMF (GpMF) and 15 mixed pMF. For the analysis of MMPs and TIMPs, the pMF groups were compared with 7 normal skin samples (NS), 10 cases of initial classical MF (cMF), 9 cases of non-transformed tumor MF (nt MFT) and 10 transformed tumor MF (t MFT). Results: The proportion of women : men was 2.44. The pMFs groups showed a longer period of time from the first symptoms to the diagnosis than the cMF group. The GpMF group had a higher incidence of pityriasis lichenoides chronica-like lesions (PLC) (79%) than the other groups. There was a high incidence of hypopigmented MF (62%) in the mixed pMF group. Histology showed typical characteristics of MF and, additionally, atrophy, telangiectasia and pigmentary alterations compatible with pMF. At immunohistochemistry the cases were predominantly CD3+, CD4+, CD7-, CD8- phenotype in all groups, and the pMF groups had a significantly lower prevalence of CD8+ phenotype than the cMF group. The GPMF group showed low positivity for clonality of the T-cell receptor at the T skin (12.5%) compared to the other groups. The MMP2 was more present in the epidermis for the cMF and pMF groups compared to MFT. In the superficial dermis, the cMF, LpMF and GpMF groups showed more MMP2 than normal skin, however there was no statistical difference between the three groups. There was no statistical difference in the presence of MMP2 in the deep dermis between the groups. The zymography showed higher MMP2 activity in the MFT group. There was no TIMP-2 expression by the normal epidermis. The epidermis of cMF and pMFs groups marked TIMP-2 in a similar way, but at a lower intensity than the MFT groups. In the superficial dermis, there was no statistical difference between the cMF and pMFs groups. TIMP-2 was more expressed in the deep dermis of the two MFT groups compared to all of the other groups. In the epidermis and superficial dermis, the MMP9 was more expressed in cMF compared to pMF groups. In the deep dermis, MMP9 expression was higher in the MFT groups, followed by cMF and finally pMF. The MMP9 activity was higher in the nt MFT group compared to other groups. TIMP-1, in epidermis, superficial dermis and deep dermis was more expressed in the cMF group compared to other groups. Discussion: The study confirmed that the pMF is an indolent form of MF and the time period between the symptoms and the diagnosis in pMF was longer than in classical MF. There were clinical differences amongst the groups of pMF. The GpMF group had a higher prevalence of PLC-like lesions than the mixed form of pMF, which had more hypochromic lesions. Histology of pMF was similar to descriptions provided in other case studies. However, the low CD8 positivity differs from previous reports. The MMP2 appeared to be a marker of activity for MF in our work, especially when their presence by immunohistochemistry was associated with the enzyme activity. The expression of MMP9 in our samples was consistent with previous data from other case studies, being more expressed in the most aggressive forms of MF and histologically more localized in most active sites of the tumor. TIMP-1 was expressed in an analogous manner to MMP9, as previously described in the literature. TIMP-2, in turn, followed the distribution pattern of MMP2. However, it was not expressed by normal skin and was more expressed by the MFT group, which did not occur with the MMP2 in immunohistochemistry. Conclusions: The expression of MMP and TIMP was correlated with the location of higher lymphocyte activity and with the aggressiveness of MF. The activity of MMP2 and MMP9 was higher in the MFT groups than the more indolent groups. It was important to split the pMF cases according to their presentation (GpMF, LpMF and mix pMF) from a clinical, laboratory and prognostic point of view

Viruses are a diverse genera of organisms adapted to thrive in many different hosts from prokaryotic to eukaryotic. We present here the structure of bacteriophage PRR1 virus-like particle (VLP), belonging to Leviviridae family. Our structure reveals calcium ions in the VLP. Metal ions are rare in the VLP among the Leviviridae and the calcium ions were found to affect VLP stability. Gene expression in Leviviridae is controlled by a specific interaction between the viral coat protein that assembles to create the VLP, and the genomic RNA. This interaction has been thoroughly studied for the levivirus MS2 but other structural data are scarce. We have solved the structure of PRR1 VLP in complex with its RNA operator stem-loop. Binding of the stem-loop in PRR1 shows similarities to MS2 but also a different arrangement of the nucleotides, in the area of the loop that we could interpret, compared to MS2. The structures of PRR1 increase our knowledge about translational control in Leviviridae and add new information about particle stability within this family. The other virus we investigated is the more infamous human pathogen, the HIV. Because of the high mutation rate of HIV new drugs are needed on a continuous basis. We describe here the structure of two new protease inhibitors bound to the HIV-1 protease and compare them with two previously published inhibitors. Due to an extended P1´site the new compounds are able to exploit a new interaction to Phe53 in the protease structure.

Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 724

Non-small-cell lung cancer (NSCLC) accounts for 80–85% of all lung cancers and is associated with significant mortality. As epidermal-growth-factor receptor (EGFR) is over-expressed in 80-90% of NSCLC, its inhibition via EGFR-Tyrosine Kinase inhibitors (EGFR-TKIs) is a main therapeutic strategy. However, patients with mutations in KRAS are resistant to EGFR-TKIs. A study in mutant KRAS-driven lung cancer in transgenic mice showed that tumor growth was dependent on the activity of focal adhesion kinase (FAK). Therefore, we hypothesized that KRAS-mutant NSCLC will be sensitive to FAK-TKIs and, given known FAK-EGFR cross-talk, FAK inhibition will sensitize KRAS-mutant NSCLC to EGFR-TKIs. We performed cell viability assays of WT versus mutant KRAS NSCLC cell lines following treatment with FAK-TKI alone or in combination with a clinically relevant EGFR-TKI. We found that KRAS-mutant cells were more sensitive to FAK-TKI than KRAS-WT NSCLC. In addition, we found that the combination treatment including FAK and EGFR TKIs resulted in reduced tumor cell viability as compared to treatment with either drug alone. This enhanced anti-tumor response could be due to FAK-TKI’s ability to down-regulate EGFR downstream targets. Our preliminary data suggests that in KRAS-mutant cells the drug combination appears to more effectively inhibit Akt activity than single drug treatment alone. This suggests an enhanced ability to impair cell survival following treatment with the drug combination. We also found that treatment with FAK TKI in KRAS mutant NSCLC cells resulted in increased activation of EGFR which was due in part to modulation of EGFR recycling and production of endogenous EGFR ligands. Thus, the combination of FAK- and EGFR-TKIs may be more effective in KRAS mutant NSCLC as treatment with EGFR-TKI overcomes the unexpected ‘side effect’ of treatment with FAK-TKI, namely activation of the EGFR pathway by this drug. The findings of our study are novel and have uncovered previously unrecognized outcomes of FAK inhibition on EGFR activity. Moreover, our data support the notion that the combination of FAK- and EGFR-TKIs could be an effective treatment for KRAS mutant NSCLC patients.

The use of a surface plasmon biosensor fills a missing link in kinetic studies of enzymes, since it measures directly the interaction between biomolecules and allows determination of parameters that are determined only indirectly in activity assays. The present thesis deals with kinetic and dynamic aspects of ligand binding to two viral enzymes: the human cytomegalovirus (HCMV) protease and the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). The improved description of interactions presented herein will contribute to the discovery and development of antiviral drugs. The biosensor method provided new insights into the interaction between serine proteases and a peptide substrate, as well as substrate-induced conformational changes of the enzymes. The direct binding assay served as a tool for characterising the binding mechanism of HCMV protease inhibitors. Kinetic details of the interaction between HIV-1 RT and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were unravelled. The recorded sensorgrams revealed several forms of complexity. A general binding model for the analysis was derived from the data, describing a two-state mechanism for the enzyme and a high- and a low-affinity interaction with the inhibitor. Interaction kinetic constants were determined for the clinically used NNRTIs and several investigational inhibitors. The established method was applied to investigate the mechanism of resistance against NNRTIs. Amino acid substitutions in the NNRTI-binding site resulted in both decreased association rates and increased dissociation rates for the inhibitors. The K103N and the L100I substitution also interfered with the formation of the binding site, thereby facilitating inhibitor binding and unbinding. Finally, thermodynamic analysis revealed that, despite the hydrophobic character of the interaction, NNRTI binding was mainly enthalpy-driven at equilibrium. Large entropy contributions in the association and dissociation indicated that binding is associated with a dynamic effect in the enzyme.

La leucémie aiguë à cellules dendritiques plasmacytoïdes (BPDCN) fait partie des cancers incurables pour lesquels les mécanismes impliqués dans la pathogénèse restent inconnus. Dans ce travail, nous avons identifié le gène NR3C1 (5q31), qui code pour le récepteur des glucocorticoïdes (GCR), et un long ARN non-codant inter-génique (appelé ici lincRNA-3q), comme étant des cibles d'altération géniques ou de dérégulation transcriptionnelles dans les BPDCN. La translocation/délétion de NR3C1 est associée avec un temps de survie extrêmement court et des activités anormales du réseau de régulation des gènes GCR, EZH2 et FOXP3. Nous avons découvert que lincRNA-3q code pour une forme nucléaire d'ARN non-codant qui est activé de façon ectopique dans les BPDCN et les AML à haut risque. Dans les cancers myéloïdes, une déplétion de lincRNA-3q induit un arrêt du cycle cellulaire qui coïncide avec la suppression des signatures d'expression génique de E2F1/Rb et des gènes spécifiques aux cellules souches leucémiques. Nos résultats démontrent qu'une inhibition des protéines à bromodomaine BET supprime sélectivement l'expression lincRNA-3q, indiquant une stratégie thérapeutique potentielle pour contrer l'activité oncogénique de cet ARN non-codant. Ce travail défini, un nouveau cadre de recherche pour comprendre la pathogénèse et la résistance au traitement dans les BPDCN
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an incurable malignancy for which disease mechanisms are unknown. Here, we identify the NR3C1 gene (5q31), encoding the glucocorticoid receptor (GCR), and a long, intergenic, non-coding RNA gene (named here lincRNA-3q), respectively, as targets for genetic alteration or transcriptional deregulation in BPDCN. NR3C1 translocation/deletion was associated to critically short survival in BPDCN and to abnormal activity of GCR, EZH2, and FOXP3 gene regulatory networks. LincRNA-3q, was found to encode a nuclear, non- coding RNA that is ectopically activated in BPDCN and high-risk AML. Depletion of lincRNA-3q in myeloid cancer cells induced cell cycle arrest, coincident to suppression of E2F1/Rb and leukemia stem cell-specific gene expression signatures. BET bromodomain protein inhibition could selectively suppress lincRNA-3q indicating a treatment strategy for counteracting oncogenic activity of this non- coding RNA. Thus, this work defines a new framework for understanding disease pathogenesis and treatment resistance in BPDCN

Introduction : Le traitement des cancers bronchiques non à petites cellules (CBNPC) EGFR mutés repose sur les inhibiteurs de tyrosine kinase (ITK) du récepteur de l’Epidermal Growth Factor (EGFR). Cependant tous les patients traités par ITK EGFR finissent par présenter une progression tumorale, du fait de mécanismes de résistance comme l’amplification du gène codant pour le récepteur tyrosine kinase MET. Il n’existe actuellement aucune donnée sur les modifications phénotypiques induites par l’activation de MET dans ce contexte. L’objectif de cette thèse est de déterminer si l’amplification de MET, lors de la résistance aux ITK EGFR dans les CBNPC EGFR mutés, confère aux cellules tumorales un phénotype plus agressif et modifie l’histoire naturelle de la maladie.Méthodes : Les capacités de prolifération, de croissance sans ancrage, de formation de sphéroïdes, de résistance à l’anoïkis et de migration ont été étudiées in vitro dans la lignée HCC827, dérivée d’un CBNPC EGFR muté, et dans sa lignée fille HCC827-GR6 (GR6) devenue résistante aux ITK EGFR via une amplification du gène MET. L’expression de la vimentine, de ZEB1, et de la E-cadherine a également été étudiée dans les deux lignées cellulaires afin d’évaluer l’impact de l’amplification de MET sur la transition épithélio-mésenchymateuse (TEM). In vivo la croissance tumorale et le potentiel métastatique ont respectivement été analysés dans des modèles murins de xénogreffe ectopique et d’injection intracardiaque. Enfin les données cliniques de patients issus de 15 centres avec un CBNPC EGFR muté métastatique, présentant une forte surexpression de MET en immunohistochimie (score 3+) ou une amplification de MET en FISH sur une re-biopsie réalisée après la progression sous ITK EGFR ont été analysées rétrospectivement. Résultats : In vitro, l’amplification de MET induisait une augmentation significative de la prolifération, de la croissance sans ancrage, de la formation de sphéroïdes, de la résistance à l’anoïkis et de la migration. En présence d’un inhibiteur de MET, le PHA-665752, ces différentes propriétés biologiques étaient réduites de façon significative dans les cellules GR6 porteuses de l’amplification de MET. Il était également mis en évidence dans les cellules GR6 une augmentation de l’expression de la vimentine et de ZEB1. In vivo, l’amplification de MET augmentait significativement la croissance tumorale et le potentiel métastatique. Un traitement par crizotinib, ITK ciblant MET, diminuait de façon significative le potentiel métastatique des cellules porteuses de l’amplification de MET. Enfin les patients atteints d’un CBNPC EGFR muté, porteur d’une amplification de MET à la résistance à l’ITK EGFR, présentaient une durée jusqu’à apparition de nouvelles métastases plus courte après progression sous ITK EGFR que les patients avec une forte surexpression de MET sans amplification génique. Conclusion L’amplification de MET dans un contexte de résistance aux ITK EGFR est associée à un phénotype tumoral plus agressif. Ces résultats plaident en faveur d’une utilisation précoce d’inhibiteurs de MET en association avec les ITK EGFR afin d’éviter l’émergence d’un clone tumoral résistant plus agressif
Introduction: Treatment of Epidermal Growth Factor Receptor (EGFR) mutated non-small cell lung cancers (NSCLC) relies on EGFR tyrosine kinase inhibitors (TKI). However, all patients treated with EGFR TKI eventually present tumor progression, due to mechanisms of resistance such as the MET amplification. There is currently no data on phenotypic changes induced by MET activation in this context. The objective of this thesis is to determine whether the MET amplification during EGFR TKI resistance in the EGFR mutated NSCLC induces a more aggressive phenotype in tumor cells and alters the natural history of the disease.Methods: Proliferation, anchorage independent growth, spheroid formation, anoïkis resistance and migration were studied in vitro in the HCC827 cell line, derived from an EGFR mutated NSCLC, and in its daughter cell line HCC827-GR6 (GR6) which became resistant to EGFR TKI through MET amplification. The expression of vimentin, ZEB1, and E-cadherin was evaluated in these cellular models in order to investigate an epithelial to mesenchymal transition (EMT) process induced by the MET amplification. In vivo, the tumor growth and the metastatic potential were analyzed by subcutaneous xenograft and intracardiac injection in mouse models. Finally, the clinical data of patients from 15 centers with a metastatic EGFR mutated NSCLC, exhibiting high MET overexpression in immunohistochemistry (score 3+) or MET amplification assessed by FISH on a re-biopsy performed after TKI EGFR progression were analyzed retrospectively.Results: In vitro, the MET amplification induced a significant increase in proliferation, anchorage independent growth, spheroid formation, anoïkis resistance and migration. Treatment with PHA-665752, a MET TKI, significantly reduced these biological properties in the GR6 cells harboring the MET amplification. An increase in the expression of vimentin and ZEB1 was also observed in the GR6 cells. In vivo, the MET amplification significantly increased the tumor growth and the metastatic potential. Treatment with crizotinib, another MET TKI, significantly decreased the metastatic potential of cells carrying MET amplification. Finally, patients with an EGFR mutated NSCLC, displayed a time to new metastases after TKI EGFR progression shorter than patients with high MET overexpression without MET amplification.Conclusion: The MET amplification during EGFR TKI resistance is associated in EGFR muted NSCLC with a more aggressive tumor phenotype. These results argue for the early use of MET inhibitors in combination with EGFR TKIs to avoid the emergence of a more aggressive resistant tumor clone

Les statines sont des médicaments hypolipémiants largement prescrits dans le cadre des maladies cardiovasculaires. Elles inhibent la synthèse endogène de cholestérol et induisent l’activation de l’expression du LDLR par le facteur de transcription SREBP-2. Ce sont des médicaments dont le bénéfice est indiscutable d’un point de vue cardiovasculaire. Néanmoins, l’action des statines est limitée par la proprotéine convertase subtilisin kexin type 9 (PCSK9), l’inhibiteur naturel du récepteur aux LDL (LDLR), qui est également sous la dépendance de SREBP-2. Ainsi, de nouvelles stratégies hypolipémiantes ciblant la forme circulante de PCSK9 ont été développées et autorisées. Ce sont les inhibiteurs de PCSK9. Bien que bénéfique, l’utilisation de statines à forte dose et à long terme induit dans certains cas l’apparition d’un diabète de type 2 chez les individus prédisposés. De plus, des variants génétiques « perte de fonction » de PCSK9 sont associés à l’apparition du diabète de type 2. Les effets des inhibiteurs de PCSK9 sur la survenue du diabète de type 2 à long terme ne sont pas connus à ce jour. Ainsi, nous avons émis l’hypothèse que l’engorgement en cholestérol des cellules β pancréatiques associé à des niveaux très élevés d’abondance du LDLR à la membrane suite à l’utilisation des statines et d’inhibiteurs de PCSK9 induit un dysfonctionnement cellulaire, une altération de la sécrétion d’insuline et à terme le diabète de type 2. Les objectifs de mes travaux de thèse ont été (i) de déterminer la modulation des taux de PCSK9 par les statines chez des individus diabétiques de type 2, (ii) d’étudier si les niveaux circulants réduits en PCSK9 seraient prédictifs de la survenue du diabète de type 2 et enfin (iii) d’évaluer l’effet des statines, de PCSK9 et des inhibiteurs de PCSK9 sur la sécrétion d’insuline par les cellules β. Au moyen de trois cohortes de patients, nous avons montré que la concentration plasmatique de PCSK9 est augmentée chez les diabétiques de types 2 et que les niveaux de PCSK9 circulants réduits sont négativement associés à une résistance à l’insuline et à une altération de la glycémie à jeun. En utilisant des sections de pancréas humains et des lignées de cellules β pancréatiques humaines, nous avons démontré pour la première fois que PCSK9 est exprimé, synthétisé et sécrété uniquement par les cellules β pancréatiques au niveau des îlots de Langerhans. Nous n’avons pas observé d’effet de PCSK9 et de ses inhibiteurs sur la sécrétion d’insuline en réponse au glucose. L’ensemble des résultats de mes travaux de thèse indiquent que l’inhibition de PCSK9 n’aura vraisemblablement pas d’effet pro-diabétogène, ce qui est rassurant pour le développement de ces nouvelles thérapies hypocholestérolémiants
Statins are lipid-lowering drugs widely prescribed to prevent cardiovascular diseases. They inhibit the endogenous synthesis of cholesterol and thereby increase LDLR gene expression by activating the SREPB-2 transcription factor. The positive effects of statins regarding cardiovascular diseases are undisputable. However, their action is limited by the proprotein convertases subtilisin kexin type 9 (PCSK9), the natural inhibitor of the LDL receptor (LDLR), which is also activated by the SREBP-2 transcription factor. As a result, novel lipid-lowering strategies targeting circulating PCSK9 have emerged and have been approved recently. These are the PCSK9 inhibitors. Despite their well-established beneficial effects, the use of high doses of statins for long-term treatments induces in rare instances the onset of type 2 diabetes in predisposed individuals. In addition, “loss of function” genetic variants of PCSK9 are associated with an increased risk of type 2 diabetes. The effects of long term use of PCSK9 inhibitors on the risk of type 2 diabetes remain to be established. Thus, we hypothesized that cholesterol overload of insulin secreting pancreatic beta cells induced by the overexpression of the LDLR at their plasma membranes following treatment with statins and PCSK9 inhibitors may cause cell dysfunction, lower insulin secretion, and ultimately type 2 diabetes. The aims of my thesis were (i) to determine the circulating levels of PCSK9 and their modulation by statins in patients with type 2 diabetes, (ii) to determine if reduced circulating PCSK9 levels are predictive of new onset type 2 diabetes and finally (iii) to investigate the effect of statins, PCSK9, and PCSK9 inhibitors on beta cell function. Using three cohorts of patients, we showed that circulating PCSK9 plasma levels are increased in patients with type 2 diabetes and that reduced circulating PCSK9 levels are negatively associated with insulin resistance and elevated fasting blood glucose. In human pancreatic sections and human pancreatic beta cell lines, we showed for the first time that PCSK9 is expressed, synthesized and secreted only by beta cells in pancreatic islets. We did not find any significant effect of PCSK9 or PCSK9 inhibitors on glucose stimulated insulin secretion. Altogether, my thesis works underpin that the use of PCSK9 inhibitors in the clinic will probably not be diabetogenic. This is reassuring regarding the development of these new lipid-lowering therapies