Orientador: Cristina Antoniali Silva / Coorientadora: Lusiane Maria Bendhack / Banca: Graziela Scalianti Ceravolo / Banca: Michele Mazzaron de Castro / Resumo: A enzima NAD(P)H oxidase (NOX) é a principal fonte de espécies reativas de oxigênio (ERO) no sistema cardiovascular e sua atividade e expressão podem ser regulada pela angiotensina (Ang) II. Demonstramos previamente que o tratamento crônico com apocinina, um inibidor de NOX, reduziu a pressão arterial e preveniu o desenvolvimento da disfunção endotelial em SHR. Estes efeitos da apocinina foram associados a redução de geração de ERO e ao aumento da biodisponibilidade de óxido nítrico em células endoteliais de SHR. Dados de nosso laboratório mostraram que o tratamento com apocinina, também reduziu o efeito pressor da Ang II em SHR. A associação entre Ang II, via receptores AT1, e o estresse oxidativo tem sido implicada na patogênese da hipertensão. Levantamos a hipótese que a apocinina, ao alterar a sinalização redox, reduz a expressão de receptores AT1 e a resposta vasoconstritora da Ang II em SHR. Neste estudo, avaliamos o efeito do tratamento crônico com apocinina sobre as respostas contráteis à Ang II em vasos sanguíneos de SHR e os mecanismos envolvidos nestes efeitos, utilizando ensaios bioquímicos, biomoleculares e funcionais. SHR foram tratados com apocinina (30 mg/Kg, v.o.) da 4ª a 10ª semana de vida e ratos Wistar foram utilizados como controle normotenso. Analisamos os efeitos da apocinina na capacidade antioxidante plasmática, expressão de NOX, geração de ERO, níveis de nitrato/ nitrito, expressão de receptores AT1 e AT2, e respostas vasoconstritor... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: NAD(P)H oxidase (NOX) is the major source of reactive oxygen species (ROS) in the cardiovascular system and its activity and expression could be regulated by angiotensin (Ang) II. We previously demonstrated that chronic treatment with apocynin, a NOX inhibitor, reduced blood pressure and prevented the development of endothelial dysfunction in SHR. These effects of apocynin have been associated with reduced generation of ROS and increased bioavailability of nitric oxide in endothelial cells of SHR. Data from our laboratory showed that treatment with apocynin also reduced the pressor effect of Ang II on SHR. The association between Ang II, via AT1 receptors, and oxidative stress has been implicated in the pathogenesis of hypertension. We hypothesized that apocynin, altering redox signaling, could reduce expression of AT1 receptors and Ang II vasoconstrictor response in SHR. In this study, we evaluated the effect of chronic treatment with apocynin on the contractile responses to Ang II in blood vessels of SHR and the mechanisms involved in the effects of these on biochemical, biomolecular and functional assays. SHR were treated with apocynin (30 mg/kg, p.o.) from the 4 th to the 10th week of life and Wistar rats were used as normotensive control. Analysis of the effects of apocynin on plasma antioxidant capacity, NOX expression, ROS generation, nitrate/ nitrite levels, expression of AT1 and AT2 receptors, and vasoconstrictor responses to Ang II on mesenteric and aortic arteries.... (Complete abstract click electronic access below) / Mestre

O microambiente tumoral é composto por células, como fibroblastos, células do sistema imune, células endoteliais e pericitos, envoltas por uma matriz extracelular, além de possuir fatores solúveis que participam da comunicação celular. Nas últimas décadas, têm-se entendido cada vez melhor seu papel na iniciação e progressão dos tumores. É de fundamental importância, portanto, entender a biologia dos seus componentes e como podem agir em favor do desenvolvimento tumoral. Diversos trabalhos demonstram que há uma associação entre a presença dos pericitos nos vasos tumorais com a agressividade e prognóstico de alguns tipos de câncer. Uma vez ativadas, além do papel estrutural, essas células modulam as atividades das células endoteliais durante a formação de novos vasos, além de adquirirem propriedades como proliferação e migração. Neste contexto, os pericitos passam a secretar fatores importantes na comunicação célula-a-célula e liberam enzimas moduladoras na matriz extracelular. A lisil oxidase (LOX) é uma das principais enzimas que atuam sobre a matriz extracelular. Já está bem descrito que, quando superexpressa em células tumorais, a LOX pode alterar a migração e invasão dessas células, promovendo a geração de metástases. Entretanto, pouco se sabe a respeito da atuação dessa enzima sobre os demais componentes celulares do estroma tumoral, como os pericitos. Sendo assim, o presente trabalho teve como objetivo principal verificar se enzima LOX é relevante para a ativação de propriedades dos pericitos que possam contribuir para suas funções pró-tumorigênicas, como migração, proliferação e formação de vasos. Os resultados foram gerados avaliando essas atividades dos pericitos após pré-tratamento de 24 horas com β-aminopropionitrile (βAPN), um inibidor irreversível da LOX. Foram utilizadas duas linhagens de pericitos derivados de tecido normal (adiposo e muscular) e duas linhagens de pericitos provenientes de tecido tumores do sistema nervoso central (neuroblastoma e ependimoma). Este composto foi capaz de diminuir a capacidade de migração das células de todas as linhagens testadas e, de maneira geral, tornou o processo de formação de estruturas tubulares in vitro menos eficiente. Entretanto, não foram observadas alterações na proliferação celular. Os dados indicam, portanto, que a enzima LOX pode ser importante para a ativação dos pericitos e, possivelmente, influenciem no seu comportamento no microambiente tumoral / The tumor microenvironment is composed of non-cancer cells, such as fibroblasts, immune cells, endothelial cells and pericytes, surrounded by an extracellular matrix, in addition to soluble factors involved in cellular crosstalk. In the last decades, it has been better understood its role in the initiation and progression of tumors. It is critical, therefore, to understand the biology of its components and how they can act in favor of tumor development. Several studies show an association between the presence of pericytes in tumor vessels with aggressiveness and prognosis of some cancers. Once activated, these cells modulate the activities of endothelial cells during the new vessels formation, and acquire properties as proliferation and migration. In this context, pericytes triggers the secretion of important factors in cell-to-cell communication and release modulating enzymes of extracellular matrix. The lysyl oxidase (LOX) is one of the main enzymes that act on the extracelular matrix. It is well described that when overexpressed in tumor cells, LOX can alter the migration and invasion of these cells, promoting the generation of metastases. However, little is known about the role of this enzyme over other cellular components of the tumor stroma, such as pericytes. Therefore, the aim of this study was to verify whether LOX enzyme is relevant to the activation of properties of the pericytes that could contribute to its pro-tumorigenic functions such as migration, proliferation and vessel formation. All the results were generated by evaluation of the activities of these pericytes after 24 hours pretreatment with β-aminopropionitrile (βAPN), an irreversible inhibitor of LOX. This study used two cell lines of pericytes derived from normal tissue (fat and muscle) and two isolated from tissue of the central nervous system. The βAPN was able to reduce the migration of cells of all tested cell lines and, in general, alter the tubular formation in vitro. However, changes in cell proliferation weren′t observed. The data showed, that the LOX family may be important for the activation of pericytes and possibly influence on their behavior in the tumor microenvironment

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-06-30T12:19:46Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Georgia Maria Ricardo Felix dos Santos_Mestrado.pdf: 1018948 bytes, checksum: eaff2e3a96759aec9a4685114c9635cb (MD5) / Made available in DSpace on 2016-06-30T12:19:46Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Georgia Maria Ricardo Felix dos Santos_Mestrado.pdf: 1018948 bytes, checksum: eaff2e3a96759aec9a4685114c9635cb (MD5) Previous issue date: 2013-08-15 / CAPEs / Elevados níveis de hiperglicemia durante a gestação pode ter consequências deletérias para prole que inclui doenças cardiovasculares (DCV) e renais. As complicações cardiovasculares do diabetes resultam de um conjunto de fatores que envolvem a hiperglicemia crônica, a formação excessiva dos produtos finais de glicação avançada (AGEs) e de oxidação proteica (AOPP) e a ativação do fator de transcrição nuclear kappaB (NF-κB), associada à liberação de citocinas pró-inflamatórias. Portanto, é de grande relevância a elucidação de mecanismos envolvidos na gênese e/ou manutenção do processo oxidativo que pode ser perpetuada na prole exposta à hiperglicemia. O objetivo deste estudo foi investigar os efeitos da exposição intra-uterina a hiperglicemia (> 200 mg/dL) sobre AGEs, AOPP, bem como a expressão proteica do NF-κB, a expressão gênica da interleucina-1 beta (IL-1ß) e Nox 4 (homólogo da subunidade da NADPH oxidase) na prole adulta. Para isto, o DG foi induzido no sétimo dia de gestação com dose única de estreptozotocina (STZ, 42mg/kg, i.p.) dissolvida em tampão citrato (veículo). Fêmeas controles receberam apenas veículo. A prole foi dividida em dois grupos de acordo com o tratamento da mãe: controle não diabético (ND) e diabetes gestacional (DG). Na idade adulta, foram realizadas as dosagens de glicose, triglicerídeos, colesterol total e HDL. A concentração plasmática de AGEs foi determinada por fluorescência e a de AOPP por espectrofotometria, os níveis de peroxidação lipídica pelas substâncias reativas ao ácido tiobarbitúrico (TBARS). A expressão proteica do NF-κB foi analisada em aortas através de Western Blot. Foi utilizada reação em cadeia de polimerase quantitativa em tempo real (RTq-PCR) para avaliar a expressão gênica da IL-1ß e Nox 4. Os níveis de glicose, triglicerídeos, colesterol total foram significativamente (p < 0,05) maiores na prole DG quando comparadas a prole ND. A prole DG apresentaram níveis elevados de AGEs (p < 0,01), AOPP (p < 0,05) e TBARS (p < 0,05). Em aorta, a expressão proteica do NF-κB foi mais elevada (p < 0,05) no grupo DG do que no ND. Adicionalmente, foi observado um aumento da expressão gênica da IL-1ß e da Nox4. O número de néfrons desses animais foi significativamente reduzido (p < 0,001). A associação de níveis elevados de estresse oxidativo e interleucina-1 beta podem ser responsáveis pela estimulação da expressão proteica do NF-κB. Juntas, essas alterações em um ambiente hiperglicêmico, com indícios de comprometimento renal, poderiam desencadear alterações vasculares. / Experimental evidences support the hypothesis that diseases can be programmed during the period intrauterine. This concept of programming fetal has been used as basis for knowledge of some chronic diseases, such the cardiovascular. Cardiovascular disease has been associated with hyperglycemia, increased level of advanced glycation end products (AGE) and of advanced oxidation products (AOPP) activate the transcription factor nuclear factor-kappaB (NF-κB) associated with inflammatory cytokine liberation. Therefore, the present study was designed to examine the alterations produced by maternal diabetes over the AGEs, AOPP, NF-κB protein expression, and mRNA expressions of IL-1β and Nox4 in adult offspring rats. Maternal diabetes was induced in female Wistar rats with a single dose of streptozotocin (STZ; 42 mg/kg, i.p.) on the 7th day of pregnancy. Control animals only received STZ vehicle. The offspring were divided into two groups: control non-diabetic (ND) and gestational diabetes (DG). Plasma levels of glucose, total cholesterol, HDL cholesterol and triglycerides were analyzed through biochemical measurements. Plasma levels of AGEs and AOPPs were detected using spectrofluorimetry and spectrophotometry, respectively. Lipid peroxidation was assessed in liver tissue by measuring thiobarbituric acid reactive substances (TBARS). The NF-κB protein expression was evaluated by Western blot. The mRNA levels of IL-1β and Nox4 were detected quantitatively by real time polymerase chain reaction (real-time PCR). In the DG offspring, it was observed that plasma levels of glucose, total cholesterol, HDL cholesterol and triglycerides were higher when compared to the control group. AGEs, AOPP and TBARS concentration were significantly higher in animals exposed to the DG compared with ND. The NF-κB protein expression was higher (p < 0,05) in aorta from DG than in ND. In addition, the expression of IL-1β and Nox4 mRNA was elevated. The number of nephron in DG was significantly reduced (p < 0,001). These results also support the involvement of oxidative stress species in IL-1β induced NF-κB expression. Together, these data suggest that hyperglycemia is a risk factor for vascular alterations.

Orientador: Jose de Assis Fonseca Faria / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-16T06:09:00Z (GMT). No. of bitstreams: 1 Cruz_AdrianoGomesda_D.pdf: 921924 bytes, checksum: ebf67e7ba3e88f34e01497fcc8032c40 (MD5) Previous issue date: 2010 / Resumo: A incorporação e a viabilidade de bactérias probióticas em alimentos ao longo do seu período de estocagem, que resultem em benefício para a saúde do consumidor, é um desafio constante para as indústrias de alimentos, requerendo a compreensão dos fatores intrínsecos e extrínsecos ao processamento. A exposição ao oxigênio apresenta-se como um fator relevante, na medida em que esse grupo microbiano apresenta metabolismo anaeróbio e/ou microaerófio, o que pode resultar em morte celular e perda da funcionalidade do produto. Este trabalho teve o objetivo de avaliar o efeito da glicose oxidase sobre o aumento da sobrevivência das bactérias probióticas em iogurte, bem como em seus parâmetros de qualidade e aceitação sensorial. Foi observado o efeito potencial da glicose oxidase na redução do oxigênio dissolvido no iogurte bem como na manutenção da sua funcionalidade, sem interferência nos parâmetros de qualidade e aceitação sensorial do produto / Abstract: The incorporation and viability of probiotic bacteria in processed food during its storage period is a constant challenge for the food industry, and thus requiring an understanding of intrinsic and extrinsic factors in relation to processing. The oxygen exposure is presented as a relevant factor, since the microbial group presents anaerobic metabolism, which can result in cell death and loss of product functionality. This study aimed to evaluate the effect of glucose oxidase as oxygen absorver for increasing the survival of probiotic bacteria in yogurt as well as its effect on the quality parameters and sensory acceptability. It was noted the potential effect of glucose oxidase in the reduction of dissolved oxygen in yogurt and in maintaining its functionality, without interference on quality parameters and sensory acceptability of the product / Doutorado / Doutor em Tecnologia de Alimentos

Magister Pharmaceuticae - MPharm / Alzheimer's disease is a progressive neurodegenerative disease characterised by low acetylcholine (ACh) levels in the hippocampus and cortex of the brain, causing symptoms like progressive memory loss, decline in language skills and other cognitive impairments to occur. The hallmarks of AD include the presence of extracellular insoluble amyloid beta plaques, intracellular neurofibrillary tangles, and the decrease in ACh concentration. The pathophysiology of AD is not well understood, however, acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and monoamine oxidases (MAO) are conspicuous role players in AD pathogenesis. Based on the cholinergic hypothesis, the AChE inhibitor donepezil was developed and has been used effectively clinically in the management of AD, with minimal side effects. Studies regarding the binding interactions of donepezil with AChE has shown that the benzyl-piperidine moiety of this compound shows substantial binding interactions at the CAS site of AChE where it blocks AChE activity. Coumarin is a compound of natural source that has shown some MAO inhibitory activity. Further studies done to clarify the potential of coumarin as a drug against AD has shown that coumarin has the capacity to bind at the PAS site of AChE, thus giving it the potential to prevent AChE induced amyloid plaque formation. Due to the multifactorial nature of AD, the drugs in the market show limited therapeutic benefits and are mainly for symptomatic relief. In order to address this limitation in AD treatment, researchers are exploring the possibility of designing a multi-target-directed-ligand (MTDL). The aim of this study was to synthesise a series of compounds out of pharmacophoric groups of donepezil and coumarin that will be able to inhibit both cholinesterases and MAO B. Four series of 5 compounds per series were synthesised. The first series of compounds consisted of the coumarin moiety to which a 1,4-dibromo benzene moiety was attached. The second series represented the coumarin moiety to which a piperidine (donepezil moiety shown to confer cholinesterase inhibitory property) was attached. The third series represented the coumarin moiety to which bromobenzyl-piperazine was attached and in the last series were compounds similar in structure to series 1 with an unsubstituted benzyl moiety as opposed to the dibromobenzyl moiety. Prior to the synthesis, molecular modelling was conducted in order to have an idea of the binding capacity of the compounds to MAO A and B and cholinesterases. In vitro biological evaluation of the compounds was done and used to determine the IC₅₀ values of the compounds. Nineteen compounds were synthesised and purified successfully as shown by their NMR, MS and IR spectra. The compounds to which dual inhibitory activity was conferred were those in series 2 and 3, of which series 2 showed the best overall inhibitory activity with IC₅₀ values within the low μM range. The compound with the best overall activity was Cp 9. Molecular modelling of Cp 9 showed that the coumarin core was located in the PAS region of AChE while the benzyl-piperidine moiety was situated in the CAS region of the enzyme. This compound orientation demonstrates the potential of Cp 9 to inhibit AChE induced amyloid beta plaque formation. Cp 9 showed no inhibitory activity towards MAO A, but showed good inhibitory activity towards MAO B with an IC₅₀ value of 0.30 μM. Its inhibitory activity towards cholinesterases also fell within the low μM range (AChE IC50 = 9.1 μM and BuChE IC₅₀ = 5.9 μM). From the results, it can be concluded that Cp 9 was able to inhibit both cholinesterase and MAO B catalytic activities at low μM concentrations. This thus means that our novel compound will not only increase ACh levels in the brain thus improving cognitive activity, but it will also have neuroprotective effect from its MAO B inhibitory property and also potentially slow down amyloid plaque formation due to AChE activity. / National Research Foundation (NRF)

This work describes the integration of a separation capillary for capillary electrophoresis (CE) with an on-column enzyme reactor for selective determination of the enzyme substrate. The enzyme reaction occurs during a capillary separation, allowing selective determination of the substrate in complex samples without the need for pre- or post- separation chemical modification of the analyte. The overall goal of this work is to develop a system in which sample introduction, separation of the analyte/substrate from other biological species, enzymatic conversion of the analyte/substrate into a detectable product, and sensitive detection are all included within a single analysis scheme. Immobilization of the enzyme is achieved by electrostatic assembly of poly(diallydimethylammonium chloride) (PDDA) followed by adsorption of a mixture of the negatively charged enzyme glucose oxidase (GOx) and anionic poly(styrenesulfonate) (PSS). The reaction of glucose with the immobilized glucose oxidase produces H2O2 which migrates the length of the capillary under the influence of electroosmotic flow and is detected amperometrically at the capillary outlet. The optimal response, kinetics, and stability for the enzyme reactor are determined through characterization of several parameters including the concentration ratio of PSS:GOx, applied separation voltage, and the inner diameter of the separation capillary. Various analyte mixtures containing the substrate and other biological species were evaluated to illustrate selective separation and determination of the substrate from other biomolecules. Optimization of this electrostatically assembled capillary enzyme reactor lead to application of these parameters to similar enzymes such as glutamate oxidase. Future application to similar enzymes like L-amino acid oxidase and possible microfluidic systems is a long-term goal of the system. / Ph. D.

No / Aromatic aldehydes are good substrates of aldehyde dehydrogenase activity but are relatively poor substrates of aldehyde oxidase and xanthine oxidase. However, the oxidation of xenobiotic-derived aromatic aldehydes by thelatter enzymes has not been studied to any great extent. The present investigation compares the relative contribution of aldehyde dehydrogenase, aldehyde oxidase and xanthine oxidase activities in the oxidation of isovanillin in separate preparations and also in freshly prepared and cryopreserved liver slices. The oxidation of isovanillin was also examined in the presence of specific inhibitors of each oxidizing enzyme. Minimal transformation of isovanillin to isovanillic acid was observed in partially purified aldehyde oxidase, which is thought to be due to residual xanthine oxidase activity. Isovanillin was rapidly metabolized to isovanillic acid by high amounts of purified xanthine oxidase, but only low amounts are present in guinea pig liver fraction. Thus the contribution of xanthine oxidase to isovanillin oxidation in guinea pig is very low. In contrast, isovanillin was rapidly catalyzed to isovanillic acid by guinea pig liver aldehyde dehydrogenase activity. The inhibitor studies revealed that isovanillin was predominantly metabolized by aldehyde dehydrogenase activity. The oxidation of xenobiotic-derived aromatic aldehydes with freshly prepared or cryopreserved liver slices has not been previously reported. In freshly prepared liver slices, isovanillin was rapidly converted to isovanillic acid, whereas the conversion was very slow in cryopreserved liver slices due to low aldehyde dehydrogenase activity. The formation of isovanillic acid was not altered by allopurinol, but considerably inhibited by disulfiram. It is therefore concluded that isovanillin is predominantly metabolized by aldehyde dehydrogenase activity, with minimal contribution from either aldehyde oxidase or xanthine oxidase.

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