O objetivo do presente trabalho foi quantificar, in vitro e in vivo, a endotoxina bacteriana (LPS) aderida a ligaduras ortodônticas elastoméricas estéticas de poliuretano e de silicone, empregando o teste Limulus Amebocyte Lysate (LAL). Para o estudo in vitro foram utilizadas quatro tipos de ligaduras elastoméricas estéticas: Sani-Ties (poliuretano) e Sili-Ties (silicone), da GAC, e Mini Single Case Ligature Stick (poliuretano) e Synergy&reg; Low-friction ligatures (silicone), da Rock Mountain, sendo 5 contaminadas com solução de endotoxina (controle positivo) e 5 não-contaminadas (controle negativo). Réplicas feitas de fio de amarrilho torcido e de aço inox fundido, de mesmo tamanho e formato das ligaduras elastoméricas, contaminadas ou não com endotoxina, foram usadas como controle. A quantificação de endotoxina foi realizada por meio do teste LAL (Kit QCL-1000&trade;), sendo os resultados expressos em unidades de endotoxina (UE/mL). No estudo in vivo, 20 pacientes de ambos os gêneros, com faixa etária entre 15 e 30 anos, que iniciaram tratamento com aparelho ortodôntico fixo na Faculdade de Odontologia de Ribeirão Preto - Universidade de São Paulo receberam, randomicamente, em quadrantes alternados, os mesmos quatro tipos de ligaduras elastoméricas utilizadas no estudo in vitro, sendo uma ligadura de cada marca inserida nos caninos superiores e inferiores (13, 23, 33 e 43), aleatoriamente. Vinte e um dias após, as ligaduras foram removidas e processadas para quantificação da endotoxina bacteriana, utilizando também o Kit QCL-1000&trade;. Todos os dados obtidos foram submetidos à análise estatística apropriada de acordo com a distribuição dos dados, por meio dos testes de Kruskal-Wallis e pós-teste de Dunn (estudo in vitro) e ANOVA de medidas repetidas e pós-teste de Tukey (estudo in vivo). Todas as análises foram efetuadas por meio do programa Graph Pad Prism 4.0, com nível de significância de 5%. De acordo com os resultados obtidos, observou-se que a endotoxina bacteriana aderiu-se a todos os materiais testados. No estudo in vitro, o grupo GAC silicone foi o que apresentou menor mediana de contaminação (1,15 UE/mL), com relação aos outros grupos (p<0,0001), os quais não apresentaram diferença estatisticamente significante quando comparados entre si (p>0,05). No estudo in vivo, de maneira semelhante ao observado no estudo in vitro, o grupo GAC silicone foi o que apresentou menor média de contaminação (0,577±0,017 UE/mL), com diferença estatisticamente significante (p<0,001) em comparação aos demais grupos. Pôde-se concluir, que a endotoxina bacteriana apresentou afinidade pelas ligaduras elastoméricas estéticas à base de silicone e poliuretano testadas. As ligaduras de silicone da marca GAC foram as que apresentaram menor quantidade de endotoxina aderida às suas superfícies / The objective of this study was to quantify, in vitro and in vivo, bacterial endotoxin (LPS) attached to esthetic elastomeric orthodontic ligatures made of polyurethane and silicone using the Limulus Amebocyte Lysate test (LAL). For the in vitro study, were used four types of aesthetic elastomeric ligatures: Sani-Ties (polyurethane) and Sili-Ties (silicone) - GAC, and Mini Single Case Ligature Stick (polyurethane) and Synergy&reg; Low-friction ligatures (silicone) - Rock Mountain; 5 of each were contaminated with endotoxin solution (positive control) and 5 non-contaminated (negative control). Replicas made of twisted wire ligatures and of cast stainless steel, of the same size and shape than the elastomeric ligatures, contaminated or not with endotoxin, were used as control. Endotoxin quantification was performed using the LAL test (QCL-1000&trade; kit), the results being expressed in EU/mL. In the in vivo study, 20 patients of both genders, with ages ranging from 15 to 30 years old, who started treatment with a fixed orthodontic appliance at the School of Dentistry of Ribeirão Preto - University of São Paulo, received randomly the same four types of elastomeric ligatures used in the in vitro study, being a ligature of each brand inserted in the upper and lower canines (UR3, UL3, LR3, LL3), randomly. Twenty-one days later, ligatures were removed and processed for quantification of bacterial endotoxin, using the same test as the in vitro study. All data were submitted to appropriate statistical analysis according to the data distribution, using KruskalWallis tests and Dunn post-test (in vitro study) and ANOVA of repeated measurements and Tukey\'s post-test (in vivo study). All analyzes were performed using the Graph Pad Prism 4.0 program, with a significance level of 5%. According to the results obtained, it was observed that the bacterial endotoxin attached to all materials tested. In the in vitro study, the GAC silicone group had the lowest median contamination (1.15 EU/mL), in relation to the other groups (p<0.0001), which did not present a statistically significant difference when compared to each other (p>0.05). In the in vivo study, similar to that observed in the in vitro study, the GAC silicone group had the lowest mean contamination (0.577 ± 0.017 EU/mL), with a statistically significant difference (p< 0.001) compared to the others groups. It could be concluded that bacterial endotoxin exhibited affinity for the tested silicone and polyurethane aesthetic elastomeric ligatures. The silicone ligatures of GAC brand were the ones that presented less amount of endotoxin attached to their surfaces

As células derivadas de fígado embrionário tanto de animais quanto de humanos tem sido cada vez mais estudas devido ao seu potencial antiinflamatório, imunomodulador e regenerativo, demonstrado as mesmas bipotencial de diferenciação em hepatócitos e colangiocitos. Na presente pesquisa utilizou-se células derivadas de fígados embrionários de ratos com 14,5 dias de gestação. As células apresentaram marcadores de células progenitoras hepáticas, bem como marcadores de células hepáticas e biliares diferenciadas confirmando, seu bipotencial. A terapia celular utilizando as células supracitadas, reduziu significativamente a progressão da fibrose hepática, diminuindo a inflamação e ainda estimulando a regeneração hepática de ratos submetidos à cirrose por ligadura do ducto biliar. As análises realizadas mediante avaliação quantitativa pela técnica de morfometria, demonstraram redução da deposição de fibras de colágeno, bem como menor proliferação de ductos biliares nos animais tratados. Os resultados foram ainda complementados por analise semiquantitativa, a qual avaliou a intensidade da necroinflamação dos tecidos hepáticos analisados, apontando menor escore de inflamação dos animais tratados. As células poderão ter efeito benéfico para o tratamento de doenças hepáticas crônicas, que estimulam a formação de fibrose. A cirrose é o estágio final comum à doenças hepáticas crônicas por causadas por fatores de diversas etiologias. Esta ocupa a decima quarta causa mundial de mortalidade em humanos, sendo que o único tratamento definitivo atualmente é transplante do órgão. Entretanto o número de transplantes está longe de suprir a demanda atual, visto que há um déficit de doadores do órgão. Terapias que possam oferecer uma alternativa de tratamento confiável, segura e acessível são bastante oportunas. Nossos resultados sugerem que as células utilizadas neste trabalho podem modular a fibrogênese, e consequentemente retardar o estabelecimento da cirrose em doenças hepáticas crônicas. / Studies on human and animal embryonic liver stem cells have been growing due to its anti-inflammatory, immunomodulatory and regenerative potential. These cells show also a bipotential do differentiate into hepatocytes and cholangiocytes. In the present study, it was used rodent embryonic liver with 14.5 of gestation. The cells presented hepatic progenitor, as well adult hepatic and biliary cells markers, confirming their bipotential. Previous studies with these cells in therapy decreased hepatic fibrosis progression in rat models submitted to cirrhosis by biliary duct ligation. Quantitative analysis was performed by morphometry showed decreased collagen fibers deposition and lower proliferation of biliary ducts in treated animals. Results were complemented with semiquantitative analysis with evaluation of necroinflammation of the analyzed hepatic tissues, in which a decreased inflammation score was observed. Cirrhosis is a common final stage for chronic hepatic diseases caused by different factors in several etiologies. It occupies the 14th world cause of mortality in human. However, the number of liver transplants is insufficient for current demand, caused by deficit in organs donors. Therapies that could offer an alternative for a reliable, safe and accessible treatment is opportune. Our results suggest that cells used in this study can modulate fibrogenesis and consequently delay the establishment of cirrhosis in chronic liver diseases.

Sepse é definida como uma resposta inflamatória sistêmica frente a um estímulo infeccioso. Na fase inicial da doença ocorre aumento da secreção de vasopressina (AVP) e na fase tardia observamos uma redução, apesar dos elevados níveis de mediadores inflamatórios e agravamento da hipotensão que são estímulos para a secreção do hormônio. O óxido nítrico (NO), produzido pela enzima óxido nítrico sintase (NOS), parece modular a secreção de AVP dependendo do contexto fisiopatológico. Durante sepse experimental nosso grupo demonstrou que as concentrações plasmáticas de nitrato, que servem como índice da produção de NO, se elevam progressivamente durante a evolução da doença, mas não há evidências de que isto ocorra também a nível central. Nosso objetivo foi avaliar temporalmente a concentração de nitrato no líquido cefalorraquidiano (LCR) e a atividade enzimática das NOS no hipotálamo de ratos submetidos à sepse por ligadura e perfuração cecal (CLP). Os procedimentos, aprovados pelo Comitê de Ética no Uso de Animais do Campus de Ribeirão Preto (CEUA-USP, protocolo nº 10.1.294.53.7), foram realizados com ratos Wistar pesando 250 ± 30 gramas, os quais foram divididos em dois grupos: CLP e operação fictícia (OF). Após 0, 2, 6 e 24 horas das cirurgias, foi feita a coleta do LCR para a determinação da concentração de nitrato pelo método de quimiluminescência NO/ozônio. Imediatamente após os animais foram decapitados para coleta de sangue utilizado para determinação de hematócrito, sódio sérico, proteína, interleucina (IL)-1?, nitrato e AVP plasmáticos. Também foram removidas as neurohipófises para análise do conteúdo de AVP e o hipotálamo para análise da atividade da NOS pelo método da Citrulina. Nos animais sépticos, as concentrações plasmáticas de AVP seguiram o padrão esperado, com aumento em 2h e 6h, retornando a níveis basais em 24h. Os estoques neurohipofisários de AVP reduziram em 2h e 6h retornando a níveis basais em 24h. O sódio sérico e as proteínas plasmáticas diminuíram 2h, 6h e 24h após CLP, e os hematócritos aumentaram nos mesmos períodos. As concentrações plasmáticas de IL-1? e nitrato aumentaram 6h e mantiveram-se elevadas 24h após CLP. Entretanto, no LCR o nitrato aumentou em 6h, mas retornou a níveis basais em 24h. A atividade hipotalâmica das NOS totais e constitutivas eram de 50 a 100 vezes maiores que as das NOS induzidas e aumentaram 6h e 24h após a CLP. Os resultados deste estudo mostram que as atividades das NOS observadas no hipotálamo ex vivo dos ratos sépticos indicam a presença de formas viáveis das enzimas, sendo a isoforma constitutiva a mais relevante para a produção de NO nesta região cerebral. Além disso, as alterações temporais das concentrações de nitrato no LCR diferem da de nitrato plasmático, revelando um perfil diferenciado da produção de NO central em relação à periférica, e que este pode estar relacionado à secreção bifásica de AVP na sepse / Sepsis is defined as a systemic inflammatory response to an infectious stimulus. In the initial phase of the disease the secretion of vasopressin (AVP) is increased and late is observed a reduction, although high levels of inflammatory mediators and worsening of the hypotension, which are stimuli for the hormone secretion. Nitric oxide (NO) produced by the enzyme nitric oxide synthase (NOS), appears to modulate the secretion of AVP depending on the pathophysiological context. During experimental sepsis our group demonstrated that the plasma nitrate concentrations, which serve as an index of NO production, increase gradually during the course of the disease. However, there is no evidence that this also occurs at the central level. Our aim was to evaluate the temporal nitrate concentration in the cerebrospinal fluid (CSF) and the enzymatic activity of NOS in the hypothalamus of rats submitted to sepsis by cecal ligation and puncture (CLP). The procedures, approved by the Ethics Committee on Animal Use of the Campus of Ribeirão Preto (CEUA - USP, Protocol nº10.1.294.53.7) were performed with Wistar rats weighing 250 ± 30 grams, which were divided into two groups: CLP and Sham operation. After 0, 2, 6 and 24 hours of surgery, CSF was collected for determining the nitrate concentration by the chemiluminescence NO/ozone method. Immediately after, the animals were decapitated, blood was collected and used for hematocrit, serum sodium, protein, interleukin (IL)-1?, nitrate and plasma AVP determination. The neurohypophysis was removed for AVP content determinations and hypothalamus for NOS activity analysis by the method of citrulline. In septic animals, plasma concentrations of AVP showed the expected pattern, an increase at 2h and 6h and returning to basal levels at 24h. The AVP neurohypophyseal stocks decreased at 2h and 6h returning to basal levels at 24h. The sodium serum and plasma proteins decreased 2h, 6h and 24h after CLP, and the hematocrit increased at the same periods. The plasma concentration of IL-1? and nitrate increased 6h and remained elevated 24h after CLP. However, in the cerebrospinal, nitrate increased at 6h but returned to basal level in 24h. The total and constitutive NOS activities were fifty to one hundred times higher than inducible NOS and were increased 6h and 24h after CLP. These results suggest that the activity of NOS observed in the hypothalamus ex vivo of rats septic indicates the presence of viable forms of enzymes, and that the constitutive isoform is the most relevant for the NO production in this brain region. Moreover the temporal alterations of the nitrate concentrations in the CSF differ from the plasma nitrate, showing a different profile of the central and peripheral NO production, which may be related to biphasic AVP secretion in the sepsis.

In recent years, DNA nanotechnology has emerged as one of the most powerful strategies for bottom-up construction of nanomaterials. Due to the high programmability of DNA molecules, their self-assembly can be rationally designed. Engineered 3D DNA crystals, as critical products from the design of DNA self-assembly, have been proposed as the structural scaffolds for organizing nano-objects into three-dimensional, macroscopic devices. However, for such applications, many obstacles need to be overcome, including the crystal stability, the characterization methodology, the revision of crystal designs as well as the modulation of crystallization kinetics. My PhD research focuses on solving these problems for engineered 3D DNA crystals to pave the way for their downstream applications.<br>In this thesis, I started by enhancing the stability of engineered 3D DNA crystals. I developed a highly efficient post-assembly modification approach to stabilize DNA crystals. Enzymatic ligation was performed inside the crystal lattice, which was designed to covalently link the sticky ends at the crystal contacts. After ligation, the crystal became a covalently bonded 3D network of DNA motifs. I investigated the stability of ligated DNA crystals under a wide range of solution conditions. Experimental data revealed that ligated DNA crystals had significantly increased stability. With these highly stabilized DNA crystals, we then demonstrated their applications in biocatalysis and protein encapsulation as examples.<br>I also established electron microscope imaging characterization methods for engineered 3D DNA crystals. For crystals from large-size DNA motifs, they are difficult to study by X-ray crystallography because of their limited diffraction resolutions to no better than 10 Å. Therefore, a direct imaging method by TEM was set up. DNA crystals were either crushed or controlled to grow into microcrystals for TEM imaging. To validate the imaging results, we compared the TEM images with predicted models of the crystal lattice. With the advance in crystal characterization, DNA crystals of varying pore size between 5~20 nm were designed, assembled, and validated by TEM imaging.<br>The post-assembly ligation was further developed to prepare a series of new materials derived from engineered 3D DNA crystals, which were inaccessible otherwise. With the directional and spatial control of ligation in DNA crystal, I prepared new DNA-based materials including DNA microtubes, complex-architecture crystals, and an unprecedented reversibly expandable, self-healing DNA crystal. The integration of weak and strong interactions in crystals enabled a lot of new opportunities for DNA crystal engineering.<br>In the final chapter, I investigated the effect of 5’-phosphorylation on DNA crystallization kinetics. I found that phosphorylation significantly enhanced the crystallization kinetics, possibly by strengthening the sticky-ended cohesion. Therefore, DNA crystals can be obtained at much lower ionic strength after phosphorylation. I also applied the result to controling the morphology of DNA crystals by tuning the crystallization kinetics along different crystallographic axes. Together with previously methods to slow down DNA crystallization, the ability to tune DNA crystallization kinetics in both ways is essential for DNA crystal engineering.

Vários estudos clínicos e experimentais relatam o aumento das concentrações plasmáticas de vasopressina (AVP) na fase inicial da sepse, como tentativa de restabelecer a pressão sanguínea que nesta fase começa a diminuir. Porém em uma fase mais tardia da doença, as concentrações do hormônio estão reduzidas mesmo sob um quadro de hipotensão progressiva, um dos principais estímulos para aumento da secreção deste hormônio. Este padrão de secreção hormonal alterada também parece ocorrer com o outro hormônio neurohipofisário, ocitocina (OT). O objetivo deste trabalho foi analisar a secreção dos hormônios AVP e OT, após estímulos osmótico e hipovolêmico em animais sobreviventes à sepse. A sepse foi induzida pelo método de ligação cecal e punção (CLP) (1 perfuração com agulha 14G) e os animais observados por 10 dias. Os sobreviventes foram submetidos à estímulo osmótico por desidratação (retirada de água para beber da gaiola) por dois dias, ou hipovolêmico por injeção intraperitoneal (i.p) de Polietileno glicol (PEG) (PEG-4000, 200mg/ml de PBS) por 90 minutos. Os animais controles foram hidratados ou receberam injeção intraperitoneal de PBS 0,01M. Após os estímulos, os animais foram decapitados para coleta de sangue e neurohipófise, para as análises de hematócrito, sódio sérico, osmolalidade plasmática, e dosagem hormonal de AVP e OT plasmática e neurohipofisária. Resultados: Os animais sobreviventes à sepse mantiveram a capacidade de responder aos estímulos com relação à secreção de AVP. Porém estes animais apresentaram uma secreção diminuída de OT após desidratação embora nenhuma alteração de secreção aparente ao estímulo volêmico. Conclusão: Animais sobreviventes à sepse apresentam alterações no padrão de resposta hormonal do eixo hipotálamoneurohipofisário ao estímulo osmótico sem aparente alteração quando o estímulo é volêmico, sugerindo que seus osmorreceptores encontram-se alterados / Many clinical and experimental studies reported the increase of plasma vasopressin (AVP) concentration in the early phase of sepsis, as an attempt to restore blood pressure that begins to decrease at this stage. However, in a later stage of the disease, the hormone concentrations are reduced even under a state of progressive hypotension, one of the major stimulus for the increase of secretion of this hormone. This pattern of impaired hormonal secretion during sepsis also seems to happen to the other pituitary hormone, oxytocin (OT). The aim of this study was to evaluate the secretion of the hormones AVP and OT after osmotic and hipovolemic stimuli in sepsis surviving animals. Sepsis was induced by cecal ligation and perforation (CLP) (1 perforation with 14G needle) and the animals observed for 10 days. The survivors were submited to osmotic stimulus by dehydration (privation of drinking water from the Cage) for two days, or hipovolemic by intraperitoneal (i.p) injection of polyethylene glycol (PEG) (PEG-4000, 200mg/ml of PBS) for 90 minutes. The control animals were hydrated or that received intraperitoneal injection of PBS 0,01M. After the stimuli, the animals were decapitated for the collection of blood and neurohypophysis, for the analysis of hematocrit, serum sodium, plasma osmolality and hormonal analysis of AVP and OT in the plasma and neurohypophysis. Results: Sepsis surviving animals maintained the capacity of answering the stimulus with AVP secretion. But these animals presented a lowered secretion of OT after dehydration, while they presented no alterations in secretion after volemic stimulus. Conclusion: Sepsis surviving animals presented alterations in the pattern of hormonal answer for the osmotic stimulus, with lowered secretion of OT, apparently there are no alterations in the pattern of a hormonal secretion after hypovolemia.

Gastrointestinal (GI) alpha-synuclein (ASN) detection may represent a clinically useful biomarker of Parkinson's disease (PD), but this has been challenged by conflicting results of recent studies employing different immunohistochemical (IHC) methods and reporting diverse morphological patterns with variable biological interpretation. To increase sensitivity and specificity, we applied three different techniques to detect different possible conformations of ASN in GI tissue derived from biopsies of the GI tract, which were obtained from a longitudinally followed, clinically well-characterized cohort of PD subjects and healthy controls (HC) (Oxford Discovery study). With IHC, we used antibodies reactive for total (T-ASN-Abs), phosphorylated (P-ASN-Abs) and oligomeric (O-ASN-Abs) ASN; with the ASN Proximity Ligation Assay (AS-PLA), we targeted oligomeric ASN species specifically; finally, with the Paraffin-Embedded Tissue Blot (PET-Blot) we aimed to detect fibrillary conformations of ASN specifically. Optimisation and validation of the PET-Blot and PLA techniques was carried out with studies on brain tissue from subjects with ASN pathology, and these experiments were used to gain insight into morphology and distribution of different conformational variants of ASN in the brain of subjects with Lewy pathology. We specified all the detected morphological staining patterns with each technique interpreting them as pathologic or non-specific. Correlation to clinical symptoms was assessed to investigate the potential predictive or diagnostic value of specific staining patterns as biomarkers. A total of 163 GI tissue blocks were collected from 51 PD patients (113 blocks) and 21 healthy controls (50 blocks). In 31 PD patients, GI biopsies had been taken before PD diagnosis (Prodromal PD group); while in 20 PD patients biopsies were obtained after PD diagnosis (Manifest PD group). The majority of these tissues blocks were from large intestine (62%), followed by small intestine (21%), stomach (10%) and oesophagus (7%). With IHC, four ASN staining patterns were detected in GI tissue (Neuritic, Ganglionic, Epithelial, and Cellular), while two distinct staining patterns were detected with AS-PLA (cellular and diffuse signal) and with AS-PET-Blot (ASN-localised and peri-crypt signal). The level of agreement between different techniques was generally low, and no single technique or staining pattern was able to reliably distinguish PD patients (Prodromal or Manifest) from HC. Overall, our study suggests that even specific detection of ASN conformational variants currently considered pathologic was not adequate for the prediction of PD. Future studies with these or other novel techniques focusing on the upper part of the GI tract could overcome current limitations in sensitivity and specificity.

Base teórica: A Síndrome Hepatopulmonar (SHP) caracteriza-se por uma tríade clínica que inclui doença hepática, anormalidades em trocas gasosas e presença de dilatações vasculares pulmonares, sendo a obstrução prolongada do ducto biliar utilizada em modelos experimentais de cirrose biliar secundária que leva a reações inflamatórias agudas, podendo, ainda, ocasionar alterações sistêmicas. Animais com cirrose induzida experimentalmente por ligadura de ducto biliar (LDB) apresentam aumento do estresse oxidativo no tecido hepático, mas a inter-relação com o tecido pulmonar ainda não está bem esclarecida. O estresse oxidativo desempenha papel central na patogênese e na progressão de doenças crônicas, e o uso de antioxidantes tem sido proposto como agente terapêutico para compensar danos sistêmicos e hepáticos. Em estudos em que a utilização da melatonina foi comparada com a utilização de outros antioxidantes conhecidos como as vitaminas C e E, tal componente mostrou-se mais eficiente na redução do estresse oxidativo/nitrosativo, demonstrando que a melatonina atua como potente antioxidante. Objetivo: A presente tese teve como objetivo investigar o efeito do antioxidante Melatonina sobre o tecido pulmonar no modelo experimental de Síndrome Hepatopulmonar por ligadura de ducto biliar. Métodos: O trabalho está dividido em três partes: Parte I- Artigo de revisão intitulado “Hepatopulmonary Syndrome: Oxidative Stress and Physical Exercise,” enviado para publicação no periódico European Medical Journal; Parte II- Artigo original intitulado “Efeitos da melatonina sobre o tecido pulmonar no modelo experimental de síndrome hepatopulmonar”, enviado para o periódico Jornal de Pneumologia; Parte III- Artigo original intitulado “Efeitos da melatonina sobre o tecido hepático e pulmonar em animais com síndrome hepatopulmonar por ligadura do ducto biliar”, a ser enviado para o periódico Digestive Diseases and Sciences. Foi utilizado o modelo de cirrose biliar secundária, induzida pelo modelo de ligadura de ducto biliar (LDB), com ratos machos Wistar, pesando ± 300 gramas, divididos em quatro grupos: CO (controle), CO+MLT, SHP e SHP+MLT. Os animais foram tratados com MLT a partir do 15° dia e com LDB até o 28° dia. No 29° dia, mediante administração de fármacos anestésicos, foram coletados sangue, fígado, pulmão e fêmur. Resultados: Nos artigos II e III, ao se avaliar as transaminases, observou-se aumento significativo nas enzimas ALT, AST e FA no grupo SHP quando comparadas aos grupos controles, o que determina a presença de doença hepática. Posteriormente à administração da 7 melatonina, houve diminuição significativa do grupo SHP+MLT quando comparado ao grupo SHP na avaliação da lipoperoxidação, atividade das enzimas Catalase (CAT), superóxido dismutase (SOD) e glutationa Peroxidade (GPx), bem como na de Nitritos/Nitratos (NO2/NO3) e no sistema inflamatório, quando da avaliação da expressão da Óxido Nítrico Sintase Induzível (iNOS) e da região p65 do fator de transcrição nuclear-kB (NF-kB). Também houve diferença na gasometria arterial e na histologia do tecido pulmonar pelas técnicas de Hematoxilina e Eosina e Picrosírius, especialmente no diâmetro dos vasos pulmonares. Ao avaliar o dano ao DNA pelo Ensaio Cometa, foi observado aumento do índice e da frequência de dano no grupo SHP, com redução após o uso da MLT. Conclusão: Os resultados sugerem efeito protetor da melatonina no tecido hepático e pulmonar, no modelo de Síndrome Hepatopulmonar por ligadura de ducto biliar. / Background: Hepatopulmonary syndrome (HPS) is clinically characterized by a combination of liver disease, gas exchange abnormalities, and pulmonary vascular dilation. Prolonged biliary duct obstruction was used in experimental models of secondary biliary cirrhosis leading to acute inflammatory reactions and possibly to systemic changes. Animals with cirrhosis experimentally induced by biliary duct ligation (BDL) showed increased oxidative stress in liver tissue, but the lung tissue has not been investigated yet. Oxidative stress has a key role in the pathogenesis and progression of chronic diseases, and antioxidants have been proposed as therapeutic agents to compensate for systemic and liver damages. In studies comparing melatonin with other known antioxidants such as vitamins C and E, melatonin was found to be more efficient in reducing oxidative/nitrosative stress, showing that it is a potent antioxidant. Objective: The present doctoral dissertation aimed to investigate the effect of the antioxidant melatonin on lung and liver tissues in an experimental model of BDL-induced HPS. Methods: This dissertation is divided into three parts: Part I consists of the review article entitled “Hepatopulmonary Syndrome: Oxidative Stress and Physical Exercise,” which was submitted to the European Medical Journal; Part II consists of the original article entitled “Efeitos da melatonina sobre o tecido pulmonar no modelo experimental de síndrome hepatopulmonar”, which was submitted to Jornal de Pneumologia; and Part III consists of the original article entitled “Effects of melatonin on liver and lung tissues of animals with bile duct ligation-induced hepatopulmonary syndrome”, which will be submitted to Digestive Diseases and Sciences. A model of secondary BDL-induced biliary cirrhosis was used in male Wistar rats weighing ± 300 g and divided into four groups: control group (CG), CG+MLT, HPS, and HPS+MLT. Animals were treated with MLT or vehicle from day 15 to day 28 after BDL. On day 29, blood, liver, lung, and femur samples were collected after the administration of anesthetic drugs. Results: As for transminases, papers II e III found that there was a significant increase in enzymes ALT, AST e FA in the HPS group compared with the control groups, which indicates the presence of liver disease. The administration of melatonin led to a significant decrease in the HPS group compared with the control groups with regard to the following variables: lipid peroxidation; catalase, superoxide dismutase, and glutathione peroxidase activity; nitrite/nitrate ratio; and inducible nitric oxide synthase and p65 region of nuclear factor kappa B. There were also differences in blood gas analysis and histology of lung tissues as assessed by hematoxylin and eosin (HE) and picrosirius red staining, especially in the diameter of pulmonary vessels. The comet assay revealed an increase the index and frequency of DNA damage in the HPS group compared with the control groups, but these parameters were reduced with the use of melatonin in the HPS+MLT group. Conclusion: Results suggest that melatonin has a protective effect in liver and lung tissues using a BLD-induce HPS model.

The potential of magnetic nanoparticle-vesicle assemblies (MNP-V) as remote controlled drug delivery platforms capable of inducing cellular responses under magnetic stimuli has been previously demonstrated in the Webb group at the University of Manchester. To create these magnetoresponsive nanomaterials biotin-avidin and Cu-histidinyl multivalent recognition were employed. This thesis describes an exploration of the potential of thiol-thioester exchange reactions (leading to native chemical ligation, NCL) to create magnetoresponsive materials, which potentially have applications in biomedicine. Firstly, iron oxide magnetic nanoparticles have been synthesised using a thermal co-precipitation method followed by chemical modification with sulfhydryl motifs for use as smart biomaterials. Knowing that the behaviour and reactivity of nanoparticles is highly influenced by their physicochemical properties, a thourough characterisation of these particles has been obtained. Secondly, during this project, several thioester derivatives have been synthesised that can be incorporated into the membranes of 800 nm liposomes. Among these, the spectrophotometric properties of synthetic lipid 38 allowed the investigation of trans-thioesterification rates with cysteinyl functionalities, both in solution and at the phospholipid membrane interface of liposomes. Product identification has been achieved using mass spectrometry and 1H-NMR spectroscopy. Finally, the conditions required to induce the release of a dye (e.g. 5(6)-CF) from MNP-V upon exposure to an AMF pulse have been established. Aurintricarboxylic acid (ATA), a general inhibitor of nucleases has been investigated as interesting payload due to its fluorescent and anti-viral properties.

<p>Proximity ligation is a recently established technique that can provide answers to questions about the concentration, localization, interactions, modifications and functions of proteins. The method enables sensitive protein measurements with a detection limit in the low femtomolar range in complex biological samples. In proximity ligation, the challenge of detecting specific proteins is converted to the analysis of specific DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins, and to form amplifiable tag sequences upon ligation when brought in proximity. Protocols for the conversion of monoclonal or polyclonal antibodies into proximity probes through the attachment of oligonucleotide sequences are described in the thesis. In addition, the thesis describes the adaptation of the proximity ligation technology for detection of microbial pathogens, analysis of interactions between proteins and nucleic acids, and of inhibition of receptor-ligand interactions. </p><p>Nucleic acid amplification allows specific detection of pathogens with single-copy sensitivity. There are many circumstances, however, when analysis of pathogen surface antigens or the antibody response can provide increased diagnostic value. Proximity ligation reactions were used to measure numbers of virus and bacteria by detection of viral or bacterial surface proteins. Detection sensitivities similar to those of nuclear acid-based detection reactions were achieved directly in infected samples for a parvovirus and for an intracellular bacterium. </p><p>Biological processes are orchestrated by interactions of proteins with molecules in their environment, and investigations of interactions between proteins and other biomolecules are thus of great importance. Protocols were established for very specific and sensitive homogeneous-phase analysis of interactions between proteins and specific nucleic acid sequences. Finally, the proximity ligation mechanism was used to monitor interactions between VEGF-A and two of its receptors, VEGFR-1 and VEGFR-2, and to characterize the effects of agents disrupting this interaction.</p>

<p>Improved methods are needed to interrogate the genome and the proteome. Methods with high selectivity, wide dynamic range, and excellent precision, capable of simultaneously analyzing many biomolecules are required to decipher cellular function. This thesis describes a molecular and microfluidic toolbox designed with those criteria in mind. It also presents a tool for graphical representation of nucleic acid sequences.</p><p>Proximity ligation is a novel protein detection method that requires dual and proximate binding of two oligonucleotide-tagged affinity reagents to a protein or protein complex in order to elicit a signal. The responses from such recognition reactions are the formation of specific nucleic acid reporter molecules that are subsequently amplified and quantitatively detected. </p><p>A scalable microfluidic platform suitable for fluorescence detection, cell culture, and actuation is also described. The platform uses rapid injection molding to produce microstructures in thermoplastic materials. By applying a thin layer of silica to the structures, a lid made of silicone rubber coated onto a thermoplastic support can be covalently bonded to generate enclosed channels.</p><p>A method is presented for precise biomolecule counting, termed “amplified single-molecule detection”. The method preserves the discrete nature of biomolecules, converting specific molecular recognition events to fluorescence-labeled micrometer-sized objects that are enumerated in microfluidic channels. </p><p>I also present a novel microarray-based detection method. To attain high selectivity and a wide dynamic range, the method is based on dual recognition with enzymatic discrimination and amplification. Upon target recognition in solution, DNA probes are subjected to thousand-fold amplification in solution, followed by selective detection on arrays and another hundred-fold amplification of reporter molecule created from the first amplification reaction. </p><p>Lastly, I describe a novel graphical representation of nucleic acid sequences using TrueType fonts that can be of value for visual inspection of DNA sequences and for teaching purposes</p>