Protein biomarkers circulating in blood hold the promise of improved diagnosis, prognosis and follow-up of treatment of disease via minimally invasive procedures. For the discovery and validation of such biomarkers, methods are needed that can facilitate parallel, highly specific and in-depth analysis of the blood proteome. The work presented in this thesis intends to develop and apply such assays, building on the concept of the proximity ligation assay (PLA). In paper I, I present an easy and non-expensive alternative for the conjugation of oligonucleotides to antibodies via biotin-streptavidin-biotin interaction. This approach can be used when large sets of antibodies and/or oligos need to be validated for their performance as probes in PLA reactions. In paper II, a solid-phase variant of PLA (SP-PLA) for the detection and quantification of proteins in blood is presented. SP-PLA exhibited an improved limit of detection compared to commercial ELISA assays by two orders of magnitude. In addition SP-PLA exhibited a broader dynamic range by at least one order of magnitude and required only 5 μl of sample, rendering the method very well suited for analyses of precious bio-banked material. Last but not least, SP-PLA was used to validate the diagnostic potential of GDF-15 as a biomarker for cardiovascular disease in a set of cardiovascular disease patients and healthy controls. Paper III discusses the development of a multiplex SP-PLA (MultiPLAy) for the simultaneous detection of 36 proteins in just 5 μl of sample. MultiPLAy exhibited an improved LOD when compared to state-of-the-art bead-based sandwich assays. Most importantly, we observed only a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that much higher levels of multiplexing will be possible. The assay was used to identify putative biomarkers in sample cohorts of colorectal cancer (CRC) and cardiovascular disease (CVD). Subsequent multivariate analysis revealed previously known diagnostic biomarkers. Furthermore, we successfully applied next-generation sequencing as a readout for the protein assays, allowing for the first time digital recording of protein profiles in blood. In paper IV, we investigated the suitability of prostasomes as blood biomarkers in patients with prostate cancer using a newly developed PLA assay (4PLA) that utilizes five binders for the detection of complex target molecules. The assay successfully detected significantly elevated levels of prostasomes in blood samples from prostate cancer patients prior to radical prostatectomy, compared to controls and men with benign biopsy results.

No description available.

Small robust affinity proteins have shown great potential for use in therapy, in vivo diagnostics, and various biotechnological applications. However, the affinity proteins often need to be modified or functionalized to be successful in many of these applications. The use of chemical synthesis for the production of the proteins can allow for site-directed functionalization not achievable by recombinant routes, including incorporation of unnatural building blocks. This thesis focuses on chemical engineering of Affibody molecules and an albumin binding domain (ABD), which both are three-helix bundle proteins of 58 and 46 amino acids, respectively, possible to synthesize using solid phase peptide synthesis (SPPS). In the first project, an alternative synthetic route for Affibody molecules using a fragment condensation approach was investigated. This was achieved by using native chemical ligation (NCL) for the condensation reaction, yielding a native peptide bond at the site of ligation. The constant third helix of Affibody molecules enables a combinatorial approach for the preparation of a panel of different Affibody molecules, demonstrated by the synthesis of three different Affibody molecules using the same helix 3 (paper I). In the next two projects, an Affibody molecule targeting the amyloid-beta peptide, involved in Alzheimer’s disease, was engineered. Initially the N-terminus of the Affibody molecule was shortened resulting in a considerably higher synthetic yield and higher binding affinity to the target peptide (paper II). This improved variant of the Affibody molecule was then further engineered in the next project, where a fluorescently silent variant was developed and successfully used as a tool to lock the amyloid-beta peptide in a β-hairpin conformation during studies of copper binding using fluorescence spectroscopy (paper III). In the last two projects, synthetic variants of ABD, interesting for use as in vivo half-life extending partners to therapeutic proteins, were engineered. In the first project the possibility to covalently link a bioactive peptide, GLP-1, to the domain was investigated. This was achieved by site-specific thioether bridge-mediated cross-linking of the molecules via a polyethylene glycol (PEG)-based spacer. The conjugate showed retained high binding affinity to human serum albumin (HSA) and a biological activity comparable to a reference GLP-1 peptide (paper IV). In the last project, the possibility to increase the proteolytic stability of ABD through intramolecular cross-linking, to facilitate its use in e.g. oral drug delivery applications, was investigated. A tethered variant of ABD showed increased thermal stability and a considerably higher proteolytic stability towards pepsin, trypsin and chymotrypsin, three important proteases found in the gastrointestinal (GI) tract (paper V). Taken together, the work presented in this thesis illustrates the potential of using chemical synthesis approaches in protein engineering. / <p>QC 20140207</p>

Proteins play important roles in most cellular functions, such as, replication, transcription regulation, signal transduction, for catalyzing chemical reaction, etc. Technologies developed to identify proteins rely either on observing their own properties such as charge, size, mass to charge ratio or sequence composition; or on using affinity reagents that recognize specific protein targets. Immunoassays utilizing functionalized affinity reagents are powerful for targeted proteomics. Among them, DNA-assisted immunoassays in which affinity reagents are labeled with DNA molecules, offer some unique advantages. In this thesis, I will present works to improve current DNA-assisted immunoassays such as proximity ligation assays (PLA), as well as to take advantage of DNA reactions to adress other problems. In paper I, a new solid support (MBC-Ts) was functionalized with antibodies and used in the solid-phase PLA for detection of VEGF. The assay using MBC-Ts was compared among the commercially available solid supports in different matrices and it was shown to exhibit enhanced limit of detection in complex matrices. In paper II, a two-step protocol was described to prepare high-quality probes used in homogeneous and in situ PLA by purifying DNA-labeled affinity reagents from unconjugated affinity reagents and excess oligonucleotides. In paper III, PLA was applied on a capillary western blotting instrument so that both the sensitivity and specificity of the original assay were improved. In paper IV, a new method was introduced to profile protein components in individual protein complexes by DNA-barcoded antibodies. This method has been used to profile protein complexes such as surface proteins on individual secreted vesicles.

One of the pressing needs in the field of disease biomarker discovery is new technologies that could allow high performance protein analysis in different types of clinical material, such as blood and solid tissues. This thesis includes four approaches that address important limitations of current technologies, thus enabling highly sensitive, specific and parallel protein measurements. Paper I describes a method for sensitive singleplex protein detection in complex biological samples, namely solid phase proximity ligation assay (SP-PLA). SP-PLA exhibited improved sensitivity compared to conventional sandwich immunoassays. We applied SP-PLA to validate the potential of GDF-15 as a biomarker for cardiovascular disease.   Paper II describes ProteinSeq, a multiplexed immunoassay based on the principle of SP-PLA, for parallel detection of 36 proteins using next-generation sequencing as readout. ProteinSeq exhibited improved sensitivity compared to multiplexed sandwich immunoassays, and the potential to achieve even higher levels of multiplexing while preserving a high sensitivity and specificity. We applied ProteinSeq to analyze 36 proteins, including one internal control, in 5 μl of plasma samples in a cohort of patients with cardiovascular disease and healthy controls. Paper III describes PLA-DTM, a strategy for recording all possible interactions between sets of proteins in clinical samples. Individual proteins and their interactions are first encoded to dual barcoded DNA by PLA, and the barcodes are interrogated by a method named dual tag microarray (DTM). We applied the method for studying interactions among protein members of the NFκB signaling pathway. Paper IV describes a novel probing strategy for analyzing individual biomolecules in solution or in situ. The technique employs a new class of probes for unfolding proximity ligation assays - uPLA probes. The probes are designed so that each probe set is sufficient in forming and replicating circular DNA reporter, without interactions among themselves when incubated with the sample. The uPLA probing strategy provides ease in the design of multiple probe sets in parallelized assays while enhancing the specificity of detection. We used the uPLA probes to detect various targets, including synthetic DNA and cancer-related transcripts in situ.

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.

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.

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.

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.

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No. of bitstreams: 1 paulohenriquediasdecarvalho.pdf: 3125617 bytes, checksum: 3e951b5c5e73d3862e1ed9b6e9e28451 (MD5) Previous issue date: 2013-07-26 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A descoberta de protótipos naturais associada às metodologias de isolamento e identificação química de constituintes a partir de novas fontes botânicas, bem como da avaliação das atividades farmacológicas e toxicológicas dessas moléculas demonstram grandes perspectivas para o desenvolvimento racional de novos fármacos. Tendo em vista a alta incidência de doenças do fígado, no Brasil e no mundo, e que existem poucos medicamentos eficazes e capazes de reverter ou reduzir a progressão destas, o isolamento e a identificação de substâncias com potencial hepatoprotetor é, hoje, uma área promissora na busca de novas substâncias bioativas. Tradicionalmente, as raízes de Glycyrrhiza sp., conhecidas como licorice, são usadas na medicina alternativa com inúmeras finalidades, dentre elas hepatoprotetora. Entretanto, até o momento, não existem relatos desta atividade vinculada à licochalcona A, uma das substâncias majoritárias nas raízes de Glycyrrhiza inflata. No presente trabalho avaliaram-se as atividades da licochalcona A em ensaios de viabilidade celular das linhagens de fibroblásto (NIH/3T3) e carcinoma hepatocelular humano (HepG-2), adesão celular de HepG-2 e em modelo de lesão hepática induzida por ligação do ducto biliar (BDL) em ratos Wistar. Além disso, desenvolveu-se uma metodologia para determinação da licochalcona A em CLAE-UV, utilizando coluna C18, fase móvel em gradiente de água acidificada (0,1% H3PO4) e metanol (50:50 – 20:80 v/v), fluxo de 1,1 mL/min e comprimento de onda para detecção em 372 nm. A licochalcona A isolada a partir do extrato seco das raízes de G. inflata foi identificada por RMN 1H e 13C. O isolamento apresentou-se satisfatório, bem como a metodologia proposta para quantificação desta substância por CLAE-UV, que apresentou excelente linearidade, precisão e exatidão. Nos experimentos in vitro, a licochalcona A não demonstrou redução significativa na viabilidade das células da linhagem HepG-2 (IC50 > 200 μM) e da NIH/3T3 (IC50 > 100 μM), bem como no experimento de adesão das células HepG-2 (IC50 > 200 μM) (p>0,05). Estes dados corroboram com aqueles encontrados no experimento in vivo, no qual a licochalcona A (50 mg/Kg) também não apresentou toxicidade ao fígado, já que os resultados encontrados não foram significativamente diferentes aos do grupo controle (p>0,05). Contudo, ela também não demonstrou capacidade de promover ou reduzir os danos hepáticos causado pelo BDL, no tempo de tratamento do estudo realizado (48 h). / The natural prototypes discovery associated with methods of chemical constituents isolation and identification from new botanical sources, as well as the evaluation of pharmacological and toxicological activities of these molecules show great prospects for the new drugs rational development. In view of the high incidence of liver disease in Brazil and the world, and there are few effective drugs and able to reverse or slow the progression of these disease, the substances isolation and identification with potential hepatoprotective today is a promising area in search for new bioactive substances. Traditionally, the roots of Glycyrrhiza sp., known as licorice, are used in alternative medicine with numerous purposes, among them hepatoprotective. However, to date, there are no reports of this activity linked to licochalcona A, one majority of the substances in the roots of Glycyrrhiza inflata. In the present study evaluated the activities of licochalcone A in cell viability assays of strains fibroblast (NIH/3T3) and human hepatocellular carcinoma (HepG-2), cell adhesion HepG-2 and model of liver injury induced by bile duct ligation (BDL) in Wistar rats. In addition, we developed a methodology for determining the licochalcone A quantitative HPLC-UV, using C18 column and a mobile phase gradient of acidified water (0.1% H3PO4) and methanol (50:50 - 20:80 v/v), flow rate of 1.1 mL/min and detection wavelength at 372 nm. The licochalcone A isolated from the dried extract of the roots of G. inflata was identified by 1H and 13C NMR. The isolation had to be satisfactory, as well as the proposed methodology for quantification of this substance by HPLC-UV, which showed excellent linearity, reproducibility and accuracy. In in vitro experiments, licochalcone A showed no significant reduction in the viability of the cell line HepG-2 (IC50 > 200 μM) and NIH/3T3 (IC50 > 100 μM), as well as in cell adhesion HepG-2 experiments (IC50 > 200 μM) (p> 0.05). These data corroborate those found in the in vivo experiment, in which the licochalcone A (50 mg/kg) also showed no toxicity to the liver, since the results were not significantly different to the control group (p>0.05). Nevertheless, it has not shown the ability to promote or reduce liver damage caused by BDL, at the treatment time of the study (48 h).