Sepsis-induced organ failure is associated with high morbidity and mortality rates. The onset of an exaggerated host response to microbial invasion and/or trauma, is believed to be the primary cause of excessive inflammation and the subsequent tissue hypoperfusion observed in patients with severe sepsis. In our mouse model of sepsis induced by cecal ligation and perforation (CLP), symptoms indicative of the disease, including diarrhea, increased ventilation and persistent hypothermia, are present at six hours after the surgery (T6). In the untreated CLP mice, mortality occurs starting at T15. As induced hypothermia has shown to exert immunomodulatory effects, this study is aimed at assessing its potential in attenuating inflammation and improving survival in experimental sepsis. Our data has shown that deep hypothermia initiated at T6, by means of cold chamber-induced cooling, prolongs survival. Plasma cytokine quantification by enzyme-linked immunosorbent assays (ELISA) also reveals that induced deep hypothermia reduces tumour necrosis factor(TNF)-α and interleukin (IL)-6 production in untreated CLP mice. In contrast, induced moderate hypothermia does not have such effect. Antibiotic (cefotaxime) and saline resuscitation initiated immediately following CLP ensures survival. However, when these supportive treatments are initiated at T6, >50% mortality is observed in the CLP mice with or without induced hypothermia. In summary, this preliminary study provides proof for a downregulated inflammatory response mediated by external cooling. However, to achieve a survival benefit, treatment strategies in addition to cooling and antibiotics may be required.

Sepsis-induced organ failure is associated with high morbidity and mortality rates. The onset of an exaggerated host response to microbial invasion and/or trauma, is believed to be the primary cause of excessive inflammation and the subsequent tissue hypoperfusion observed in patients with severe sepsis. In our mouse model of sepsis induced by cecal ligation and perforation (CLP), symptoms indicative of the disease, including diarrhea, increased ventilation and persistent hypothermia, are present at six hours after the surgery (T6). In the untreated CLP mice, mortality occurs starting at T15. As induced hypothermia has shown to exert immunomodulatory effects, this study is aimed at assessing its potential in attenuating inflammation and improving survival in experimental sepsis. Our data has shown that deep hypothermia initiated at T6, by means of cold chamber-induced cooling, prolongs survival. Plasma cytokine quantification by enzyme-linked immunosorbent assays (ELISA) also reveals that induced deep hypothermia reduces tumour necrosis factor(TNF)-α and interleukin (IL)-6 production in untreated CLP mice. In contrast, induced moderate hypothermia does not have such effect. Antibiotic (cefotaxime) and saline resuscitation initiated immediately following CLP ensures survival. However, when these supportive treatments are initiated at T6, >50% mortality is observed in the CLP mice with or without induced hypothermia. In summary, this preliminary study provides proof for a downregulated inflammatory response mediated by external cooling. However, to achieve a survival benefit, treatment strategies in addition to cooling and antibiotics may be required.

Esophageal varices are expanded veins of the submucosa that develop in patients with portal hypertension. They develop as collaterals between the portal vein and the superior vena cava. Varices are seen when the HPVG rises >12mmHG and can lead to a life-threatening bleeding episode. Endoscopic band ligation (EBL) is the treatment of choice of acute variceal bleeding. It is also performed for primary and secondary prophylaxis of bleeding from esophageal varices. After EBL, patients are at risk of postinterventional bleeding. Therefore, patients are often hospitalized until endoscopy proves all applied ligation bands have dropped off. At present, there is no standardized algorithm for surveillance of patients after EBL. Furthermore, risk factors for bleeding complications after EBL are poorly evaluated. The available studies mostly investigated patient collectives .after endoscopic sclerotherapy. The aim of this study was to investigate bleeding behaviour after EBL, to make recommendations for in- and out-patient surveillance after EBL and to analyze independent risk factors for bleeding complications after EBL.

Η ηπατική εγκεφαλοπάθεια είναι ένα πολύπλοκο νευροψυχιατρικό σύνδρομο που έχει συσχετισθεί με οξείες και χρόνιες ηπατοπάθειες. Τα τελευταία χρόνια συσσωρεύονται πληροφορίες που εμπλέκουν το οξειδωτικό στρες (φορτίο) ως παράγοντα-κλειδί στην παθογένεση της ηπατικής εγκεφαλοπάθειας σε μελέτες που χρησιμοποιούν ως μετρούμενους δείκτες την υπεροξείδωση λιπιδίων και το οξειδοαναγωγικό ζεύγος της γλουταθειόνης (GSH/GSSG). Στην παρούσα μελέτη χρησιμοποιήθηκε ένα μοντέλο πειραματικού αποφρακτικού ίκτερου με απολίνωση του χοληδόχου πόρου. Αρσενικοί αρουραίοι χωρίστηκαν σε ομάδες ελέγχου, ψευδώς χειρουργημένων, και σε ομάδες απολίνωσης χοληδόχου πόρου που είτε θυσιάστηκαν την 5η ημέρα, είτε τη 10η ημέρα, ή τους χορηγήθηκαν αντιοξειδωτικοί παράγοντες (NAC, ALP, Vit-E). Στη συνέχεια, μελετήθηκε η θειολική οξειδοαναγωγική κατάσταση στον εγκέφαλο των αρουραίων, και μάλιστα ανά περιοχές (εγκεφαλικός φλοιός, στέλεχος, παρεγκεφαλίδα), καθώς και η επίδραση των επιλεγμένων αντιοξειδωτικών παραγόντων σε αυτήν. Χρησιμοποιήθηκε για πρώτη φορά μια πιο αντιπροσωπευτική εκτίμηση του οξειδωτικού στρες, καθώς ποσοτικοποιήθηκαν συγκεκριμένοι δείκτες υψηλού (GSSG, NPSSR, NPSSC, PSSR, PSSC, PSSP, υπεροξείδια λιπιδίων) και χαμηλού (GSH, CSH, PSH) οξειδωτικού στρες. Τα αποτελέσματά μας δείχνουν αύξηση των πρώτων και μείωση των τελευταίων σε όλες τις εγκεφαλικές περιοχές καταδεικνύοντας έτσι την παρουσία αυξημένου οξειδωτικού φορτίου στον αποφρακτικό ίκτερο. Το σημαντικότερο αποτέλεσμα αυτής της μελέτης είναι ότι κατέδειξε πρώιμα σημεία οξειδωτικού στρες στον εγκέφαλο ήδη από την 5η ημέρα μετά την απολίνωση του χοληδόχου πόρου. Οι μεταβολές των βιοχημικών δεικτών, και αυτό αφορά σε όλους τους δείκτες και σε όλες τις εγκεφαλικές περιοχές, αρχίζουν να φαίνονται από την 5η ημέρα και γίνονται στατιστικά σημαντικές τη 10η ημέρα από την απολίνωση του χοληδόχου πόρου. Διαπιστώσαμε επιπλέον ότι η GSH είχε περίπου διπλάσιες τιμές στον εγκεφαλικό φλοιό από ό, τι στο στέλεχος και την παρεγκεφαλίδα, και ότι στο στέλεχος παρατηρήθηκε μια δραματική αύξηση των επιπέδων των NPSSR τη 10η ημέρα μετά την απολίνωση του χοληδόχου πόρου, τα οποία όμως παρέμειναν χαμηλά στις άλλες δύο περιοχές. Καθώς είναι γνωστό πως το οξειδωτικό στρες έχει ενοχοποιηθεί στην παθογένεση διαφόρων νευροεκφυλιστικών παθήσεων στον άνθρωπο, τα ευρήματα αυτά θα μπορούσαν να συσχετισθούν με διαφορές στη φυσιολογία και τη βιοχημεία των περιοχών αυτών και ενδεχομένως να σχετίζονται με τον τρόπο που το οξειδωτικό στρες εκφράζεται στην παθοφυσιολογία και άλλων νοσολογικών καταστάσεων όπως η πλάγια αμυοτροφική σκλήρυνση, η νόσος Parkinson, και η νόσος Alzheimer. Έχει ενδιαφέρον το γεγονός ότι τα βασικά γάγγλια και οι πυρήνες του στελέχους είναι θέσεις εκλεκτικής βλάβης στην εγκεφαλοπάθεια από χολερυθρίνη στο νεογνικό ίκτερο. Στη δεύτερη πειραματική φάση, στους ικτερικούς αρουραίους χορηγήσαμε αντιοξειδωτικούς παράγοντες, που έχουν επανειλημμένα μελετηθεί τόσο in vitro όσο και σε ζωικά μοντέλα, σε μια προσπάθεια να αναστρέψουμε τις διαταραχές που είχαν παρατηρηθεί. Ένα πρώτο εύρημα ήταν η ευεργετική δράση στην υπεροξείδωση των λιπιδίων, που ποσοτικοποιήθηκε με τον υπολογισμό της MDA, στις ομάδες και των τριών αντιοξειδωτών αλλά κυρίως στις ALP και Vit-E. Και στις τρεις ομάδες που έλαβαν αντιοξειδωτικά, αντίθετα με την ομάδα απολίνωσης χοληδόχου πόρου, δεν παρατηρήθηκε εξάντληση του συνολικού κυτταρικού περιεχόμενου γλουταθειόνης. Επιπλέον, και στις τρεις ομάδες αντιοξειδωτικών δεν παρατηρήθηκε αύξηση των NPSSR στο εγκεφαλικό στέλεχος, γεγονός που υποδηλώνει ότι η συσσώρευση των μη-πρωτεϊνικών δισουλφιδίων στο στέλεχος εμποδίστηκε από τους αντιοξειδωτικούς παράγοντες. Η ανισορροπία των πρωτεϊνικών θειολών, όπως αυτή φάνηκε από τη μείωση της PSH και την αύξηση του PSSP, αναστράφηκε σημαντικά μόνο στην ομάδα NAC στην οποία η PSH αυξήθηκε στα φυσιολογικά επίπεδα. Εν συντομία, η παρούσα μελέτη είναι η πρώτη που καταδεικνύει με σαφήνεια το οξειδωτικό στρες στον εγκέφαλο στο μοντέλο του αποφρακτικού ίκτερου και μάλιστα αρκετά πρώιμα. Χρησιμοποιεί μια συστοιχία βιοχημικών δεικτών που περιγράφουν την θειολική αναγωγική κατάσταση και την υπεροξείδωση των λιπιδίων και μελετά τις ευεργετικές επιδράσεις γνωστών αντιοξειδωτικών παραγόντων στον πειραματικό αποφρακτικό ίκτερο. Τα αποτελέσματά της θα μπορούσαν να προσφέρουν κάποια βοήθεια στην κατανόηση ορισμένων μηχανισμών της ηπατικής εγκεφαλοπάθειας στους ανθρώπους. Μελλοντικές έρευνες θα απαντήσουν ερωτήματα σχετικά με τα γενεσιουργά αίτια του οξειδωτικού στρες, την ίδια την παρουσία των ελευθέρων ριζών και την παθοφυσιολογία του φαινομένου. / Hepatic encephalopathy is a complex neuropsychiatric syndrome that has been associated with acute and chronic liver diseases. Accumulated evidence over the last several years has implicated oxidative stress a key factor in the pathogenesis of hepatic encephalopathy. These studies utilize measurements of lipid peroxidation products and glutathione (GSH) and its oxidized disulfide GSSG. A model of experimental obstructive jaundice after ligation of the biliary duct has been used in the present study. Male Wistar rats were divided into control, sham operated and bile duct ligated groups that were sacrificed either on the 5th or the 10th day, or they have been treated with antioxidant agents (NAC, ALP, Vit-E). Subsequently, the thiol redox state of various areas (cortex, midbrain and cerebellum) of the rat brain and the effect of selected antioxidants were studied. For the first time specific markers of high oxidative stress (GSSG, NPSSR, NPSSC, PSSR, PSSC, PSSP, lipid peroxides) and low oxidative stress (GSH, CSH, PSH) were quantified providing a more detailed assessment of the phenomenon. Our results show increase in the first and decrease in the latter group of markers in all studied brain areas, therefore demonstrating high oxidative stress in the obstructive jaundice. The major impact of the present study is the demonstration of early signs of oxidative stress in the brain. Using this battery of biochemical markers, deviations from control and sham animals occurred as early as 5 days following bile duct ligation; by the 10th day the majority of these changes became statistically significant. It was also observed that GSH values in cerebral cortex were twice as high as those in midbrain and cerebellum and a dramatic increase in the levels of NPSSR on the 10th day after bile duct ligation in midbrain that was not observed in the other brain areas. These findings could be attributed to specificities of metabolic or biochemical status of neurons and astrocytes and alterations of blood-brain barrier permeability in different brain areas and probably should be taken into account in further studies, since, as we know, oxidative stress has been implicated in the pathogenesis of many human diseases like Parkinson’s , Alzheimer’s and Amyotrophic Lateral Sclerosis (ALS). It is of interest that basal ganglia and brainstem nuclei are, as well, the sites of selective damage in bilirubin encephalopathy in jaundiced neonates. Jaundiced rats were treated with agents that have frequently been used in vitro and in vivo for their antioxidant effects, in an effort to reverse the observed alterations in redox state. In the treated groups, there was no decrease in the total cell glutathione content unlike the bile duct ligated rats. There was also no significant difference in the levels of lipid peroxidation as compared with control and sham groups. The imbalance of protein thiols demonstrated by the decrease of PSH and the increase of PSSP was considerably reversed only in the NAC group. In all treated groups, no NPSSR increase was found suggesting that the antioxidant agents suppressed the accumulation of non-protein disulfides in the midbrain. In brief, this experimental study demonstrates the oxidative profile of the brain associated with obstructive jaundice at an early and later stage. A battery of biochemical markers that define the thiol redox state is utilized and the beneficial effects of known antioxidants are examined. The evidence presented supports the concept that oxidative stress is neither a uniform matter affecting brain in a general way nor that any antioxidant agent could prevent damage by enhancing equally well different defence system. It is also likely that oxidative stress is one of the important mechanisms of jaundice-induced encephalopathy. Further studies could provide with more evidence on the pathogenetic mechanisms and generative causes of the oxidative stress in obstructive jaundice.

Proteins are fundamental structural, enzymatic and regulatory components of cells. Analysis of proteins, such as by measuring their concentrations, characterizing their modifications, and detecting their interactions, provides insights in how biological systems work physiologically or pathologically at the molecular level. To perform such analysis, molecular tools with good sensitivity, specificity, high multiplexing and throughput capacity are needed. In this thesis, four different assays were developed and applied to detect and profile proteins and protein complexes in human body fluids, and in cells or tissues. These assays are based on targeting proteins or protein complexes by oligonucleotide-conjugated antibodies, and subsequent proximity dependent enzymatic reactions involving the attached DNA reporter sequences. In paper I, a solid-phase proximity ligation assay (SP-PLA) was applied to detect synthetic and endogenous amyloid beta protofibrils. The SP-PLA provided better sensitivity and increased dynamic range than a traditional enzyme-linked immunosorbent assay (ELISA). In paper II, in situ PLA was applied to investigate the correlation between MARK2-dependent phosphorylation of tau and Alzheimer’s disease. Greater numbers of MARK2-tau interactions and of phosphorylated tau proteins were observed in brain tissues from Alzheimer’s patients than in healthy controls. In paper III, a multiplex SP-PLA was applied to identify protein biomarker candidates in amyotrophic lateral sclerosis (ALS) disease and in the analgesic mechanism of spinal cord stimulation (SCS). Among 47 proteins in human cerebrospinal fluid (CSF) samples, four were found at significantly lower concentrations (p-values < 0.001) in the samples from ALS patients compared to those from healthy controls (follistatin, IL-1α, IL-1β, and KLK5). No significant changes of the analyzed proteins were found in the CSF samples of neuropathic pain patients in   the stimulated vs. non-stimulated condition using SCS. In paper IV, a new technology termed the proximity barcoding assay (PBA) was developed to profile individual protein complexes. The performance of PBA was demonstrated on artificially assembled streptavidin-biotin oligonucleotide complexes. PBA was also proven to be capable of profiling transcriptional pre-initiation complexes from nuclear extract of a hepatic cell line.

Sepsis-induced organ failure is associated with high morbidity and mortality rates. The onset of an exaggerated host response to microbial invasion and/or trauma, is believed to be the primary cause of excessive inflammation and the subsequent tissue hypoperfusion observed in patients with severe sepsis. In our mouse model of sepsis induced by cecal ligation and perforation (CLP), symptoms indicative of the disease, including diarrhea, increased ventilation and persistent hypothermia, are present at six hours after the surgery (T6). In the untreated CLP mice, mortality occurs starting at T15. As induced hypothermia has shown to exert immunomodulatory effects, this study is aimed at assessing its potential in attenuating inflammation and improving survival in experimental sepsis. Our data has shown that deep hypothermia initiated at T6, by means of cold chamber-induced cooling, prolongs survival. Plasma cytokine quantification by enzyme-linked immunosorbent assays (ELISA) also reveals that induced deep hypothermia reduces tumour necrosis factor(TNF)-α and interleukin (IL)-6 production in untreated CLP mice. In contrast, induced moderate hypothermia does not have such effect. Antibiotic (cefotaxime) and saline resuscitation initiated immediately following CLP ensures survival. However, when these supportive treatments are initiated at T6, >50% mortality is observed in the CLP mice with or without induced hypothermia. In summary, this preliminary study provides proof for a downregulated inflammatory response mediated by external cooling. However, to achieve a survival benefit, treatment strategies in addition to cooling and antibiotics may be required.

Background: Endoscopic band ligation (EBL) is generally accepted as the treatment of choice for bleeding from esophageal varices. It is also used for secondary prophylaxis of esophageal variceal hemorrhage. However, there is no data or guidelines concerning endoscopic control of ligation ulcers. We conducted a retrospective study of EBL procedures analyzing bleeding complications after EBL. Methods: We retrospectively analyzed data from patients who underwent EBL. We analyzed several data points, including indication for the procedure, bleeding events and the time interval between EBL and bleeding. Results: 255 patients and 387 ligation sessions were included in the analysis. We observed an overall bleeding rate after EBL of 7.8%. Bleeding events after elective treatment (3.9%) were significantly lower than those after treatment for acute variceal hemorrhage (12.1%). The number of bleeding events from ligation ulcers and variceal rebleeding was 14 and 15, respectively. The bleeding rate from the ligation site in the group who underwent emergency ligation was 7.1% and 0.5% in the group who underwent elective ligation. Incidence of variceal rebleeding did not vary significantly. Seventy-five percent of all bleeding episodes after elective treatment occurred within four days after EBL. 20/22 of bleeding events after emergency ligation occured within 11 days after treatment. Elective EBL has a lower risk of bleeding from treatment-induced ulceration than emergency ligation. Conclusions: Patients who underwent EBL for treatment of acute variceal bleeding should be kept under medical surveillance for 11 days. After elective EBL, it may be reasonable to restrict the period of surveillance to four days or even perform the procedure in an out-patient setting.

Intra- and intercellular information is communicated by posttranslational modifications (PTMs) and protein-protein interactions, transducing information over cell membranes and to the nucleus. A cells capability to respond to stimuli by several highly complex and dynamic signaling networks provides the basis for rapid responses and is fundamental for the cellular collaborations required in a multicellular organism. Having received diverse stimuli, being positioned at various stages of the cell cycle or, for the case of cancer, containing altered genetic background, each cell in a population is slightly different from its neighbor. However, bulk analyses of interactions will only reveal an average, but not the true variation within a population. Thus studies of interacting endogenous biomolecules in situ are essential to acquire a comprehensive view of cellular functions and communication. In situ proximity ligation assay (in situ PLA) was developed to investigate individual endogenous protein-protein interactions in fixed cells and tissues and was later applied for detection for PTMs. Progression of signals in a pathway can branch out in different directions and induce expression of different target genes. Hence simultaneous measurement of protein activity and gene expression provides a tool to determine the balance and progression of these signaling events. To obtain this in situ PLA was combined with padlock probes, providing an assay that can interrogate both PTMs and mRNA expression at a single cell level. Thereby different nodes of the signaling pathway as well as drug effects on different types of molecules could be investigated simultaneously. In addition to regulation of gene expression, protein-DNA interactions present a mechanism to manage accessibility of the genomic DNA in an inheritable manner, providing the basis for lineage commitment, via e.g. histone PTMs. To enable analyses of protein-DNA interactions in situ we developed a method that utilizes the proximity dependence of PLA and the sequence selectivity of padlock probes. This thesis presents new methods providing researchers with a set of tools to address cellular functions and communication in complex microenvironments, to improve disease diagnostics and to contribute to hopefully finding cures.

Improved analytical methods provide new opportunities for both biological research and medical applications. This thesis describes several novel molecular techniques for nucleic acid and protein analysis based on detection or sequencing of amplified single molecules (ASMs). ASMs were generated from padlock probe assay and proximity ligation assay (PLA) through a series of molecular processes. In Paper I, a simple colorimetric readout strategy for detection of ASMs generated from padlock probe assay was used for highly sensitive detection of RNA virus, showing the potential of using padlock probes in the point-of-care diagnostics. In Paper II, digital quantification of ASMs, which were generated from padlock probe assay and PLA through circle-to-circle amplification (C2CA), was used for rapid and sensitive detection of nucleic acids and proteins, aiming for applications in biodefense. In Paper III, digital quantification of ASMs that were generated from PLA without C2CA was shown to be able to improve the precision and sensitivity of PLA when compared to the conventional real-time PCR readout. In Paper IV, a non-optical approach for detection of ASMs generated from PLA was used for sensitive detection of bacterial spores. ASMs were detected through sensing oligonucleotide-functionalized magnetic nanobeads that were trapped within them. Finally, based on in situ sequencing of ASMs generated via padlock probe assay, a novel method that enabled sequencing of individual mRNA molecules in their natural context was established and presented in Paper V. Highly multiplex detection of mRNA molecules was also achieved based on in situ sequencing. In situ sequencing allows studies of mRNA molecules from different aspects that cannot be accessed by current in situ hybridization techniques, providing possibilities for discovery of new information from the complexity of transcriptome. Therefore, it has a great potential to become a useful tool for gene expression research and disease diagnostics.

Orientador: Rogério Antonio Oliveira / Resumo: A cirrose é uma doença hepática assintomática que, muitas vezes, é descoberta quando o quadro é irreversível. Por isso, o tratamento consiste em uma série de medidas para controlar o avanço da enfermidade, visto que a principal consequência da cirrose é o aumento da pressão na veia portal, que por sua vez, acarreta no surgimento de varizes e no seu respectivo rompimento, podendo ser fatal. Estudos relacionados à esta doença são muito importantes, pois a análise estatística é uma ferramenta que permite auxiliar na tomada de decisões nos procedimentos médicos e acompanhamento de pacientes. Um método estatístico bastante explorado nas ciências biomédicas é a análise de sobrevivência, que consiste em descrever o tempo de um evento inicial até a ocorrência de um outro de interesse. No entanto, existem situações em que uma proporção da amostra não vivencia o desfecho de interesse, mesmo que acompanhado por um período longo de tempo. Nestes casos, tais observações são dita imunes ao desfecho de interesse e as metodologias tradicionais de análise de sobrevivência não são indicadas. Logo, os modelos de fração de cura ou de longa duração, desenvolvido a partir do modelo de mistura, são os utilizados nestas situações. Neste trabalho diverso modelos foram considerados para analisar os tempos de vida de pacientes submetidos à Ligadura Elástica de Varizes Esofágicas, ao qual foram anotados os tempos até o óbito durante o acompanhamento de 129 pacientes do Hospital das Clínicas da Faculdade... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Cirrhosis is an asymptomatic liver disease that is often discovered when the patient's condition is irreversible. Therefore, the treatment consists of a series of measures to control the progression of the disease, since the main consequence of the cirrhosis is the increase of the portal venous pressure, which causes the appearance of varices and their respective rupture may be fatal. Studies related to that disease are very important, so the statistical analysis is a tool that helps to make decisions in medical procedures and patient follow-up. The most applied statistical method in the biomedical sciences is survival analysis, which consists of describing the time of occurrence until the event of interest. However, there are situations in which a proportion of the sample does not experience the interest outcome, even if they are accompanied by a long period of time. In such cases, such observations are said to be immune to the outcome of interest and traditional survival analysis methodologies are not appropriated. Therefore, the care fraction or long duration models can be used in these situations because they incorporate mixtures of models to solve the complexity inherent in the actual study. In this work, some statistical models were considered to analyze the survival times of patients, after surgery of Endoscopic Band Ligation of the Esophageal Varices, such as Exponential, Gamma and Weibull models. The data is related to survival times of 129 patients, who were treated... (Complete abstract click electronic access below) / Mestre