Resultados obtidos anteriormente pelo nosso grupo mostraram que a proteína de fase aguda amilóide sérica A (SAA) é um potente estímulo para a expressão de mRNA e liberação de TNF-α, IL-1-β e IL-8 em leucócitos humanos, além de atuar como priming para a liberação de espécies reativas de oxigênio (EROs) por neutrófilos. Nosso objetivo, nesse trabalho, foi mostrar a presença de SAA em exsudatos e definir sua origem, além de verificar sua atividade pró-inflamatória in vivo. Para tanto, utilizamos soro e exsudatos pleurais de 32 pacientes com pneumonia. Mostramos primeíramente a presença da SAA no material inflamatório através de SDS-PAGE, immunoblotting e HPLC. A quantificação de SAA nas amostras foi realizada por ELISA. Nestas amostras também foram determinadas as concentrações de proteína total, proteína C reativa (PCR), apo A-I, apo A-II, apo B, colesterol total, triglicérides, TNF-α, IL-1-β e IL-8. A análise integrada dos nossos resultados indica que há uma passagem preferencial da HDL para o foco inflamatório, quando comparada as demais lipoproteínas. A SAA presente em exsudatos é originada do soro e deve sofrer intensa degradação ou associação com células. O efeito da SAA no exsudato é pró-inflamatório, sendo que esta proteína poderia ser um dos alvos para as enzimas proteolíticas e EROs presentes em exsudatos. Acreditamos que este trabalho contribua significamente para a compreensão do, ainda incerto, papel da SAA no processo inflamatório e dá nova abrangência para as funções da HDL e sua participação na reposta imune. / Previous results from our lab showed that the acute phase protein serum amyloid A (SAA) is a potent stimulus for the expression of mRNA and secretion of TNF-α, IL-1-β and IL-8 from human leukocytes. Furthermore SAA primes neutrophils for the generation of reactive oxygen species (ROS). Our goal here was to show the presence of SAA in exudates and define its origin, besides the verification of its proinflammatory activity in vivo. To achieve this goal we used serum and pleural exudates from 32 patients with pneumonia. At first, we showed the presence of SAA in the exudate through SDS-PAGE, immunoblotting and HPLC. SAA was quantified by ELISA. Besides SAA, we also determined the concentrations of total protein, C reactive protein, apo A-I, apo A-II, apo B, cholesterol, triglyceride, TNF-α, IL-1-β e IL-8. The integrate analysis of our results indicates that there is a preferential leakage of HDL to the inflammatory focus when compared to other lipoproteins. SAA present in exudates is originated from serum and may be intensively degraded or associated to cells. The effect of SAA in the exudate is proinflammatory and this protein may be a target for proteolytic enzymes and for ROS present in exudates. We believed that this work adds new insights to the, yet undefined, role of SAA in the inflammatory process and gives a broader compreension to the functions of HDL and its participation in the on the immune response.

Les nanomédecines injectées par voie intraveineuse interagissent avec les éléments biologiques qui les entourent dans le compartiment sanguin. Parmi ces interactions, celles avec les protéines sanguines se révèlent être très importantes dans le devenir de ces nanovecteurs, leur conférant une identité biologique influençant leur chemin jusqu’au tissu et aux cellules cibles. La compréhension et le contrôle de ces phénomènes reste un enjeu crucial dans le développement des nanomédecines. Des méthodes permettant une étude facilitée de ces interactions sont nécessaires à cet égard. Les travaux de cette thèse ont eu pour but de développer des méthodes, utilisables en routine, permettant une caractérisation fine des nanomédecines et de leurs interactions avec les protéines plasmatiques, applicables dans un contexte clinique. Ils s’inscrivent dans un projet intitulé « Nano Innovation for CancEr » (NICE, BPI France) regroupant un consortium de partenaires industriels en développement clinique de nanomédecines.Dans un premier temps, un travail bibliographique sur les méthodes actuellement mises en œuvre pour une telle caractérisation ont pu mettre en avant deux limitations majeures. (i) D’une part, la complexité des méthodes actuelles disponibles pour lesquelles la spécificité des équipements et l’expertise requise limitent une utilisation à large échelle. (ii) D’autre part, les propriétés aujourd’hui caractérisées en routine (taille, morphologie globale, charge) ne sont que grossières comparées à la finesse des processus biologiques qui interagissent et « analysent » les nanovecteurs une fois introduits dans le milieu biologique. Ces deux aspects limitent aujourd’hui un développement plus sûr des nanomédecines pour une bonne reproductibilité en clinique et garantir des essais de contrôle qualité fiables.Au cours de nos travaux, nous avons développé des méthodes permettant de répondre en partie à la problématique posée par la caractérisation des nanomédecines. Une méthode d’analyse à haut débit de l’activation du système du complément par immunoélectrophorèse en deux dimensions a été développée et validée. Elle permet l’analyse reproductible de l’activation de la protéine C3. Elle est applicable à l’étude de l’effet de la présence de nanoparticules dans le sérum humain et leur degré d’action sur la cascade du complément. Cette méthode a été utilisée pour mener une étude plus fondamentale du mécanisme de l’activation du système du complément en regard de l’architecture de la surface de nanoparticules.Une deuxième méthode d’étude de l’activation du complément produit par des nanomédecine a été proposée sur la base de la résonnance plasmonique de surface (SPR). Une puce permettant un screening automatisé de l’activation du complément a été développée. L’application de cette méthode comparée à d’autres méthodes d’études de l’activation du système du complément (Immunoélectrophorèse 2D, ELISA) a permis d’identifier des biais lors de leur application à l’évaluation des nanomédecines.Enfin, une approche originale de caractérisation de la surface de nanoparticules a été proposée utilisant des protéines pour sonder la capacité de la surface des nanoparticules à adsorber ou repousser ces dernières. Dans cette méthode, l’électrophorèse capillaire est utilisée comme outil analytique permettant une analyse directe de l’échantillon sans séparation préalable des nanomédecines.Les méthodes développées au cours de ces travaux peuvent être appliquées à la caractérisation de nanomédecines et proposées comme des méthodes de contrôle en routine de façon plus générale. Un développement de la caractérisation dans ce sens constitue l’un des leviers pour une translation plus fructueuse des nanomédecines entrant en phase clinique. / Nanomedicines injected intravenously interact with surrounding biological elements in the bloodstream. Among these interactions, those with blood proteins turn out to be very important regarding the becoming of the nanovectors. They acquire a biological identity upon interaction with proteins which influence their path to target tissue and cells. The understanding and mastering of these phenomena remains a crucial issue in nanomedicine development. Methods allowing an easier study of these interactions are needed. The aim of these PhD thesis was to develop such methods, usable on a routine basis in a clinical context, allowing a fine characterization of nanomedicines and their interactions with plasmatic proteins. This PhD is part of the project “Nano Innovation for CancEr” (NICE, BPI France), gathering a consortium of industrials partners developing clinical nanomedicines.In a first time, a bibliographic study about current methods used for such a characterization could identify two major limitations. (i) On one hand, the complexity of current available methods for which the equipment specificity and required expertise prevent their use at a large scale. (ii) On the other hand, properties today characterized on a daily basis (size, morphology, charge) are too rough compared to the sharpness of biological processes who interact and “analyze” the nanovectors introduced in biological media. These two aspects are limiting a safer development of nanomedicines as well as a good reproducibility of their action in clinics.During this thesis, we developed methods allowing a beginning of answer to the wide problematic of nanomedicine characterization. A method for a high throughput analysis of complement activation by nanomedicines via 2D immunoelectrophoresis was developed and validated. It allows the reproducible analysis of protein C3 fragmentation. This method is applicable to the study of the impact of nanoparticles in human serum and their degree of action on the complement cascade. This method has been used for a more fundamental study on complement activation pathways activated according to the architecture of nanoparticles surface.A second method for the study of complement activation produced by nanoparticles has been proposed using surface plasmon resonance (SPR). A chip allowing an automated screening of complement activation has been developed. This method was compared to other methods for complement activation study (2D immunoelectrophoresis, ELISA) and allowed the identification of bias during nanomedicine evaluation.Finally, an original approach for the characterization of nanomedicine’s surface architecture using proteins as molecular probes has been proposed. In this method, capillary electrophoresis has been used as analytical tool to allow a direct analysis of sample without preliminary nanoparticle removal step.Methods developed during this work can be applied to the characterization of nanomedicines and proposed as routine methods for quality control. A development of nanomedicines characterization in this direction constitute one of the lever for a more fruitful translation of nanomedicines entering in clinical phase.

A Dissulfeto Isomerase Proteica (PDI) é uma chaperona ditiol-dissulfeto oxidoredutase da superfamília tiorredoxina que catalisa o enovelamento de proteínas secretadas ou de membrana por meio da introdução, redução ou isomerização de pontes dissulfeto. A PDI é primariamente localizada no lúmen do retículo endoplasmático, no entanto a presença de uma pequena fração da PDI na superfície celular e no meio extracelular tem sido documentada em diversos tipos celulares. Particularmente em plaquetas e células endoteliais, a PDI epi/pericelular (pecPDI) está envolvida em diversos processos incluindo ativação de plaquetas/trombose, infecções virais e remodelamento vascular. A ausência de PDI (e outras tiol isomerases) na circulação tem sido proposta como mecanismo para prevenir trombose na ausência de lesão vascular. No entanto, esta questão permanece obscura e existe pouca informação sobre a concentração circulante da PDI e outras tiol isomerases vasculares. Neste estudo, investigamos a ocorrência e implicações fisiológicas de um pool circulante de PDI em indivíduos saudáveis e validamos um ensaio para detecção da PDI. Os resultados mostraram um pool detectável de PDI no plasma por ELISA, confirmados por imunoprecipitação e ensaio de atividade (inibição da redução da sonda di-eosina-GSSG pela rutina, um inibidor específico da PDI). A concentração de PDI no plasma (mediana=330 pg/mL) indicam uma alta variabibilidade interindividual, com valores muito baixos/indetectáveis (plasmas pobres em PDI [PP-PDI], definidos como <= 330 pg/mL) até valores superiores a 1000 pg/mL (plasmas ricos em PDI [PR-PDI], designados como > 330 pg/mL). Por outro lado, um resultado importante foi o fato de que valores de PDI mostraram variabilidade intraindividual muito baixa ao longo do tempo, detectada através de medidas repetidas em diferentes ocasiões e/ou condições. A fração de PDI presente em micropartículas plasmáticas foi variável, mas em geral pequena em relação ao pool total de PDI. O pool da PDI no plasma está majoritariamente reduzido (60-80%) sem diferenças entre os grupos PP-PDI e PR-PDI. Importante, os valores de PDI associaram-se a distintos perfis proteômicos plasmáticos. Enquanto os PR-PDI se associaram preferencialmente a proteínas relacionadas a diferenciação celular, processamento de proteínas, funções housekeepings, entre outras, os PP-PDI mostraram expressão diferencial de proteínas associadas a coagulação, respostas inflamatórias e imunoativação. A atividade de plaquetas medida por agregação foi semelhante entre os indivíduos com PP-PDI vs. PR-PDI. No entanto, a PDI solúvel foi diminuída após agregação plaquetária na maioria dos indivíduos em ambos os grupos, sugerindo captura devida a exposição de moléculas adesivas. Em outras séries de experimentos, mostramos que tais perfis proteômicos plasmáticos se correlacionaram ao fenótipo e função endotelial. Células endoteliais em cultura incubadas com PP-PDI ou PR-PDI recapitularam padrões de expressão gênica e de secreção de proteínas similares aos perfis plasmáticos correspondentes. Além disso, as assinaturas proteômicas identificadas em ambos os tipos de plasma traduziram-se em distintas respostas funcionais endoteliais. Os PP-PDI promoveram comprometimento da adesão de células endoteliais à fibronectina e perturbaram o padrão de migração celular associado à reparação de lesão endotelial. Em contraste, os PR-PDI não afetaram significativamente a adesão celular e sustentaram um padrão de migração organizado. Em outra população de pacientes com eventos cardiovasculares, os valores de PDI no plasma (mediana= 35 pg/mL) foram significativamente inferiores aos de indivíduos saudáveis. Em conclusão, o pool detectável de PDI presente no plasma se associou a distintos perfis proteômicos e parece se comportar como um indicador/ marcador de assinaturas proteômicas relacionadas à função e sinalização endotelial. Este é o primeiro estudo descrevendo valores circulantes de PDI diretamente relacionados a distintos fenótipos endoteliais / Protein disulfide isomerase (PDI) is a dithiol-disulfide oxidoreductase chaperone from thioredoxin superfamily which catalyzes introduction, reduction or isomerization of disulfide bonds in nascent proteins, typically destined to extracellular secretion or membrane insertion. PDI is primarily located into the endoplasmic reticulum; however, there are clear evidences for the presence of a small PDI fraction at the cell surface and extracellular milieu in several cell types. Particularly in platelets and endothelial cells, such peri/epicellular pool of PDIA1 (pecPDI) is involved in distinct processes including platelet activation/thrombosis, viral infection and vascular remodeling. The absence of PDI (and other thiol isomerases) from circulating plasma has been proposed as a mechanism to prevent thrombogenesis in the absence of vascular injury. However, this question remains unclear, as there is little information on the circulating levels of PDI and other vascular thiol isomerases. Here we investigated the occurrence and physiological significance of a circulating pool of PDI in healthy humans. We validated an assay for detecting PDI in plasma of healthy individuals. The results showed a detectable pool of plasma PDI by ELISA, confirmed by immunoprecipitation and activity assay (dieosin-GSSG inhibitable by rutin, a specific PDI inhibitor). PDI levels (median= 330 pg/mL) exhibited high interindividual variability, ranging from undetectable/low (PDI-poor plasma, defined as <= 330 pg/mL) until 1000 pg/mL (PDI-rich plasma, > 330 pg/mL). Remarkably, opposite to interindividual variability, the intra-individual variability was quite low, so that values assessed under distinct conditions over time were close and reproducible. The majority (60-80%) of plasma PDI is in the reduced state, without any difference among individuals with PDIpoor and PDI-rich plasma. Importantly, plasma PDI levels could discriminate between distinct plasma proteome signatures, with PDI-rich plasma differentially expressing proteins related to cell differentiation, protein processing, housekeeping functions and others, while PDI-poor plasma differentially displayed proteins associated with coagulation, inflammatory responses and immunoactivation. Platelet activity assessed by aggregation was similar between PDI-poor vs. PDI-rich plasma. However, soluble PDI was decreased after platelet activation in both groups, suggesting sequestration of plateletderived PDI by its potential substrates. In other set of experiments, we showed that such protein signatures closely correlated with endothelial function and phenotype, since cultured endothelial cells incubated with PDI-poor or PDI-rich plasma recapitulated gene expression and secretome patterns in line with their corresponding plasma signatures. Furthermore, such signatures translated into functional responses, with PDI-poor plasma promoting impairment of endothelial adhesion to fibronectin and a disturbed pattern of wound-associated migration and recovery area. In contrast, PDI-rich plasma did not significantly affect cell adhesion and supported organized endothelial migration. In another dataset, patients with cardiovascular events had lower PDI levels (median= 35 pg/mL) vs. healthy individuals. In conclusion, a PDI pool detectable in plasma from healthy individuals is associated with distinct proteomic profiles and seems to behave as an indicator/marker of proteomic signatures related with endothelial function and signaling. This is the first study describing PDI levels as reporters of specific plasma proteome signatures directly promoting contrasting endothelial phenotypes and functional responses

The marked increase in surface-to-volume ratio associated with microscale devices for hemodialysis leads to problems with hemocompatibility and blood flow distribution that are more challenging to manage than those encountered at the conventional scale. In this work, stable surface modifications with pendant polyethylene oxide (PEO) chains were produced on polycarbonate microchannel and polyacrylonitrile membrane materials used in construction of microchannel hemodialyzer test articles. These coatings were evaluated in relation to protein repulsion, impact on urea permeability through the membrane, and impact on bubble retention through single-channel test articles. PEO layers were prepared by radiolytic grafting of PEO-PBD-PEO (PBD = polybutadiene) triblock copolymers to microchannel and membrane materials. Protein adsorption was detected by measurement of surface-bound enzyme activity following contact of uncoated and PEO-coated surfaces with ��-galactosidase. Protein adsorption was decreased on PEO-coated polycarbonate and polydimethyl siloxane (PDMS) materials by 80% when compared to the level recorded on uncoated materials. Protein adsorption on membrane materials was not decreased with PEO-PBD-PEO treatment; a PEI (polyethylene imide) layer exists on the AN69 ST membrane which is intended to trap heparin during membrane pre-treatment. It is still unclear how this PEI layer interacts with PEO-PBD-PEO. Neither the PEO-PBD-PEO triblocks nor the irradiation process was observed to have any effect on polyacrylonitrile membrane permeability to urea, nor did the presence of additional fibrinogen and bovine serum albumin (BSA) in the urea filtrate. The PEO-PBD-PEO treatment was not able to visibly reduce bubble retention during flow through single-channel polycarbonate test articles, however, the rough surfaces of the laser-etched polycarbonate microchannels may be causing this bubble retention. This surface treatment holds promise as a means for imparting safe, efficacious coatings to blood processing equipment that ensure good hemocompatibility and blood flow distribution, with no adverse effects on mass transfer. / Graduation date: 2013

A RBP urinária tem seu papel bem definido como marcador de progressão de doença renal em tubulopatias, em glomerulopatias e em pacientes transplantados. No entanto, seu papel em DRC lato senso foi pouco estudado. Por sua vez, a dosagem de RBP sérica, caracterizada recentemente como biomarcador de resistência insulínica, também não teve seu papel esclarecido em população portadora de DRC. O objetivo do presente estudo foi avaliar a relação entre a RBP (urinária e sérica) e a função renal, assim como sua relação com fatores de risco cardiovascular em população de DRC. Para tanto, foram analisados os dados da linha de base da Coorte PROGREDIR, constituída por 454 participantes portadores de DRC, oriundos do Hospital das Clínicas, São Paulo. Inicialmente, além de estar inversamente relacionada às medidas de depuração de creatinina, a RBP urinária mostrou-se relacionada a diversos fatores de risco cardiovascular e variáveis associadas à função renal, como proteinúria, metabolismo ósseo, anemia, acidose, albumina, RPB sérica, Hb glicada, HOMA, lípides, velocidade de onda de pulso (VOP), átrio esquerdo (ECO AE), diâmetro diastólico do ventrículo esquerdo (ECO-Ddve), diâmetro sistólico do ventrículo esquerdo (ECO-Dsve) e fração de ejeção (ECO-Fe). Entretanto, após diversos modelos de regressão, permaneceram como variáveis independentemente associadas à RBP urinária a função renal, a pressão arterial sistólica, a albuminúria, a acidose e a medida do AE. Esse resultado se manteve quando o modelo foi repetido mediante estratificação por albuminúria, sugerindo que mesmo em população normoalbuminúrica a RBP urinária correlacione-se inversamente com função renal. Além disso, a relação inversa de RBP urinária com dilatação cardíaca sugere que, em população com DRC já estabelecida, a RBP urinária possa ter um papel em identificar mecanismos etiológicos, possivelmente por distinguir mecanismos hemodinâmicos daqueles onde há uma patologia renal intrínseca. Por sua vez, a RBP sérica relacionou-se inversamente à função renal e idade, e positivamente a triglicérides, albumina e potássio. Curiosamente, a RBP sérica não se mostrou associada às medidas de metabolismo de carboidrato, sugerindo que seu papel como biomarcador de resistência insulínica seja atenuado na DRC. Também não foram encontradas relações entre RBP urinária ou sérica e calcificação coronária ou espessura de carótidas. Nossos resultados sugerem que a RBP urinária deva ser melhor explorada como marcador de função renal e, possivelmente, como marcador de risco de progressão da DRC / The role of urinary RBP as a biomarker of tubular injury and CKD progression in tubulopathies, glomerulopathies and in transplantation is well established. However, its role in CKD is less studied. In addition, serum RBP has been recently characterized as an insulin resistance marker, but controversial results have been shown. The aim of the study was to evaluate the association of both urinary RBP and serum RBP with kidney function and other variables related to the uremic syndrome and cardiovascular risk in a CKD population. We used the baseline data from the PROGREDIR Cohort, which comprehends 454 participants with CKD, recruited from Hospital das Clínicas, Sao Paulo. In univariate analysis, urinary RBP was inversely related to renal function. In addition, it was also related to albuminuria, SBP, anemia, mineral metabolism, acidosis, albumin, serum RPB, glycated hemoglobin, HOMA, lipids, pulse-wave velocity, left atrium diameter, left ventricle diastolic diameter, left ventricle systolic diameter and ejection fraction. However, in the multivariate analysis, only SBP, albuminuria, acidosis, left atrium diameter and renal function remained significantly associated to urinary RBP. After stratification for albuminuria levels, the same relationship was observed, suggesting that even in the normoalbuminuric population urinary RBP is significantly related to renal function. Interestingly, the inverse association between urinary RBP and cardiac dilation suggests that urinary RBP may play a role in identifying mechanisms related to CKD, by differentiating vascular/cardio-renal conditions versus more intrinsic kidney disease and possibly tubule-interstitial fibrosis. The serum RBP was positively related to renal function, triglycerides, albumin, age and potassium, but not to measurements of carbohydrate metabolism. No relationship between urinary or serum RBP and coronary calcification or carotid thickness was found. Our results suggest that urinary RBP should have its role as a marker of CKD and CKD progression further explored

Chen, Zhang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (p. 109-114). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.III / ACKNOWLEDGEMENTS --- p.IV / PUBLICATIONS --- p.VI / CONTRIBUTORS --- p.VII / TABLE OF CONTENTS --- p.VIII / LIST OF TABLES --- p.XIII / LIST OF FIGURES --- p.XIV / LIST OF ABBREVIATIONS --- p.XVI / Chapter SECTION I : --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- PRENATAL DIAGNOSIS OF FETAL TRISOMY BY NEXT GENERATION SEQUENCING TECHNOLOGY --- p.2 / Chapter 1.1 --- FETAL TRISOMY --- p.2 / Chapter 1.2 --- CONVENTIONAL PRENATAL DIAGNOSIS OF FETAL TRISOMIES --- p.3 / Chapter 1.3 --- CELL FREE FETAL D N A AND ITS APPLICATION IN PRENATAL DIAGNOSIS --- p.5 / Chapter 1.4 --- NEXT GENERATION SEQUENCING TECHNOLOGY --- p.5 / Chapter 1.5 --- SUBSTANTIAL BIAS IN THE NEXT GENERATION SEQUENCING PLATFORM --- p.9 / Chapter 1.6 --- PRENATAL DIAGNOSIS OF TRISOMY BY NEXT GENERATION SEQUENCING --- p.10 / Chapter 1.7 --- AIMS OF THIS THESIS --- p.11 / Chapter SECTION I I : --- MATERIALS AND METHODS --- p.13 / Chapter CHAPTER 2: --- METHODS FOR NONINVASIVE PRENATAL DIAGNOSIS OF FETAL TRISOMY MATERNAL PLASMA DNA SEQUENCING --- p.14 / Chapter 2.1 --- STUDY DESIGN AND PARTICIPANTS --- p.14 / Chapter 2.1.1 --- Ethics Statement --- p.14 / Chapter 2.1.2 --- "Study design, setting and participants" --- p.14 / Chapter 2.2 --- MATERNAL PLASMA D N A SEQUENCING --- p.17 / Chapter 2.3 --- SEQUENCING DATA ANALYSIS --- p.18 / Chapter SECTION I I I : --- TRISOMY 13 AND 18 DETECTION BY THE T21 BIOINFORMATICS ANALYSIS PIPELINE --- p.21 / Chapter CHAPTER 3: --- THE T21 BIOINFORMATICS ANALYSIS PIPELINE FOR TRISOMY 13 AND 18 DETECTION --- p.22 / Chapter 3.1 --- INTRODUCTION --- p.22 / Chapter 3.2 --- METHODS --- p.23 / Chapter 3.2.1 --- Bioinformatics analysis pipeline for trisomy 13 and 18 detection --- p.23 / Chapter 3.3 --- RESULTS --- p.23 / Chapter 3.3.1 --- Performance of the T21 bioinformatics analysis pipeline for trisomy 13 and 18 detection --- p.23 / Chapter 3.3.2 --- The precision of quantifying chrl 3 and chrl 8 --- p.27 / Chapter 3.4 --- DISCUSSION --- p.29 / Chapter SECTION IV : --- IMPROVING THE T21 BIOINFORMATICS ANALYSIS PIPELINE FOR TRISOMY 13 AND 18 DETECTION --- p.30 / Chapter CHAPTER 4: --- IMPROVING THE ALIGNMENT --- p.31 / Chapter 4.1 --- INTRODUCTION --- p.31 / Chapter 4.2 --- METHODS --- p.32 / Chapter 4.2.1 --- Allowing mismatches in the index sequences --- p.32 / Chapter 4.2.2 --- Calculating the mappability of the human reference genome --- p.33 / Chapter 4.2.3 --- Aligning reads to the non-repeat masked human reference genome --- p.34 / Chapter 4.2.4 --- Trisomy 13 and 18 detection --- p.34 / Chapter 4.3 --- RESULTS --- p.34 / Chapter 4.3.1 --- Increasing read numbers by allowing mismatches in the index sequences --- p.34 / Chapter 4.3.2 --- Increasing read numbers by using the non-masked reference genome for alignment . --- p.38 / Chapter 4.3.3 --- Allowing mismatches in the read alignment --- p.42 / Chapter 4.3.4 --- The performance of trisomy 13 and 18 detection after improving the alignment --- p.47 / Chapter 4.4 --- DISCUSSION --- p.50 / Chapter CHAPTER 5: --- REDUCING THE GC BIAS BY CORRECTION OF READ COUNTS --- p.53 / Chapter 5.1 --- INTRODUCTION --- p.53 / Chapter 5.2 --- METHODS --- p.54 / Chapter 5.2.1 --- Read alignment --- p.54 / Chapter 5.2.2 --- Calculating the correlation between GC content and read counts --- p.55 / Chapter 5.2.3 --- GC correction in read counts --- p.55 / Chapter 5.2.4 --- Trisomy 13 and 18 detection --- p.56 / Chapter 5.3 --- RESULTS --- p.56 / Chapter 5.3.1 --- GC bias in plasma DNA sequencing --- p.56 / Chapter 5.3.2 --- Correcting the GC bias in read counts by linear regression --- p.59 / Chapter 5.3.3 --- Correcting the GC bias in read counts by LOESS regression --- p.65 / Chapter 5.3.4 --- Bin size --- p.72 / Chapter 5.4 --- DISCUSSION --- p.75 / Chapter CHAPTER 6: --- REDUCING THE GC BIAS BY MODIFYING THE GENOMIC REPRESENTATION CALCULATION --- p.77 / Chapter 6.1 --- INTRODUCTION --- p.77 / Chapter 6.2 --- METHODS --- p.78 / Chapter 6.2.1 --- Modifying the genomic representation calculation --- p.78 / Chapter 6.2.2 --- Trisomy 13 and 18 detection --- p.78 / Chapter 6.2.3 --- Combining GC correction and modified genomic representation --- p.78 / Chapter 6.3 --- RESULTS --- p.79 / Chapter 6.3.1 --- Reducing the GC bias by modifying genomic representation calculation --- p.79 / Chapter 6.3.2 --- Combining GC correction and modified genomic representation --- p.86 / Chapter 6.4 --- DISCUSSION --- p.89 / Chapter CHAPTER 7: --- IMPROVING THE STATISTICS FOR TRISOMY 13 AND 18 DETECTION --- p.91 / Chapter 7.1 --- INTRODUCTION --- p.91 / Chapter 7.2 --- METHODS --- p.92 / Chapter 7.2.1 --- Comparing chrl 3 or chrl8 with other chromosomes within the sample --- p.92 / Chapter 7.2.2 --- Comparing chrl 3 or chrl 8 with the artificial chromosomes --- p.92 / Chapter 7.3 --- RESULTS --- p.93 / Chapter 7.3.1 --- Determining the trisomy 13 and 18 status by comparing chromosomes within the samples --- p.93 / Chapter 7.3.2 --- Determining the trisomy 13 and 18 status by comparing chrl3 or chrl 8 with artificial chromosomes --- p.97 / Chapter 7.4 --- DISCUSSION --- p.100 / Chapter SECTION V : --- CONCLUDING REMARKS --- p.102 / Chapter CHAPTER 8: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.103 / Chapter 8.1 --- THE PERFORMANCE OF THE T21 BIOINFORMATICS ANALYSIS PIPELINE DEVELOPED FOR TRISOMY 21 DETECTION IS SUBOPTIMAL FOR TRISOMY 13 AND 18 DETECTION --- p.103 / Chapter 8.2 --- THE ALIGNMENT COULD BE IMPROVED BY ALLOWING ONE MISMATCH IN THE INDEX AND USING THE NON-REPEAT MASKED HUMAN REFERENCE GENOME AS THE ALIGNMENT REFERENCE --- p.104 / Chapter 8.3 --- THE PRECISION OF QUANTIFYING CHR13 AND CHR18 COULD BE IMPROVED BY THE G C CORRECTION OR THE MODIFIED GENOMIC REPRESENTATION --- p.104 / Chapter 8.4 --- THE STATISTICS FOR TRISOMY 13 AND 18 DETECTION COULD BE IMPROVED BY COMPARING CHR13 OR CHR18 WITH ARTIFICIAL CHROMOSOMES WITHIN THE SAMPLE --- p.105 / Chapter 8.5 --- PROSPECTS FOR FUTURE WORK --- p.106 / REFERENCE --- p.109

<p>The objective of this thesis was to investigate and characterize the interaction between blood proteins and different surfaces with emphasis on protein adsorption to bioceramics and model surfaces. Special effort was made to monitor the spontaneous and selective adsorption of proteins from human plasma and to examine the orientation, conformation and functional behavior of single proteins after adsorption. </p><p>Five different ceramic biomaterials: alumina (Al₂O₃), zirconia (ZrO₂), hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) and two glass-ceramics, AP40 (SiO₂-CaO-Na₂O-P₂O₅-MgO-K₂O-CaF₂) and RKKP (AP40 with Ta₂O₃-La₂O₃), were exposed to human plasma and their protein binding capacities and affinities for specific proteins were studied by chromatography, protein assays, two-dimensional gel electrophoresis and Western blotting. The studies showed that all materials adsorbed approximately the same high amount of plasma proteins and that they therefore should be fully covered by proteins in an <i>in vivo</i> setting. The adsorbed proteins were different for most materials which could explain their previously observed different levels of tissue integration <i>in vivo</i>. </p><p>Four of the proteins that behaved differently, ceruloplasmin, prothrombin, α₂-HS-glycoprotein and α₁-antichymotrypsin, were selected for characterization with atomic force microscopy and ellipsometry. The studies, which were performed on ultraflat silicon wafers (silica), showed that the proteins oriented themselves with their long axis parallel to the surface or as in case of ceruloplasmin with one of its larger sides towards the surface. All of them had globular shapes but other conformational details were not resolved. Furthermore, prothrombin (none of the others) formed multilayers at high proteins concentrations. </p><p>The functional behaviour of the adsorbed proteins, referring to their cell binding and cell spreading capacity on silica and a positive cell adhesion reference surface (Thermanox®), was affected by the underlying substrate. Ceruloplasmin, α₂-HS-glycoprotein and α₁-antichymotrypsin stimulated cell attachment to silica, but suppressed attachment to Thermanox®. Prothrombin stimulated cell attachment to both surfaces. The attachment was in most cases mediated both by cell membrane-receptors (integrins) and by non-specific interactions between the cell and the material. </p><p>This thesis showed that the compositional mixture, orientation, conformation and functional behavior of the adsorbed proteins are determined by the properties of the underlying surface and if these parameters are controlled very different cellular responses can be induced.</p>

The objective of this thesis was to investigate and characterize the interaction between blood proteins and different surfaces with emphasis on protein adsorption to bioceramics and model surfaces. Special effort was made to monitor the spontaneous and selective adsorption of proteins from human plasma and to examine the orientation, conformation and functional behavior of single proteins after adsorption. Five different ceramic biomaterials: alumina (Al2O3), zirconia (ZrO2), hydroxyapatite (Ca10(PO4)6(OH)2) and two glass-ceramics, AP40 (SiO2-CaO-Na2O-P2O5-MgO-K2O-CaF2) and RKKP (AP40 with Ta2O3-La2O3), were exposed to human plasma and their protein binding capacities and affinities for specific proteins were studied by chromatography, protein assays, two-dimensional gel electrophoresis and Western blotting. The studies showed that all materials adsorbed approximately the same high amount of plasma proteins and that they therefore should be fully covered by proteins in an in vivo setting. The adsorbed proteins were different for most materials which could explain their previously observed different levels of tissue integration in vivo. Four of the proteins that behaved differently, ceruloplasmin, prothrombin, α2-HS-glycoprotein and α1-antichymotrypsin, were selected for characterization with atomic force microscopy and ellipsometry. The studies, which were performed on ultraflat silicon wafers (silica), showed that the proteins oriented themselves with their long axis parallel to the surface or as in case of ceruloplasmin with one of its larger sides towards the surface. All of them had globular shapes but other conformational details were not resolved. Furthermore, prothrombin (none of the others) formed multilayers at high proteins concentrations. The functional behaviour of the adsorbed proteins, referring to their cell binding and cell spreading capacity on silica and a positive cell adhesion reference surface (Thermanox®), was affected by the underlying substrate. Ceruloplasmin, α2-HS-glycoprotein and α1-antichymotrypsin stimulated cell attachment to silica, but suppressed attachment to Thermanox®. Prothrombin stimulated cell attachment to both surfaces. The attachment was in most cases mediated both by cell membrane-receptors (integrins) and by non-specific interactions between the cell and the material. This thesis showed that the compositional mixture, orientation, conformation and functional behavior of the adsorbed proteins are determined by the properties of the underlying surface and if these parameters are controlled very different cellular responses can be induced.

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 5 uppsatser. Även tryckt utgåva.

A RBP urinária tem seu papel bem definido como marcador de progressão de doença renal em tubulopatias, em glomerulopatias e em pacientes transplantados. No entanto, seu papel em DRC lato senso foi pouco estudado. Por sua vez, a dosagem de RBP sérica, caracterizada recentemente como biomarcador de resistência insulínica, também não teve seu papel esclarecido em população portadora de DRC. O objetivo do presente estudo foi avaliar a relação entre a RBP (urinária e sérica) e a função renal, assim como sua relação com fatores de risco cardiovascular em população de DRC. Para tanto, foram analisados os dados da linha de base da Coorte PROGREDIR, constituída por 454 participantes portadores de DRC, oriundos do Hospital das Clínicas, São Paulo. Inicialmente, além de estar inversamente relacionada às medidas de depuração de creatinina, a RBP urinária mostrou-se relacionada a diversos fatores de risco cardiovascular e variáveis associadas à função renal, como proteinúria, metabolismo ósseo, anemia, acidose, albumina, RPB sérica, Hb glicada, HOMA, lípides, velocidade de onda de pulso (VOP), átrio esquerdo (ECO AE), diâmetro diastólico do ventrículo esquerdo (ECO-Ddve), diâmetro sistólico do ventrículo esquerdo (ECO-Dsve) e fração de ejeção (ECO-Fe). Entretanto, após diversos modelos de regressão, permaneceram como variáveis independentemente associadas à RBP urinária a função renal, a pressão arterial sistólica, a albuminúria, a acidose e a medida do AE. Esse resultado se manteve quando o modelo foi repetido mediante estratificação por albuminúria, sugerindo que mesmo em população normoalbuminúrica a RBP urinária correlacione-se inversamente com função renal. Além disso, a relação inversa de RBP urinária com dilatação cardíaca sugere que, em população com DRC já estabelecida, a RBP urinária possa ter um papel em identificar mecanismos etiológicos, possivelmente por distinguir mecanismos hemodinâmicos daqueles onde há uma patologia renal intrínseca. Por sua vez, a RBP sérica relacionou-se inversamente à função renal e idade, e positivamente a triglicérides, albumina e potássio. Curiosamente, a RBP sérica não se mostrou associada às medidas de metabolismo de carboidrato, sugerindo que seu papel como biomarcador de resistência insulínica seja atenuado na DRC. Também não foram encontradas relações entre RBP urinária ou sérica e calcificação coronária ou espessura de carótidas. Nossos resultados sugerem que a RBP urinária deva ser melhor explorada como marcador de função renal e, possivelmente, como marcador de risco de progressão da DRC / The role of urinary RBP as a biomarker of tubular injury and CKD progression in tubulopathies, glomerulopathies and in transplantation is well established. However, its role in CKD is less studied. In addition, serum RBP has been recently characterized as an insulin resistance marker, but controversial results have been shown. The aim of the study was to evaluate the association of both urinary RBP and serum RBP with kidney function and other variables related to the uremic syndrome and cardiovascular risk in a CKD population. We used the baseline data from the PROGREDIR Cohort, which comprehends 454 participants with CKD, recruited from Hospital das Clínicas, Sao Paulo. In univariate analysis, urinary RBP was inversely related to renal function. In addition, it was also related to albuminuria, SBP, anemia, mineral metabolism, acidosis, albumin, serum RPB, glycated hemoglobin, HOMA, lipids, pulse-wave velocity, left atrium diameter, left ventricle diastolic diameter, left ventricle systolic diameter and ejection fraction. However, in the multivariate analysis, only SBP, albuminuria, acidosis, left atrium diameter and renal function remained significantly associated to urinary RBP. After stratification for albuminuria levels, the same relationship was observed, suggesting that even in the normoalbuminuric population urinary RBP is significantly related to renal function. Interestingly, the inverse association between urinary RBP and cardiac dilation suggests that urinary RBP may play a role in identifying mechanisms related to CKD, by differentiating vascular/cardio-renal conditions versus more intrinsic kidney disease and possibly tubule-interstitial fibrosis. The serum RBP was positively related to renal function, triglycerides, albumin, age and potassium, but not to measurements of carbohydrate metabolism. No relationship between urinary or serum RBP and coronary calcification or carotid thickness was found. Our results suggest that urinary RBP should have its role as a marker of CKD and CKD progression further explored