The early identification of septically infected newborn infants is important for ensuring good outcomes. Blood cell differentiations are helpful, but they are often time consuming and inaccurate. In this study, we evaluated the use of automatic white blood cell differentiations by flow cytometry for the diagnosis of neonatal sepsis. Episodes of suspected infection in neonates were retrospectively classified into two groups, unlikely infection (UI, levels of Interleukin-6 < 400 pg/mL or CRP within 48 h < 10 mg/L), n = 101 and probable infection (PI, Interleukin-6 ≥ 400 pg/mL or CRP within 48 h ≥ 10 mg/L), n = 98. Complete blood cell counts were performed by Sysmex XN-9000® using flow cytometry. Relative and absolute proportions of immature granulocytes were evaluated. Unexpectedly, the absolute count of immature granulocytes was significantly lower in the group of PI compared to UI neonates. Similar results were found when analysing the relative proportion of immature granulocytes among all neutrophil granulocytes. On the other hand, manually counted immature to total (I/T) ratios of granulocytes were higher in PI than in UI infants. Therefore, we conclude that differentiations of granulocytes by Sysmex XN-9000® can be used to distinguish between infected and uninfected neonates if the results are interpreted according to our findings. A low count of immature granulocytes as determined by Sysmex XN-9000® may indicate neonatal infection.

The delivery of drugs into solid tumors is not trivial due to obstructions in the tumor microenvironment. Innovative drug delivery vehicles are currently being designed to overcome this challenge. In this research, computational fluid dynamics (CFD) simulations were used to evaluate the behavior of several drug delivery vectors in tumor capillaries—specifically motile bacteria, non-motile bacteria, and nanoparticles. Red blood cells, bacteria, and nanoparticles were imposed in the flow using the immersed boundary method. A human capillary model was developed using a novel method of handling deformable red blood cells (RBC). The capillary model was validated with experimental data from the literature. A stochastic model of bacteria motility was defined based on experimentally observed run and tumble behavior. The capillary and bacteria models were combined to simulate the intracapillary transport of bacteria. Non-motile bacteria and nanoparticles of 200 nm, 300 nm, and 405 nm were also simulated in capillary flow for comparison to motile bacteria. Motile bacteria tended to swim into the plasma layer near the capillary wall, while non-motile bacteria tended to get caught in the bolus flow between the RBCs. The nanoparticles were more impacted by Brownian motion and small scale fluid fluctuations, so they did not trend toward a single region of the flow. Motile bacteria were found to have the longest residence time in a 1 mm long capillary as well as the highest average radial velocity. This suggests motile bacteria may enter the interstitium at a higher rate than non-motile bacteria or nanoparticles of diameters between 200–405 nm. / Master of Science / The last 50 years have brought significant advancements in cancer treatment. Despite progress, cancer still remains one of the leading causes of death. In 2016, an estimated 1.7 million new cases of cancer will be diagnosed, and nearly 600,000 people will die from the disease in the United States alone. This is due to numerous unsolved challenges in the field of cancer research. The present study looks at one of these challenges—specially the delivery of drugs into a solid tumor. Several biological factors prohibit chemotherapy drugs from fully penetrating tumors. This prevents the drugs from completely killing the cancer, and can lead to ineffective treatment or recurrence. Innovative new techniques to help drugs better penetrate tumors are under development. One such technique is to harness bacteria to carry drugs inside of tumors. The goal of the present research is to evaluate the behavior of drug carrying bacteria with computer simulations. Blood vessels carry things in and out of tumors. The smallest blood vessels, the capillaries, are the location at which bacteria enter the tumor. The computer simulations found potential for swimming bacteria to enter the tumor at greater rates than other methods of drug delivery. Behavior of bacteria in capillaries is important, but just one of many aspects of this treatment strategy so research is ongoing. Beyond the simulations run for this study, the computer software developed during this project could also have other applications in engineering and biology research.

Les facteurs génétiques peuvent apporter des réponses à plusieurs questions que nous nous posons sur les traits humains, les maladies et la réaction aux médicaments, entre autres. Avec le temps, le développement continu d'outils d'analyse génétique nous a permis d'examiner ces facteurs et de trouver des explications pertinentes. Cette thèse explore plusieurs méthodes et outils génétiques, tels que le séquençage pan-exomique et le génotypage sur puce, dans un contexte d'analyse familial et populationnel pour étudier ces facteurs génétiques qui jouent un rôle dans une maladie rare, la cardiomyopathie dilatée (DCM), et dans deux traits complexes soient les globules rouges et les plaquettes. DCM est une maladie rare qui est définie par un ventricule gauche dilaté et une dysfonction systolique. Environ 30% des cas de DCM sont héréditaires, et plus de 50 gènes ont été associés à un rôle dans la pathogénicité de DCM. Le dépistage génétique est un outil de référence dans la gestion clinique de DCM familiale. Par contre, pour la majorité des patients, les tests génétiques ne parviennent pas à identifier une mutation causale dans un gène candidat. Les cellules sanguines remplissent une variété de fonctions biologiques, incluant le transport de l'oxygène, les fonctions immunologiques, ainsi que la guérison de plaies. Les niveaux de ces cellules et leurs paramètres auxiliaires sont mesurés par un test sanguin, et une différence avec les valeurs optimales peut signifier certains troubles. De plus, ces traits sont étudiés méticuleusement dans le contexte des maladies cardiovasculaires (CVD) où différents niveaux sont associés avec un risque variable de CVD ou sont des prédicteurs de complications de CVD. iii J'ai examiné la DCM et les traits sanguins avec comme objectif de découvrir des nouvelles associations de mutations génétiques. Pour la DCM, j'ai évalué la pertinence d'un séquençage pan-exomique dans un environnement clinique. Je rapporte plusieurs nouvelles mutations dans des gènes candidats (DSP, LMNA, MYH7, MYPN, RBM20, TNNT2) et des mutations nonsenses dans deux gènes nouvellement associés (TTN et BAG3), et je démontre que les mutations nonsenses influencent la maladie d'une manière différente des autres mutations causales. Je rapporte aussi une mutation dans un nouveau gène, FLNC, qui cause une forme rare et distincte de cardiomyopathie. Pour l'étude des traits complexes, dans le grand consortium Blood Cell Consortium (BCX), j'ai utilisé l’exomechip pour disséquer le rôle des variantes rares et communes dans les globules rouges et les plaquettes. J'ai identifié 16 nouvelles régions génomiques associées avec les globules rouges et 15 avec les plaquettes, parmi lesquelles se retrouvent plusieurs variantes de basses fréquences (MAP1A, HNF4A, ITGA2B, APOH), et j'ai démontré un chevauchement significatif de régions associées avec d'autres traits, incluant les lipides. Mes résultats sur la DCM ont mis en évidence le rôle de plusieurs gènes candidats, et suggèrent un traitement différent au niveau de la gestion clinique des patients qui portent des mutations dans BAG3 et FLNC. En ce qui concerne les traits sanguins, mes résultats contribuent à enrichir le répertoire de régions associées avec ces traits, soulignant l'importance de l'utilisation de grands ensembles de données pour détecter les variantes rares ou de basses fréquences. La découverte de gènes dans les maladies rares et les traits complexes contribue à la compréhension des mécanismes sous-jacents qui ultimement favorisera de meilleurs diagnostics, gestions et traitements de maladies. / Genetic factors hold within them the answers to many questions we have on human traits, disease, and drug response among others. With time, the continuously advancing genetic tools have enabled us to examine those factors and provided and continue to provide astonishing answers. This thesis utilizes various methods of genetic tools such as exome sequencing and chip-based genotyping data in the context of both family and population-based analyses to interrogate the genetic factors that play a role in a rare disease, dilated cardiomyopathy (DCM), and in two complex traits, red blood cells and platelets. DCM is a rare disease that is defined by a dilated left ventricle and systolic dysfunction. It is estimated that 30% of DCM cases are hereditary and more than 50 genes have been linked to play a role in the pathogenesis of DCM. Genetic screening of known genes is a gold standard tool in the clinical management of familial DCM. However, in the majority of probands, genetic testing fails to identify the causal mutation. Blood cells play a variety of biological functions including oxygen transport, immunological functions, and wound healing. Levels of these cells and their associated indices are measured by a blood test, and deviation from optimal values may indicate certain disorders. Additionally, these traits are heavily studied in the context of cardiovascular disease (CVD) where different levels associate with a variable risk of CVD or are predictors of CVD complications or outcomes (for example, a higher level of white blood cells or lower level of hemoglobin). I examined both DCM and blood cell traits and aimed to discover new mutations and variants that are associated with each. For DCM, I evaluated the value of whole exome vi sequencing in a clinical setting, and I report a number of novel mutations in candidate genes (DSP, LMNA, MYH7, MYPN, RBM20, TNNT2) and truncating mutations in two newly established genes, TTN and BAG3, and I demonstrate that truncating mutations in the latter influence disease differently than other causal mutations. I also report a mutation in a novel gene, FLNC that causes a rare and distinct form of cardiomyopathy. In examining complex traits, I dissected the role of common and rare variants in red blood cells and platelets within a large consortium, the Blood Cell Consortium (BCX) using the ExomeChip, and identified 16 novel loci associated with red blood cell traits and 15 with platelet traits, some of which harbored low-frequency variants (MAP1A, HNF4A, ITGA2B, APOH), and demonstrated a substantial overlap with other phenotypes predominantly lipids. My results on DCM establish the role of a number of candidate genes in this disorder and suggest a different course of clinical management for patients that carry mutations in BAG3 and FLNC. As for blood cell traits, my results contributed to expanding the repertoire of loci associated with red blood cell and platelet traits and illustrate the importance of using large datasets to discover low-frequency or rare variants. Gene discovery in rare disease and complex traits gives insight into the underlying mechanisms which ultimately contributes to a better diagnosis, management, and treatment of disease.

Le paludisme est causé par un parasite appelé Plasmodium falciparum, transmis lors de la piqûre d'un moustique. Au stade sanguin, ce parasite unicellulaire, de forme ovoïde, envahit les globules rouges, s'y multiplie avant d'être libéré pour une nouvelle invasion à la fin d'un cycle de 48 heures. Ce travail de thèse porte sur le mouvement du parasite au cours du processus d'invasion. L'étape préalable à la pénétration du parasite dans sa cellule hôte est le mouvement de réorientation permettant de mettre en contact son complexe apical avec la membrane de la cellule hôte. Afin de comprendre comment le parasite génère les mouvements nécessaires à cette réorientation sans l'aide de flagelle, de cil ou de déformation, notre approche est d'observer et de décrire le mouvement des parasites sur un substrat rigide, au travers d'une analyse détaillée des trajectoires du parasite. Nous observons que le parasite explore tous les degrés de liberté qui lui sont accessibles compte tenu de son attachement au substrat: translation et rotation dans le plan et réorientation de sa partie apicale. Nous avons identifié trois types de mouvement: confiné, dirigé et circulaire. Nous caractérisons ces trajectoires et mouvements en utilisant une analyse de corrélation et en discutant les mécanismes possibles à l'origine de ces trajectoires particulières. Enfin, nous examinons le rôle des constituants du cytosquelette sur le mouvement du parasite, en affectant spécifiquement les filaments d'actine et les microtubules. Les conséquences de la polymérisation de ces structures sur le mouvement du parasites sont discutées. / Malaria is caused by a parasite called Plasmodium falciparum, transmitted via mosquito's bites. At the blood stage, these unicellular ovoidal parasites invade red blood cells (RBCs), multiply and are released at the end of a 48h cycle, ready for new invasions. This work is focused on the motion of the parasite during the invasion process. To penetrate into the host cell, the parasite reorient its apical part towards the RBC membrane. For this purpose, the parasite generates different movements that allow him to find the correct position to form a specific junction to invade the cell. To understand how the parasite is able to move and reorient without the aid of cilia, flagella or deformations, we performed a detailed analysis of the parasite trajectories and orientation on rigid substrate. We observe that the substrate-attached parasite explores all degrees of freedom with in-plane rotation, translation and flipping. Three types of motion have been identified: confined, directed circular . We characterize these trajectories and motions using correlation analysis and we discuss the possible mechanisms that could explain these peculiar trajectories. Finally, to determine the role of the cytoskeleton components in the parasite motion, specific structures such as the actin filaments and the microtubules have been specifically affected. We will describe and discuss the consequences of depolymerizing or stabilizing these structures.

Introdução: As transfusões de sangue e as Reações transfusionais (RT) têm tido grande destaque nas discussões e estudos da hemoterapia atual, devido a necessidade e relevância para a prática transfusional e na busca em qualificar as transfusões e refinar a classificação das RT. As reações transfusionais febris não hemolíticas (RTFNH) apresentam um crescente no número de notificações e despertam a necessidade de mais estudos. Durante a estocagem dos hemocomponentes, ocorrem uma série de alterações morfológicas, aumento de potássio (K+) extracelular, hemólise e aumento de hemoglobina (Hb) sobrenadante. Analisar a qualidade e viabilidade do hemocomponente pode nos levar a verificar os fatores preditores de uma RT, procurando minimizar os riscos e selecionar um hemocomponente de melhor qualidade ao paciente. Objetivos: Avaliar potenciais fatores etiológicos na precipitação das RTFNH por meio da mensuração na concentração de sódio (Na+) e K+ no sobrenadante, a contagem leucocitária por mcL, o cultural e o Hematócrito (Ht) e Hb da bolsa de concentrado de hemácias (CH) envolvidas, comparando estes parâmetros em relação a um grupo controle de bolsas de CH. Analisar e comparar o perfil dos pacientes envolvidos com a RTFNH e do grupo controle e, estimar a frequência de culturais coletados positivos e os germes envolvidos. Metodologia: Estudo de caso-controle com seleção de amostras a partir de notificações de suspeita de RTFNH ao Serviço de Hemoterapia de um Hospital Universitário de Porto Alegre - RS, no período de setembro de 2015 a setembro de 2016. O grupo controle foi selecionado a partir da mesma população de bolsas, sendo pareadas por tipagem sanguínea e data de vencimento do hemocomponente, numa proporção de 1:2,1. Resultados: o total incluído foi de 124 bolsas, sendo 39(30,5%) do grupo RT e 85(69,5%) do grupo controle, onde uma série de variáveis foram avaliadas. A média de dias de estocagem das bolsas foi de 10,7(DP=6,7) dias, sendo que no grupo RT 12,1(DP=8,1), foi significativamente maior que no grupo controle 10(DP=5,8) com (P=0,037). Também quando avaliamos as dosagens de Ht as médias verificadas foram de 68,3(DP=7,27), sendo no grupo RT 71(DP=81) e 67(DP=6,5) no grupo controle e, na comparação dos grupos, observamos um P<0,001. Dessa forma, a cada dia a mais de estocagem e, a cada ponto a mais no HT da bolsa, há um aumento na chance de aparecimento de RTFNH. Conclusões: a lesão de estocagem é uma temática importante no momento da oferta de hemocomponentes ao paciente, principalmente aos pacientes em tratamento oncológico de tumores sólidos. A avaliação do HT e do tempo de estocagem da bolsa demonstraram ter relevância estatística e clínica na predição de aparecimento de RTFNH. O manejo de estoque adequado para poder haver essa oferta se faz necessário. Novos estudos serão necessários para verificarmos os mecanismos desencadeantes da RTFNH comparado com o Ht da bolsa e, também estudos relacionados à utilização de pré medicação nas transfusões. / Introduction: Blood transfusions and the transfusion reactions (TR) have had great emphasis in current hemotherapy discussions and studies, due to its importance in transfusion practice and with the aim of qualifying the transfusions and refining TR classifications. The non-hemolytic febrile transfusion reaction (NHFTR) show an increasing number of notifications and arouse the necessity for further studies. During the storage of blood products a series of morphologic alterations occur, such as extracellular potassium (K+) increase, hemolysis and supernatant Hemoglobin (Hb) increase. Analyzing the blood product quality and availability may lead us to verifying predictive factors of a TR, seeking to minimize the risks and select a blood product of a superior quality for the patient. Objective: Evaluate potential etiological factors in the NHFTR precipitation through sodium (Na+) concentration measurement and K+ in the supernatant, the leukocyte count by mcL, the cultural and the Hematocrit (Ht),and Hb of erythrocyte concentrate bag (EC) involved, comparing those parameters in relation to a control group of EC blood bags. Analyze and compare the profile of the patients involved with a NHFTR to the control group and estimate the frequency of positive cultures collected and the germs involved. Methodology: Case-control study with sampling selections from a notification of NHFTR suspicion at a Hemotherapy Service in a College Hospital in Porto Alegre, RS, during the period from September 2015 to September 2016, where the control-group was selected from the same blood bag population, being grouped by blood type and blood product expiry date, in proportion 1:2.1. Results: Were studied 124 blood bags, being 39(39,5%) from the TR group and 85(69,5%) from the control group, where a series of invariables were evaluated. The mean of blood bag storage was 8.5 days, 10,7(PD=6,7) in the TR group and 10(DP=5,8) in the control group, and when compared they showed a P=0.037. Moreover, when we analyzed the Ht dosage, it was verified an mean of 68,3(DP=7,27), in the TR group and 71(DP=81), 67(DP=6,5) in the control group and, comparing both groups, we observed a P=<0.001. Therefore, with each additional storage day and, with each additional point in the Ht bool bag, the chance of NHFTR appearance increases. Conclusions: Storage injury is an important topic at the moment of the offer of blood components to the patient, especially to the ones with ongoing oncological treatments for solid tumors. The HT evaluation and the storage time of the blood bag demonstrate clinical and statistical relevance in the prediction of NHFTR appearance. The management of adequate storage is fundamental for the offer’s availability. Further studies are needed to verify the triggering mechanisms of NHFTR compared to the Ht of the bag, as well as studies associated with the use of premedication in transfusions.

Introdução: O Red blood cell distribution width (RDW) é um preditor de mortalidade em pacientes criticamente enfermos. A associação do RDW na alta da UTI com o risco de readmissão à UTI não é conhecida. Nós fizemos este estudo com o objetivo de investigar a associação entre a presença de anisocitose na alta da UTI e o risco de readmissão à UTI ou óbito inesperado na enfermaria. Métodos: Estudo de coorte retrospectivo que incluiu 813 pacientes com alta da UTI para a enfermaria em um hospital terciário de Porto Alegre, Brasil. A variável de interesse foi o RDW coletado no momento da alta da UTI. Anisocitose foi definida como RDW > 16%. Desfechos de interesse foram readmissão à UTI, óbito inesperado na enfermaria e óbito hospitalar. Hazard ratios (HR) foram estimadas usando o Modelo de Riscos proporcionais de Cox. Variáveis com P < 0.1 na análise univariada foram incluídas na análise multivariada para ajuste. Resultados: Anisocitose na alta da UTI está independentemente associada com readmissão à UTI ou óbito inesperado na enfermaria (HR: 1,682; IC 95% 1,219 – 2,322; P = 0,002). Outras variáveis associadas com este desfecho foram: idade, escore Sequential Organ Failure Assessment (SOFA) na alta da UTI e traqueostomia. Resultados significativos semelhantes foram obtidos após exclusão dos óbitos inesperados na enfermaria (HR: 2,031; IC 95% 1,428 – 2,889; P< 0,001) e para óbito hospitalar (HR: 1,716; IC 95% 1,141 – 2,580; P = 0,01). Conclusões: Anisocitose no momento da alta da UTI está independentemente associada com readmissão à UTI e óbito hospitalar. / Introduction: Red blood cell distribution width (RDW) is a predictor of mortality in critically ill patients. The relationship between the RDW at ICU discharge and the risk of ICU readmission is unknown. The purpose of this study was to investigate the association between the presence of anisocytosis at ICU discharge and the risk of ICU readmission or unexpected death in the ward. Methods: This retrospective cohort study included 813 patients discharged alive from the ICU to the ward in a tertiary hospital in Porto Alegre, Brazil. The target variable was the RDW collected at the time of ICU discharge. Anisocytosis was defined as an RDW > 16%. Outcomes of interest included readmission to the ICU, unexpected death in the ward and in-hospital death. Hazard ratios (HR) were estimated using the Cox proportional hazards model. Variables with a value of P < 0.1 in the univariate analysis were included in the multivariate analysis for adjustment. Results: Anisocytosis at ICU discharge was independently associated with readmission to the ICU or unexpected death in the ward (HR: 1.682; 95% CI 1.219-2.322; P = 0.002). Other variables associated with this outcome included age, Sequential Organ Failure Assessment (SOFA) score at ICU discharge and tracheostomy. Similar significant results were obtained after the exclusion of unexpected deaths in the ward (HR 2.031; CI 1.428 – 2.889; P < 0.001) and for in-hospital deaths (HR 1.716; 95% CI 1.141-2.580; P = 0.01). Conclusions: Anisocytosis at ICU discharge is independently associated with ICU readmission and in-hospital death.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-05-15T17:37:00Z No. of bitstreams: 1 GieseleSilva.pdf: 1262459 bytes, checksum: 4110f17f9c7f709f8e6e08765eeae663 (MD5) / Made available in DSpace on 2017-05-15T17:37:00Z (GMT). No. of bitstreams: 1 GieseleSilva.pdf: 1262459 bytes, checksum: 4110f17f9c7f709f8e6e08765eeae663 (MD5) Previous issue date: 2017-02-01 / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) / Objective: Early Childhood Caries (ECC) has been associated with systemic conditions such as obesity and other nutritional deficiencies. These associations have not been adequately studied yet. The present study aimed at evaluating systemic conditions that may be underlying the severity of caries in early childhood. The aim of this study was to evaluate the serum levels of interleukin- 1β (IL-1β), interleukin (IL-6), tumor necrosis factor-α (TNF-α) and lipocalin associated with human neutrophil gelatinase (NGAL) associated with higher caries severity in early childhood (number of cavities), in a theoretical model adjusted for social variables, BMI Z score and consumption of beverages with added sugar; and Chapter II aimed at comparing the mean hemoglobin, neutrophils and eosinophils in children with Severe Early Childhood Cavities (S-ECC) and carie-free children and also to analyze the association between these components of the hemogram with the presence of S-ECC, in models adjusted for socioeconomic variables and for the consumption of sugar rich beverages. Methods: A case-control study was carried out in São Luis, Maranhão, Brazil, from June 2015 to September 2016. The sample was obtained from the randomization of the original sample from a historical cohort; with 152 children aged between 4 and 5 years old, 72 cases (children with ECC) and 80 controls (caries free). The data were obtained through a structured and validated questionnaire containing socioeconomic, demographic and maternal schooling questions applied to mothers or caregivers; a Food Frequency Questionnaire (FFQ) and anthropometric examination, to evaluate the nutritional status; oral clinical examination of children for assessment of decayed teeth (ceo-d index); blood tests and reading of serum inflammatory markers and blood count. For Article 1, a Poisson regression model was suggested to evaluate the association between social factors, the BMI Z score (Body Mass Index), sugar consumption and severity of ECC; and for the analysis of the association between serum levels of IL-1β, IL-6, TNF-α and NGAL and severity of ECC with adjusted model for social variables, BMI Z score and consumption of added sugar beverages. The average ratio (AR) and the respective confidence intervals (95% CI) were estimated at a significance level of 5%. For Article 2, a comparison test of the hemogram (hemoglobin, neutrophils and eosinophils) of children with and without S-ECC was performed, and Logistic Regression models were applied to test the association between Levels of hemoglobin, neutrophils, and eosinophils and presence of S-ECC and adjusted for family income, mother's schooling, sex and consumption of sugar-rich beverages, with estimated coefficients in OR, 95% confidence intervals, Significance level of 5%. Results: In chapter 1, the highest tertiles of the serum levels of IL-6 (2nd tercil- AR = 1,55, CI = 1,14-2,10, p = 0.005, 3rd tercil- AR = 1,54, CI = 13-2.09, p = 0.006), TNF-α (3rd tercil- RM = 1.33, CI = 1.00-1.78, p = 0.040) and NGAL (2nd tercil- AR = 1.79, CI = 1.10-2.90, p ≤ 0.001, 3rd tercil- AR = 2.04, CI = 1.26- 3.30, p = 0.003) were associated with the highest severity of caries in early infancy, even after adjusting for BMI, however after adjusting the model for the consumption of beverages with added sugar, the associations were maintained for IL-6 the strength of association of NGAL was reduced, and for TNF-α there was no association. In Chapter 2, lower mean hemoglobin levels (p = 0.036), higher mean neutrophils (p = 0.040) and eosinophils (p = 0.034) were found in children with S-ECC compared to caries-free ; in the regression models adjusted for economic and socioeconomic variables, higher levels of hemoglobin were protective for SECC (OR 0.64; IC=0,41-0,97; p=0,040) while higher levels of neutrophils were indicators of risk of S-ECC (OR 1.03; IC=1,02-1,06; p=0,040), and after adjustment for frequency of consumption of sugar-rich beverages, the strength of association of these components with the S-ECC was reduced, suggesting that the higher frequency of consumption of these sugars are a common factor that bind blood-to-blood changes to S-ECC. Conclusion: Higher serum levels of IL-6, TNF-α and NGAL are associated with higher caries severity in children, suggesting the presence of underlying systemic inflammation and excessive consumption of added sugars seem to be implicated in the relationship between higher serum levels of TNF-α and NGAL with Severity of caries in children. Higher levels of neutrophils and eosinophils and lower levels of hemoglobin have been shown in children with S-ECC suggesting that systemic alterations such as iron deficiency and inflammation should be investigated in the presence of S-ECC by multidisciplinary health teams. / Objetivo: A Cárie na Primeira Infância (CPI) tem sido associada a condições sistêmicas como a obesidade e outras carências nutricionais; no entanto, as evidências ainda são frágeis. O presente estudo propôs-se a avaliar condições sistêmicas que possam estar subjacentes à gravidade de cárie na primeira infância. Assim sendo, esta tese foi dividida em dois capítulos: O capítulo I teve como objetivo avaliar os níveis séricos de interleucina- 1β (IL-1β), interleucina (IL- 6), fator de necrose tumoral- α (TNF-α) e lipocalina associada à gelatinase dos neutrófilos humanos (NGAL) associados a maior gravidade da cárie na primeira infância (número de cavidades), em modelo teórico ajustado para as variáveis socioeconômicas e demográficas, escore Z do IMC e consumo de bebidas com açúcar de adição; e o capítulo II teve como objetivo comparar as médias de hemoglobina, neutrófilos e eosinófilos em crianças com Cárie Grave na Primeira Infância (CPI-G) e livres de cárie e analisar a associação entre esses componentes do hemograma com a presença da CPI-G, em modelos ajustados para variáveis socioeconômicas e demográficas e para o consumo de bebidas ricas em açúcar. Métodos: Estudo caso-controle foi realizado em São Luis, Maranhão, Brasil, no período de junho de 2015 à setembro de 2016. A amostra foi obtida a partir da aleatorização da amostra original de uma coorte histórica; sendo selecionadas 152 crianças, entre 4 e 5 anos de idade, 72 casos (crianças com CPI) e 80 controles (crianças livres de cárie). Os dados foram obtidos por meio de um questionário estruturado e validado contendo questões socioeconômicas, demográfica aplicado às mães ou responsáveis; de um Questionário de Frequência Alimentar (QFA) e de exame antropométrico, para avaliação do estado nutricional; exame clínico bucal das crianças para aferição de dentes cariados (índice ceo-d); exames de sangue e leitura de marcadores inflamatórios séricos e do hemograma. Para o Artigo 1, foi sugerido modelo de regressão de Poisson para avaliar: a associação entre os fatores sociais, o escore Z do IMC (Índice de Massa Corpórea), consumo de açúcar e gravidade de CPI; e para análise da associação entre os níveis séricos de IL-1β, IL-6, TNF-α e NGAL e gravidade de CPI com modelo ajustado para variáveis socioeconômicas e demográfica, escore Z do IMC e consumo de bebidas com açúcar de adição. Estimou-se a razão das médias (RM) e os respectivos intervalos de confiança (IC 95%), a um nível de significância de 5%. Para o Artigo 2 foi feito um teste de comparação de médias (Teste T não pareado) das variáveis do hemograma (hemoglobina, neutrófilos e eosinófilos) em crianças com CPI-G e livres de cárie, e aplicados modelos de Regressão Logística para testar a associação entre os níveis de hemoglobina, de neutrófilos, e eosinófilos e presença de CPI-G e ajustados para renda familiar, escolaridade da mãe, sexo e consumo de bebidas ricas em açúcar, com os coeficientes estimados em OR, intervalos de confiança de 95%, a um nível de significância de 5%. Resultados: No capítulo 1, maiores tercis dos níveis séricos de IL-6 (2º tercil- RM= 1,55, IC= 1,14-2,10, p = 0,005; 3º tercil- RM=1,54, IC=1,13-2,09, p = 0,006), de TNF-α (3º tercil- RM=1,33, IC=1,00-1,78, p=0,040) e de NGAL (2º tercil- RM=1,79, IC= 1,10-2,90, p ≤ 0,001; 3º tercil- RM=2,04, IC=1,26-3,30, p = 0,003) se associaram com a maior gravidade da cárie na primeira infância, e se mantiveram associados mesmo após ajuste para IMC, entretanto após ajuste do modelo para o consumo de bebidas com açúcar de adição, as associações foram mantidas para IL-6, a força de associação do NGAL foi reduzida, e para TNF-α não houve associação. No capítulo 2, menores médias de hemoglobina (p=0,036), maiores médias de neutrófilos (p=0,040) e de eosinófilos (p=0,034) foram encontradas em crianças com CPI-G comparadas às livres de cárie; nos modelos de regressão ajustados para variáveis socioeconômicas, maiores níveis de hemoglobina foram protetores à CPI-G (OR 0,64; IC=0,41-0,97; p=0,040) enquanto maiores níveis de neutrófilos foram indicadores de risco da CPI-G (OR 1,03; IC=1,02-1,06; p=0,040), e após ajuste para a frequência de consumo de bebidas ricas em açúcares, a força de associação desses componentes do hemograma com a CPI-G foi reduzida, sugerindo que a maior frequência de consumo desses açúcares seja fator comum às alterações do hemograma em relação à CPI-G. Conclusão: maiores níveis séricos de IL-6, TNF-α e NGAL estão associados à maior gravidade de cárie em crianças, sugerindo presença de inflamação sistêmica subjacente e o excessivo consumo de açúcares de adição parece estar implicado na relação entre maiores níveis séricos TNF-α e NGAL com gravidade da cárie em crianças. Maiores níveis de neutrófilos e de eosinófilos e menores de hemoglobina foram mostrados em crianças com a CPI-G sugerindo que alterações sistêmicas como deficiência de ferro e inflamação devam ser pesquisadas na presença de CPI-G por equipes multidisciplinares de saúde.

Red blood cells (RBC) have been shown to mediate plaque development seen in coronary artery disease (CAD). This study determined whether differences in RBC fatty acid (FA) composition were related to CAD risk. FAs were extracted from RBCs of 38 individuals who have undergone cardiac catheterization, 9 of whom had obstructive CAD, and analyzed via gas chromatography. Ferric reducing ability of plasma (FRAP) assay was used to determine oxidative stress. Food frequency questionnaires were used to correlate RBC omega-3 FA to daily intake of omega-3 FA. No correlation was found between RBC content and intake of omega-3 FA. FRAP values and RBC FA composition did not differ between the 2 groups with exception of the saturated FA, palmitic acid (p=0.018). These results suggest that RBC FA composition may differ between individuals with or at risk for CAD. Additional research is needed to validate this biomarker as a predictor of CAD.

Particle analysis using multiwavelength UV-visible spectroscopy provides the potential for extracting quantitative red blood cell information, such as hemoglobin concentration, cell size, and cell count. However, if there is a significant presence of hypochromism as a result of the concentrated hemoglobin (physiological value of 33%), successful quantification of red cell values would require a correction. Hypochromism has been traditionally defined as a decrease in absorption relative to the values expected from the Beer-Lambert Law due to electronic interactions of chromophores residing in close proximity of one another. This phenomenon has been suggested to be present in macroscopic systems composed of strong chromophores such as nucleic acids, chlorophyll, and hemoglobin. The study presented in this dissertation examines the presence of hypochromism in red blood cells as a part of a larger goal to qualitatively and quantatively characterize red blood cells using multiwavelength UV-visible spectroscopy. The strategy of the study was three-fold: 1) to determine the instrumental configuration that would provide the most complete information in the acquired spectra, 2) to develop an experimental model system in which the hemoglobin content in red blood cells could be modified to various concentrations, and 3) to implement an interpretation model based on light scattering theory (which accounts for both the scattering and absorption components of the optical density spectrum) to provide quantitative information for the experimental system. By this process, hypochromicity was redefined into two categories with molecular hypochromicity representing the traditional definition and macroscopic hypochromicity being an attenuation of the absorption component due to a scattering-related effect. Successful simulations of experimental red cell spectra containing various amounts of hemoglobin were obtained using the theoretical model. Furthermore, successful quantitative interpretation of the red blood cell spectra was achieved in the context of corpuscular hemoglobin concentration, corpuscular volume, and cell count solely by accounting for the scattering and absorption effects of the particle, indicating that molecular hypochromicity was insignificant in this macroscopic system.

Cette thèse vise à évaluer deux techniques différentes pour la distinction d'échantillons biologiques mécaniquement altérés. Les deux approches ont été mises au point et étudiées avec un objectif à long terme qui est l'application de ces méthodes au diagnostic basé sur le phénotype mécanique. Bien que, nous avons élaboré ces approches dans le cas de globules rouges (GR), comme leurs propriétés mécaniques peuvent être affectées par des pathologies importantes, tous les concepts développés dans ce travail peuvent être adaptés à d'autres types de cellules.Nous avons caractérisé les propriétés mécaniques de GR à l'aide d'une technique microfluidique passive. L'accent a été mis sur la relaxation de globules rouges sains (GRs) à la sortie d'un canal à largeur variante et la dépendance de la réponse mécanique aux conditions expérimentales a été démontrée. Nous avons rapporté deux modes de relaxation du GR en fonction des paramètres de l'écoulement, et nous avons utilisé l'interface entre ces deux modes pour remonter aux propriétés mécaniques du GR. Lors de la seconde approche, nous avons présenté les résultats obtenus au cours de l'étude du comportement mécanique des GRs à l'aide de l'électrodeformation. Nous avons étudié l'influence des paramètres expérimentaux sur la réponse cellulaire et conclu sur des conditions optimisées pour la sollicitation des cellules sans les altérer. Pour finir, nous avons fait le point sur l'évaluation de ces deux techniques dans la discrimination des échantillons mécaniquement défaillants des sujets sains. Les avantages et inconvénients des deux approches ont été discutés / This thesis aims at evaluating two different techniques for the distinction of mechanically impaired biological samples. Both approaches were developed and investigated keeping in mind the long term objective which is the application of these approaches to diagnosis based on cell mechanical phenotype. Although, we developed these approaches on Red Blood Cells as as their mechanical properties can be affected by important pathologies, all the concepts developed in this work can be adapted to other cell types.We characterized mechanically RBCs using a passive microfluidic technique. We focused on the shape relaxation of healthy Red Blood Cells (hRBCs) flowing out of an oscillating width channel and demonstrated the dependency of their mechanical response upon the experimental conditions. We reported the existence of two relaxation modes for RBCs depending on the flow parameters, we used the interface between the two modes to extract the mechanical properties of RBC.Using a second approach, we presented the results obtained during the study of the mechanical behavior of hRBCs using electrodeformation assays. We investigated the influence of the solicitation parameters on the cell response and concluded on optimized conditions for cell solicitation without altering them. Finally, we evaluated both techniques in the discrimination of rigidified samples from healthy couterparts. Advantages and drawbacks of both techniques were discussed