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.

Cette thèse traite de la dynamique de cellules sanguines dans la microcirculation. Cette appellation regroupe les deux thématiques de mon travail. La première est l'étude du mouvement de globules rouges soumis à un écoulement de cisaillement. Prenant la suite des travaux réalisés par Manouk Abkarian, Magalie Faivre et Annie Viallat, nous avons étudié le mouvement de cellules dans un flux oscillant et mis en évidence l'apparition de chaos (Dupire J. et al, PRL 104,168101 (2010)). Nous avons ensuite repris l'étude sous écoulement constant pour comprendre les régimes de mouvement encore non étudiés (article accepté à PNAS). Tous ces travaux se basent sur un modèle à forme ellipsoïdale constante (type Keller & Skalak) auquel a été rajouté un terme tenant compte de l'élasticité de la membrane. Pour mieux modéliser la mémoire de forme, nous avons recalculé les équations du modèle en tenant compte d'une nouvelle forme non contrainte du cytosquelette élastique. Elle nous permet entre autres d'ajuster le modèle aux données expérimentales en utilisant des valeurs de viscosité et de module élastique de cisaillement compatibles avec la littérature. Le deuxième partie traite de l'étude du mouvement de globules blancs dans un réseau de canaux microfluidiques. Ce réseau est régulier et possède des dimensions biomimétiques. Nous étudions comment la rhéologie des cellules influe sur leur mouvement à travers le dispositif. Nous montrons que l'entrée des cellules, et donc leur première déformation, peut être utilisée pour obtenir des informations sur leur rhéologie (viscosité, élasticité, tension). / This thesis deals with dynamics of blood cells in microflow. This title regroups two aspects of my work. The first one studies the movement of red blood cells (RBC) under flow. Continuing the work done by M. Abkarian, M. Faivre and A. Viallat, we looked at RBCs in an oscillating shear flow and showed the presence of chaos in the motion (Dupire J. et al, PRL 104,168101 (2010) ). Then we continued the study of RBC under constant flow to understand the regime of motion that were still to elucidate (PNAS, accepted for publication). These works use a ellipsoidal fixed shape model (based on Keller and Skalak's) to which we add an elastic membrane term. To take into account the shape memory, we calculated again the equations of motion considering a new stress-free shape of the elastic cytoskeleton. It allows us to fit the model on the experimental data using viscosity and elasticity coefficient compatible with the litterature. The second part deals with the motion of white blood cell (WBC) in a microfluidic channel network. The device has a regular geometry and has biomimetic shape characteristics matching the human lung mean values. We aim to study how the cell's rheology is related to their motion through the device. We show how the entry of the cell, and thus their first deformation, can be used to obtain information about a single cell rheology (viscosity, elasticity, tension). The motion is then decomposed in 2 phases : a transient regime right after the entrance and a final stationary regime. We study these regimes in terms of cellular deformation and wall friction.

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.

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

La malària és encara avui en dia una malaltia que causa aproximadament un milió de morts a l'any a tot el món. La seva eradicació suposa un gran repte per a la humanitat i per a la comunitat científica, en particular. El cultiu in vitro del paràsit és essencial per al desenvolupament de nous medicaments. Els mètodes de cultiu actuals es basen en l'heurística i requereixen millores.En aquesta tesi es presenta una aproximació teòrica al procés d'infecció a eritròcits en cultius in vitro amb Plasmodium falciparum, un dels protozous paràsits causants de la malària. El treball està centrat en la construcció i avaluació de models d'una complexitat adequada per tractar els problemes específics detectats pels experts en l'àmbit, i inclou també la formulació d'algorismes de simulació i el disseny de protocols experimentals.Aquest tipus de treball requereix de la col·laboració multidisciplinària. La visió dels experts en malària es complementa amb la modelització i simulació, que permet la comprovació dels supòsits preestablerts, la comprensió de fenòmens observats i la millora dels mètodes de cultiu actuals. Així doncs, cal establir i desenvolupar eines que permetin crear, analitzar i compartir models amb grups que estudien la malària des d'altres perspectives. En aquesta tesi, s'ha optat per la modelització basada en l'individu (IbM) i orientada a la reproducció de múltiples patrons (PoM). El model s'ha formulat seguint l'ODD, un protocol estàndard en el camp de l'ecologia teòrica, que s'ha adaptat a la representació de comunitats microbianes.Els models basats en l'individu (IbMs) defineixen un conjunt de normes que regeixen el comportament de cada cèl·lula i les seves interaccions amb les altres cèl·lules i amb el seu entorn immediat. A partir d'aquestes regles, i tenint en compte una certa diversitat dins de la població i un cert grau d'aleatorietat en els processos individuals, els IbMs mostren explícitament el comportament emergent del sistema en conjunt. Complementàriament, s'han aplicat conceptes propis de la termodinàmica per tal d'entendrel'aparició de patrons macroscòpics a partir de l'estructura de la població (per exemple de la distribució de les fases d'infecció entre els glòbuls vermells infectats).Aquesta recerca ha comportat la la creació i aplicació del model i simulador INDISIM-RBC, que ha demostrat ser una bona eina per millorar la comprensió dels cultius estudiats. Es tracta d'un model mecanicista, basat en l'individu, que reprodueix quantitativament els patrons observats en cultius reals a diferents nivells de descripció, i que en prediu el comportament sota determinades condicions.Hem demostrat que INDISIM-RBC pot ser emprat per a estudiar en detall alguns aspectes del cultiu del paràsit causant de la malària que calia aclarir. Permet realitzar experiments virtuals i així impulsar noves línies de recerca i explorar noves tècniques de cultiu. En particular, INDISIM-RBC s'ha utilitzat per millorar els protocols experimentals actuals del cultius estàtics, definint la geometria òptima de l'hematòcrit i els protocols de subcultiu més adequats per als cultius continus.El treball realitzat en malària s'ha comparat amb la investigació duta a terme pel grup de recerca em relació amb d'altres comunitats microbianes. D'aquesta manera, podem estudiar les propietats emergents dels sistemes microbians en general en relació als efectes de la individualitat de la cèl·lula, la diversitat de les poblacions, l'heterogeneïtat en el medi, o el caràcter local de les interaccions, entre d'altres. Aquesta visió general proporciona eines conceptuals que poden ser emprades per refinar l'anàlisi dels processos d'infecció sota estudi. / Malaria is still a major burden that causes approximately one million deaths annually worldwide. Its eradication supposes a great challenge to the humanity and to the scientific community, in particular. In vitro cultivation of the parasite is essential for the development of new drugs. Current culture methods are based on heuristics and demand for specific improvements.The present thesis is a theoretical approach to in vitro cultivation of the protozoan parasite Plasmodium falciparum infecting human red blood cells. It mainly focuses on the process of building a model of appropriate complexity to deal with the specific demands above mentioned, but it also includes the formulation and implementation of algorithms, and the design and execution of experimental trials.This kind of work requires multidisciplinary collaboration: the insight of the experts in malaria research is complemented with modeling and simulation, which allows for checking settled assumptions, increasing the understanding on the system and improving the current culturing methods.The use of tools for building, analyzing and sharing models is an imperative to this end. In this thesis, Pattern-oriented Modeling (PoM) has been adopted as the most appropriate way for raising of models and the ODD protocol (Objectives, Design Concepts and Details) has been proposed as the standard tool for communicating them.Individual-based Modeling (IbM) has been used to tackle malaria culture systems. IbMs define a set of rules governing each cell, its interactions with others and with its immediate surroundings. From this set of rules, and taking into account diversity within the population and a certain degree of randomness in the individual processes, IbMs explicitly show the emerging behavior of the system as a whole. Methods from statistical thermodynamics have been applied to understand the emergence of macroscopic patterns from the population structure (e.g. distribution of infection stages among infected red blood cells).The research resulted in the development of the model and simulator INDISIM-RBC, which has proved to be a good tool to improve understanding of the cultures under study. It is a mechanistically rich individual-based model and it quantitatively reproduces and predicts several patterns observed in real cultures at different levels of description.We demonstrated that INDISIM-RBC can be used to study in detail several aspects of malaria cultivation that remained unclear, as well as to perform virtual experiments. Consequently, it can be used to open novel lines of research and to examine potential experimental techniques. INDISIM-RBC has also been used to improve the current experimental culturing protocols in static cultivation by obtaining the optimal geometry of the hematocrit layer and subcultivation periods in the continuous cultures.This study on malaria has been compared to the research carried out by the group regarding other microbial communities. Thereby studying general emerging properties of microbial systems in general, with regard to the effect of cell individuality, heterogeneity and diversity, the local nature of interactions; and biological and spatial complexity. In doing so, the acquired holistic view has been used to develop tools that allow for a better characterization and study of the infection process, in particular.

Babesia microti is a tickborne hemoprotozoan parasite that causes the disease babesiosis in humans. Babesia microti Apical Membrane Antigen-1 (AMA-1) is a micronemal protein suspected to play a role in erythrocyte invasion. To investigate interaction between AMA-1 and the host cell, the ectodomain region of the B. microti ama-1 gene was cloned into an expression vector, expressed as a histidine-tagged fusion protein, and used to probe red blood cell membrane proteins in far Western blot assays. The B. microti ama-1 ectodomain, which excludes the signal peptide and the transmembrane region of the open reading frame, was amplified from a cloned gene sequence. The AMA-1 ectodomain is a membrane bound polypeptide that extends into the extracellular space and is most likely to interact or initiate interaction with the host red blood cell surface receptor(s). The amplicon was ligated into a protein expression vector to produce a 58.1 kDa recombinant His-tagged fusion protein, which was confirmed by Western blot analysis. The recombinant B. microti AMA-1 fusion protein was enriched on nickel affinity columns and then used to probe mouse, human and horse red blood cell membrane proteins in far Western blot assays. Babesia microti AMA-1 consistently reacted strongly with a protein migrating at 49 kDa. A similar reaction occurred between the B. microti AMA-1 and horse red blood cell membrane proteins, suggesting that similar interacting proteins of this size are shared by red blood cells from the three species. The B. microti AMA-1 may bind to red blood cell membrane sialic-acid groups, as shown for other Babesia spp. This may explain the signal at the 49 kDa position observed between B. microti AMA-1 and red blood cell membrane proteins from three different species. Further studies may determine if the binding epitopes of the red blood cell binding partner at this position vary and contribute to the specificity of each parasite AMA-1 for their respective host cells.

In order to simulate cellular blood, a coarse-grained spectrin-link (SL) red blood cell (RBC) membrane model is coupled with a lattice-Boltzmann (LB) based suspension solver. The LB method resolves the hydrodynamics governed by the Navier--Stokes equations while the SL method accurately models the deformation of RBCs under numerous configurations. This method has been parallelized using Message Passing Interface (MPI) protocols for the simulation of dense suspensions of RBCs characteristic of whole blood on world-class computing resources. Simulations were performed to study rheological effects in unbounded shear using the Lees-Edwards boundary condition with good agreement with rotational viscometer results from literature. The particle-phase normal-stress tensor was analyzed and demonstrated a change in sign of the particle-phase pressure from low to high shear rates due to RBCs transitioning from a compressive state to a tensile state in the flow direction. Non-Newtonian effects such as viscosity shear thinning were observed for shear rates ranging from 14-440 inverse seconds as well as the strong dependence on hematocrit at low shear rates. An increase in membrane bending energy was shown to be an important factor for determining the average orientation of RBCs, which ultimately affects the suspension viscosity. The shear stress on platelets was observed to be higher than the average shear stress in blood, which emphasizes the importance of modeling platelets as finite particles. Hagen-Poiseuille flow simulations were performed in rigid vessels for investigating the change in cell-depleted layer thickness with shear rate, the Fåhraeus-Linqvist effect, and the process of platelet margination. The process of platelet margination was shown to be sensitive to platelet shape. Specifically, it is shown that lower aspect ratio particles migrate more rapidly than thin disks. Margination rate is shown to increase with hematocrit, due to the larger number of RBC-platelet interactions, and with the increase in suspending fluid viscosity.