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.

Anion exchanger 1 (AE1) is a red cell membrane glycoprotein that associates with cytoskeletal protein 4.2 in a complex bridging the cell membrane to the cytoskeleton. Disruption of this linkage results in unstable erythrocytes and hereditary spherocytosis (HS). Three HS mutations (E40K, G130R and P327R) in the cytoplasmic domain of AE1 (cdAE1) result in a decreased level of protein 4.2 in the red cell yet maintain normal amounts of AE1. Biophysical analyses showed the HS mutations had little effect on the structure and conformational stability of the isolated domain. However, the conformation of the cytoplasmic domain of the kidney anion exchanger, lacking the first 65 amino acids including a central -strand, was thermally destabilized relative to cdAE1 and had a more open structure. In transfected human embryonic kidney (HEK)-293 cells the HS mutants had similar expression levels as wild-type AE1, and protein 4.2 expression level was not dependent on the presence of AE1. Protein 4.2 localized to the plasma membrane with wild-type AE1, the HS mutants of AE1, the membrane domain of AE1 and kidney AE1, and to the ER with Southeast Asian ovalocytosis AE1. A fatty acylation mutant of protein 4.2, G2A/C173A, could not localize to the plasma membrane in the absence of AE1. Subcellular fractionation showed wild-type and G2A/C173A protein 4.2 were mostly associated with the cytoskeleton. Co-immunoprecipitation and Ni-NTA pull-down assays revealed impaired binding of protein 4.2 to HS mutants compared to AE1, while the membrane domain of AE1 was unable to bind protein 4.2. These studies show that HS mutations in cdAE1 cause impaired binding of protein 4.2, without causing gross structural changes in the domain. The mutations change the binding surface on cdAE1 by the introduction of positive charges into an otherwise acidic domain. This binding impairment may render protein 4.2 more susceptible to degradation or loss during red cell development.

The thesis is based on three studies in which data on children with acute lymhpblastic leukemia (ALL) treated in Lithuania or the Nordic countries were analyzed. Study I. Epidemiological and survival data of 459 children treated in Lithuania in 1992-2012 were analyzed. Children with ALL were included for the first time into international clinical trial that was conducted by pediatric oncologists in the Nordic countries. This resulted in survival improval of childhood ALL by 20% reaching results reported by large international childhood ALL study groups. Study II. Study of 2636 childhood ALL patients treated in the Nordic countries in 1992-2008 revealed significant differences in white blood cell (WBC) distribution both among different biological ALL subsets defined by immunophenotype or cytogenetical aberrations of leukemic blasts, and among patients of different age or gender. WBC remained as a risk factor to have a significantly poorer prognosis for patients with a WBC ≥100 x 109/L, but only among slow-responding patients. Study III. A population-based multicenter study of 221 children aged <15 y. with ALL and WBC ≥200x109/L at diagnosis treated in Lithuania and the Nordic countries were analyzed for early mortality and the impact of initial treatment strategy to survival. The Nordic/Baltic guidelines for initial treatment of ALL patients with hyperleukocytosis and a high risk for the development of tumor lysis syndrome (WBC ≥100 x 109/L) were conducted. / Disertacijoje nagrinėjami Lietuvoje ir Šiaurės šalyse 1992-2012 m. ūmine limfoblastine leukemija (ŪLL) sirgusių vaikų duomenys. I tyrimas. Surinkti ir išanalizuoti 1992-2012 m. Lietuvoje gydytų ŪLL sirgusių vaikų (N=459) epidemiologija ir gydymo rezultatai, įsitraukta į tarptautinio (kartu su Šiaurės šalių vaikų onkohematologais) vaikų ŪLL gydymo protokolą. ŪLL sergančių vaikų išgyvenamumas Lietuvoje pagerėjo apie 20% ir pasiekė tarptautinį lygį. II tyrimas. Išanalizavus Šiaurės šalyse 1992-2008 m. dėl ŪLL gydytų vaikų duomenis (N=2363) nustatytos leukeminių blastų citogenetinės aberacijos ir ligonio biologiniai veiksniai, lemiantys skirtingą LS diagnozuojant ŪLL. Nustatyta, kad LS buvo reikšmingas išgyvenamumo rizikos veiksnys tiek pre-B ŪLL, tiek T-ŪLL ligoniams, kuriems pradinis LS buvo ≥100 x 109/L, tačiau tik tais atvejais, kai atsakas į gydymą buvo lėtas. III tyrimas. Populiaciniame tyrime išanalizuoti 1992-2011 m. Lietuvoje ar Šiaurės šalyse ŪLL sirgusių vaikų, kurių pradinis LS≥200 x 109/L, duomenys (N=221). Nustatyta, kad didelis leukocitų skaičius, o ne naviko lizės sindromas buvo pagrindinis ankstyvo mirtingumo rizikos veiksnys. Uždelstas specifinis ŪLL gydymas ar sumažintos pradinės chemopreparatų dozės galėjo turėti neigiamos reikšmės ankstyvų mirčių išsivystymui. Sudarytos bendros Baltijos ir Šiaurės šalių centrams skirtos rekomendacijos dėl pradinės ligonių su ŪLL ir hiperleukocitoze bei didele naviko lizės išsivystymo rizika (LS ≥100 x 109/L), gydymo taktikos... [toliau žr. visą tekstą]

Disertacijoje nagrinėjami Lietuvoje ir Šiaurės šalyse 1992-2012 m. ūmine limfoblastine leukemija (ŪLL) sirgusių vaikų duomenys. I tyrimas. Surinkti ir išanalizuoti 1992-2012 m. Lietuvoje gydytų ŪLL sirgusių vaikų (N=459) epidemiologija ir gydymo rezultatai, įsitraukta į tarptautinio (kartu su Šiaurės šalių vaikų onkohematologais) vaikų ŪLL gydymo protokolą. ŪLL sergančių vaikų išgyvenamumas Lietuvoje pagerėjo apie 20% ir pasiekė tarptautinį lygį. II tyrimas. Išanalizavus Šiaurės šalyse 1992-2008 m. dėl ŪLL gydytų vaikų duomenis (N=2363) nustatytos leukeminių blastų citogenetinės aberacijos ir ligonio biologiniai veiksniai, lemiantys skirtingą LS diagnozuojant ŪLL. Nustatyta, kad LS buvo reikšmingas išgyvenamumo rizikos veiksnys tiek pre-B ŪLL, tiek T-ŪLL ligoniams, kuriems pradinis LS buvo ≥100 x 109/L, tačiau tik tais atvejais, kai atsakas į gydymą buvo lėtas. III tyrimas. Populiaciniame tyrime išanalizuoti 1992-2011 m. Lietuvoje ar Šiaurės šalyse ŪLL sirgusių vaikų, kurių pradinis LS≥200 x 109/L, duomenys (N=221). Nustatyta, kad didelis leukocitų skaičius, o ne naviko lizės sindromas buvo pagrindinis ankstyvo mirtingumo rizikos veiksnys. Uždelstas specifinis ŪLL gydymas ar sumažintos pradinės chemopreparatų dozės galėjo turėti neigiamos reikšmės ankstyvų mirčių išsivystymui. Sudarytos bendros Baltijos ir Šiaurės šalių centrams skirtos rekomendacijos dėl pradinės ligonių su ŪLL ir hiperleukocitoze bei didele naviko lizės išsivystymo rizika (LS ≥100 x 109/L), gydymo taktikos... [toliau žr. visą tekstą] / The thesis is based on three studies in which data on children with acute lymhpblastic leukemia (ALL) treated in Lithuania or the Nordic countries were analyzed. Study I. Epidemiological and survival data of 459 children treated in Lithuania in 1992-2012 were analyzed. Children with ALL were included for the first time into international clinical trial that was conducted by pediatric oncologists in the Nordic countries. This resulted in survival improval of childhood ALL by 20% reaching results reported by large international childhood ALL study groups. Study II. Study of 2636 childhood ALL patients treated in the Nordic countries in 1992-2008 revealed significant differences in white blood cell (WBC) distribution both among different biological ALL subsets defined by immunophenotype or cytogenetical aberrations of leukemic blasts, and among patients of different age or gender. WBC remained as a risk factor to have a significantly poorer prognosis for patients with a WBC ≥100 x 109/L, but only among slow-responding patients. Study III. A population-based multicenter study of 221 children aged <15 y. with ALL and WBC ≥200x109/L at diagnosis treated in Lithuania and the Nordic countries were analyzed for early mortality and the impact of initial treatment strategy to survival. The Nordic/Baltic guidelines for initial treatment of ALL patients with hyperleukocytosis and a high risk for the development of tumor lysis syndrome (WBC ≥100 x 109/L) were conducted.

Stem and progenitor cell fate (self-renewal, proliferation, survival, differentiation) is tightly controlled by niche factors and the interplay of these factors is particularly important to comprehend for the development of stem cell therapies. During erythropoiesis, erythroid progenitors at the colony forming unit-erythroid (CFU-E) stage are responsive to both stem cell factor (SCF) and erythropoietin (EPO); however, the joint action of SCF and EPO in these cells and the underlying mechanisms remain to be defined. In this study, quantitative data on the activation of signaling pathways and gene expression profiles provided definitive evidence for two parallel but complementary mechanisms that resulted in enhanced generation of red blood cells from mouse bone marrow-derived CFU-E culture in the presence of SCF and EPO. First, SCF and EPO signaling intersected within the extracellular signal-regulated kinase (ERK) pathway and the sustained ERK activation was required for the maximal changes in the expression levels of genes that are involved in the proliferation and survival of CFU-Es. Second, the apparent competition between SCF and EPO in regulating c-Kit expression was found to have a dramatic impact on the terminal differentiation of CFU-Es. The latter mechanism was, for the first time, reported in a primary cell system. In addition, a fetal liver-derived conditioned medium further enhanced the survival and proliferation of bone marrow CFU-Es in the presence of SCF and EPO by not only increasing the ERK signaling duration but also, the amplitude. The agents present in the conditioned media possess significant clinical potential to stimulate erythropoiesis both in vivo and in vitro. In conclusion, our study has provided novel insights into the mechanisms by which combinations of niche factors control the fate of erythroid progenitors at a unique transitional stage and highlighted the important role of the ERK signaling dynamics in adult erythropoiesis.

Stem and progenitor cell fate (self-renewal, proliferation, survival, differentiation) is tightly controlled by niche factors and the interplay of these factors is particularly important to comprehend for the development of stem cell therapies. During erythropoiesis, erythroid progenitors at the colony forming unit-erythroid (CFU-E) stage are responsive to both stem cell factor (SCF) and erythropoietin (EPO); however, the joint action of SCF and EPO in these cells and the underlying mechanisms remain to be defined. In this study, quantitative data on the activation of signaling pathways and gene expression profiles provided definitive evidence for two parallel but complementary mechanisms that resulted in enhanced generation of red blood cells from mouse bone marrow-derived CFU-E culture in the presence of SCF and EPO. First, SCF and EPO signaling intersected within the extracellular signal-regulated kinase (ERK) pathway and the sustained ERK activation was required for the maximal changes in the expression levels of genes that are involved in the proliferation and survival of CFU-Es. Second, the apparent competition between SCF and EPO in regulating c-Kit expression was found to have a dramatic impact on the terminal differentiation of CFU-Es. The latter mechanism was, for the first time, reported in a primary cell system. In addition, a fetal liver-derived conditioned medium further enhanced the survival and proliferation of bone marrow CFU-Es in the presence of SCF and EPO by not only increasing the ERK signaling duration but also, the amplitude. The agents present in the conditioned media possess significant clinical potential to stimulate erythropoiesis both in vivo and in vitro. In conclusion, our study has provided novel insights into the mechanisms by which combinations of niche factors control the fate of erythroid progenitors at a unique transitional stage and highlighted the important role of the ERK signaling dynamics in adult erythropoiesis.

Anion exchanger 1 (AE1) is a red cell membrane glycoprotein that associates with cytoskeletal protein 4.2 in a complex bridging the cell membrane to the cytoskeleton. Disruption of this linkage results in unstable erythrocytes and hereditary spherocytosis (HS). Three HS mutations (E40K, G130R and P327R) in the cytoplasmic domain of AE1 (cdAE1) result in a decreased level of protein 4.2 in the red cell yet maintain normal amounts of AE1. Biophysical analyses showed the HS mutations had little effect on the structure and conformational stability of the isolated domain. However, the conformation of the cytoplasmic domain of the kidney anion exchanger, lacking the first 65 amino acids including a central -strand, was thermally destabilized relative to cdAE1 and had a more open structure. In transfected human embryonic kidney (HEK)-293 cells the HS mutants had similar expression levels as wild-type AE1, and protein 4.2 expression level was not dependent on the presence of AE1. Protein 4.2 localized to the plasma membrane with wild-type AE1, the HS mutants of AE1, the membrane domain of AE1 and kidney AE1, and to the ER with Southeast Asian ovalocytosis AE1. A fatty acylation mutant of protein 4.2, G2A/C173A, could not localize to the plasma membrane in the absence of AE1. Subcellular fractionation showed wild-type and G2A/C173A protein 4.2 were mostly associated with the cytoskeleton. Co-immunoprecipitation and Ni-NTA pull-down assays revealed impaired binding of protein 4.2 to HS mutants compared to AE1, while the membrane domain of AE1 was unable to bind protein 4.2. These studies show that HS mutations in cdAE1 cause impaired binding of protein 4.2, without causing gross structural changes in the domain. The mutations change the binding surface on cdAE1 by the introduction of positive charges into an otherwise acidic domain. This binding impairment may render protein 4.2 more susceptible to degradation or loss during red cell development.

La microcirculation sanguine est un système constitué de réseaux complexesde vaisseaux sanguins de diamètres micrométriques. C’est le lieu privilégié deséchanges de gaz et de nutriments entre le sang et les tissus.Les écoulements dans ces réseaux sont gouvernés par les propriétés des constituantsdu sang c’est-à-dire des cellules en suspension dans du plasma et plus particulièrementdes globules rouges qui sont les cellules majoritaires dans le sang.Selon les conditions de l’écoulement, les globules rouges, qui sont des particulesdéformables, peuvent présenter différents types de formes et de dynamiques quiinfluencent la rhéologie de la suspension. Les interactions hydrodynamiques entreglobules rouges et avec les parois dans les vaisseaux confinés influencent égalementles écoulements à travers des phénomènes de diffusion mais aussi de structurationdes globules au sein de la suspension. Il a notamment été montré que des couchesde plasma dépourvues de globules rouges près des parois des vaisseaux sanguins induisentune diminution de la viscosité effective de la suspension lorsqu’on diminuele diamètre du vaisseau. Par ailleurs, des structurations en file ont également étéobservées dans la microcirculation sanguine avec des conséquences probables surla rhéologie. Au cours de ces travaux de thèse, nous avons investigué les propriétésrhéologiques de suspensions de globules rouges en micro-écoulement grâce à uneméthode de rhéométrie microfluidique. Nous avons focalisé notre attention sur larelation entre la rhéologie et la structuration de la suspension dans un canal, liéeau confinement ainsi qu’aux régimes dynamiques des globules en écoulement.Dans certains cas pathologiques comme la drépanocytose où les propriétés desglobules rouges peuvent être modifiées, les écoulements dans la microcirculationpeuvent être perturbés et conduire à des crises vaso-occlusives dont les mécanismesphysiques restent mal compris. Nous avons exploré la dynamique de formationd’occlusions et leurs évolutions dans des réseaux de canaux micrométriques modèlesavec des suspensions de globules rouges dont les propriétés ont été modifiées,révélant ainsi une dynamique complexe où l’adhésion et des effets d’obstructionsinterviennent. / Blood microcirculation consists in blood flowing in complex microvessel networks.Gas and nutrient exchanges between blood and tissues occur in these networks.Microcirculatory blood flows are governed by the properties of blood components,mainly red blood cells suspended in plasma. Red blood cells are deformableparticles which can exibit different shapes and motion dynamics that influence therheology. Hydrodynamic interactions between red blood cells and with walls of theconfined channels lead to diffusion and structuration in the suspension that alsoaffects the rheology. Plasma cell-free layers near walls observed in the microcirculationinduce a decrease of the effective viscosity with decreasing vessel diameter.Other types of structuration like layering of red blood cells have been observed inthe microcirculation with possible rheological consequences. In the present work,we investigated the rheology of confined red blood cell suspensions and focusedon the link between rheology and structuration of the suspension thanks to amicrofluidic viscosimeter.The sickle cell disease which modifies the properties of red blood cells leadsto flow disorders with the formation of occlusions in the narrow capillaries ofthe microcirculation. We explored the formation and the evolution of occlusionsin simplified networks of microchannels when properties of red blood cells aremodified, revealing complex dynamics where adhesion and jamming effect occur.