Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A pré-eclâmpsia (PE) é classificada em de início (EOPE) e tardio (LOPE) quando presente antes ou após 34 semanas de gestação, respectivamente. Este estudo transversal investigou as diferenças e possíveis associações existentes entre os perfis antropométricos, hemodinâmicos, hematológicos e bioquímicos na pré-eclâmpsia entre os grupos EOPE e LOPE. O estudo incluiu 65 voluntárias admitidas em um hospital universitário no Brasil sendo 29 normotensos (grupo C) e 36 com pré-eclâmpsia (13 com EOPE e 23 com LOPE). O grupo LOPE apresentou um aumento significante de peso e aumento limítrofe no índice de massa corporal ao final da gestação em relação aos outros grupos, o que é compatível com a origem metabólica, associada à obesidade, atribuída a esta forma da doença. As mulheres grávidas do EOPE apresentaram uma redução limítrofe no número de eritrócitos e uma diminuição signi-ficante no número de plaquetas e um aumento significante de reticulócitos, ferro sérico e fer-ritina quando comparados ao grupo C e LOPE. O grupo EOPE demonstrou um aumento significante na estabilidade osmótica dos eritrócitos em relação aos demais gru-pos. A análise hemodinâmica por ultrasonografia Doppler da artéria oftálmica mostrou que ambos os grupos de mulheres grávidas com PE apresentaram alterações compatíveis com a ocorrência de hiperfluxo no território orbital. Essas alterações hemodinâmicas foram associa-das a alterações nos índices hematimétricos. / Preeclampsia (PE) is classified in early- (EOPE) and late-onset PE (LOPE) when pre-sent before or after 34 gestation weeks, respectively. This transversal study aimed to investi-gate the differences and possible associations existing in the anthropometric, hemodynamic, hematologic and biochemical profiles of late- and early-onset preeclampsia. The study in-cluded 65 volunteers admitted to a tertiary hospital in Brazil, 29 normotensive and 36 with preeclampsia (13 with EOPE and 23 with LOPE). Pregnant women with LOPE presented greater weight gain and borderline increase in body mass index at the end of gestation in rela-tion to the other groups, which is compatible with the metabolic origin, associated with obesi-ty, attributed to this form of the disease. Pregnant women with EOPE presented a borderline reduction in the number of erythrocytes and a significant decrease in the number of platelets, in addition to a significant increase in reticulocytes, serum iron and ferritin when compared to normotensive pregnant women and pregnant women with LOPE. A significant increase in osmotic stability of erythrocytes was observed in the EOPE group in relation to groups. He-modynamic analysis by Doppler ultrasonography of the ophthalmic artery showed that both groups of pregnant women with PE presented alterations compatible with the occurrence of hyperflow in the orbital territory. These hemodynamic changes were associated with changes in hematimetric indices / Tese (Doutorado)

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / A insuficiência renal crônica (IRC) e a hipertensão arterial sistêmica (HAS) são patologias com alta morbidade e mortalidade, consumindo grandes verbas de saúde pública. A disfunção endotelial presente tanto na IRC, como na hipertensão, contribui para a manutenção de elevada resistência periférica, favorecendo complicações como a aterosclerose. Esta disfunção endotelial é parte de um estado pró-trombótico, levando à ocorrência de eventos cardiovasculares, principal causa de morte nestas patologias. O óxido nítrico (NO) tem um papel importante na modulação da atividade plaquetária. Anormalidades na síntese e/ou inativação do NO são descritas tanto na insuficiência renal crônica como na hipertensão. Estudos prévios demonstraram uma redução do influxo de L-arginina em eritrócitos e plaquetas de pacientes hipertensos e em um modelo animal de hipertensão. Além disso, em IRC, nosso grupo mostrou uma ativação da via L-arginina-NO em plaquetas. O objetivo dessa tese é avaliar a via L-arginina-NO na HAS e em diferentes estágios da IRC, bem como investigar o ciclo da uréia, e a presença de marcadores de estresse nesses pacientes. De acordo com o presente estudo pôde-se verificar que não houve alteração na síntese de NO em eritrócitos na hipertensão, todavia ocorre uma ativação do ciclo da uréia, que pode ser dada pelo aumento do influxo de L-arginina eritrocitário previamente demonstrado. Não foi demonstrada diferença significativa na peroxidação lipídica sistêmica, em plaquetas ou eritrócitos na HAS. Em plaquetas, no entanto, houve uma redução da atividade da NO sintase (NOS), que não foi acompanhada por alteração da expressão das isoformas da NOS, da arginase, da fosfodiesterase 5 (PDE5) ou da guanilato ciclase (GC) solúvel. Essa redução na síntese de NO em plaquetas pode ser explicada por um menor influxo de L-arginina que está presente na hipertensão. Os eritrócitos de pacientes renais crônicos em hemodiálise mostraram um maior influxo de L-arginina associado a um aumento da expressão e da atividade da arginase, não havendo diferença na atividade da NOS. Além disso, apesar e não ter sido mostrado alteração nos marcadores de peroxidação lipídica em eritrócitos e plaquetas, foi detectado um aumento dos mesmos no soro de pacientes com IRC em hemodiálise. Por outro lado, as plaquetas dos mesmos pacientes apresentaram uma maior expressão da eNOS, da iNOS e da GC solúvel, acompanhada de uma redução da atividade da arginase, o que pode justificar a disfunção plaquetária que existe nesses pacientes. Plaquetas de pacientes portadores de IRC em tratamento conservador mostraram um aumento da atividade da NOS associado com maior expressão tanto da iNOS como da eNOS. Curiosamente foram detectados menores concentrações de 35-monofosfato de guanosina cíclica (GMPc), não havendo no entanto, diferença nos padrões de agregação plaquetária induzida por colágeno ou adenosina difosfato (ADP). As descobertas aqui apresentadas certamente contribuirão para uma melhor compreensão da fisiopatologia da HAS e da IRC. / Chronic renal failure (CRF) and essential hypertension (EH) are diseases associated with high rates of morbidity and mortality, consuming huge amounts of money from the public health system. The endothelial dysfunction existent in both diseases, CRF and EH, contributes to the maintenance of the high peripheral resistance, and contribute to circulatory complications such as atherosclerosis. This endothelial dysfunction is part of a pro-thrombotic state, leading to cardiovascular events, which are the major cause of death in these disorders. Nitric oxide (NO) plays an important role in the modulation of platelet function. Abnormalities of NO synthesis or inactivation are described in CRF and EH. It was previously reported an inhibition of L-arginine transport in erythrocytes of hypertensive patients and in an animal model of hypertension. Moreover, we have also demonstrated an activation of L-arginine-NO pathway in platelets taken from uraemic patients. The aim of the present thesis is to investigate L-arginine-NO pathway in arterial hypertension and in different stages of chronic renal failure. It will also be evaluated urea cycle and the presence of oxidative stress markers in these patients. According to the present study it was not detected any alteration in erythrocytres NO synthesis in hypertension, however, there was an activation of urea cycle, which could be explained by an increase in L-arginine influx. The present study has not demonstrated significative difference in markers of lipid peroxidation in the serum, platelets or erythrocytes in hypertension. In platelets however, there was an inhibition of NO synthase (NOS) activity without any alterations of NOS isoforms, arginase, phosphodiesterase 5 (PDE5) or soluble guanylyl cyclase (sGC) expression. This reduction of NO synthesis may be explained by a lower influx of L-arginine that is present on hypertension. Erythrocytes from chronic renal failure patients under haemodyalysis have shown an increased influx of L-arginine associated with a higher expression and activity of arginase with no difference in NOS activity. Therefore, although it was not shown abnormalities of lipid per oxidation markers in erythrocytes and platelets, it was detected increased levels of these markers in the serum of chronic renal failure patients under hemodyalysis. On the other hand, platelets from the same patients exhibited increased expression of eNOS, iNOS and soluble guanylyl cyclase associated with reduced arginase activity, which can explain the platelet dysfunction observed in these patients. Platelets taken from patients with chronic renal failure under conservative treatment have shown increased NOS activity associated with higher expression of both iNOS and eNOS. Curiously, it was been detected a lower concentration of cyclic guanosine monophosphate (cGMP), although there was no difference in the patterns of platelet aggregation induced by collagen or adenosine diphosphate (ADP). The findings reported in this study may contribute to a better understanding of EH and CRF physiopathology.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / A insuficiência renal crônica (IRC) e a hipertensão arterial sistêmica (HAS) são patologias com alta morbidade e mortalidade, consumindo grandes verbas de saúde pública. A disfunção endotelial presente tanto na IRC, como na hipertensão, contribui para a manutenção de elevada resistência periférica, favorecendo complicações como a aterosclerose. Esta disfunção endotelial é parte de um estado pró-trombótico, levando à ocorrência de eventos cardiovasculares, principal causa de morte nestas patologias. O óxido nítrico (NO) tem um papel importante na modulação da atividade plaquetária. Anormalidades na síntese e/ou inativação do NO são descritas tanto na insuficiência renal crônica como na hipertensão. Estudos prévios demonstraram uma redução do influxo de L-arginina em eritrócitos e plaquetas de pacientes hipertensos e em um modelo animal de hipertensão. Além disso, em IRC, nosso grupo mostrou uma ativação da via L-arginina-NO em plaquetas. O objetivo dessa tese é avaliar a via L-arginina-NO na HAS e em diferentes estágios da IRC, bem como investigar o ciclo da uréia, e a presença de marcadores de estresse nesses pacientes. De acordo com o presente estudo pôde-se verificar que não houve alteração na síntese de NO em eritrócitos na hipertensão, todavia ocorre uma ativação do ciclo da uréia, que pode ser dada pelo aumento do influxo de L-arginina eritrocitário previamente demonstrado. Não foi demonstrada diferença significativa na peroxidação lipídica sistêmica, em plaquetas ou eritrócitos na HAS. Em plaquetas, no entanto, houve uma redução da atividade da NO sintase (NOS), que não foi acompanhada por alteração da expressão das isoformas da NOS, da arginase, da fosfodiesterase 5 (PDE5) ou da guanilato ciclase (GC) solúvel. Essa redução na síntese de NO em plaquetas pode ser explicada por um menor influxo de L-arginina que está presente na hipertensão. Os eritrócitos de pacientes renais crônicos em hemodiálise mostraram um maior influxo de L-arginina associado a um aumento da expressão e da atividade da arginase, não havendo diferença na atividade da NOS. Além disso, apesar e não ter sido mostrado alteração nos marcadores de peroxidação lipídica em eritrócitos e plaquetas, foi detectado um aumento dos mesmos no soro de pacientes com IRC em hemodiálise. Por outro lado, as plaquetas dos mesmos pacientes apresentaram uma maior expressão da eNOS, da iNOS e da GC solúvel, acompanhada de uma redução da atividade da arginase, o que pode justificar a disfunção plaquetária que existe nesses pacientes. Plaquetas de pacientes portadores de IRC em tratamento conservador mostraram um aumento da atividade da NOS associado com maior expressão tanto da iNOS como da eNOS. Curiosamente foram detectados menores concentrações de 35-monofosfato de guanosina cíclica (GMPc), não havendo no entanto, diferença nos padrões de agregação plaquetária induzida por colágeno ou adenosina difosfato (ADP). As descobertas aqui apresentadas certamente contribuirão para uma melhor compreensão da fisiopatologia da HAS e da IRC. / Chronic renal failure (CRF) and essential hypertension (EH) are diseases associated with high rates of morbidity and mortality, consuming huge amounts of money from the public health system. The endothelial dysfunction existent in both diseases, CRF and EH, contributes to the maintenance of the high peripheral resistance, and contribute to circulatory complications such as atherosclerosis. This endothelial dysfunction is part of a pro-thrombotic state, leading to cardiovascular events, which are the major cause of death in these disorders. Nitric oxide (NO) plays an important role in the modulation of platelet function. Abnormalities of NO synthesis or inactivation are described in CRF and EH. It was previously reported an inhibition of L-arginine transport in erythrocytes of hypertensive patients and in an animal model of hypertension. Moreover, we have also demonstrated an activation of L-arginine-NO pathway in platelets taken from uraemic patients. The aim of the present thesis is to investigate L-arginine-NO pathway in arterial hypertension and in different stages of chronic renal failure. It will also be evaluated urea cycle and the presence of oxidative stress markers in these patients. According to the present study it was not detected any alteration in erythrocytres NO synthesis in hypertension, however, there was an activation of urea cycle, which could be explained by an increase in L-arginine influx. The present study has not demonstrated significative difference in markers of lipid peroxidation in the serum, platelets or erythrocytes in hypertension. In platelets however, there was an inhibition of NO synthase (NOS) activity without any alterations of NOS isoforms, arginase, phosphodiesterase 5 (PDE5) or soluble guanylyl cyclase (sGC) expression. This reduction of NO synthesis may be explained by a lower influx of L-arginine that is present on hypertension. Erythrocytes from chronic renal failure patients under haemodyalysis have shown an increased influx of L-arginine associated with a higher expression and activity of arginase with no difference in NOS activity. Therefore, although it was not shown abnormalities of lipid per oxidation markers in erythrocytes and platelets, it was detected increased levels of these markers in the serum of chronic renal failure patients under hemodyalysis. On the other hand, platelets from the same patients exhibited increased expression of eNOS, iNOS and soluble guanylyl cyclase associated with reduced arginase activity, which can explain the platelet dysfunction observed in these patients. Platelets taken from patients with chronic renal failure under conservative treatment have shown increased NOS activity associated with higher expression of both iNOS and eNOS. Curiously, it was been detected a lower concentration of cyclic guanosine monophosphate (cGMP), although there was no difference in the patterns of platelet aggregation induced by collagen or adenosine diphosphate (ADP). The findings reported in this study may contribute to a better understanding of EH and CRF physiopathology.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CHAPTER II: The stability of the erythrocyte membrane, which is essential for maintenance of cell functions, occurs in a critical region of fluidity, which depends largely on its composition and the composition and characteristics of the medium. As the composition of the erythrocyte membrane is influenced by several blood variables, the stability of the erythrocyte membrane must have relations with them. The present study aimed to evaluate, by bivariate and multivariate statistical analyses, the correlations and causal relationships between hematological and biochemical variables and the stability of the erythrocyte membrane against the chaotropic action of ethanol. The validity of this type of analysis depends on the homogeneity of the population and on the variability of the studied parameters, conditions that can be filled by patients who undergo bariatric surgery by the technique of Roux-en-Y gastric bypass, since they will suffer feeding restrictions that have great impact on their blood composition. Pathway analysis revealed that an increase in hemoglobin leads to decreased stability of the cell, probably through a process mediated by an increase in MCV. Furthermore, an increase in the MCH leads to an increase in the erythrocyte membrane stability, probably because higher values of MCH are associated to smaller quantities of RBC and larger contact area between the cell membrane and ethanol present in the medium. CHAPTER III: The need to treat obesity, a growing worldwide public health problem, has led to an increase in performing bariatric surgery, particularly the Roux-en-Y gastric bypass. The sudden change in eating habits, resulting from this type of surgery, leads to abrupt changes in the body. This study analyzed the correlation between the osmotic stability of erythrocytes and various biochemical and hematological indices in a population consisting of 24 female volunteers, before and at four different times after surgery, distributed along eight weeks, what allowed the generation of 120 sampling points. The osmotic stability of erythrocytes proved to be of great importance for understanding the meaning of the redcell distribution width (RDW), because the stability variables (1/H50 and dX) were positively correlated with this hematological index. However, the stability variables and RDW seem to suffer different influences from other variables, as the LDL-cholesterol (LDL-C), because only RDW has increased throughout time. Indeed, the stability of variable 1/H50 showed positive correlation with the blood levels of LDL-C, which declined throughout time. Path analysis showed that the body mass index (BMI) has an indirect effect, mediated by RDW, on the osmotic stability of erythrocytes. The correlations that the osmotic stability variables presented with RDW may help to understand the origin of the predictive ability of this hematological index in relation to various pathological conditions. / CAPÍTULO II: A estabilidade de membrana de eritrócitos, que é essencial para a manutenção da função dessas células, ocorre em uma região crítica de fluidez, que depende largamente de sua composição e das características do meio. Como a composição da membrana do eritrócito é influenciada por muitas variáveis sanguíneas, a estabilidade de membrana do eritrócito deve ter relações com elas. O presente estudo objetivou avaliar, por análises estatísticas bivariadas e multivariadas, as correlações e relações causais entre variáveis hematológicas e bioquímicas e a estabilidade de membrana de eritrócitos conta a ação caotrópica do etanol. A validade deste tipo de análise depende da homogeneidade da população e da variabilidade dos parâmetros estudados, condições que podem ser satisfeitas por pacientes que sofrem cirurgia bariátrica pela técnica do desvio gástrico em Y-de-Roux, uma vez que eles passam por restrições alimentares que têm grande impacto sobre a composição sanguínea deles. A análise de caminho revelou que um aumento na concentração de hemoglobina leva a uma diminuição da estabilidade da célula, provavelmente através de um processo mediado por um aumento no volume corpuscular médio (MCV). Além disso, um aumento na hemoglobina corpuscular media (MCH) leva a um aumento na estabilidade de membrana do eritrócito, provavelmente porque valores elevados de MCH são associados a menores quantidades de células vermelhas (RBC) e maiores áreas de contato entre a membrana da célula e o etanol presente no meio. CAPÍTULO III: A necessidade de tratar a obesidade, um crescente problema de saúde pública em todo o mundo, tem levado a um aumento na execução de cirurgia bariátrica, particularmente o desvio gástrico pelo Y-de-Roux. A súbita mudança nos hábitos alimentares, resultante deste tipo de cirurgia, leva a mudanças abruptas no corpo. Este estudo analisou a correlação entre a estabilidade osmótica de eritrócitos e vários índices hematológicos e bioquímicos em uma população constituída de 24 participantes do sexo feminino, antes e em quatro diferentes momentos após a cirurgia, distribuídos ao longo de oito semanas, o que permitiu a geração de 120 pontos amostrais. A estabilidade osmótica de eritrócitos mostrou ser de grande importância para a compreensão do significado da distribuição de volumes das células vermelhas do sangue (RDW), porque as variáveis de estabilidade (1/H50 and dX) foram positivamente correlacionadas com este índice hematológico. Entretanto, as variáveis de estabilidade e o RDW parecem sofrer diferentes influências de outras variáveis, como o LDLcolesterol (LDL-C), porque somente o RDW aumentou ao longo do tempo após a cirurgia. Realmente, a variável de estabilidade 1/H50 apresentou correlação positiva com os níveis sanguíneos de LDL-C, os quais diminuíram ao longo do tempo. A análise de caminho mostrou que o índice de massa corporal (BMI) tem um efeito indireto, mediado pelo RDW, sobre a estabilidade osmótica de eritrócitos. As correlações que as variáveis de estabilidade osmótica apresentaram com RDW podem ajudar na compreensão da origem da habilidade preditiva que este índice hematológico tem em relação a várias condições patológicas. / Doutor em Genética e Bioquímica

L’attention scientifique s’est récemment portée sur l’identification des mécanismes proximaux sous-tendant les compromis évolutifs;tels que les compromis existant entre croissance/reproduction et longévité. La production d’espèces réactives de l’oxygène (ROS )a été suggérée comme un candidat potentiel ,de par sa liaison étroite au métabolisme énergétique (sous-­produits du fonctionnement mitochondrial) et son caractère inévitable. Si la production de ROS excède le niveau des défenses antioxydantes, une situation de stress oxydant va en résulter et a été associé au vieillissement . Puisque la mitochondrie n’est pas uniquement la centrale énergétique de la cellule mais aussi le principal producteur de ROS, cette thèse s’est attachée à clarifier les relations entre métabolisme énergétique , fonctionnement mitochondrial et stress oxydant ; avec des études concernant l’impact d’activités coûteuses en énergie (croissance, reproduction, thermogénèse) sur l’équilibre de la balance oxydative. / In recent years, scientific attention has turned towards the identification of the mechanisms underlying the trade-­‐offs occurring between growth rate/reproductive investment and longevity. Amongst these mechanisms, the production of reactive oxygen species (ROS) appears to be a key factor due both to its universal and inevitable nature. ROS are by-­‐products of energy processing by the mitochondria. If ROS production exceeds the capacity of the various antioxidant systems, oxidative stress will occur, and the accumulation of oxidative damage over time is thought to be a potential cause of ageing. Since mitochondria are not only the powerhouse of animal cells but also the main producer of ROS, this PhD thesis aimed to clarify the relationships between mitochondrial uncoupling state (i.e. efficiency to produce ATP), energy metabolism and oxidative stress. I investigated the impact of energy-­‐demanding activities such as thermogenesis, reproduction and growth on oxidative homeostasis.

The experiment discussed in the next chapter was to confirm the aforementioned bystander effect. In the first experiment we separated hosting and non-hosting mRBCs by the percol purification method and then measured the corner frequencies of them. The mean fc of the distribution is almost the same, and this confirms the effect of the parasite on the non-hosting mRBC. In the next experiment, we have incubated nRBCs in the spent media and measured the corner frequency at six-hours intervals to see how the fc changed with the incubation time. The results showed that within 24 hours, the fc of the incubated nRBCs increases to the level of the iRBCs. The fact that nRBCs are getting affected by the spent media indicates that some substances must be released in the spent media which alter the physical properties of the nRBCs. This kind of effect on non-host mRBCs was previously observed by some earlier works [Dondorp97, Sabolovic91a, Bambardekar08]. It has also been recently shown that the rosetting of the host mRBCs to the non-host mRBCs is also activated by the substances released in the medium [Handunnetti89, Wahlgren89], which are also somewhat similar to the bystander effect observed by us. In addition to this, there are reports which suggest that sickle cell disease also shows binding properties [Roseff08, Zhang12] which may be due to the substances released in the medium. So it was already observed that the released substances induced changes in the properties of RBCs, but our study gives a direct confirmation of the same. The next study was to find out the released substances which were responsible for the observed changes above. We incubated infected and uninfected RBCs in different drugs. Then, we measured them to see what kind of changes occur in the corner frequency of the incubated RBCs. The corner frequency of normal RBCs incubated in db-cAMP shows the maximum change. So the released substance that is responsible for the bystander effect may be due to the db-cAMP. All the experiments above were done using samples cultured only in the lab. Since the environment of the blood taken directly from the patient may differ from the one that is cultured in the lab, it is natural to find out if similar kinds of changes can be observed in the clinical sample or not. The study in chapter 6 was targeted to find out the same. We took clinical samples from BMRI for patients having a confirmed malaria infection by both P. falciparum and P. vivax. This also provided us the opportunity to work with the P. vivax infected sample as it is very difficult to culture them in the lab. The results shown in this chapter clearly indicate that similar kinds of changes occur in the clinical sample also. It is worth noting that even though P. vivax infects only immature RBCs (reticulocytes), changes were also observed in P. vivax samples. This gives us another strong confirmation about the previously observed bystander effect. This also indicates that this technique can be used as a tool to diagnose malaria. Although we cannot differentiate between P. falciparum and P. vivax, this technique combined with other well established techniques can give us more confirmation. So, in all the experiment above we have shown an easy and novel technique which can be used to differentiate between normal and malaria-infected RBCs. We have also observed the bystander effect and tried to find out the released substances which are responsible for this effect. We have shown that this technique can use the bystander effect of malaria to identify malaria. It has also been shown that the RBCs taken from the patient sample also show the same changes as the cultured samples, which gives us the possibility that this technique can be used as a diagnostic tool combined with other technique. This technique can also be used in experiments like the effects of drugs and to find out drugs for diseases like malaria. Future outlook 1. We have observed the changes only for malaria. There may be other diseases like sickle cell anemia which can also alter the corner frequency of the distribution of RBCs. We have to find out the specificity of the observed changes. 1 We can directly measure the elasticity of RBCs using dual traps in optical tweezers to find out the effect of different infections and drugs on the rigidity of RBCs and compare the with the data above. 2 We can also study other cells using the same method to see if we can find out any difference between healthy and unhealthy cells.

The current treatment for hepatitis C virus (HCV) infection – combination therapy with pegylated interferon and ribavirin – elicits sustained responses in only ~50% of the patients treated. Greater cumulative exposure to ribavirin increases response to interferon-ribavirin combination therapy. A key limitation, however, is the toxic sideeffect of ribavirin, hemolytic anemia, which often necessitates a reduction of ribavirin dosage and compromises treatment response. Maximizing treatment response thus requires striking a balance between the antiviral and hemolytic activities of ribavirin. Current models of viral kinetics describe the enhancement of treatment response due to ribavirin. Ribavirin-induced anemia, however, remains poorly understood and precludes rational optimization of combination therapy. Here, we develop a new mathematical model of the population dynamics of erythrocytes that quantitatively describes ribavirin-induced anemia in HCV patients. Based on the assumption that ribavirin accumulation decreases erythrocyte lifespan in a dose-dependent manner, model predictions capture several independent experimental observations of the accumulation of ribavirin in erythrocytes and the resulting decline of hemoglobin in HCV patients undergoing combination therapy, estimate the reduced erythrocyte lifespan in patients and describe inter-patient variations in the severity of ribavirin-induced anemia. Further, model predictions estimate the threshold ribavirin exposure beyond which anemia becomes intolerable and suggest guidelines for the usage of growth hormones. A small fraction of the population (~30%) with polymorphisms in the ITPA gene shows protection from ribavirin-induced anemia. The optimum dosage of ribavirin that can be tolerated is then dependent on the ITPA polymorphisms. Coupled with a previous population pharmacokinetic study, our model yields a facile formula for estimating the optimum dosage given a patient’s weight, creatinine clearance, pretreatment hemoglobin levels, and ITPA polymorphism. The reduced lifespan we predict is in agreement with independent measurements from breath tests as well as estimates derived from in vitro studies of ATP depletion. The latter estimates also agree with the extent of ATP depletion due to ribavirin that we predict from a detailed analysis of the nucleoside metabolism in erythrocytes. Our model thus facilitates in conjunction with models of viral kinetics the rational identification of treatment protocols. Our formula for optimum dose presents an avenue for personalizing ribavirin dosage. By keeping anemia tolerable, the predicted optimal dosage may improve adherence, reduce the need for drug monitoring, and increase response rates.

Einleitung: Vitamin B12 hat wichtige Funktionen im Energiestoffwechsel sowie bei der Erythropoese. Beide Funktionskreise werden bei Hochleistungskühen besonders beansprucht und können bei Belastungen und ungenügender Vitamin-B12-Versorgung Ausgangspunkt für klinische Störungen werden. Ziele der Untersuchungen: In den vorliegenden Studien wurde der Fragestellung nachgegangen, wie sich die Vitamin-B12-Konzentration im Blutserum von Milchkühen in der Frühlaktation verhält und welche Zusammenhänge zu Stoffwechselparametern, dem Erythrogramm sowie dem Gesundheitsstatus der Kühe bestehen. Des Weiteren wurde geprüft, inwieweit der postpartale Stoffwechsel und der Gesundheitsstatus durch orale Vitamin-B12-Substitutionen stabilisiert werden können. Material und Methoden: Die Untersuchungen zur Vitamin-B12-Statuserhebung erfolgten an 157 Kühen der Rasse Holstein Friesian. Blutproben zur Stoffwechselanalytik wurden 2-6 Tage p.p. sowie 4-5 Wochen p.p. entnommen. Parallel dazu wurden klinische Daten zu Leistung und Gesundheitsstatus bis 3 Monate p.p. erhoben. In einem zweiten Versuch wurden die Kühe in 2 Gruppen eingeteilt, wobei die Versuchsgruppe (65 Kühe) eine orale Vitamin-B12-Substitution in Höhe von 0,5 g Cyanocobalamin/Kuh/Tag 4-6 Wochen a.p. beginnend bis zur Kalbung erhielt. 71 Kühe, die das stallübliche Mineralfutter erhielten, dienten als Kontrollgruppe. Auch hier erfolgten die Blutkontrollen 2-6 Tage p.p. sowie 4-5 Wochen p.p. Es wurden die Milchleistung sowie auftretende Erkrankungen dokumentiert. Die Blutentnahme erfolgte aus der Vena caudalis mediana. Neben den hämatologischen Untersuchungen wurden folgende Parameter aus dem Serum bestimmt: Freie Fettsäuren (FFS), Betahydroxybutyrat (BHB), Glukose, Bilirubin, Cholesterol, Gamma-Glutamyltransferase (GGT), Creatinkinase (CK), Harnstoff, Calcium, Eisen, anorganisches Phosphat (Pi), Cyanocobalamin und Cobalt. Ergebnisse: Die Vitamin-B12-Konzentration zeigt eine signifikante Laktationsdynamik. Alle untersuchten Kühe hatten 4 Wochen p. p. gegenüber 2–6 Tage p. p. erniedrigte Vitamin-B12-Konzentrationen (p ≤ 0,05). Bei den p.p. kranken Kühen sank gegenüber den gesunden Kühen die Vitamin-B12-Konzentration weniger stark ab (p ≤ 0,05), d.h., höhere Vitamin-B12-Konzentrationen können auf klinische Probleme hinweisen. Gesunde sowie auch p.p. kranke Kühe wiesen 2–6 Tage p. p. höhere Werte für die Parameter Erythrozytenzahl, Hämatokrit und Hämoglobinkonzentration auf als 4 Wochen p. p. Die BHB-, FFS- und Bilirubin-Konzentrationen waren bei allen Kühen 2–6 Tage p. p. infolge der partusbedingten Lipolysesteigerung erhöht (p ≤ 0,05). Bei allen Kühen korrelierte die Aktivität der cholestaseanzeigenden GGT eng mit der Vitamin-B12-Konzentration (p ≤ 0,01). Aufgrund dieser engen Korrelation mit der GGT-Aktivität sowie der Bilirubinkonzentration kann Vitamin B12 bei einer Serumkonzentration ≥ 227 ng/l bei Kühen cholestatische Stoffwechselbelastungen anzeigen. Nach Vitamin-B12-Substitution blieben in der Versuchsgruppe 60 % und in der Kontrollgruppe 47,9 % der Kühe in der Frühlaktation gesund. In der Kontrollgruppe hatten 12,7 % eine Nachgeburtsverhaltung und 40,8 % eine Mastitis, in der Versuchsgruppe betrugen die Anteile 21,5 % sowie 26,2 %. Mit x̃ = 320 pg/ml war die Vitamin-B12-Konzentration 2-6 Tage p.p. in der Vitamin-B12-substituierten Gruppe gegenüber x̃ = 224 pg/ml in der Kontrollgruppe gesichert höher. Auch vier Wochen p.p. war die Differenz noch signifikant. Cobalt, die Parameter des Leber- und Energiestoffwechsels sowie Harnstoff und CK unterschieden sich zwischen der Vitamin-B12-substituierten Gruppe und der Kontrollgruppe nicht gesichert. Die Erythrozytenzahlen sowie die Hämoglobin-Konzentrationen waren in der Vitamin-B12-substituierten Gruppe gesichert höher. Der Milchfettgehalt (%) war bei den Vitamin-B12-substituierten Kühen gegenüber den Kontrollkühen signifikant erhöht (p = 0,022), die Milchleistung unterschied sich unwesentlich. Schlussfolgerungen: Signifikant höhere Vitamin-B12- und Hämoglobin-Konzentrationen, höhere Erythrozytenzahlen sowie geringere Morbidität sprechen für positive Effekte der Vitamin-B12-Substitution. Anhand der Parameter des Leber-Energiestoffwechsels sowie der Milchleistung ließ sich dies nicht bestätigen. Cholestase stört die Vitamin-B12-Bewertung im Blut bzw. ist bei der Interpretation zu beachten. / Introduction: Vitamin B12 has important functions in energy metabolism and erythropoiesis. Both functional groups are particularly stressed in high-yielding dairy cows and can be a starting point for clinical disorders under stress and insufficient vitamin B12 supply. Objective: The studies presented were designed to ascertain the characteristics of the serum vitamin B12 concentration of dairy cows in early lactation and to check the relations with metabolic parameters, the erythrogram as well as the health status of the cows. Furthermore, it was examined to what extent the postpartum metabolism can be stabilized by oral vitamin B12 substitutions. Material and methods: The investigations on vitamin B12 status survey were carried out on 157 cows of the Holstein Friesian breed. Blood samples were taken for metabolic analysis at 2-6 days p.p. and at 4-5 weeks p.p. In parallel, clinical findings on the milk yield and the health status were compiled up to 3 months p.p. In a second trial the cows were divided into 2 groups in which the experimental group (65 cows) received an oral vitamin B12 substitution in the amount of 0.5 g cyanocobalamin/cow/day starting 4-6 weeks a.p. up to calving. 71 cows, which recieved the common mineral feed, served as control group. Again the blood tests were performed at 2-6 days p.p. and at 4-5 weeks p.p. The milk yield and emerging diseases were documented. The blood samples werde taken from the Vena caudalis mediana. In addition to haematological investigations the following parameteres were measured: Non-esterified fatty acids (NEFA), beta-hydroxybutyrate (BHB), glucose, bilirubin, cholesterol, gamma-glutamyl transferase (GGT), creatinkinase (CK), urea, calcium, ferric, anorganic phosphor, cyanocobalamin and cobalt. Results: The vitamin B12 concentration shows a significant dynamic in lactation. All examined cows had decreased vitamin B12 concentrations at 4 weeks p. p. compared to 2-6 days p. p. (p ≤ 0.05). In the p.p. morbid cows the vitamin B12 concentration fell less than in the healthy cows (p ≤ 0.05), which means higher vitamin B12 concentrations may indicate clinical problems. Healthy as well as p.p. morbid cows showed higher values for the parameters erythrocyte count, hematocrit and hemoglobin concentration at 2-6 days p.p. than 4 weeks p.p. BHB, FFS, and bilirubin levels were increased in all cows at 2-6 days p.p. as a result of partus related rise in lipolysis (p ≤ 0.05). In all cows, the activity of the GGT, which indicates cholestasis, was closely correlated with the vitamin B12 concentration p ≤ 0.01). Because of this close correlation with GGT activity and bilirubin concentration, vitamin B12 may show cholestatic metabolic stress in cows at a serum concentration ≥ 227 ng/l. After vitamin B12 substitution 60 % of the cows in the experimental group and 47.9 % of the cows in the control group remained healthy during early lactation. In the control group 12.7 % had a Retentio secundinarum and 40.8 % had a mastitis, in the experimental group the proportions were 21.5 % and 26.2 %. At two to six days p.p. the vitamin B12 concentration in the vitamin B12 substituted group was significant higher (x̃ = 320 pg/ml) than in the control group (x̃ = 224 pg/ml). This difference was still significant at four weeks p.p. Cobalt, parameters of liver and energy metabolism as well as urea and CK did not differ significantly between the vitamin B12 substituted group and the control group. Erythrocyte counts and hemoglobin concentrations were significant higher in the vitamin B12 substituted group. Milk fat content (%) was significant higher in the vitamin B12 substituted group compared to the control group (p = 0.022), the milk yield did not differ significantly. Conclusions: Significant higher vitamin B12 and hemoglobin concentrations, higher erythrocyte counts as well as lower morbidity speak for positive effects of vitamin B12 substitution. Based on the parameters of hepatic and energy metabolism as well as milk yield this could not be confirmed. Cholestasis interferes with the evaluation of vitamin B12 in the blood respectively should be considered in the interpretation.

Ce mémoire concerne la modélisation mathématique de l’érythropoïèse, à savoir le processus de production des érythrocytes (ou globules rouges) et sa régulation par l’érythropoïétine, une hormone de contrôle. Nous proposons une extension d’un modèle d’érythropoïèse tenant compte du vieillissement des cellules matures. D’abord, nous considérons un modèle structuré en maturité avec condition limite mouvante, dont la dynamique est capturée par des équations d’advection. Biologiquement, la condition limite mouvante signifie que la durée de vie maximale varie afin qu’il y ait toujours un flux constant de cellules éliminées. Par la suite, des hypothèses sur la biologie sont introduites pour simplifier ce modèle et le ramener à un système de trois équations différentielles à retard pour la population totale, la concentration d’hormones ainsi que la durée de vie maximale. Un système alternatif composé de deux équations avec deux retards constants est obtenu en supposant que la durée de vie maximale soit fixe. Enfin, un nouveau modèle est introduit, lequel comporte un taux de mortalité augmentant exponentiellement en fonction du niveau de maturité des érythrocytes. Une analyse de stabilité linéaire permet de détecter des bifurcations de Hopf simple et double émergeant des variations du gain dans la boucle de feedback et de paramètres associés à la fonction de survie. Des simulations numériques suggèrent aussi une perte de stabilité causée par des interactions entre deux modes linéaires et l’existence d’un tore de dimension deux dans l’espace de phase autour de la solution stationnaire. / This thesis addresses erythropoiesis mathematical modeling, which is the process of erythrocytes production and its regulation by erythropeitin. We propose an erythropoiesis model extension which includes aging of mature cells. First, we consider an age-structured model with moving boundary condition, whose dynamics are represented by advection equations. Biologically, the moving boundary condition means that the maximal lifespan varies to account for a constant degraded cells flux. Then, hypotheses are introduced to simplify and transform the model into a system of three delay differential equations for the total population, the hormone concentration and the maximal lifespan. An alternative model composed of two equations with two constant delays is obtained by supposing that the maximal lifespan is constant. Finally, a new model is introduced, which includes an exponential death rate depending on erythrocytes maturity level. A linear stability analysis allows to detect simple and double Hopf bifurcations emerging from variations of the gain in the feedback loop and from parameters associated to the survival function. Numerical simulations also suggest a loss of stability caused by interactions between two linear modes and the existence of a two dimensional torus in the phase space close to the stationary solution.