Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The high sickle cell gene frequency has been hypothesised to be related to the protective advantage against malaria disease among heterozygous individuals. In this thesis, we study the interaction between the dynamics of malaria and sickle cell gene. The main aim is to investigate the impact of malaria treatment on the frequency of sickle cell gene. For this, we develop a mathematical model that describes the interactions between malaria and sickle cell gene under malaria treatment. The model includes both homozygous for the normal gene (AA) and heterozygous for sickle cell gene (AS) and assumes that AS individuals are not treated since they do not show clinical symptoms. We first analyse the model without malaria treatment, using singular perturbation techniques, basing on the fact that epidemiological and demographical dynamics occur on two different time scales (fast and slow dynamics). Our analysis on the fast time scale shows that high sickle cell gene frequency leads to high endemic levels for longer duration of parasitemia among heterozygous individuals. However, if the duration of parasitemia is reduced then high sickle cell gene frequency is associated with low endemic levels. We also note that on the slow time scale, the invasion ability of sickle cell gene is dependent on the malaria epidemiological parameters. The invasion coefficient given as the difference in the weighted death rates of AA and AS individuals is used as a measure to determine the establishment of sickle cell gene in the population. Results show that, the gene may establish itself if the weighted death rate of AA individuals is greater than that of AS individuals otherwise it fails. We note that, high mortality of AA individuals leads to establishment of sickle cell gene in the population. Then we analysed the model with treatment, our results indicate that the frequency of sickle cell gene decreases with an increase in the recovery rate of AA individuals. We thus conclude that eradication of malaria disease will lead to a reduction in sickle cell gene frequency. / AFRIKAANSE OPSOMMING: Daar word veronderstel dat die hoë sekelsel geenfrekwensie onder heterosigotiese individue verwant is aan die beskermende voordeel teen malaria siekte. In hierdie verhandeling ondersoek ons die wisselwerking tussen die dinamika van malaria en die sekelsel geen. Die hoofdoel is om die invloed van malaria behandeling op die frekwensie van die sekelsel geen te ondersoek. Hiervoor het ons ‘n wiskundige model ontwikkel, wat die wisselwerking tussen die dinamika van malaria en die sekelsel geen met malaria behandeling, beskryf. Die model sluit beide homosigotiese vir die normale geen (AA) en heterosigotiese vir die sekelsel geen (AS) in, en neem aan dat AS individue nie behandel is nie omdat hulle nie die eerste kliniese simptome getoon het nie. Ons ontleed eers die model sonder malaria behandeling, deur gebruik te maak van enkelvoudige pertubasie tegnieke, wat gegrond is op die feit dat epidemiologiese en demografiese dinamika plaasvind op twee verskillende tydskale (vinnige en stadige dinamika). Ons ontleding op die vinnige tydskaal dui dat hoë sekelsel geenfrekwensie onder heterosigotiese individue lei tot hoë endemiese vlakke vir ‘n langer duur van parasitemie. Nietemin, as die duur van parasitemie afneem, dan word hoë sekelsel geenfrekwensie verbind met lae endemiese vlakke. Ons neem ook waar dat op die stadige skaal die indringingsvermoë van die sekelsel afhanklik is van malaria se epidemiologiese parameters. Die indringingskoëffisiënt wat bereken word as die verskil van die geweegde sterftekoerse van AA en AS individue, word gebruik as ‘n maatstaf om die vestiging van die sekelsel geen in die bevolking te bepaal. Resultate toon dat die geen homself kan vestig as die geweegde sterftekoers van AA individue groter is as di e van die AS individue, andersins misluk dit. Ons let op dat hoë mortaliteit van AA individue lei tot die vestiging van die sekelsel geen in die bevolking. Daarna het ons die model wat behandeling insluit ge-analiseer en ons resultate toon dat die frekwensie van die sekelsel geen afneem met ‘n toename in die herstelkoers van AA individue. Ons kom dus tot die gevolgtrekking dat die uitwissing van malaria siekte sal lei tot die afname in sekelsel geenfrekwensie.

<p>Malaria, caused by the apicomplexan parasite Plasmodium, is a disease which affects up to 500 million people each year. Historically, malaria infection has been combated both through the control of its vector, the Anopheles mosquito, and use of a variety of drugs, such as quinine (1800s) and chloroquine (1900s). However, with the evolution of resistance to the majority of available anti-malarial drugs, current approaches have settled upon combinatorial therapies. The most effective of these currently are ACTs (Artemisinin Combination Therapies - Artemisinin derivatives combined with a number of other drugs). However reports of Artemisinin resistance are continuing to emerge, suggesting that new approaches and increased understanding of the Plasmodium parasite is required.</p><p> Beginning with the complete sequencing of Plasmodium falciparum genome and continuing with comprehensive profiling of both the parasite's proteome and transcriptome, various genomic approaches applied in the study of malaria have led to significant new insights into the underlying biology of this parasite. While these new findings have greatly increased our understanding of genetic regulation within the malaria parasite, they largely have not yet translated into new therapeutic approaches. For this reason, considerable attention has been paid to the study of human genetic disorders which convey resistance to malaria, in the hopes that elucidating the mechanisms behind these resistances might lead to increased understanding of the parasite's biology and thus novel therapeutic approaches.</p><p> Sickle cell (HbS) erythrocytes are well known to resist malaria infection. However, the molecular basis of this resistance, long been recognized as multifactorial, contains elements which remain poorly understood. Here we show that the dysregulated erythrocytic microRNA composition, present in both HbAS and HbSS erythrocytes, is a significant determinant of resistance against the malaria parasite Plasmodium falciparum. During the intraerythrocytic lifecycle of P. falciparum, a subset of erythrocyte microRNAs translocate into the parasite. Two microRNAs, miR-451 and let-7i, were highly enriched in HbAS and HbSS erythrocytes and these miRNAs, along with miR-223, negatively regulated parasite growth. Surprisingly, we found that miR-451 and let-7i integrated into essential parasite mRNAs and, via impaired ribosomal loading, resulted in translational inhibition of the target mRNA. Hence, sickle cell erythrocytes exhibit cell-intrinsic resistance to malaria in part through an atypical microRNA activity which may present a novel host defense strategy against complex eukaryotic pathogens. In addition, the formation of these chimeric transcripts even in normal host erythrocytes illustrates a unique parasitic post-transcriptional adaptation to the host-cell environment.</p> / Dissertation

The enormous mortality burden exerted by P. falciparum malaria has evolutionarily selected for red blood cell (RBC) polymorphisms which confer protection against the severe manifestations of this disease. Although the epidemiological protection by these polymorphisms has been well-established for the past half-century, the mechanisms underlying this protection are still being uncovered. Recent studies implicate impaired cytoadherence to microvascular endothelial cells (MVECs) due to reduced surface levels and altered display of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) as a mechanism of protection against severe malaria by sickle hemoglobin (Hb) S and HbC. Consequently, in this thesis, I have described three separate, but related investigations into whether hemoglobins S and C influence a parasite’s cytoadherence binding phenotype (Chapter 3), the PfEMP1 variants that parasites express in vivo (Chapter 4), and the IgG recognition of PfEMP1 domains in Malian children (Chapter 5). We found that parasites from HbAS children show statistically insignificant increased binding to MVECs and that parasites did not express a restricted subset of var genes in HbAS and HbAC children. Compared to HbAA and HbAC children, HbAS children demonstrated a slower rate of acquisition of IgG responses to a repertoire of PfEMP1 domains. These findings suggest that, although hemoglobin type influences the binding phenotype of P. falciparum isolates and the acquisition of PfEMP1-specific IgG responses, other factors more likely determine the expressed var gene repertoire within parasites than hemoglobin type.

Sickle cell disease (SCD) is one of the most common genetic diseases worldwide. HbS polymerisation causes altered red blood cell (RBC) rheology and fragility, increase in blood viscosity with blockage of small blood vessels, and RBC membrane permeability changes. Excessive levels of cell-free Hb, high autoxidation of Hb, contribute to the production of reactive oxygen species (ROS) in SCD patients. In this work, oxidants showed direct and indirect effects on the main cation permeability pathways involved in dehydration of HbS/S RBCs - Psickle, the Gardos channel and the KCl cotransporter (KCC) - and thus on RBC volume causing polymersation. Psickle and Gardos channel activities showed significant correlation, consistent with the hypothesis that Ca2+ entry via Psickle causes activation of the Ca2+-dependent K+ channel. Treatment of SCD remains inadequate relying on the blood transfusion and supportive therapy depending on the organ affected. In the present study antioxidants and aromatic aldehydes showed some promising results towards future alternative treatments for SCD. Antioxidants showed inhibitory effects on the cation permeability pathways leading to inhibition of polymerisation and haemolysis and thus maintained RBC volume. Aromatic aldehydes interact with HbS and are usually given to increase oxygen affinity, thereby reducing its tendency to polymerise. GBT1118 had a marked inhibitory effect on all three cation permeability pathways. It reduced sickling, Psickle and Gardos activity. It inhibited KCC by affecting the regulatory protein phosphorylation cascade. It maintained RBC hydration, and stabilised RBCs. Historically Oman was the principal trading port of the Persian Gulf region, resulting in the complex mix of social and ethnic backgrounds. In 1989 a second mutation in the β chain of Hb, at position β121 was found in an Omani patient in addition to the usual HbS mutation at the β6 position, and termed HbS-Oman. At low percentage of HbS-Oman patients show severe SCD symptoms. Despite RBCs containing at most 25% HbS-Oman, there was high sickling percentage and K+ permeability showed many features similar to those seen in homozygous HbS/S patients. The presence of α thalassaemia was protective and represents an obvious potential prognostic marker for this rare SCD genotype. Overall, the present work contributes to elucidation of the pathogenesis of SCD, suggests approaches to the development of novel therapies and increases our understanding of a rare SCD genotype, HbS-Oman.

Orientador: Milton Artur Ruiz / Coorientador: Claudia Regina Bonini Domingos / Banca: Silma Maria Alves de Melo / Banca: Isabeth da Fonseca Estevão / Resumo: A anemia falciforme (AF) afeta milhões de pessoas em todo o mundo e está associada a altas taxas de morbidade e mortalidade. Apresenta uma série de manifestações fenotípicas, que são influenciadas por fatores genéticos e ambientais, resultando em fenótipos diversificados, e um tratamento bastante eficaz tem sido o uso de hidroxiureia (HU), que ameniza os sintomas e a necessidade de transfusão sanguínea e hospitalização. Estudos de associação de genomas já demonstraram que polimorfismos genéticos podem desempenhar influência no perfil clínico dos pacientes, assim como na resposta destes à medicação. Os polimofismos -308G/A no gene TNFA e -509C/T no gene TGFB aumentam a produção das suas respectivas citocinas que atuam principalmente em vias inflamatórias e são fortes candidatos a estarem envolvidos na ocorrência de episódios vaso-oclusivos característicos da doença. Dessa forma, o objetivo do trabalho foi verificar a frequência desses polimorfismos em portadores da AF, com e sem o uso de HU, e possível relação com a gravidade das manifestações clínicas da doença. Foram obtidas 588 amostras de sangue periférico de pacientes com doença falciforme em acompanhamento no HEMORIO. A partir destas, foram separados, aleatoriamente, 240 pacientes com AF, cujo genótipo foi confirmado por procedimentos laboratoriais clássicos e moleculares. Estes foram genotipados para os polimorfismos -308G/A (TNFA) e -509C/T (TGFB) por PCR-RFLP. Os dados hematológicos e clínicos parciais de 118, dos 240 pacientes, foram obtidos por questionário e consulta aos prontuários médicos e banco de dados. A frequência do polimorfismo -308G/A foi de 0,83 em homozigose e 17,92% em heterozigose. Para o polimorfismo -509C/T, foi de 6,25% e 48,33%, respectivamente. Não foi observada associação entre o polimorfismo -308G/A e as manifestações clínicas... (Resumo completo, clicar acesso eletrônico aba / Abstract: Sickle cell anemia (SCA) affects millions of people worldwide and is associated with high morbidity and mortality. This affection shows various phenotypic manifestations, which are influenced by genetic and environmental factors, resulting in many phenotypes, and the most effective treatment has been the use of hydroxyurea (HU), which improves the symptoms and the requirement for blood transfusion and hospitalization. Genome association studies have shown that genetic polymorphisms may play role on the clinical profile of patients, as well as in response to these medications. The -308G/A and -509C/T polymorphisms, in TNFA and TGFB genes respectively, increase production of their cytokines, which act on inflammatory pathways and are strong candidates to be involved in the occurrence of vaso-occlusive episodes. The aim of this study was to determine the frequency of these polymorphisms in patients with AF, with and without HU utilization, and possible relationship with the severity of clinical manifestations of disease. We obtained 588 peripheral blood samples of patients with sickle cell disease at HEMORIO. From these, were separated at random 240 patients with AF, whose genotype was confirmed by classical and molecular laboratory procedures. They were genotyped for polymorphisms-308G/A (TNFA) and-509C/T (TGFB) by PCR-RFLP. The hematological and clinical data of 118 of the 240 patients, were obtained by questionnaire and medical records and database. The frequency of polymorphism -308G/A was 0.83% in homozygous and 17.92% in heterozygous. For the polymorphism -509C/T, was 6,25% and 48.33% respectively. No association between polymorphism -308G/A and clinical manifestations in patients was found. Concerning the polymorphism-509C/T, the mutant allele (T) proved to be a risk... (Complete abstract click electronic access below) / Mestre

This research study was focused on two broad areas of exploration. The first area deals with the identification of various factors affecting a family when a family member has the anemia or symptomatic form of sickle cell disease. Data obtained from a personally administered questionnaire (Form A), enabled the researchers to determine if genetic counseling had been offered and received, and if this counseling was considered helpful by the respondents. In addition to this, data was collected on several demographic variables, including sex and age of patient, family income, religion, education, ethnic group, living arrangements and occupation, and response to and knowledge of sickle cell anemia.

Sickle cell trait (SCT), sickle cell disease’s carrier status, is a common genetic variant found in many people of African, South Asian, Middle Eastern and Mediterranean descent. While overall considered a benign carrier status, it has been associated with an increased risk of several diseases, including exertional rhabdomyolysis (ER), and chronic kidney disease. While epidemiological evidence links SCT with ER, the actual pathophysiological mechanism less understood. Additionally, while there is an increased prevalence of atrial fibrillation (AF) documented in people with sickle cell disease, studies in individuals with SCT are lacking. The objectives of this thesis are twofold: The first chapter is a literature review of studies to examine the physiological mechanisms linking SCT and exertional rhabdomyolysis. The second chapter is original research into the associations of SCT with AF. The first chapter reviews studies that identify aggravating factors that may promote ER. It then reviews observed pathophysiological changes in people with SCT that may increase the risk of ER. It summarizes studies that assess mitigating factors that decrease the risk of ER. It then presents a postulated pathway of mechanisms that associate SCT with ER. The second chapter uses data from African-American participants in the REasons for Geographic and Racial Differences in Stroke (REGARDS) study to assess the association of SCT with prevalent AF (by electrocardiogram or medical history) using logistic regression models adjusting for age, sex, income, education, history of stroke, myocardial infarction, diabetes, hypertension, and chronic kidney disease. In 10,409 participants with baseline ECG data and genotyping, 778 (7.5%) had SCT and 811 (7.8%) had prevalent AF. After adjusting for age, sex, education and income, SCT was associated with AF, OR 1.32 (95% CI 1.03-1.70). SCT remained associated with prevalent AF after adjusting for potential factors on the causal pathway such as hypertension and chronic kidney disease suggesting alternate mechanisms for the increased risk. SCT was associated with a higher prevalence of AF and a non-significantly higher incident AF over a 9.2 year period independent of AF risk factors.

In this study we present feasibility of a mobile monitoring and reporting system that would provide an accurate unbiased screening tool to systematically analyze opioid adherence in Sickle cell disease patients. In addition, the software simultaneously measures pain. The Mobile Applications Rating Scale: a new and validated tool for assessing the quality of health mobile apps for engagement, functionality, aesthetics, information quality, subjective quality, relevance and overall impact was administered post usage to evaluate the application. A total of 28 patients were recruited to review and test the software at one sitting. The majority of the population found the application to be relevant for their care. Patients were also asked to report on the completeness of information within the app, the majority (96%) reported on the application’s completeness while 4% estimated the information to be minimal or overwhelming. The quality of information as it pertains to sickle cell patients was overwhelimingly reported to be relevant (91.7%); only 8.3% found the application to be poorly relevant to sickle cell disease. The application’s performance was positively rated while the ease of its use positively rated at 91.7%. Most participants (85.7%) found the application to be interesting to use while 74% found it entertaining. All users found the application’s navigation to be logical and accurate with consistent and intuitive gestural design. We conclude that surveyed patients believe it is feasible to use a smartphone application specifically targeted to monitor opioid use and behavior in patients with sickle cell disease (SCD)-associated pain

O avanço de novas tecnologias tem conduzido à geração de grandes volumes de dados biológicos, provenientes, por exemplo, de sequenciamento de genomas, expressão de genes e proteínas, estrutura de proteínas e RNAs, análise de imagens, formulários eletrônicos e exames médicos. Com o intuito de transformar esses volumosos conjuntos de dados brutos em informação e conhecimento que sejam compreensíveis e interpretáveis, técnicas de mineração de dados têm sido aplicadas no estudos de diversos processos biológicos, como a predição de genes, funções de genes, fenótipos, módulos regulatórios, estrutura de proteínas, função de proteínas e descoberta de interações moleculares. Cada conjunto de dados tem suas particularidades, demandando o emprego de distintas metodologias de análises e algoritmos de reconhecimento de padrões, como Florestas Aleatórias, Redes Neurais, Deep Learning, Modelo Oculto de Markov, Máquina de Vetores de Suporte, K-médias e Análise de Componentes Principais. A escolha do algoritmo a ser utilizado é influenciada por fatores como o tipo dos dados, a forma como são gerados, sua natureza, suas características e o objetivo do estudo. Assim, este trabalho teve como objetivo explorar técnicas de reconhecimento de padrões e estatística aplicadas a um conjunto de dados biológicos envolvendo pacientes com anemia falciforme, para extração de informação e conhecimento sobre os processos, fenômenos e sistemas biológicos envolvidos na doença. Foram realizadas análises de um conjunto de dados diverso, proveniente de registros clínicos, entrevistas com pacientes, exames clínicos e sequenciamento de polimorfismos de nucleotídeo único. Os dados demandam diferentes abordagens de análises, exploração e revelação da estrutura de dados intrínseca. Em uma análise inicial, foram aplicados algoritmos de reconhecimento de padrões a dados clínicos de pacientes com anemia falciforme, com o objetivo de obter grupos contendo pacientes similares. Os algoritmos PCAMix, PAM e TwoStep clustering foram capazes de gerar grupos homogêneos de pacientes, sendo que estes grupos apresentam distintas características clínicas e diferentes níveis de gravidade da doença quando comparados entre si. Os resultados indicam que características como idade, níveis de bilirrubina, histórico de transfusões, dor aguda da anemia falciforme, síndrome torácica aguda, acidente vascular cerebral, infarto cerebral silencioso, ataque isquêmico transitório, úlcera de pernas, moyamoya, ferritina, contagem de reti- culócitos, retinopatias, ataques epiléticos e hemossiderose transfusional são importantes para a definição de grupos homogêneos de pacientes, que apresentem distintos níveis de gravidade de anemia falciforme quando comparados entre si. Adicionalmente à análise de agrupamento, o conjunto de pacientes com histórico de priapismo, uma das complicações da anemia falciforme, foi estudado. O objetivo desta análise foi caracterizar clinicamente os pacientes com histórico de priapismo, e investigar fatores genéticos que alterassem o risco da doença. Observou-se que o priapismo ocorreu mais frequentemente em pacientes com genótipo HbSS, estando associado a idades mais avançadas e à ocorrência de hipertensão pulmonar e necrose avascular. Dois novos SNPs foram associados à ocorrência de priapismo, bem como houve indicativo de replicação da associação do gene TGFBR3 ao risco da doença. / Technology has been producing large biological datasets of genome sequences, gene and protein expression, RNA and protein structure, images, electronic questionnaires and laboratory test results. In order to extract information and knowledge from these large datasets, data mining techniques have been used in the investigation of a wide range of biological processes, with the goal of predicting gene, gene function, phenotype, regulatory modules, molecular interaction, protein function and protein structure. Each dataset has different characteristics and demands the application of different statistical methodologies and pattern recognition algorithms, such as Random Forests, Neural Networks, Deep Learning, Markov Hidden Model, Support Vector Machine, K-means and Principal Component Analysis. The choice of the algorithm depends on data type, data generation, data characteristics and goal of the study. Therefore, the goal of this work was to explore pattern recognition and statistical techniques in a biological dataset on sickle cell disease patients, in order to extract information and knowledge about the biological systems, processes and mechanisms associated with the disease. A diverse dataset was analyzed, containing data from medical records, patient interviews, laboratory tests and single nucleotide polymorphisms. The dataset requires a variety of analysis approaches, in order to explore and reveal the hidden data structure. In an initial investigation, pattern recognition algorithms were used in the analysis of clinical data from sickle cell patients, in order to obtain clusters containing similar patients. PCAMix, PAM and TwoStep clustering algorithms generated homogeneous clusters of patients that display different clinical characteristics and different levels of disease severity. The results show that age, bilirubin levels, transfusion history, vaso-occlusive pain episodes, acute chest syndrome, infarctive stroke, hemorrhagic stroke, ischemic attack, leg ulcers, moyamoya, ferritin, reticulocyte count, retinopathy, seizures and transfusional hemosiderosis are important to define homogeneous patient clusters, with distinct levels of sickle cell severity. Additionally, the patients with history of priapism, a sickle cell related complication, were studied. The goal of the study was to characterize patients with priapism history and investigate genetic factors that modify the risks of the disease. Priapism more frequently occurred among patients with HbSS genotype and was associated with older age and occurrence of pulmonary hypertension and avascular necrosis. Two novel SNPs were associated with priapism and there was evidence of replication of a previously reported association of TGFBR3 with priapism risk.

Linus Pauling incorporated hemoglobin and a disease of the blood, sickle cell anemia, into many of his researches between the mid-1930s and mid-1970s. In the early 1930s Pauling became interested in organic chemistry and named hemoglobin as one of the first biochemical substances that he planned to analyze. In 1935 he published his first paper on hemoglobin, which determined the structure of the four hemes in hemoglobin. Pauling continued to study the structure of hemoglobin until the early 1950s when he proposed that it was an alpha-helix. In 1945 Pauling learned about sickle cell anemia and published an important paper in 1949 with Harvey A. Itano, S.J. Singer, and Ibert C. Wells titled "Sickle Cell Anemia, a Molecular Disease." Pauling investigated hemoglobin into the mid-1970s when he tried to find an orthomolecular therapy for sickle cell anemia. From the mid-1950s to early 1970s, Pauling also used sickle cell anemia to promote negative eugenics, point out the possible mutagenic effects caused by nuclear weapons testing, and propose an evolutionary theory. Additionally, in the final year of his life, Pauling wrote two forewords for books on sickle cell anemia, which were published in 1994, the year he died. Hemoglobin and sickle cell anemia can be considered a theme within Pauling's work. He often returned to normal and abnormal hemoglobin as his primary substance for examination, and his familiarity with hemoglobin and sickle cell anemia inspired new research. / Graduation date: 2004