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Choi Wai-wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 120-138). / Abstracts in English and Chinese. / Statement --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / Abstract (Chinese Version) --- p.v / Table of Contents --- p.vii / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- "Hemoglobin ´ؤ Structures, Types and Functions" --- p.1 / Chapter 1.1.1 --- Structures of Hemoglobin --- p.1 / Chapter 1.1.2 --- Types of Hemoglobin --- p.2 / Chapter 1.1.3 --- Functions of Hemoglobin --- p.3 / Chapter 1.2 --- Human Globin Genes and Their Regulation --- p.5 / Chapter 1.2.1 --- Organization of the Human Globin Genes --- p.5 / Chapter 1.2.2 --- Regulation of Globin Gene Expression --- p.6 / Chapter 1.2.2.1 --- The Locus Control Region (LCR) --- p.6 / Chapter 1.2.2.2 --- Cis-Regulatory Elements --- p.7 / Chapter 1.2.2.2.1 --- Promoters --- p.7 / Chapter 1.2.2.2.2 --- Enhancers --- p.7 / Chapter 1.2.2.2.3 --- Silencers --- p.8 / Chapter 1.2.2.3 --- Trans-Acting Factors --- p.8 / Chapter 1.2.2.3.1 --- GATA Family --- p.9 / Chapter 1.2.2.3.2 --- Kruppel-like Factors --- p.9 / Chapter 1.2.2.3.3 --- Nuclear Factor-Erythroid (NF-E) --- p.9 / Chapter 1.2.2.4 --- Chromatin Remodelling --- p.10 / Chapter 1.2.2.5 --- Intergenic Sequences --- p.11 / Chapter 1.3 --- Mechanisms of Hemoglobin Switching --- p.12 / Chapter 1.3.1 --- Autonomous Silencing --- p.12 / Chapter 1.3.2 --- LCR and Globin Gene Interaction --- p.12 / Chapter 1.4 --- Hemoglobinopathies --- p.14 / Chapter 1.4.1 --- α -thalassemia --- p.14 / Chapter 1.4.2 --- β -thalassemia --- p.14 / Chapter 1.4.3 --- Sickle Cell Anemia --- p.16 / Chapter 1.5 --- Therapies for β-thalassemia --- p.16 / Chapter 1.5.1 --- Blood Transfusion --- p.16 / Chapter 1.5.2 --- Bone Marrow Transplantation --- p.17 / Chapter 1.5.3. --- Gene Therapy --- p.17 / Chapter 1.6 --- Gene Switch Therapy --- p.18 / Chapter "1.6,1" --- Pharmacological Induction of HbF --- p.18 / Chapter 1.6.1.1 --- Hydroxyurea --- p.19 / Chapter 1.6.1.2 --- Butyrate --- p.20 / Chapter 1.6.1.3 --- Summary --- p.21 / Chapter 1.7 --- Objectives --- p.22 / Chapter Chapter 2 --- Induction of HbF by LC978 in K562 / Chapter 2.1 --- Introduction --- p.23 / Chapter 2.2 --- Materials --- p.26 / Chapter 2.2.1 --- Chemicals and Reagents --- p.26 / Chapter 2.2.2 --- Kits --- p.27 / Chapter 2.2.3 --- Buffers and Solutions --- p.27 / Chapter 2.2.4 --- Primers --- p.30 / Chapter 2.2.5 --- Equipment and Other Consumables --- p.30 / Chapter 2.2.6 --- Maintenance of K562 --- p.31 / Chapter 2.2.7 --- Handling and Treatment of utilities for RNA isolation --- p.31 / Chapter 2.3 --- Methods --- p.32 / Chapter 2.3.1 --- Dose-response and time-response study of LC978 in K562 by TMB assay --- p.32 / Chapter 2.3.2 --- Detection of γ -Globin Gene Expression in LC978-induced K562 by RT-PCR --- p.33 / Chapter 2.3.3 --- Fetal Hemoglobin Analysis by Human Fetal Hemoglobin (HbF) ELISA Quantitation Kit --- p.36 / Chapter 2.3.4 --- Statistical Analysis --- p.38 / Chapter 2.4 --- Results --- p.39 / Chapter 2.4.1 --- Dose-response and time-response study of LC978 in K562 by TMB assay --- p.39 / Chapter 2.4.2 --- Detection of γ -Globin Gene Expression in LC978-induced K562 by RT-PCR --- p.45 / Chapter 2.4.3 --- Fetal Hemoglobin Analysis by Human Fetal Hemoglobin (HbF) ELISA Quantitation Kit --- p.48 / Chapter 2.5 --- Discussions --- p.51 / Chapter Chapter 3 --- Signal Transduction Pathways Modulated by LC978 / Chapter 3.1 --- Introduction --- p.54 / Chapter 3.2 --- Materials --- p.57 / Chapter 3.2.1 --- Chemicals and Reagents --- p.57 / Chapter 3.2.2 --- Kits --- p.57 / Chapter 3.2.3 --- Buffers and Solutions --- p.58 / Chapter 3.2.4 --- Primers --- p.59 / Chapter 3.2.5 --- Equipment and Other Consumables --- p.60 / Chapter 3.2.6 --- Maintenance of K562 --- p.60 / Chapter 3.2.7 --- Handling and Treatment of utilities for RNA isolation --- p.60 / Chapter 3.3 --- Methods --- p.61 / Chapter 3.3.1 --- Identification of Signaling Pathways by Microarray --- p.61 / Chapter 3.3.2 --- Real-time RT-PCR --- p.65 / Chapter 3.4 --- Results --- p.67 / Chapter 3.4.1 --- Identification of Signaling Pathways by Microarray --- p.67 / Chapter 3.4.2 --- Real-time RT-PCR --- p.74 / Chapter 3.5 --- Discussions --- p.80 / Chapter Chapter 4 --- MAPK pathways and HbF induction by LC978 / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.2 --- Materials --- p.87 / Chapter 4.2.1 --- Chemicals and Reagents --- p.87 / Chapter 4.2.2 --- Kits --- p.88 / Chapter 4.2.3 --- Buffers and Solutions --- p.88 / Chapter 4.2.4 --- Equipment and Other Consumables --- p.90 / Chapter 4.2.5 --- Maintenance of K562 --- p.90 / Chapter 4.3 --- Methods --- p.91 / Chapter 4.3.1 --- "Roles of three MAPKs ´ؤ ERK, JNK and p38 in LC978-mediated γ -globin gene induction in K562 using CASE´ёØ Kits" --- p.91 / Chapter 4.3.2 --- Effect of p38 inhibitor SB203580 on HbF induction --- p.94 / Chapter 4.3.3 --- Statistical Analysis --- p.97 / Chapter 4.4 --- Results --- p.98 / Chapter 4.4.1 --- "Roles of three MAPKs - ERK, JNK and p38 in LC978-mediated γ -globin gene induction in K562 using CASETM Kits" --- p.98 / Chapter 4.4.2 --- Effect of p38 inhibitor SB203580 on HbF induction --- p.106 / Chapter 4.5 --- Discussions --- p.110 / Chapter Chapter 5 --- Summary and Prospects / Appendix / References

Der Diabetes mellitus zählt zu den Hauptursachen einer terminalen Niereninsuffizienz. Aufgrund der veränderten Erythropoiese ist die Verwendung des HbA1c für die Beurteilung der Stoffwechseleinstellung bei Diabetikern mit einer chronischen Nierenerkrankung im Stadium 5 nach KDOQI immer noch in Frage gestellt. Die vorliegende Arbeit untersuchte die HbA1c-Variabilität in Abhängigkeit der ESA-Dosis und des ESA-Präparates bei Hämodialysepatienten. Zum Einsatz kamen entweder das klassische Epoetin-Analogon Erythropoietin α oder ein Wirkstoff der neueren Generation Methoxy-Polyethylenglycol-Epoetin beta. Es wurden 102 Hämodialysepatienten, darunter 41 Diabetiker und 61 Nicht-Diabetiker, über einen Zeitraum von neun Monaten beobachtet. Von den Untersuchungsteilnehmern erhielten 48 Patienten Epoetin α und 54 Patienten Methoxy-Polyethylenglycol-Epoetin beta. Unsere Daten konnten zeigen, dass es zu einer Beeinflussung des HbA1c durch die Therapie mit ESA-Präparaten kommt. Das als alternativer Stoffwechselparameter fungierende Fructosamin unterlag keiner derartigen Beeinflussung, korrelierte jedoch nur ungenügend mit den höheren Blutzuckerwerten, sodass seine Anwendbarkeit bei Diabetikern ebenfalls erheblich einschränkt ist. In der Gegenüberstellung der beiden ESA-Präparate erzielten beide Wirkstoffe vergleichbare Ergebnisse bezüglich des Erreichens der Hämoglobin-Zielwerte. Methoxy-Polyethylenglycol-Epoetin beta zeigte im Gegensatz zu Epoetin α eine seltener notwendige Dosisanpassung und längere Applikationsintervalle. Aus der vorliegenden Arbeit geht hervor, dass bei Hämodialysepatienten das HbA1c kritisch und mit Bedacht auf mögliche Einflussfaktoren interpretiert werden sollte. Es ist dennoch derzeit der konstanteste Parameter in der Stoffwechselverlaufskontrolle bei Diabetes mellitus.

Hemoglobin-based oxygen carriers for use as transfusion fluids have emerged as a leading technology directed at stemming shortages of a safe blood supply and providing a readily available resuscitation fluid in various trauma situations. The purpose of this investigation was to determine the effects of isovolemic hemodilution with Hespan and a hemoglobin-based oxygen carrier (HBOC) on erythrocyte-associated transients (EATs) in capillary PO2. The particulate nature of blood flow in capillaries, when observed from a stationary observation point, results in fluctuations of PO2 as alternating red blood cells and plasma gaps move through the detection region. Therefore, through experimental methods which provided the necessary temporal and spatial resolution required to make such measurements, EATs can be observed and corresponding PO2 fluctuations can be determined. The spinotrapezius muscle in sixteen Sprague-Dawley rats was exteriorized for intravital microscopy measurements in capillaries. Capillary PO2 was measured using Pd-porphyrin phosphorescence quenching microcopy. The hemodiluents used in isovolemic hemodilution included Hespan, a non-oxygen carrying plasma expander, and Oxyglobin®, a HBOC. Two isovolemic hemodilution steps were performed, reducing the systemic hematocrit to an average of 27.5% after the first step and 13.5% after the second step. Results showed that erythrocyte-associated transients in PO2 can be observed in the rat spinotrapezius with significant differences occurring between red blood cell and plasma gap PO2 under control conditions, isovolemic hemodilution with Oxyglobin after step one, and isovolemic hemodilution with Hespan after step two. This study concludes that EATs are observable and PO2 transients relating to EATs can be measured in the rat spinotrapezius muscle. Furthermore, it can be concluded that the HBOC Oxyglobin caused a decrease in erythrocyte-associated capillary PO2 transients, as well as a general decrease in capillary PO2. In addition, this study concludes that erythrocyte-associated capillary PO2 transients can best be observed under control conditions and after step two of isovolemic hemodilution with Hespan.

O arranjo oligomérico e as mudanças na estrutura polipeptídica podem influenciar fortemente na velocidade de auto-oxidação das hemoproteínas, assim como os diferentes nucleófilos que atuam neste processo em função do pH. No presente trabalho, a velocidade de auto-oxidação da hemoglobina extracelular gigante de Glossoscolex paulistus (HbGp) em seu estado íntegro e do monômero d isolado é estudada em função do pH e na presença do ligante cianeto. O comportamento da curva cinética é dependente do pH, apresentando-se monofásico ou bifásico dependendo do estado de oligomerização da proteína. Assim, nos valores de pH que geram a dissociação oligomérica, a curva cinética foi bifásica. Na faixa de pH 5,9-7,3, que apresenta a proteína íntegra, foi verificado um comportamento cinético mono-exponencial. Em meio alcalino, a auto-oxidação da hemoglobina íntegra na presença dos ligantes hidroxila e/ou cianeto apresenta comportamento complexo, descrito pela combinação de duas cinéticas de primeira-ordem. O processo lento ocorre devido à auto-oxidação do monômero d e o processo rápido é atribuído ao trímero abc. Em pH 7,0, a cinética é mono-exponencial, indicando uma estrutura oligomérica altamente preservada. Em meio ácido, tanto a hemoglobina íntegra como o monômero d apresentam a auto-oxidação catalisada por próton. É observada, através das constantes de velocidade de auto-oxidação da hemoglobina íntegra na presença de cianeto, uma cooperatividade no processo rápido devido ao trímero da HbGp. Além disso, a influência do surfactante dodecil sulfato de sódio na auto-oxidação da HbGp é avaliada. Uma influência mais efetiva do SDS é constatada em pH 7,0, enquanto que, em pH 9,0, a dissociação oligomérica é a influência predominante. Provavelmente, esta diferença está relacionada ao ponto isoelétrico ácido da HbGp, que favorece a interação entre SDS e HbGp em pH 7,0, quando comparado ao pH 9,0. O mecanismo de auto-oxidação e as correlações entre a cinética e o arranjo oligomérico são discutidos em detalhes no presente trabalho. / The oligomeric assembly and the changes in the polypeptidic structure can to influence in the autoxidation rate of the heme proteins, as well as the different nucleophiles, which act in this process as a function of pH. In the present work, the autoxidation rate of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in integral state and of the isolated d monomer is studied as a function of pH and in the presence of the cyanide ligand. The kinetic decay behavior is dependent of pH, presenting mono-exponential or biexponential character, depending of the oligomeric state of the protein. Thus, in the pH values that originated the oligomeric dissociation, the kinetic decay was i-exponential. In the pH range 5.9 - 7.3, which show the whole protein, a monoexponential kinetic behavior was verified. In alkaline medium, the whole hemoglobin autoxidation in the presence of the hydroxyl and/or cyanide presents complex behavior described by the combination of two first order kinetics. The slow process occurs due to the d monomer autoxidation and the fast process is attributed to abc trimer. At pH 7.0, the kinetic is monoexponential,indicating a highly conserved oligomeric structure. In acid medium, both, the whole hemoglobin and the d monomer presented the proton-catalyzed autoxidation. A cooperativity in the fast process due to trimer of the HbGp can be observed, through the autoxidation rate constants of the whole hemoglobin in the presence of cyanide. Furthermore, the influence of the anionic surfactant sodium dodecyl sulfate (SDS) in the HbGp autoxidation was evaluated. At pH 7.0, it was verified a more effective influence for SDS, while at pH 9.0, the oligomeric dissociation was the main influence. Probably, this difference is related to the acid isoelectric point of the HbGp, which favors the interaction between SDS and HbGp at pH 7.0, as compared with the pH 9.0. The autoxidation mechanism and the correlation between the kinetic and the oligomeric arrangement are discussed in details in the present work.

There is a disproportionate burden of disease (measured in daily-adjusted life years, or DALYs) in low-income countries. Much of this disparity is due to infectious diseases: 53% of DALYs in Africa are due to infectious diseases, compared with only 3% in the American continents. This disparity is largely due to differences in electrical and transport infrastructure as well as access to skilled personnel and monetary resources. Current diagnostic solutions are primarily designed for high-resource settings and therefore these solutions cannot be easily translated to a lower-resource setting. In order to tackle this health disparity, new solutions must be designed specifically for a lower-resource setting. In this dissertation, we take a translational approach to engineering appropriate diagnostics for resource-limited settings. First, we develop a handheld smartphone accessory to perform an assay similar to enzyme-linked immunosorbent assay (ELISA), traditionally a laboratory-based test. In 15 minutes, it provides an objective diagnostic readout important for minimal training, while using an average of 1.6mW of power and costing only $34. We further develop the device to provide a quantitative hemoglobin measurement simultaneously with an HIV immunoassay, for use in antenatal care screening. The multiplexing two assay types that are clinically relevant has the potential to streamline workflow. While specifications can be demonstrated in the laboratory, the true test of the device must be performed in the field. We brought our smartphone accessory to three health centers in Kigali, Rwanda to be used by healthcare workers with no prior experience in ELISA. After a short 30 minute training, the healthcare workers were able to obtain diagnostic results comparable to other immunoassays run under field conditions. With a simple and user-friendly design, we sought to further expand the usage of our device as a self-testing device, having patients test themselves. Lastly, we explore manufacturable thermoplastics as a material for a microfluidic diagnostic for nucleic acid detection. The sum of this work aims to gain insight into methods of design, testing, and implementation of translational design.

nÃo hà / A diabetes à considerada um fator de risco significativo para a ocorrÃncia de doenÃas periodontais. Entretanto, à necessÃrio que estudos a respeito deste assunto seja realizado em diferentes populaÃÃes, com diferentes caracterÃsticas. O objetivo deste estudo transversal foi avaliar os parÃmetros clÃnicos periodontais de diabÃticos do tipo 2 com diferentes padrÃes de controle glicÃmico. Foram selecionados portadores desta doenÃa, de ambos os gÃneros, residentes na sede do municÃpio de Sobral - CearÃ. Estes deveriam ser nÃo-fumantes, com idade igual ou superior a 40 anos, possuir pelo menos 6 dentes na arcada dentÃria e fazer uso de medicaÃÃo hipoglicemiante. Os indivÃduos foram submetidos ao exame clÃnico periodontal: Ãndice de placa visÃvel (IP), sangramento gengival (IG), sangramento à sondagem (SS), profundidade de sondagem (PS) e recessÃo gengival (RG), realizado por um examinador previamente calibrado. Os indivÃduos foram divididos em trÃs grupos de acordo os nÃveis de hemoglobina glicada â Hb1Ac (Controlados - C: Hb1Ac ≤ 7%, n=103; Descontrolados - D: 7,1% ≤ Hb1Ac ≤ 9%, n= 60; Elevado Descontrole = E: Hb1Ac ≥ 9,1%, n=22). NÃo foram observadas diferenÃas significantes em relaÃÃo Ãs mÃdias de idade em anos, mÃdia de dentes presentes, IP, IG e SS. Verificou-se diferenÃa significante entre os grupos para as mÃdias de Hb1Ac e tempo de diagnÃstico da doenÃa. NÃo foi verificada associaÃÃo estatisticamente significante entre elevados nÃveis glicÃmicos e a maior presenÃa de dentes e de sÃtios periodontais com PS ≥ 6 mm. Entretanto quando foram analisados apenas os indivÃduos que apresentaram 20 ou mais dentes isso foi observado. Assim, pÃde-se concluir que o pobre controle glicÃmico dos diabÃticos do tipo 2, foi associado a maior presenÃa de periodontite apenas nos indivÃduos com elevado nÃmero de dentes. / Diabetes is a significant risk factor for the occurrence of periodontal diseases. Studying the relationship of both diseases in different populations with heterogeneous characteristics are still necessary to better understand them. The aim of this cross-sectional study was to evaluate the clinical periodontal parameters of type 2 diabetes patients with different levels of glycemic control. There were selected type 2 diabetics residing in the urban area of Sobral, Ceara. They must be non-smokers, aging 40 years or more and presenting at least and 6 teeth in their mouth. All had to be using any medication to control the glycemic level. Subjects were assigned to three groups based on their respective glycated hemoglobin levels - Hb1Ac (Control - C: Hb1Ac ≤ 7%, n=103; Moderate control - M: 7,1% ≤ Hb1Ac ≤ 9%, n= 60; Poor control = P: Hb1Ac ≥ 9,1%, n=22). The following clinical data were obtained from all patients: Plaque Index (PI), Gingival Index (GI), bleeding on probing (BOP), probing depth (PD) and gingival recession (GR). The mean age, number of teeth, PI, GI and BOP did not show any significance between groups. But this was observed for Hb1Ac mean levels and time of diabetes diagnosis. The presence of at least one periodontal site with PD ≥ 6 mm was considered for the diagnosis of periodontitis. There was no association between the increase of the glycemic level and the presence of periodontitis. However, data from patients presenting at least 20 teeth showed a significant association between periodontal diseases and higher glycemic levels. It can be concluded that the poor glycemic control was associated to the presence of periodontitis only in subjects with high number of teeth.

A evolução in vitro é um método laboratorial criado para a evolução de moléculas, principalmente de proteínas. Por meio de mutações, o método busca novas propriedades de moléculas, objetivando criar novas proteínas e, com isso, intensificar o estudo e a cura de doenças, pelo desenvolvimento de novos fármacos. O grande desafio na evolução in vitro é criar o maior número possível de moléculas de proteínas que atinjam propriedades desejadas, uma vez que apenas uma fração infinitesimal das diversidades geradas utilizando-se seqüências de DNA é aproveitada. Para se obter moléculas com funcionalidade adequada por meio dessa técnica, é requerido muito tempo e aporte financeiro. Com o objetivo de avaliar computacionalmente a funcionalidade de proteínas variantes a partir das seqüências de aminoácidos buscando reduzir o custo e o tempo desprendido em laboratório, este trabalho propõe o uso de técnicas de computação inteligentes (evolução in silicio), baseadas em aprendizado de máquina e computação evolutiva. Para o emprego de técnicas de AM, bancos de dados com elevado número de informações são fundamentais. Neste sentido, escolheu-se investigar as moléculas mutantes de hemoglobina, uma vez que a quantidade de informações disponíveis sobre a mesma é bastante extensa na literatura. Os resultados obtidos mostram que é possível desenvolver algoritmos eficientes para determinar a funcionalidade de variantes de hemoglobina. Com esses resultados, busca-se contribuir no desenvolvimento de técnicas de evolução dirigida com suporte computacional / In vitro evolution is a laboratorial method developed to molecule evolution mainly proteins. By producing mutations, this method looks for new molecule properties, aiming achieve new proteins for the development of drugs for diseases. The great challenge of in vitro evolution is the development of the highest possible number of molecules that reaches desired properties. This objective is a great challenge to be transposed, since only one infinitesimal fraction of generated proteins using DNA sequencies is usefull to obtain molecules with the desired function. Besides high financial support and time are required to apply this technique. With the objective of evaluating computacionaly and functionality of proteins mutants starting from aminoacids sequences looking for to reduce the cost and the time loosened at laboratory, this work proposes the use of intelligent computation techniques based on learning of it conspires and evolutionary computation. On the other hand, when machine learning techniques are used, it is fundamental to access data mining with high number of information. In order to reduce these difficulties, this work proposes a machine learning (ML) based on approach to evaluate computationaly hemoglobin variants. ML techniques require, in general, large data base. In order to supply this requirement, hemoglobin variants were used because there is a large number of hemoglobin variants available in the literature. The obtained results shown that is possible to develop efficient algorithms to determine hemoglobin variant function. These results can contribute for development of molecule evolution techniques

Abstract Introduction Sickle cell trait carriers may experience sickling events, which can cause severe health problems. Some sickle cell haplotypes contain genetic modifiers that are associated with increased levels of fetal hemoglobin, which is resistant to sickling. The aim of this study is to determine if sickle cell trait individuals who do not carry these modifiers are more likely to experience sickling episodes than those who do carry the modifiers. Methods: Participants were eligible for inclusion in this study if they were male, 18 years of age or older, a sickle cell trait carrier, and had previously played any level of organized football. Participants were recruited via Facebook, www.clinicaltrials.gov, e-mail, phone calls, and word of mouth. They were asked to complete a survey and return a buccal swab for genetic analysis to look for alleles associated with fetal hemoglobin persistence. To date, no genetic analyses have been run. Data from the surveys was analyzed using Fisher's Exact Test with the SAS 9.2 software. Results: Twenty participants were included in this phase of the study and all returned both the survey and buccal swab. Five of the 20 participants had been diagnosed with exertional sickling, 2 with heat illness, and 12 had experienced dehydration. Conclusion: Data in this study is purely observational, as no genetic analyses have been performed at this point. Early results indicate that the probability a player feels their muscle pain lasts longer than their peers' is greater among those who feel it takes their muscles longer to recover than their peers'.