5 Abstract Aims Determination of gene expression of HBG1 gamma globin in myelodysplastic syndrome (MDS) patients in CD34+ pluripotent hæmatopoietic cells and connection of HBG1 gene expression with various subtypes of MDS. Furthermore, detection of single nucleotide polymorphisms rs 4671393 and rs 11886868 in these patients and in healthy Czech population donors and to determine whether a connection exists between the occurrence of the above polymorphisms and HBG1 gene over-expression, as demonstrated in some hæmatological disorders. Samples The source of genetic material to identify gene expression were 80 HBG1 RNA samples isolated from the pluripotent hematopoietic CD34+ cells of MDS patients. As a sample of healthy controls, 6 samples of commercially purchased CD 34+ cells from the Lonza com- pany were used. The source of genetic material for the detection of polymorphisms were 140 DNA samples isolated from purified granulocytes of MDS patients and 49 samples of DNA isolated from peripheral blood granulocytes from healthy Czech population donors. Methods Real-Time PCR was used to determine HBG1 gene expression and detection of single nu- cleotide polymorphisms. Taqman Gene Expression Assay was used to determine the level of expression and the results were evaluated using the comparative ΔΔCT method....

Analytical methods used clinically in Sweden for detection and quantification of fetal hemoglobin (HbF) in maternal blood are either the microscopic method Kleinhauer Betkes test (KBT) or high performance liquid chromatography. A more modern alternative to detect and quantify HbF+ erythrocytes is flow cytometry. The aim of this project was therefore to evaluate the commercial kit "Fetal Cell Count kit" using flow cytometry. The kit used two antibodies; one directed against the specific γ-chain of HbF protein and the other directed against the intracellular enzyme carbanhydrase (CA), which is found in all erythrocytes in adults. The resulting data showed good precision, sensitivity and linearity. A reference interval based on male blood donors was determined to <0.1 % HbF+ erythrocytes and <1.3 %F-cells. The kit is well suited to detect and quantify F-cells. It could be used as a important tool to follow-up patients withβ-thalassemia and sickle cell anemia. However the kit was not as useful for detection and quantification of HbF+ erytrocytes in fetomaternal hemorrhage induced by Rhimmunization.

Swine wastewater is one of the major pollutions in Taiwan. The abundant nutrition and organic matters in it may lead eutrophication of water body. Meanwhile, the low dissolved oxygen level and high suspended solids concentration may also make it more difficult to handle the wastewater treatment. At present facility of swine wastewater in Taiwan is the three-process treatment which includes the solid-liquid separation, anaerobic digestion, and activated sludge system. Even though the three-process treatment is widely used, the efficiency of sediment and filter effectively to remove the nutrition, e.g., ammonia and phosphate is still in question. On the other hand, the activated sludge system is a difficult technique that the swine farmers can¡¦t easily to operate. Based on these reasons, an efficient swine wastewater treatment process should be established. In this study, we replace the activated sludge system in three-process treatment with the horizontal biofilter (HBF) and subsurface system constructed wetland (SFSCW) to assess the practicability of improving the traditional process. HBF is a fix-biofilm system which is superior to the suspended growth of activated sludge system by its simple operation and no need to return the sludge from the final clarifier. Porosity in the gravel of SFSCW and the root zone of Canna indica can also help to remove the nutrients from the outflow of HBF. The tested HRT (hydraulic retention time) has been controlled at about 30 hours and results show that SS, BOD and COD removal efficiencies as 84.07%, 86.48%, and 68.45%, respectively. HBF and SFSCW provided approximate 70~80% and 10~20% removal efficiencies, respectively. This design of combining HBF and SFSCW system has high potential to treat the swine wastewater, and adding the backwash unit may further facilitate the operation in the future.

名古屋大学博士学位論文 学位の種類:博士(医療技術学) (課程) 学位授与年月日:平成20年9月30日

Le premier objectif de cette thèse était de déterminer les effets isolés et combinés de l'alpha thalassémie, des polymorphismes inducteurs (QTLs) de l'HbF et du génotype G6PD dans un contexte d'évolution naturelle de la drépanocytose (Etudes 1 et 2). L'étude 1 a permis d'évaluer pour la première fois les fréquences alléliques de ces gènes modificateurs chez 301 enfants sénégalais SS. Contrairement aux autres populations africaines, le Variant Betica de la G6PD était majoritaire par rapport au variant A(-). De plus, 12% de notre cohorte avait un déficit en G6PD combiné à une absence d'alpha-thalassémie. Ces patients-là seront à privilégier pour la réalisation d'un Doppler transcrânien. Les résultats obtenus dans l'étude 2 nous ont permis de conclure que l'alpha-thalassémie et les QTLs de l'HbF sont interdépendants et ne doivent pas être étudiés séparément pour une prédiction clinique précise. En effet, une combinaison d'alpha-thalassémie avec au moins 2 QTLs de l'HbF est nécessaire pour retarder de manière significative la première complication de la maladie. Cependant, une alpha-thalassémie homozygote, même associée à 3 à 6 QTLs de l'HbF, augmente la fréquence des CVO pendant l'enfance. Par conséquent, une alpha-thalassémie hétérozygote avec au moins deux QTL HbF constituerait le génotype le plus favorable relativement à la survenue des CVO. Le deuxième objectif de cette thèse était d'étudier les interrelations entre le stress oxydant (phénotype et génotype) et la sévérité clinique de la maladie (Etudes 3 à 4). La drépanocytose est caractérisée par un stress oxydatif élevé pouvant expliquer une partie des manifestations cliniques. Nos résultats ont montré que l'alpha-thalassémie homozygote semble diminuer le stress oxydatif, ce qui contribuerait à son effet protecteur sur certaines complications du sous-phénotype hémolytique. En outre, les patients qui ont le moins d'hospitalisations et de CVO semblent présenter une meilleure défense antioxydante (activités catalase et GPx augmentées). Dans l'étude 4 nous avons étudié 4 SNPs de gènes du stress oxydant (rs4880 du gène SOD2, rs207454 du gène XO, rs233322 du gène MPO et rs35652124 du gène NFE2L2). Le SNP rs4880 aurait un effet favorable au niveau biologique (réticulocytose moindre, activité GPx augmentée) mais sans traduction clinique associée. Il en est de même pour rs233322 qui serait associé à une hémolyse et à un stress oxydatif (AOPP) plus importants. En revanche, une tendance à un effet protecteur de rs207454 vis-à-vis de certaines complications (hospitalisations, ostéonécrose, sepsis, STA) a été observée. Notre travail contribue à la compréhension de l'impact des gènes modificateurs dans la drépanocytose. Il pourrait donc permettre, via une sélection positive des patients à risque, d'améliorer la prise en charge de la maladie dans les pays où les traitements de fond (hydroxyurée, doppler transcrânien, échanges transfusionnels) ne peuvent être proposés à tous / The primary objective of this thesis was to determine the isolated and combined effects of alpha-thalassemia, inductors polymorphisms (QTLs) of HbF and genotype G6PD in a context of natural progression of sickle cell disease (Studies 1 and 2). Study 1 was undertaken to evaluate for the first time the allelic frequencies of these modifiers genes in 301 Senegalese SS children. Unlike other African populations, the G6PD Betica Variant was predominant over the A (-) variant. In addition, 12% of our cohort had G6PD deficiency combined with no alpha-thalassemia. These patients will be favoured for the realization of a transcranial doppler. The results obtained in Study 2 allowed us to conclude that alpha thalassemia and QTLs of HbF are interdependent and should not be studied separately for accurate clinical prediction. Indeed, a combination of alpha thalassemia with at least 2 QTLs of HbF is required to significantly delay the first complication of the disease. However, a homozygous alpha thalassemia, even associated with 3 to 6 QTLs of HbF, increases the frequency of CVOs during childhood. Therefore, a heterozygous alpha-thalassemia with at least two QTL HbFs would be the most favourable genotype for the occurrence of CVOs. The second objective of this thesis was to study the interrelationships between oxidative stress and the clinical severity of the disease (Studies 3 to 4). Sickle cell disease is characterized by high oxidative stress that may explain some of the clinical manifestations. Our results showed that homozygous alpha-thalassemia appears to reduce oxidative stress, which would contribute to its protective effect on certain complications of the hemolytic sub-phenotype. In addition, patients with the least hospitalization and CVO appear to have better antioxidant defense (catalase and GPx activities increased). In Study 4 we studied 4 SNPs of oxidative stress genes (rs4880 of the SOD2 gene, rs207454 of the XO gene, rs233322 of the MPO gene and rs35652124 of the NFE2L2 gene). The rs4880 SNP would have a favourable effect on the biological level (less reticulocytosis, increased GPx activity) but without associated clinical translation. The same is true for rs233322, which is associated with greater haemolysis and oxidative stress (AOPP). On the other hand, a tendency to a protective effect of rs207454 for some complications (hospitalizations, osteonecrosis, sepsis, STA) was observed. Our work contributes to the understanding of the impact of modifiers genes in sickle cell disease. It could therefore, through a positive selection of at-risk patients, improve the management of the disease in countries where the basic treatments (hydroxyurea, transcranial doppler, blood transfusion) cannot be offered to all

Sickle cell disease is a disabling chronic autosomal recessive blood disease characterized by abnormal hemoglobin, pain crises, and frequent emergency department visits. Adherence to hydroxyurea therapy has been shown to improve these patient outcomes. Guided by the theory of comfort, the purpose of this project was to determine if an educational intervention would increase adherence to hydroxyurea therapy in pediatric patients between 2 and 17 years of age recruited from an urban university hospital hematology clinic. The RE-AIM model was used to support the translation of evidence and the change process. An educational video produced by AFLAC was viewed by patients' parents 4 weeks after enrollment into this pretest/posttest design project. A total of 22 African-American parent participants completed the 8-item Morisky Medication Adherence Scale at baseline and again at 8 weeks to assess hydroxyurea adherence. The Short Test of Functional Health Literacy in Adults tool was used to assess parents' health learning needs; all parents met the adequate literacy level at baseline. Using t test statistics, no statistically significant differences were found pretest to posttest on the Morisky Medication Adherence Scale scores, mean corpuscular hemoglobin, and fetal hemoglobin percentages. Wilcoxon Signed Rank tests showed no significant differences in emergency room visits nor number of pain crisis. Although no significant changes emerged in short-term hematologic findings, emergency room visits, and pain crises, social change in the health care setting was promoted by confirming parents were able to understand education and a high level of hydroxyurea adherence was maintained; literature indicated that long-term adherence to hydroxyurea limits severe attacks.

Τα αυξημένα επίπεδα εμβρυϊκής αιμοσφαιρίνης (HbF) μετριάζουν την βαρύτητα των διαταραχών που αφορούν στην β-σφαιρίνη, δηλαδή τη δρεπανοκυτταρική αναιμία (SCD) και την β-μεσογειακή αναιμία, που αποτελούν σημαντικές αιτίες παγκόσμιας νοσηρότητας και θνησιμότητας. Γι’ αυτό το λόγο είναι μακροχρόνιο το ενδιαφέρον για την ανάπτυξη θεραπευτικών προσεγγίσεων για την επαγωγή της παραγωγής HbF. Η αναζήτηση μορίων που ρυθμίζουν την μετάβαση από την έκφραση της εμβρυϊκής (HbF) στην έκφραση της αιμοσφαιρίνης των ενηλίκων (HbA) και που συντελούν στην διατήρηση της αποσιώπησης ή αντίθετα στην ενεργοποίηση της έκφρασης της HbF στους ανθρώπους αποτελεί πολυετές αντικείμενο έρευνας με σκοπό την στόχευση αυτών των παραγόντων για την επαγωγή της HbF (Sankaran et al. 2011). Έτσι, εκτός από τα cis-ρυθμιστικά στοιχεία, έχουν εντοπιστεί και trans-ρυθμιστικά στοιχεία, τα οποία αποτελούν κυρίως μεταγραφικούς παράγοντες. Όμως υπάρχουν και γονιδιακοί τόποι εκτός του β-συμπλέγματος που φαίνεται να επιδούν στην ρύθμιση της έκφρασης των γονιδίων του β-γονιδιακού τόπου. Τέτοιοι είναι οι τόποι που συνδέονται με «ποσοτικά γνωρίσματα» (Quantitative trait loci-QTL). Η χρωμοσωμική περιοχή 6q23 έχει σε διάφορες μελέτες προσδιοριστεί ως QTL, που συνδέεται με την μεταβολή των επιπέδων της HbF σε ασθενείς με SCD. (Close et al. 2004, Thein et al. 2007, Wyszynski et al. 2004). Ο σκοπός της παρούσας μελέτης είναι διττός: A. Ο εντοπισμός μονονουκλεοτιδικών πολυμορφισμών (SNP) εντός των γονιδίων MAP3K5 και PDE7B του QTL στην 6q23 χρωμοσωμική περιοχή, που να σχετίζονται με αυξημένα επίπεδα HbF. B. Η αξιολόγηση των SNP αυτών ως φαρμακογονιδιωματικών δεικτών, που να σχετίζονται με την μεταβλητότητα των επιπέδων της HbF ως απόκριση στη θεραπεία με HU. / Hemoglobinopathies, particularly β-thalassemia and sickle cell disease (SCD), are major health problems, in which quantitative or qualitative defects in hemoglobin production occur, respectively. Under normal circumstances, different types of hemoglobin (Hb) are produced during embryonic, fetal, and adult life. At birth, fetal hemoglobin (HbF), in particular, composes 80–90% of the total hemoglobin synthesized, but it gradually decreases to approximately 1% by 10 months in infancy as its synthesis is restricted to a small subset of erythrocytes termed ‘F cells’ [Patrinos&Grosveld, 2008]. The first studies searching for regulators of HbF expression were conducted on individuals with heterocellular hereditary persistence of HbF (HPFH) – i.e. increase of HbF levels unevenly distributed among ‘F cells’ – and suggested the absence of linkage between the determinant of the HbF levels and the β-globin gene cluster, back then named “non-α globin cluster” [Gianni et al., 1983]. Later, while seeking for genetic elements associated with elevated HbF levels in healthy adults, several cis-acting variants on the β-globin gene complex were unraveled, including the XmnI-Gγ (HBG2) gene promoter polymorphism [Gilman et al., 1985]. Ιn addition, variants unlinked to the β-locus (trans-acting), such as quantitative trait loci (QTLs) on Xp22 [Dover et al., 1992] and 6q23 [Craig et al., 1996] became known soon after. Initially, a study on an extensive, inbred kindred of Asian Indian origin with heterocellular HPFH revealed that a key locus controlling HPFH resides on chromosome 6q, which was fine-mapped to 6q22.3–23.1 [Craig et al., 1996]. Among the first positional candidate genes in the 6q23 region, assumed to possibly explain this QTL, were the MYB proto-oncogene and the eukaryotic release factor-similar HBS1L, as well as the mitogen-activated protein kinase kinase kinase 5 (MAP3K5) [Game et al., 2000]. In addition, genes within this region are associated with response to hydroxyurea (HU) treatment based on elevated HbF levels, in SCD patients; however, the mechanism by which this chromosome 6q22-23 QTL influences HbF levels in the context of HU treatment remains unknown [Ma et al., 2007] and very few, if any, studies have addressed this question. In continuing the global effort of scrutinizing the 6q23 region for variants accounting for the modulation of HbF production, we investigated a possible association of SNPs residing within the MAP3K5 and PDE7B genes with elevated HbF levels in β-thalassemia intermediate or major patients and normal (non-thalassemic) individuals. We also examined a cohort of 38 heterozygous SCD/β-thalassemia patients who had undergone HU therapy, in order to clarify whether there is a correlation of these SNPs with HU treatment response in patients of Hellenic origin.

In the present work, novel dewatering aids and a novel centrifuge configuration were developed and applied for the purpose of dewatering fine particles. Three different types dewatering reagents were tested in different filtration and centrifugation units. These chemicals included low-HLB surfactants, naturally occurring lipids, and modified lipids. Most of these reagents are insoluble in water; therefore, they were used in solutions of appropriate solvents, such as light hydrocarbon oils and short-chain alcohols. The role of these reagents was to increase the hydrophobicity of the coal and selected mineral particles (chalcopyrite, sphalerite, galena, talc, clay, phosphate, PCC and silica) for the dewatering. In the presence of these reagents, the water contact angles on the coal samples were increased up to 90o. According to the Laplace equation, an increase in contact angle with the surfactant addition should decrease the capillary pressure in a filter cake, which should in turn increase the rate of dewatering and help reduce the cake moisture. The use of the novel dewatering aids causes a decrease in the surface tension of water and an increase in the porosity of the cake, both of which also contribute to improved dewatering. A series of batch-scale dewatering tests were conducted on a variety of the coal and mineral samples using the novel dewatering aids. The results obtained with a Buchner funnel and air pressure filters showed that cake moistures could be reduced substantially, the extent of which depends on the particle size, cake thickness, drying time, reagent dosage, conditioning time, reagent type, sample aging, water chemistry, etc. It was determined that use of the novel dewatering aids could reduce the cake formation time by a significant degree due to the increased kinetics of dewatering. At the same time, the use of the dewatering aids reduced the cake moistures by allowing the water trapped in smaller capillaries of the filter cake. It was found that final cake moistures could be reduced by 50% of what can be normally achieved without using the reagents. However, the moisture reduction becomes difficult with increasing cake thickness. This problem can be minimized by applying a mechanical vibration to the cake, spraying a short-chain alcohol on the cake and by adding a small amount of an appropriate coagulant, such as alum and CaCl2 to the coal and mineral slurries. The novel dewatering aids were also tested using several different continuous filters, including a drum filter, disc filter and horizontal belt filter (HBF). The results obtained with these continuous filtration devices were consistent with those obtained from the batch filters. Depending on the coal and mineral samples and the type of the reagent, 40 to 60% reductions in moisture were readily achieved. When using vacuum disc filters, the cake thickness increased substantially in the presence of the novel dewatering aids, which could be attributed to the increased kinetics of dewatering. A dual vacuum system was developed in the present work in order to be able to control the cake thickness, which was necessary to achieve lower cake moistures. It was based on using a lower vacuum pressure during the cake formation time, while a full vacuum pressure was used during the drying cycle time. Thus, use of the dual vacuum system allowed the disc filter to be used in conjunction with the novel dewatering aids. Its performance was similar to that of HBF, which is designed to control cake thickness and cake formation time independently. The effectiveness of using the novel dewatering aids were also tested in a full-continuous pilot plant, in which coal samples were cleaned by a flotation column before the flotation product was subjected to the disc filter. The tests were conducted with and without using novel dewatering aids. These results were consistent with those obtained from the laboratory and batch-scale tests. The novel centrifuge developed in the present work was a unit, which combined a gravity force and air pressure. The new centrifuge was based on increasing the pressure drop across the filter cake formed on the surface of the medium (centrifuge wall). This provision made it possible to take advantage of Darcy s law and improve the removal of capillary water, which should help lower the cake moisture. A series of tests were conducted on several fine coal and mineral particles and obtained more than 50% moisture reduction even at very fine particle size (2 mm x 0). Based on the test results obtained in the present work, two proof-of-concept (POC) plants have been designed. The first was for the recovery of cyclone overflows that are currently being discarded in Virginia, and the other was for the recovery of fines from a pond in southern West Virginia. The former was designed based on the results of the plant tests conducted in the present work. Cost vs. benefit analyses were conducted on the two POC plants. The results showed very favorable internal rates of return when using the novel dewatering aids. Surface chemistry studies were conducted on the coal samples based on the results obtained in the present investigation. These consisted mainly of the surface characterization of the coal samples (surface mineral composition, surface area, zeta potential, x-ray photoelectron microscopy (XPS)), acid-base interactions of the solids and liquids, dewatering kinetic tests, contact angle measurements of the coal samples and surface force measurements using AFM. In addition, carbon coating on a silica plate using palsed laser deposition (PLD) and Langmuir-Blodgett (LB) film deposition tests were conducted on the sample to better understand the surfactant adsorption and dewatering processes. The test results showed that the moisture reductions on the fine particles agree well with the surface chemistry results. / Ph. D.