Human alkaline phosphatase (ALP) comprises four isozymes, viz liver/bone/ kidney or tissue unspecific (AP), intestinal (LAP), placental (PLAP) and germ cell or PLAP-like alkaline phosphatase, with their main expression in specific tissues as indicated by their names. The isozymes are coded by different genes, but they are closely related, with more than 50% amino acid sequence homologies. Their biological function is unclear. In certain malignant and benign diseases, serum elevations of one or more of the isozymes occur, which is of diagnostic importance. In this study, the special expression of the intestinal isozyme in human tissues and sera, in normal as well as in pathological conditions, has been investigated by use of isozyme specific monoclonal antibodies. Monoclonal antibodies against the AP, IAP and PLAP isozymes were prepared, and specific assays developed, based on these monoclonal antibodies and the catalytic activity of the isozymes. By use of these assays the basal levels of all three isozymes were examined in selected normal organs. The isozymes were found to be expressed in measurable amounts in all the examined organs. IAP was immunohistochemically localized to the epithelial cells of membranes lining the ducts and tubules of the kidney, liver, pancreas and small intestine. Normal human serum contained all three isozymes. The AP isozyme constituted about 90% of the total ALP activity, the IAP isozyme less than 10% and the PLAP isozyme about 1%. Considerable interindividual variations of the serum IAP activity were observed. The serum activities of the IAP isozyme were related to the individual ABO blood group and secretor status. Non-secretors had low levels of IAP activity amounting to about one tenth of the activity in sera from blood group B or 0 secretors, while blood group A secretors had serum IAP activities in the same order as non-secretors. High individual day to day variations were observed. Fat absorption caused serum IAP to increase significantly for all persons, but it was rapidly cleared from the blood. We found that the release of IAP into the blood was linked to lipid absorption, but removal from the blood was not linked to lipoprotein clearance. Certain tumors of the testis expressed elevated levels of all three ALP isozymes. The highest activitiy of LAP was observed in one yolk sac tumor, in agreement with the endodermal origin of this tumor. In seminoma tissue the AP and PLAP isozymes were significantly, and IAP moderately elevated. Cirrhosis of the liver caused significantly increased serum levels of IAP besides the AP isozyme. In inflammatory diseases of the small intestine, normal serum IAP activities were observed. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1992, härtill 7 uppsatser.</p> / digitalisering@umu

Diss. Linköping : Univ., 2003.

Diss. (sammanfattning) Linköping : Univ., 2004. / Härtill 5 uppsatser.

Orientador: Lilian Maria de Castilho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T19:56:37Z (GMT). No. of bitstreams: 1 Amaral_DaphneRenataTavares_M.pdf: 1431812 bytes, checksum: 82f80c8b6c347220a22a8345ee9be187 (MD5) Previous issue date: 2010 / Resumo: A análise de plasma materno para a determinação do genótipo RHD fetal é uma importante ferramenta no acompanhamento de gestantes RhD-negativo, especialmente em pacientes aloimunizadas. Este trabalho verificou a acurácia da genotipagem RHD fetal pela análise do plasma materno em uma população do Brasil. Foram analisadas 88 gestantes RhD-negativo entre 11 e 39 semanas de gestação, com idade mediana de 28 anos. Treze (14,78%) pacientes encontravam-se aloimunizadas com anti-D. Foram utilizados primers e sondas para detecção do gene RHD (exons 4, 5 e 10) por PCR em tempo real. Como controle interno, utilizou-se um conjunto de primers e sondas para identificar o genes SRY e CCR5. Sangue periférico ou sangue de cordão umbilical dos respectivos neonatos foram coletados durante o parto para a realização da fenotipagem RhD. A genotipagem RHD convencional foi realizada em todas as 88 amostras de DNA materno. Oitenta e três (94,32%) gestantes apresentaram a deleção do gene RHD e em 5 (5,68%) amostras foram identificadas variantes do gene RHD (3 RHD e 2 DFR). A genotipagem RHD convencional foi também realizada em 17 amostras de DNA paternas. Quinze amostras (88,24%) foram genotipadas como RHD+ (5 RHD+/RHD+ e 10 RHD+/RHD-) e 2 (11,76%), como RHD-. Cinqüenta e oito (65,91%) fetos foram genotipados como RHD+. Vinte e sete (30,68%) amostras apresentaram ausência completa do gene RHD e três fetos apresentaram amplificação apenas para o exon 10, demonstrando a presença de uma possível variante RHD ou RHD-CE-Ds. Todos os resultados da genotipagem RHD fetal foram concordantes com a fenotipagem neonatal incluindo os 3 fetos com a variante RHD, fenotipados como RhD-. Nossos resultados indicam que a genotipagem RHD fetal através da análise do plasma materno amplificando 3 regiões do gene RHD (exons 4, 5 e 10) é adequada para a aplicação clínica. Este protocolo pode-se tornar prática em um futuro próximo / Abstract: Maternal plasma analysis for determination of the fetal RHD status is an important tool for the management of RhD-negative pregnant, specially alloimunized women. We assessed the accuracy of fetal RHD genotyping by analysis of maternal plasma in a multi-ethnic population. We analyzed plasma samples from 88 RhD-negative pregnant women between 11 and 39 weeks of gestation, median age of 28 years old to determine the fetal RHD genotype. This population was from Southeastern Brazil with high mixed ethnic background. Thirteen patients (14,78%) had anti-D alloantibody. We used TaqMan primers and probes to detect exons 4, 5 and 10 of RHD, by real-time PCR. As internal controls, we used primers/probes sets to SRY and CCR5. Peripheral or umbilical cord bloods from respective neonates were collected during delivery and hemagglutination was performed. Conventional RHD genotyping was realized in all pregnant. Eighty-three patients had a deletion of RHD gene and five samples were identified RHD variants (3 RHD and 2 DFR). The conventional RHD genotyping was also performed on 17 DNA samples from fathers. Fifteen samples were genotyped as RHD+ (5 RHD+/RHD+ and 10 RHD+/RHD-) and 2 RHD-negative. Fifty-eight (65,91%) fetuses were genotyped as RHD+. Twenty-seven (30,68%) samples showed completely absence of RHD and three fetuses showed amplification only for the exon 10, demonstrating the presence of a possible variant (RHD or RHD-CE-Ds). All fetal RHD results agreed with the neonatal typing including the 3 fetuses with RHD variant, phenotyped as RhD-negative. Thus, the accuracy of the fetal RHD genotyping in this population was 100%. The earliest pregnancy in which fetal RHD was detected was 11 weeks. Our findings indicate that the accuracy of fetal RHD genotyping from maternal plasma using 3 regions (exons 4, 5 and 10) can be sufficient for clinical application in a multi-ethnic population. This knowledge helped us on the development of a feasible protocol for fetal RHD genotyping on DNA from maternal plasma in our population and should become practice in the near future / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

O sistema sanguíneo ABO é o mais importante grupo sanguíneo na medicina transfusional. Atualmente, a determinação do tipo sanguíneo dos doadores de sangue é feita através de testes sorológicos rotineiros de laboratório, porém outros testes realizados com o DNA humano obtido de amostra de sangue, tornam-se complementos valiosos para a determinação correta do grupo sanguíneo do doador e do receptor, aumentando a segurança transfusional. Estudos realizados com o grupo sanguíneo A3 e A3B demonstraram que este subgrupo possui um alto grau de heterogeneidade, uma vez que diversos eventos moleculares foram associados a ele, embora apenas um pequeno número de amostras tenha sido testado até hoje. Nesse trabalho, foi investigada a frequência e os tipos de eventos moleculares do grupo sanguíneo A3 e A3B em um grupo de 100 doadores de sangue. Para seleção desse grupo foram analisadas 12.283 amostras de doadores de sangue saudáveis de ambos os sexos do grupo sanguíneo A e AB obtidas na Fundação Pró-Sangue/Hemocentro de São Paulo, pelos métodos teste em tubo e gel teste. Obtiveram-se 13 amostras A3 e 87 amostras A3B. Após extração e quantificação do DNA genômico das amostras utilizou-se amplificação do DNA pela técnica de reação da polimerase em cadeia alelo específico (PCR-ASP) para as regiões 467C>T, 829G>A, 871G>A e 1060C>A (del C) do exon 7 do gene ABO. Os resultados de amplificação das regiões estudadas apontam para um novo genótipo, aqui denominado A30*/ , presente em 30,7% das amostras A3 e em 58,6% das amostras A3B (A30*/B10*) (necessária a confirmação por sequenciamento); detectou-se também o genótipo A302/A301 em 30,7% das amostras A3 e o genótipo A302/B104 em 17,1% das amostras A3B; outros genótipos já descritos para subgrupo A3 (A301/A301, A302/A302, uma amostra de cada) foram também verificados bem como presença da mutação na região 871G>A em 9 amostras A3 ; verificou-se também que mutações nas regiões 467 C>T e 1060 C>A (del C), anteriormente descritas somente em indivíduos A2 e A2B, são muito frequentes em indivíduos A3 e A3B. / ABO blood system is the most important blood group in transfusional medicine. Actualy, the blood group type in blood donors and receptors is determined by serological laboratorial tests, complemented by human DNA blood tests that assure the correct blood group determination for the blood donor and receptor, optimizing transfusional safety. Studies on blood subgroup A3 e A3B demonstrated a large subgroup heterogenity, with various associated molecular changes in small sample groups tested. At present study, it was proposed investigation about the frequency and the types of molecular alterations occuring among 100 blood donors samples phenotyped as A3 e A3B. These samples are selected after tub and gel tests analysis of 12.283 A and AB samples of healthy blood donors of both sex from Fundação Pró-Sangue/Hemocentro de São Paulo. Thirteen A3 and 87 A3B samples were selected, each sample genomic DNA was extracted and quantified and it was performed DNA amplification by alellic specific polimerase chain reaction (PCR-ASP). It was studied exon 7 ABO gene mutations 467C>T, 829G>A, 871G>A and 1060C>A (del C). Amplification results pointed that a new genotipe is present at these A3 and A3B subgroup, named in this study as the A30*/ genotipe (sequencing confirmation needed); these genotipe is present in 30.7% A3 samples and in 58.6% A3B samples (A30*/B10*). However, it was detected the ulterior decrived genotipes: A302 present in 30.7% A3 samples (A302/A301) and in 17.1% A3B samples (A302/B104); for subgroup A3 it was observed the genotipes A301/A301, A302/A302 (one sample each) and the presence of 871G>A mutation at 9 A3 samples; 467 C>T and 1060 C>A (del C) exon 7 ABO gene mutations descrived at A2 e A2B samples, are very frequents at these A3 e A3B samples tested.

Orientador: Joyce Maria Annichinno-Bizzacchi / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T02:29:39Z (GMT). No. of bitstreams: 1 Mello_TayanaBezerraTeixeira_D.pdf: 2343820 bytes, checksum: be3d8e4b057cd84ec6771c67b8b1407d (MD5) Previous issue date: 2006 / Resumo: Nos últimos anos tem sido demonstrada uma associação entre níveis elevados de certos fatores da coagulação e risco de tromboembolismo venoso (TEV). Entretanto, o número de estudos é pequeno e a maioria estão restritos a populações caucasóides. Neste estudo caso-controle emparelhado por idade, sexo e etnia investigamos os níveis plasmáticos do fibrinogênio, FVII, FVIII, FIX, FX, FXI, FvW e grupo sanguíneo (GS) ABO no risco trombótico. Foram analisados 175 pacientes com TEV (122 mulheres e 53 homens), com idade mediana de 36 anos (13-63), acompanhados no Ambulatório de Hemostasia da Unicamp no período de julho de 2002 a julho de 2005. Na análise univariada, níveis elevados de FVIII (OR: 5,3 IC95% 2,9-9,6), FvW (OR: 4,9 IC95% 2,7-8,9), FIX (OR: 2,4 IC95% 1,3-4,4), FXI (OR: 2,1 IC95% 1,1-4,0) e GS não O (OR: 3,0 IC95% 1,9-4,6) foram fatores de risco para TEV. O FVIII, FvW e GS não O foram associados ao risco tanto em membro inferior como em sítio incomum da doença. A análise de todas estas variáveis, segundo critério de seleção ¿stepwise¿, mostrou o FVIII (OR: 3,1 IC95% 1,6-6,0), FvW (OR:2,8 IC95% 1,4-5,4) e o GS não-O (OR:2,2 IC95% 1,3-3,5) como fatores de risco independentes para TEV e que o FIX e FXI agiram sinergicamente a estas variáveis no acréscimo do risco trombótico. Risco de recorrência foi conferido por elevações do FIX (OR: 4,7 IC95% 1,8-11,9) e FXI (OR: 3,4 IC95% 1,8-8,7). Os determinantes dos níveis plasmáticos do FVIII não estão totalmente esclarecidos. Como a LRP (Low density lipoprotein receptor related protein) tem um papel no catabolismo do FVIII, foram pesquisados os polimorfimos C200T, o A775P e o D2080N no gene codificador dessa proteína, como fator de risco para TEV e a influência dos mesmos sobre os níveis do FVIII e FvW. Não houve diferença nas prevalências destes polimorfismos entre pacientes e controles. No entanto, no grupo-controle, o genótipo DN, do polimorfismo D2080N, foi associado a menores concentrações do FVIII (77,4 UI/dl) e FvW (70,2 UI/dl), quando comparado ao genótipo DD (127 UI/dl e 108,4 UI/dl, respectivamente p<0,05). Em conclusão, nesta população brasileira miscigenada, o FVIII, FvW , FIX, FXI e GS não O foram associados ao risco de TEV e o polimorfismo D2080N, no gene da LRP, interferiu com os níveis plasmáticos de FVIII e FvW / Abstract: During the last years an association between high levels of certain coagulation factors and the risk for venous thromboembolism (VTE) has been demonstrated. The number of studies, however, is small, and most of them are restricted to Caucasian populations. In this case control study, matched for age, sex and ethnicity, we investigated the plasma levels of fibrinogen, FVII, FVIII, FIX, FX, FXI, vWF and the ABO blood group (BG) in the thrombotic risk. It was analyzed 175 patients with VTE (122 women and 53 men), median age 36 years (13-63), followed at the hemostasis outpatient clinic at the State University of Campinas ¿ UNICAMP, between July 2002 and July 2005. In univariate analysis, elevated levels of FVIII (OR: 5.3 95%CI 2.9-9.6), FvW (OR: 4.9 95%CI 2.7-8.9), FIX (OR: 2.4 95%CI 1.3-4.4), FXI (OR: 2.1 95%CI 1.1-4.0) and non O BG (OR: 3.0 95%CI 1.9-4.6) were risk factors for VTE. FVIII, FvW and non O BG were associated with the risk both in lower limbs and unusual sites of disease. Analysis of all these variables by stepwise selection criteria showed FVIII (OR:3.1 95%CI 1.6-6.0), FvW (OR:2.8 95%CI 1.4-5.4) and non O BG (OR:2.2 95%CI 1.3-3.5) as independent risk factors for VTE, and FIX and FXI increased synergistically the risk of thrombosis of these variables. Risk of recurrence was conferred by FIX (OR:4.7 IC95% 1.8-11.9) and FXI (OR:3.4 IC95% 1.8-8.7). The FVIII plasma levels determinants are not well established. Since LRP (Low density lipoprotein receptor related protein) has a role in the catabolism of FVIII, we evaluated the C200T, A775P and, D2080N polymorphisms in the gene coding of this protein, as risk factor for VTE and their influence upon the levels of FVIII and vWF. There was no difference regarding the prevalence of these polymorphisms between patients and controls. However, in the control group, the DN genotype, of the D2080N polymorphism, was associated with lower concentrations of FVIII (77.4 UI/dl) and vWF (70.2 UI/dl), when compared to DD genotype (127 UI/dl e 108.4 UI/dl, respectively p<0.05). In conclusion, in these Brazilian miscigenous population, increased levels of FVIII, FvW, FIX, FXI and non O BG were associated with VTE risk and the D2080N polymorphism, in the LRP gene, influenced plasma levels of FVIII and vWF / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

<p>Uvod: Pojam &quot;sekretor&rdquo; ili &bdquo;nesekretor&quot; se odnosi na sposobnost pojedinca da luči antigene krvnih grupa ABO sistema u telesnim tečnostima. Određivanje ABO krvne grupe i sekretornog statusa, testom inhibicije hemaglutinacije pljuvačke i Lewis fenotipa na eritrocitima su važni u kliničkoj i forenzičkoj medicini, u odnosu na etiopatogenezu mnogih bolesti. Nesekretorstvo ABO krvnogrupne supstance je udruženo sa če&scaron;ćom pojavom autoimunih inflamatornih oboljenja među kojima su i seronegativne spondiloartropatije. Veća učestalost seronegativnih spondiloartropatija među osobama koje imaju HLA-B27 antigen predstavlja polaznu osnovu stanovi&scaron;ta da genetski faktori u kombinaciji sa faktorima sredine utiču na pojavu seronegativnih spondiloartropatija u genetski predisponiranih individua. Teza istražuje značaj testa inhibicije hemaglutinacije plijuvačke i određivanja ABO i Lewis fenotipa na eritrocitim u dijagnostici seronegativnih spondiloartropatija. Ciljevi i hipoteze: Cilj istraživanja je bio da se utvrdi učestalost nesekretora i sekretora ABO krvnogrupne supstance i Lewis fenotipa u grupi obolelih od seronegativnih spondiloartropatija i izvr&scaron;iti poređenje rezultata u odnosu na grupu zdravih ispitanka. Pretpostavljeno je da postoji značajno veći broj nesekretora ABO krvnogrupne supstance u obolelih od seronegativnih spondiloartropatija u odnosu na zdrave ispitanike. Pretpostavljeno je i da postoji značajno veća učestalost nesekretora ABO krvnogrupne supstance u obolelih od seronegativnih spondiloartropatija sa negativnim HLA-B27 antigenom. Pretpostavljeno je da kod osoba obolelih od seronegativnih spondiloartropatija dolazi do promene Lewis fenotipa na eritrocitima u odnosu na sekretorni status u pljuvačci. Metode: Sprovedena je longitudinalna prospektibvna studija. Ispitanici stariji od &scaron;est godina oba pola podeljeni su u dve randomizovane grupe. Eksperimentalnu grupu sačinjavalo je 110 ispitanika sa dijagnozom oboljenja iz grupe seronegativnih spondiloartropatija. Kontrolnu grupu sačinjavalo je 103 dobrovoljna davaoca krvi, bez dijaghnoze oboljenja iz grupe seronegativnih spondiloartropatija. Ispitanicima kontrolne i eksperimentalne grupe određena je pripadnosti ABO krvnogrupnom sistemu, sekretorni status i Lewis fenotip, dok je osobama eksperimentalne grupe određen i fenotip HLA-B27. Uključujući kriterijumi osim navedenog bili su da ispitivane osobe ženskog pola nisu trudinice i da ispitanici obe grupe nisu primali transfuziju krvi tri meseca pre uključivanja u istraživanje. Rezultati: Rezultati istraživanja ukazuju da nesekretori ABO krvnogrupne supstance imaju 1,63 puta veći rizik (veću učestalost), oboljevanja od seronegativnih spondiloartropatija u odnosu na zdravu populaciju ispitanika, kao i da smanjena ekspresija Lewis (b) antigena predstavlja doprinoseći faktor razvoja seronegativnih spondiloartropatija. Ustanovljeno je da pod uticajem seronegativnih spondiloartropatija dolazi do izmene Lewis fenotipa na eritrocitima obolelih. Verovatnoća dokazivanja oboljenja iz grupe seronegativnih spondilartropatija među obolelima veća je za 11% kod nesekretora fenotipa HLA-B27- u odnosu na obolele nesekretore fenotipa HLA-B27+. Zaključak: Sekretorni status i Lewis fenotip predstavljaju zasebne dijagnostičke biohemijske markere nezavisne od HLA-B27 antigena koji doprinose ranijem otkrivanju osoba koje imaju predispoziciju razvoja oboljenja iz grupe seronegativnih spondiloartropatija.</p> / <p>Introduction:The term secretory state referes to ability of individual to secrete ABO blood group antigens in body fluids. Determination of the ABO blood group antigens and secretory status by hemagglutionation inhibition test using saliva as well as Lewis phenotype on erythrocytes are important in clinical and forensic medicine, in relation to the etiopathogenesis of many diseases. Non secretory status of ABO blood groupantegens is related with higher incidence of autoimmune inflammatory disease which include seronegative spondyloarthropathyes. Increased frequency of seronegative spondyloarthropathies among people who have the HLA-B27 antigen is starting point of the view that genetic factors in combination with environmental factors influence the occurence of seronegative spondyloarthropathies in genetically predisposed individuals. The tesis explores the significance of the hemagglutionation inhibition test of saliva and determination of ABO and Lewis antigens in diagnostics of seronegativespondyloarthropathyes. Goals and hypothesis: The aim of this study was to determine the frequency of non secretors and secretors of ABO blood group antigens and Lewis phenotype in a group of patient with seronegative spondyloarthropathies and make comparsion to the healthy examined group. It was assumed that there is a significantly higher number of non secretors of ABO blood group antigens among the patients with seronegative spondyloarthropathies compared to healthy examined persons. It was assumed that there is a significantly higher number of non secretors of ABO blood group antigens among the patients with seronegative spondyloarthropathies who do not have HLA-B27 antigen. It was assumed that in patients with seronegative spondyloarthropathies Lewis phenotype on erythrocits can be changed in relation to the secretory status in the saliva. Methods: We performed a prospective longitudinal study. Respondants older than six years of both gender were divided into two randomized groups. Experimental group is consisted of 110 patients diagnosed with seronegative spondyloarthropathy. The control group consisted of 103 blood donors who did not have diagnosed disease from the group of seronegative spondyloarthropathies. To the subjects of the control and experimental groups was determined ABO blood group antigens, secretory status and Lewis phenotype, while to the subjects of experimental group also was designated HLA-B27 phenotype. Including criteria other than the above were that among female respondants were not pregnant, and that both groups of respondants did not receive a blood transfusion three months before joining the study. Results: The resuts of the study showed that non secretors of ABO blood group antigens have a 1,63 times higher rise (higher incidence) of developing disease from the group of seronegative spondyloarthropathies compared to a healthy population of subjects, as well as that decreased expression of Lewis antigens (b) represents a contributing factor for development of seronegativespondyloarthropathies. It was found that under the influence of seronegative spondyloarthropathies there are changes in Lewis phenotype on erythrocytes of patients. It was found that under the influence of seronegative spondyloarthropathies there are changes in Lewis phenotype on erythrocytes of patients. Probability for confirmation seronegative spondyloarthropathies is higher for 11% among non secretors who have HLA-B27 negative phenotype in comparison to non secretors who have HLA-B27 positive phenotype. Conclusion: Secretory status and Lewis phenotype are separate diagnostic biochemical markers independent of HLA-B27 antigen that contribute to the early detection of people who have a predisposition of the disease from the group of seronegative spondyloarthropathies.</p>

Over a third of the world’s population lives at risk of potentially severe Plasmodium vivax malaria. Unique aspects of this parasite’s biology and interactions with its human host make it harder to control and eliminate than the better studied Plasmodium falciparum parasite. Spatial mapping of two human genetic polymorphisms were developed to support evidence-based targeting of control interventions and therapies. First, to enumerate and map the population at risk of P. vivax infection (PvPAR), the prevalence of this parasite’s human blood cell receptor – the Duffy antigen – was mapped globally. Duffy negative individuals are resistant to infection, and this map provided the means to objectively model the low endemicity of P. vivax across Africa. The Duffy maps helped resolve that only 3% of the global PvPAR was from Africa. The second major research focus was to map the spatial distribution of glucose-6-phosphate dehydrogenase enzyme deficiency (G6PDd), the genetic condition which predisposes individuals to potentially life-threatening haemolysis from primaquine therapy. Despite this drug’s vital role in being the only treatment of relapsing P. vivax parasites, risks of G6PDd-associated haemolysis result in significant under-use of primaquine. G6PDd was found to be widespread, with an estimated frequency of 8.0% (50% CI: 7.4-8.8%) across malarious regions. Third, it was important to represent more detailed descriptions of the genetic diversity underpinning this enzyme disorder, which ranges in phenotype from expressing mild to life-threatening primaquine-induced haemolysis. These variants’ spatial distributions were mapped globally and showed strikingly conspicuous distributions, with widespread A- dominance across Africa, predominance of the Mediterranean variant from the Middle East across to India, and east of India diversifying into a different and diverse array of variants, showing heterogeneity both at regional and community levels. Fourth, the G6PDd prevalence and severity maps were synthesised into a framework assessing the spatial variability of overall risk from G6PDd to primaquine therapy. This found that risks from G6PDd were too widespread and potentially severe to sanction primaquine treatment without prior G6PDd screening, particularly across Asia where the majority of the population are Duffy positive and G6PDd was common and severe. Finally, the conclusions from these studies were discussed and recommendations made for essential further research needed to support current efforts into P. vivax control.

Les travaux présentés dans cette thèse s’intéressent à l’immobilisation de biomolécules pour le développement d’outils d’analyse multiparamétrique pour la caractérisation d’échantillons biologiques et le diagnostic, sur un support de type biopuce couplé à une détection colorimétrique.Un premier axe de recherche concerne le développement de tests d’hybridation d’acides nucléiques et d’immunotests à haut-débit automatisés sur plaque de filtration. Cette méthode a permis la mise au point d’un test de génotypage automatisé pour le dépistage transfusionnel haut-débit (génotypage érythrocytaire étendu) en collaboration avec l’Établissement Français du Sang Rhône-Alpes (EFS-RA). Il permet d’analyser 96 échantillons en quatre heures, et de caractériser six génotypes par échantillon. Cet outil a fait l’objet d’une validation sur un panel de 293 donneurs.La seconde partie des travaux présentés s’intéresse au développement d’un procédé d’immobilisation d’oligonucléotides sur un polymère particulier (PolyshrinkTM) pour l’élaboration d’un système d’analyse miniaturisé. Plusieurs stratégies d’activation ont été envisagées et ont abouti à la mise au point d’une technique d’immobilisation d’oligonucleotides in situ dans des plots d’hydrogel. La méthode de fabrication permet d’obtenir une matrice de plots d’hydrogel de 60 µm de diamètre et d’une hauteur de 6 µm en moyenne. En outre, il a été démontré que les oligonucléotides immobilisés dans les plots pouvaient détecter de façon quantitative et sélective les cibles complémentaires présentes dans l’échantillon analysé en utilisant une détection par colorimétrie ou par chimiluminescence. / The work reported in this thesis focuses on biomolecules immobilization for the development of multiparametric analysis tools on a biochip coupled with a colorimetric detection, applied to the characterization of biological samples and to diagnosis.The first concern was the development of high-throughput automated hybridization tests and immunotests on a filtration plate. This method led to the elaboration of an automated platform for extended blood group genotyping in collaboration with the Etablissement Français du Sang Rhône-Alpes (EFS-RA). It enables to analyze 96 samples in four hours and to characterize six genotypes per sample. Its analytical performances were validated on a panel of 293 blood donors.The second part of this work aimed to elaborate a new strategy for oligonucleotide immobilization on an innovative polymer (PolyshrinkTM) for the development of miniaturized analysis systems. Several approaches were evaluated and led to an in-situ immobilization of oligonucleotides in hydrogel dots technique. This method leads to 6 µm hydrogel dots with a diameter of 60 µm. Moreover it was demonstrated that such immobilized oligonucleotides were able to detect targets specifically and quantitatively using either a chemiluminescent or a colorimetric detection.

INTRODUÇÃO: A determinação do genótipo RHD fetal no plasma materno é um teste de diagnóstico pré-natal não invasivo oferecido a gestantes RhD negativo que apresentam potencial de sensibilização e/ou Doença Hemolítica Perinatal. Atualmente, este exame é realizado de rotina em diversos países, mas não no Brasil. A Clínica Obstétrica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) oferece atendimento terciário a gestantes RhD negativo, com monitorização dos títulos de anticorpos irregulares, administração da imunoglobulina anti-D e/ou terapêutica fetal, quando necessários. OBJETIVO: Avaliar a acurácia do teste semiautomatizado para determinação do genótipo RHD fetal no plasma materno. METODOLOGIA: Foram coletadas prospectivamente amostras de sangue de 220 gestantes RhD negativo, com idade gestacional entre 8-28 semanas. O plasma foi obtido em no máximo 2 horas após a coleta, e uma alíquota de 1 mL foi submetida à extração de ácidos nucléicos no equipamento automatizado MagNA Pure Compact (Roche), empregando o kit Large Volume. O DNA extraído foi submetido a PCR em tempo real (Step One Plus - Applied Biosystems), usando o protocolo do grupo SAFE, que tem como alvos os éxons 5 e 7 do gene RHD. RESULTADOS: Ocorreu exclusão de 35 amostras devido a problemas pré-analíticos, aborto ou desconhecimento do fenótipo do recém-nascido. Entre as 185 amostras analisadas, 130 (70,2%) foram genotipadas como RHD+ e 55 (29,8%) RHD-. Os resultados obtidos foram comparados com a fenotipagem do cordão umbilical, e houve concordância completa (100%). Sete amostras exibiram amplificação exclusiva para o éxon 7. Essas amostras foram submetidas aos protocolos em PCR convencional, e PCR em tempo real específico para o pseudogene RHD. Ambos os ensaios apresentaram os mesmos resultados: cinco positivos e dois negativos. Nesses mesmos 7 casos, após extração da camada de leucócitos materna, os protocolos foram repetidos, e o resultado confirmou que cinco mães eram RHD. As duas amostras com resultado negativo foram submetidas ao protocolo Multiplex, envolvendo os éxons 3-9 do gene RHD, com resultados negativos, confirmando que as mães são verdadeiramente RHD- portanto o sinal do éxon 7 é provindo dos fetos que são D variantes. CONCLUSÃO: O método para a determinação do RHD fetal no plasma materno descrito demonstrou ser rápido, de fácil execução, alta precisão e reprodutível, além de indicar possíveis variantes RHD em nossa população / BACKGROUND: Fetal RHD genotype determination in maternal plasma is a noninvasive prenatal diagnostic test performed in RhD negative pregnant women at risk of alloimmunization and/or Hemolytic Disease of Fetus and Newborn. Currently, this test is routinely performed in many countries but not in Brazil. The Department of Obstetrics at Hospital das Clínicas, São Paulo University Medical School provides tertiary antenatal care for RhD negative pregnant women including anti-D immunoglobulin administration, antibody levels monitoring and intrauterine treatment if necessary. AIMS: To validate the accuracy of a semi-automated test for fetal RHD genotype determination in maternal plasma. METHODS: Two-hundred and twenty blood samples were prospectively collected between 8 and 28 weeks of gestational age. Plasma processing was performed within 2 hours after blood collection, and nucleic acids were extracted from 1mL aliquots with an automated extraction platform (MagNA Pure Compact Roche) and the Large Volume kit. RHD gene exons 5 and 7 were amplified with real-time PCR (Step One Plus - Applied Biosystems) using the SAFE group protocol. RESULTS: Thirty-five samples were excluded due to pre-analytical problems, miscarriage and missing follow-up. In the remaining 185 samples, 130 (70.2%) were genotyped as RhD+ and 55 (29.8%) RhD-. Comparison with umbilical cord blood group phenotype showed 100% concordance. Seven samples showed amplification for exon 7 only. These were further investigated with conventional and real-time PCR with an specific protocol for RHD? pseudogene: 5 were positive and 2, negative. In these 7 cases, maternal buffy-coat DNA analysis also confirmed that 5 women were RHD?. In the remaining 2 cases, a multiplex protocol directed at RHD gene exons 3-9 confirmed that both mothers were truly RhD negative so exon 7 signal comes from the fetuses, further found to harbor D variants. CONCLUSION: The present study demonstrates that fetal RHD determination in maternal plasma is a fast, easy-to-perform and reproducible technique with high accuracy in our population. Moreover, it helps in the identification of possible RHD variants in our population