Os testes pré-transfusionais são o conjunto de técnicas imuno-hematológicas aplicadas nas amostras do receptor e do doador com a finalidade de fornecer uma unidade de hemácias compatível e segura para a transfusão. Além da fenotipagem ABO e RhD obrigatória nos testes pré-transfusionais, a determinação do perfil antigênico de outros antígenos eritrocitários de importância clínica, melhora a segurança transfusional, especialmente para pacientes previamente aloimunizados ou candidatos à transfusões crônicas. Para tais pacientes, conhecer o fenótipo eritrocitário permite a seleção de concentrados de hemácias com o mesmo fenótipo, o que previne reações transfusionais hemolíticas, assim como nova aloimunização. Indivíduos que apresentam as hemácias sensibilizadas \"in vivo\" por anticorpos anti-eritrocitários, como ocorre na anemia hemolítica autoimune (AHAI), terão o teste direto da antiglobulina (TDA) positivo, dificultando a realização da fenotipagem. O TDA positivo provoca resultados falsos positivos especialmente quando são utilizados para a fenotipagem soros comerciais da classe IgG que exigem a fase de antiglobulina humana, ou teste indireto da antiglobulina (TIA), para leitura. Em 1982 Edwards, Moulds e Judd propuseram a utilização da técnica da cloroquina para dissociar os complexos de antígenoanticorpo preservando a membrana eritrocitária e permitindo a fenotipagem. Atualmente é a técnica mais utilizada nos centros brasileiros. A técnica da glicina-ácida/EDTA foi primeiramente descrita por Louie, Jieng e Zeroulis em 1986, porém é pouco utilizada por não haver os reagentes prontos comercialmente no mercado brasileiro. O presente estudo avaliou a técnica da glicina-ácida-EDTA com reagentes preparados \"in house\" em paralelo à técnica da cloroquina, que é atualmente a técnica aplicada no Hemocentro de Ribeirão Preto. Para isso, amostras de hemácias de doadores (n=50) com fenótipo conhecido foram sensibilizadas com anticorpos humanos de especificidade conhecida e ambas as técnicas foram aplicadas. Foi verificado ainda o efeito da técnica sobre a expressão dos antígenos eritrocitários (Fya, Fyb, Jka, Jkb, S e s) através da fenotipagem das amostras que tiveram o TDA negativado. A viabiliadade dos anticorpos presentes no eluato recuperado após tratamento com a glicina- ácida/EDTA também foi testada. A glicina-ácida/EDTA foi efetiva em negativar o TDA em 33 amostras (66%), comparável com a cloroquina que negativou 28 amostras (56%). O eluato apresentou-se viável no tratamento com a glicina-ácida/EDTA e não foi constatada destruição dos antígenos eritrocitários após o tratamento. Concluímos que a técnica da glicina- ácida/EDTA pode ser utilizada como uma opção no tratamento de hemácias com TDA positivo com a vantagem de seu tempo de execução ser inferior ao da cloroquina (2 minutos x 30 a 120 minutos), o que lhe torna útil em situações de maior urgência. Quando ambas as técnicas são utilizadas em hemácias diferentes de uma mesma amostra a efetividade está significativamente aumentada (p<0,05). / The pre-transfusion tests are the group of immunohematology techniques applied in the receptor and donor samples in order to provide a unit of red blood cells compatible and safe for transfusion. In addition to the obligatory ABO and RhD phenotyping in the pretransfusion tests, the determination of the antigenic profile of other red cells antigens of clinical importance improves transfusion safety, especially for patients previously alloimmunized or candidates for chronic transfusion. For such patients, knowing the phenotype allows the selection of red blood cell concentrates with the same phenotype, which prevents hemolytic transfusion reactions, as well as new alloimmunization. Individuals who have antibody-coated red blood cells sensitized in vivo, such as in autoimmune hemolytic anemia (AHAI), will have a direct antiglobulin test (DAT) positive, making it difficult to perform phenotyping. Positive DAT causes false positive results especially when commercially available IgG serums which require the human antiglobulin phase or indirect antiglobulin test (IAT) for reading are used for phenotyping. In 1982 Edwards, Moul ds and Judd proposed the use of the chloroquine technique to dissociate antigen-antibody complexes by preserving the erythrocyte membrane and allowing phenotyping. It is currently the most used technique in Brazilian centers. The EDTA/glycine-acid technique was first described by Louie, Jieng and Zeroulis in 1986, but it is little used because commercially available reagents are not available in the Brazilian market. The present study evaluated the EDTA/glycine-acid technique with reagents prepared in house in parallel to the chloroquine technique, which is currently the technique applied at the Hemocentro de Ribeirão Preto. For this, donor red cells samples (n = 50) with known phenotype were sensitized with human antibodies of known specificity and both techniques were applied. The effect of the technique on the expression of erythrocyte antigens (Fya, Fyb, Jka, Jkb, S and s) was also verified by the phenotyping of the samples that had negative DAT. The viability of the antibodies present in the recovered eluate after treatment with EDTA/glycine-acid. A was also tested EDTA/glycine-acid was effective in negatively affecting DAT in 33 samples (66%), comparable to chloroquine that negatived 28 samples (56%). The eluate was viable in treatment with EDTA/glycine-acid and no destruction of erythrocyte antigens after treatment. We conclude that the EDTA/ glycine-acid technique can be used as an option in the treatment of red cells with TDA positive, with the advantage that its execution time is inferior to that of chloroquine (2 minutes x 30 to 120 minutes), which makes it useful in emergency situations. When both techniques are used in different erythrocytes of the same sample, the effectiveness is significantly increased (p <0.05).

A mudança anual de penas é um componente crítico do ciclo biológico de todas as espécies de aves e pode modificar as respostas imunológicas individuais, sendo que suas consequências são pouco estudadas. Neste contexto, uma vez que são escassos os estudos acerca do perfil clínico e parâmetros imunológicos de pinguins-de-Magalhães em diferentes fases da muda, este estudo propôs avaliar determinados aspectos relacionados a esses perfis, em animais mantidos em ambiente controlado. Foram estudados 21 indivíduos adultos (dez machos e 11 fêmeas) e os parâmetros avaliados foram: teste de hipersensibilidade cutânea tardia à fitohemaglutinina (FHA) com a avaliação histopatológica de biópsias que incluíram a análise qualitativa e quantitativa dos tipos celulares presentes na reação e no controle às 6, 24 e 48 horas; parâmetros clínicos (massa corpórea e hematologia); fagocitose e burst oxidativo; fenotipagem e resposta linfoproliferativa. O teste de FHA demonstrou diferença significativa na espessura da membrana interdigital de ambas as patas inoculadas com FHA e com o controle (PBS) quando comparadas antes da inoculação e antes da biópsia às 24 e 48 horas. A análise histomorfométrica revelou não haver diferenças significativas na frequência relativa de granulócitos (heterófilos/ eosinófilos), linfócitos, macrófagos ou trombócitos entre as patas inoculadas com FHA e PBS em nenhum dos momentos analisados. Este resultado sugere que a diferença na espessura da membrana interdigital das patas inoculadas seja em decorrência do edema e não do infiltrado inflamatório. Para analisar os efeitos do ciclo da muda nos parâmetros clínicos e hematológicos, os animais foram divididos em três grupos: pré-muda (-5 a 0 dias da muda), muda (10&ordm; ao 21&ordm; dia da muda) e padrão basal (&gt;120 dias da muda). Os resultados mostraram que em relação ao momento basal: houve aumento significativo na massa corpórea dos animais no momento da pré-muda; diminuição significativa de proteínas totais, hematócrito, eritrócitos totais e leucócitos totais na pré-muda e muda. Não houve diferença no número total de monócitos e heterófilos, contudo, houve eosinofilia e linfopenia na pré-muda e muda, respectivamente. Com relação aos parâmetros imunológicos, houve aumento no burst oxidativo gerado pelo estímulo biológico (Staphylococcus aureus) na pré-muda em relação à muda e ao padrão basal; ocorreu aumento do burst oxidativo gerado pelo estímulo químico (éster de forbol) na pré-muda em relação à muda. Em relação à fagocitose, houve diminuição desta capacidade dos leucócitos dos animais na pré-muda e muda em relação ao padrão basal quando o agente foi bacteriano; entretanto, o mesmo não foi observado para a fagocitose de leveduras (Zymosan A); houve diminuição de linfócitos T CD4+ circulantes na pré-muda e muda em relação ao padrão basal e o índice de proliferação de linfócitos aumentou durante o período de muda em relação ao basal. Esses resultados tomados em conjunto sugerem que as mudanças fisiológicas observadas durante o ciclo da muda interferem na modulação de parâmetros imunológicos e são sugestivas de serem consequência do prejuízo energético sofrido, mesmo em animais mantidos em ambientes controlados / The annual replacement of feathers is a critical component of the biological cycle of all bird species. Molt can modify their individual immune response, and the consequences of this are not well known. Within this context, and the few studies on the clinical profile and immunological parameters of Magellanic penguins at different stages of their molt, this study aimed to evaluate certain aspects related to the clinical profiles of animals kept in a controlled environment. A total of 21 adult individuals (ten males and 11 females) was analyzed and the parameters evaluated in this study were: delayed-type hypersensitivity test to phytohemagglutinin (PHA) based on the histopathological evaluation of foot web biopsies that included qualitative and quantitative analysis of cell types present in the essay and control at 6, 24 and 48 hours; clinical parameters (body mass and hematology); phagocytosis and oxidative burst; phenotyping and lymphoproliferative response. The PHA test showed significant differences in the thickness of the webbing of both feet inoculated with PHA and control (PBS), when compared before inoculation and before biopsy at 24 and 48 hours. Histomorphometric analysis revealed no significant differences in the relative frequencies of granulocytes (heterophils/ eosinophils), lymphocytes, macrophages, thrombocytes, or between the feet inoculated with PHA and with PBS in any of the samples. This result suggests that the difference in thickness of the foot webbing is due to edema and not an inflammatory infiltrate. To analyze the effects of the molting cycle on clinical and hematological parameters, the birds were divided into three groups: pre-molt (from -5 days to the start of the molt, day 0), molt (10th to 21st days of molt) and baseline (&gt;120 days after the completion of the molt). The results showed that in comparison with baseline values: a significant increase in body mass occurred during pre-molt; and a significant decrease in total protein, hematocrit, total erythrocytes and total leukocytes occurred during pre-molt and molt. There was no difference in the total numbers of monocytes and heterophils, however, there were eosinophilia and lymphopenia during pre-molt and molt, respectively. There was an increase in the oxidative burst generated by the biological stimulus (Staphylococcus aureus) in the pre-molt compared to molt and baseline levels; and there was an increased oxidative burst generated by the chemical stimuli (phorbol esters) in the pre-molt in relation to the molt. There was a decreased capacity of phagocytosis for the leukocytes in animals during pre-molt and molt compared to baseline when the agent was bacterial; however, this was not observed for the phagocytosis of yeast (Zymosan A). There was a decrease of circulating CD4+ T lymphocytes in the pre-molt and molt compared to baseline, and the lymphocyte proliferation index increased during the molt compared to baseline. The results suggest that the physiological changes observed during the molt cycle does interfere with the modulation of immune parameters, and are suggestive of resulting as a consequence of from the high energy demands of molt, even for birds kept in controlled environments

The rare disease pulmonary arterial hypertension (PAH) results in high blood pressure in the lung caused by narrowing of lung arteries. Genes causative in PAH were discovered through family studies and very often harbour rare variants. However, the genetic cause in heritable (31%) and idiopathic (79%) PAH cases is not yet known but are speculated to be caused by rare variants. Advances in high-throughput sequencing (HTS) technologies made it possible to detect variants in 98% of the human genome. A drop in sequencing costs made it feasible to sequence 10,000 individuals including 1,250 subjects diagnosed with PAH and relatives as part of the NIHR Bioresource - Rare (BR-RD) disease study. This large cohort allows the genome-wide identification of rare variants to discover novel causative genes associated with PAH in a case-control study to advance our understanding of the underlying aetiology. In the first part of my thesis, I establish a phenotype capture system that allows research nurses to record clinical measurements and other patient related information of PAH patients recruited to the NIHR BR-RD study. The implemented extensions provide a programmatic data transfer and an automated data release pipeline for analysis ready data. The second part is dedicated to the discovery of novel disease genes in PAH. I focus on one well characterised PAH disease gene to establish variant filter strategies to enrich for rare disease causing variants. I apply these filter strategies to all known PAH disease genes and describe the phenotypic differences based on clinically relevant values. Genome-wide results from different filter strategies are tested for association with PAH. I describe the findings of the rare variant association tests and provide a detailed interrogation of two novel disease genes. The last part describes the data characteristics of variant information, available non SQL (NoSQL) implementations and evaluates the suitability and scalability of distributed compute frameworks to store and analyse population scale variation data. Based on the evaluation, I implement a variant analysis platform that incrementally merges samples, annotates variants and enables the analysis of 10,000 individuals in minutes. An incremental design for variant merging and annotation has not been described before. Using the framework, I develop a quality score to reduce technical variation and other biases. The result from the rare variant association test is compared with traditional methods.

Field data collection involves time and money consuming processes, additionally carrying possible measurement errors. With the technological advance in the last years, low cost remote sensing tools have emerged to facilitate procedures for in-field measurements, being one of the most known techniques the use of multispectral cameras coupled to RPA. These tools are complemented by the implementation of procedures in GIS and image-processing software, from which are developed methodologies leading to extract target values from a certain original set of data. In this work, multispectral images were used in two case studies: (1) for yield estimation in pea plots for breeding research, and (2) for plant counting in an apple nursery planted directly on the soil; both fields are located in Washington State, USA. In the first case, a reliable and replicable methodology for yield estimation was created as a high throughput phenotyping technique; while in the second case an algorithm capable of identifying the number of apple plants with more than 95% accuracy was developed. In both studies, remote sensing is used as an efficient and practical way to improve field operations under the specified conditions of each case. / A coleta de dados de campo envolve processos de grande consumo em tempo e dinheiro, ademais de levar o risco de possíveis erros de medição. Com o avanço tecnológico nos últimos anos, surgiram ferramentas de sensoriamento remoto de baixo custo para facilitar procedimentos de medição em campo, sendo uma das técnicas mais conhecidas o uso de câmeras multiespectrales acopladas a um ARP. Essas ferramentas são complementadas pela implementação de procedimentos em programas SIG e de processamento de imagens, a partir dos quais são desenvolvidas metodologias que visam extrair valores alvo desde um determinado conjunto original de dados. Neste trabalho, foram utilizadas imagens multiespectrais no desenvolvimento de dois estudos de caso: (1) para estimativa de produtividade em parcelas para pesquisa de ervilha, e (2) para contagem de plantas em um viveiro de maçã plantado diretamente no solo; ambos os campos localizados no estado de Washington, EUA. No primeiro caso, foi criada uma metodologia confiável e replicável para estimativa de produtividade como técnica de fenotipagem de alto rendimento; enquanto no segundo caso, foi desenvolvido um algoritmo capaz de identificar o número de plantas de maçã com mais de 95% de exatidão. Em ambos os estudos, o sensoriamento remoto é usado como uma ferramenta eficiente e prática na melhora de operações de campo.

In a very simplified view, the plant leaf growth can be reduced to two processes, cell division and cell expansion, accompanied by expansion of their surrounding cell walls. The vacuole, as being the largest compartment of the plant cell, plays a major role in controlling the water balance of the plant. This is achieved by regulating the osmotic pressure, through import and export of solutes over the vacuolar membrane (the tonoplast) and by controlling the water channels, the aquaporins. Together with the control of cell wall relaxation, vacuolar osmotic pressure regulation is thought to play an important role in cell expansion, directly by providing cell volume and indirectly by providing ion and pH homestasis for the cytosoplasm. In this thesis the role of tonoplast protein coding genes in cell expansion in the model plant Arabidopsis thaliana is studied and genes which play a putative role in growth are identified. Since there is, to date, no clearly identified protein localization signal for the tonoplast, there is no possibility to perform genome-wide prediction of proteins localized to this compartment. Thus, a series of recent proteomic studies of the tonoplast were used to compile a list of cross-membrane tonoplast protein coding genes (117 genes), and other growth-related genes from notably the growth regulating factor (GRF) and expansin families were included (26 genes). For these genes a platform for high-throughput reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) was developed by selecting specific primer pairs. To this end, a software tool (called QuantPrime, see http://www.quantprime.de) was developed that automatically designs such primers and tests their specificity in silico against whole transcriptomes and genomes, to avoid cross-hybridizations causing unspecific amplification. The RT-qPCR platform was used in an expression study in order to identify candidate growth related genes. Here, a growth-associative spatio-temporal leaf sampling strategy was used, targeting growing regions at high expansion developmental stages and comparing them to samples taken from non-expanding regions or stages of low expansion. Candidate growth related genes were identified after applying a template-based scoring analysis on the expression data, ranking the genes according to their association with leaf expansion. To analyze the functional involvement of these genes in leaf growth on a macroscopic scale, knockout mutants of the candidate growth related genes were screened for growth phenotypes. To this end, a system for non-invasive automated leaf growth phenotyping was established, based on a commercially available image capture and analysis system. A software package was developed for detailed developmental stage annotation of the images captured with the system, and an analysis pipeline was constructed for automated data pre-processing and statistical testing, including modeling and graph generation, for various growth-related phenotypes. Using this system, 24 knockout mutant lines were analyzed, and significant growth phenotypes were found for five different genes. / Sehr vereinfacht gesagt kann Blattwachstum auf zwei Prozesse reduziert werden, Zellteilung und Zellexpansion, gefolgt von Zellwandexpansion. Die Vakuole, das größte Organell der Zelle, übt durch die Kontrolle des Wasserhaushaltes der Pflanze eine wichtige Funktion im Zusammenhang mit der Zellexpansion aus. Dies geschieht durch die Regulierung des osmotischen Druckes, durch Import und Export von organischen und anorganischen Ionen über die Vakuolenmembran (den Tonoplast) und durch die Kontrolle ihrer Wasserkanäle (der Aquaporine). Es wird angenommen, dass die Regulierung des vakuolären osmotischen Druckes eine große Rolle bei der Zellexpansion spielt, da der osmotische Druck die Stärke der mechanischen Kraft des Tonoplast auf die Plasmamembran und die Zellwand bestimmt. In dieser Dissertation wird die Rolle von Tonoplastproteinen und ihrer Gene auf die Zellexpansion anhand der Modellpflanze Arabidopsis thaliana (Ackerschmalwand) untersucht, und Kandidaten für wachstumsrelevante Gene werden identifiziert. Da bisher noch kein Signal für die Lokalisierung von Proteinen im Tonoplast identifiziert wurde, gibt es keine Möglichkeit, genomweite Voraussagen über solche Proteinlokalisierungen zu machen. Daher haben wir eine Reihe von aktuellen Proteom-Studien genutzt, um eine Liste von 117 Genen, die für transmembrane tonoplastproteinkodierende Gene kodieren, zusammenzustellen. Zusätzlich wurden andere wachstumsrelevante Gene und Zellzyklus-Gene in die Liste aufgenommen (38 Gene). Die Expression der Gene während der Blattentwicklung sollte mittels einer sensitiven Technik, der quantitativen Polymerasekettenreaktion (qPCR), untersucht werden. Um rasch die für dieses Verfahren notwendigen Oligonukleotide zu entwerfen, wurde ein Computerprogramm („QuantPrime“) entwickelt. Das Programm entwirft automatisch solche Oligonukleotide und überprüft deren Spezifizität in silico auf Ebene der Transkriptome und Genome um Kreuz-Hybridisierungen zu vermeiden, die zu unspezifischen Amplifikationen führen würden. Die qPCR-Plattform wurde in einer Expressions-Studie eingesetzt, um wachstumsrelevante Gen-Kandidaten zu identifizieren. Um wachstumsaktive und nichtaktive Prozesse vergleichen zu können, wurden Proben von unterschiedlichen Bereichen des Blattes zu unterschiedlichen Wachstumsstadien beprobt. Eine musterbasierte Expressionsdatenanalyse wurde eingesetzt, um die Gene hinischtlich ihrer Assoziation mit der Blattexpansionen in eine Rangordnung zu bringen. Die Gene mit dem höchsten Rang wurden als Kandidaten für weitere Experimente ausgewählt. Um die funktionelle Beteiligung dieser Gene auf einer makroskopischen Ebene zu untersuchen, wurden Knockout-Mutanten für die Gen-Kandidaten hinsichtlich ihres Wachstums analysiert. Zu diesem Zweck wurde ein System für die automatisierte Phänotypisierung des Blattwachstums etabliert. Zum einen wurde ein Programm-Paket für detaillierte Annotation von Wachstumsstadien und zum anderen ein Analyse-Paket für automatisierte Datenvorbereitung und statistische Tests entwickelt. Das Analyse-Paket erlaubt die Modellierung und graphische Darstellung verschiedener wachstumsrelevanter Phänotypen. Mit Hilfe dieses Systems wurden 24 Knockout-Mutanten untersucht und signifikante Phänotypen wurden für fünf verschiedene Gene gefunden.

The zebrafish larva is an ideal candidate for in vivo high-throughput screening: it is a small vertebrate, it is optically transparent, possesses complex organs, and is easy to culture. In addition, genetic mutants and models of human diseases are widely available. Despite these attractive features there are no tools capable of screening at sufficient throughput and resolution to fully exploit the zebrafish. Here, I present a collection of technologies that enable high-throughput studies on zebrafish larvae at cellular resolution. / Engineering and Applied Sciences

Most administered drugs are metabolised in the liver by Phase I enzymes and more importantly by the cytochrome P450 (CYP) system. The extent of first-pass metabolism is important in determining whether the drug will have therapeutic or adverse effects after being administered to a patient. To date the CYP family has been shown to consist of 74 families denoted as CYPl to CYP118, and only a few families are significantly involved in drug metabolism. CYP3A4 is the most important isoenzyme followed by CYP2D6, CYP2C9, and CYP2C19 with a small contribution by CYP2E1, CYP2A6, and CYPlA4. CYP2D6 and CYP3A4 enzyme isoforms have been well established to exhibit interethnic and interindividual variability with regard to drug metabolising capacity. Mutation on the gene coding for a metabolising enzyme is a major cause of variation in drug metabolism. This mutation gives rise to allelic variants producing enzymes with altered metabolising activity. The presence of an allele with decreased metabolic activity in an individual gives rise to the poor metabolising (PM) phenotype. When the PM phenotype occurs at a frequency of more than 1% within a given population, then the term genetic polymorphism applies. The aberrant metabolic capacity translates into variable drug responses of more than 20-fold, leading to different susceptibility to sub-therapeutic effects or adverse drug reactions. A significant number of drugs, such as the B-adrenergic blockers, antidepressants, antipsychotic and antiarrhythmic agents, are entirely or partly metabolised by CYP2D6 and CYP3A4. Genetic polymorphism is especially important for drugs with a narrow therapeutic/toxicity window. Phenotyping involves the use of a probe drug that is administered to the subject, followed by determination of the parent drug and its metabolites in the urine. The aim of this study was to develop and validate an HPLC method for phenotypic determination of the CYP3A4 and CYP2D6 enzymes, followed by the application of the assay in a random heterogeneous population of males. Dextromethorphan (DXM) was used as an in vivo probe for simultaneous determination of the phenotypic expression of CYP2D6 and CYP3A4. An HPLC method coupled with a fluorescence detector was developed for the phenotypic determination of CYP2D6 and CYP3A4 iso-enzymes as determined by the concentration of dextromethorphan/dextrophan (DXM/DX) and dextromethorphan/3methoxy-morphinan (DXM/3MM) metabolic ratios respectively. The compounds were separated on a phenyl column (150 x 4,6 mm, 5-um particle size) serially connected to nitrile column (250 x 4,6 mm, 5-um particle size) using mobile phase of 80% (1.5% glacial acetic acid and 0.1% triethyl amine in distilled water) and 20% acetonitrile. Solid phase extraction was used to extract the analytes from urine samples using silica cartridges. The suitability of the method was demonstrated in a preliminary study with sixteen healthy Caucasian males. After a single oral 30 mg DXM dose, the volunteers were required to collect all urine samples voided 8 hours post oral dose. DXM/3HM and DXM/DX metabolic ratios were determined from collected urine samples. The method was validated for DXM and DX at a concentration range of 0.25 - 30 ug/ml, and at 0.025 - 3 ug/ml for 3MM. Calibration curves were linear with R2 values of at-least 0.999 for all compounds of interest. Recoveries were 97%, 93%, and 65% for DX, DXM and 3MM, respectively. The method was reproducible with intra-day precision having coefficients of variation percentage (CV%) of less than 17% for all analytes. Inter-day precision had a CV% of less than 14% for all analytes. The limit of detection was 30 ug/ml for all compounds. All volunteers were classified with an extensive metaboliser (EM) phenotype. In conclusion the method described is suitable for polymorphic determination of CYP2D6 and CYP3A4 in a population study, and may have value in further studies planned at investigating the critical issue of racial genetic polymorphism in ethnic groups in South Africa. / Thesis (M.Sc. (Pharm.))--North-West University, Potchefstroom Campus, 2004.

No description available.

Most administered drugs are metabolised in the liver by Phase I enzymes and more importantly by the cytochrome P450 (CYP) system. The extent of first-pass metabolism is important in determining whether the drug will have therapeutic or adverse effects after being administered to a patient. To date the CYP family has been shown to consist of 74 families denoted as CYPl to CYP118, and only a few families are significantly involved in drug metabolism. CYP3A4 is the most important isoenzyme followed by CYP2D6, CYP2C9, and CYP2C19 with a small contribution by CYP2E1, CYP2A6, and CYPlA4. CYP2D6 and CYP3A4 enzyme isoforms have been well established to exhibit interethnic and interindividual variability with regard to drug metabolising capacity. Mutation on the gene coding for a metabolising enzyme is a major cause of variation in drug metabolism. This mutation gives rise to allelic variants producing enzymes with altered metabolising activity. The presence of an allele with decreased metabolic activity in an individual gives rise to the poor metabolising (PM) phenotype. When the PM phenotype occurs at a frequency of more than 1% within a given population, then the term genetic polymorphism applies. The aberrant metabolic capacity translates into variable drug responses of more than 20-fold, leading to different susceptibility to sub-therapeutic effects or adverse drug reactions. A significant number of drugs, such as the B-adrenergic blockers, antidepressants, antipsychotic and antiarrhythmic agents, are entirely or partly metabolised by CYP2D6 and CYP3A4. Genetic polymorphism is especially important for drugs with a narrow therapeutic/toxicity window. Phenotyping involves the use of a probe drug that is administered to the subject, followed by determination of the parent drug and its metabolites in the urine. The aim of this study was to develop and validate an HPLC method for phenotypic determination of the CYP3A4 and CYP2D6 enzymes, followed by the application of the assay in a random heterogeneous population of males. Dextromethorphan (DXM) was used as an in vivo probe for simultaneous determination of the phenotypic expression of CYP2D6 and CYP3A4. An HPLC method coupled with a fluorescence detector was developed for the phenotypic determination of CYP2D6 and CYP3A4 iso-enzymes as determined by the concentration of dextromethorphan/dextrophan (DXM/DX) and dextromethorphan/3methoxy-morphinan (DXM/3MM) metabolic ratios respectively. The compounds were separated on a phenyl column (150 x 4,6 mm, 5-um particle size) serially connected to nitrile column (250 x 4,6 mm, 5-um particle size) using mobile phase of 80% (1.5% glacial acetic acid and 0.1% triethyl amine in distilled water) and 20% acetonitrile. Solid phase extraction was used to extract the analytes from urine samples using silica cartridges. The suitability of the method was demonstrated in a preliminary study with sixteen healthy Caucasian males. After a single oral 30 mg DXM dose, the volunteers were required to collect all urine samples voided 8 hours post oral dose. DXM/3HM and DXM/DX metabolic ratios were determined from collected urine samples. The method was validated for DXM and DX at a concentration range of 0.25 - 30 ug/ml, and at 0.025 - 3 ug/ml for 3MM. Calibration curves were linear with R2 values of at-least 0.999 for all compounds of interest. Recoveries were 97%, 93%, and 65% for DX, DXM and 3MM, respectively. The method was reproducible with intra-day precision having coefficients of variation percentage (CV%) of less than 17% for all analytes. Inter-day precision had a CV% of less than 14% for all analytes. The limit of detection was 30 ug/ml for all compounds. All volunteers were classified with an extensive metaboliser (EM) phenotype. In conclusion the method described is suitable for polymorphic determination of CYP2D6 and CYP3A4 in a population study, and may have value in further studies planned at investigating the critical issue of racial genetic polymorphism in ethnic groups in South Africa. / Thesis (M.Sc. (Pharm.))--North-West University, Potchefstroom Campus, 2004.

Nors yra žinoma, kad pacientams, kuriems neseniai atliktos eritrocitų transfuzijos, kraujo grupių nustatymo hemagliutinacijos reakcija rezultatai gali būti nepatikimi dėl kraujyje cirkuliuojančių donoro ertrocitų, tačiau literatūroje nėra aiškių rekomendacijų, kuriais atvejais reikėtų naudoti molekulinius tyrimo metodus. Serologinis Duffy ir Kidd sistemų antigenų ir antikūnų prieš juos nustatymas – viena svarbiausių imunohematologinių problemų transfuzinėje medicinoje. Šiame darbe pirmą kartą Lietuvoje atlikti moksliniai tyrimai imunohematologijos srityje ir panaudoti genetiniai Duffy ir Kidd antigenų sistemų tyrimo metodai. Siekiant įvertinti Duffy ir Kidd antigenų sistemų fenotipavimo ir genotipavimo reikšmę, mes nagrinėjome klinikinių, demografinių, imunohematologinių bei su transfuzija susijusių veiksnių įtaką tyrimų rezultatams. Mūsų tyrimo metu nustatytas didesnis nei 30 proc. nesutapimų dažnis tarp Duffy ir Kidd antigenų sistemų fenotipavimo ir genotipavimo rezultatų patvirtino genetinių tyrimų naudą, atliekant dažnas eritrocitų transfuzijas. Atliekant šį darbą, pirmą kartą Lietuvoje nustatytas Duffy ir Kidd antigenų sistemų fenotipų paplitimas. Nustatytas laiko tarpas, per kurį įprastai klinikinėje praktikoje naudojamų serologinių tyrimų (hemagliutinacijos reakcijos) rezultatai gali būti patikimi, bei pateiktos atitinkamos rekomendacijos didina šio tyrimo praktinę vertę ir yra novatoriška šiuolaikinėje transfuzinėje medicinoje. / Accurate phenotyping of multitransfused patients is often complicated - mostly due to the presence of circulating transfused donor’s RBCs in the recipient’s blood, leading to discrepancies in the assessment of test results. The question when genotyping including Duffy and Kidd systems should be used for patients undergoing chronic RBC transfusions is still being discussed. Serological testing and evaluation of the antigens and antibodies of Duffy and Kidd systems are among the main problems in multitransfused patients. The research on immunohematology and blood group genetics has been caried out for the first time in Lithuania. A high rate (more than 30%) of disagreements between the results of phenotyping and genotyping in our study demonstrates the benefit of DNA-based testing for chronically-transfused patients. In order to estimate the value of phenotyping and genotyping of Duffy and Kidd antigen systems in patients undergoing long-term RBC transfusions, the impact of demographic, clinical, immunohaematological or transfusion-related factors, on the discrepancy of the results, was investigated. Time frame that could be appropriate to obtain reliable results of conventionaly used serologic tests (hemmagglutination reaction) after the last transfusion was established as well as appropriate recommendations were made. We believe that these studies could be helpful for clinical practice as well as in decreasing the risk of transfusion of red blood cells.