As cascavéis são classificadas como grupo monofilético contendo dois gêneros descritos ao grupo: Crotalus ssp. e Sistrurus ssp., os quais surgiram no México a aproximadamente 20 milhões de anos, colonizando então, praticamente todo o continente americano. Fatores como dieta, dimorfismo sexual, ontogenia, mutações e distribuição geográfica podem influenciar na composição dos venenos e consequentemente no envenenamento. O presente trabalho tem como objetivo caracterizar o perfil proteico, bem como as propriedades enzimáticas e imunológicas dos venenos de algumas espécies e subespécies de Crotalus ssp. (C. atrox, C. scutulatus scutulatus, C. viridis viridis, C. vegrandis, C. durissus cascavella, C. d. collilineatus e C. d. terrificus). Os resultados indicaram pouca variabilidade entre os perfis eletroforéticos dos venenos, contudo as diferenças foram na concentração relativa das proteínas. A análises proteômica identificou alguns componentes dos venenos e serinopeptidases, metalopeptidases e fosfolipases A2 foram as mais abundantes. Além disso, por zimografia, observou-se que todos os venenos analisados apresentaram atividade proteolítica e que os venenos norte-americanos em todos os zimogramas foram mais hidrolíticos. Em caseína, a atividade enzimática dos venenos foi menos intensa comparado aos outros substratos. Em relação às gelatinases das amostras estudadas, pôde ser observado inibição da atividade enzimática induzida por alguns componentes utilizando EDTA, principalmente nos venenos de C. atrox e C. vegrandis. Em relação à inibição das serinopeptidases, foi observado que todas as gelatinases dos venenos crotálicos apresentaram inibição total ou parcial da atividade hidrolítica. Houve variabilidade entre as hialuronidases encontradas dos venenos crotálicos, tanto em relação à massa das enzimas e intensidade da degradação, quanto em diferentes pHs. Nos ensaios enzimáticos quantitativos (azocaseinolítico fosfolipásico e peptidásico) os venenos Norte Americanos demonstraram conter mais proteases em relação aos venenos Sul Americanos. Por Western Blotting, as amostras reagiram com os anticorpos presentes nos soros anti-crotálico e anti-botrópico, apresentando reatividade antigênica cruzada entre as amostras homólogas e heterólogas. Além disso, houve imunoreatividade entre o soro anti-jararagina e alguns componentes de todos os venenos crotálicos norte-americanos. / The rattlesnakes are classified as a monophyletic group containing two genera referring to the group: Crotalus ssp. and Sistrurus ssp., which arose in Mexico 20 millions of years ago, colonizing then, practically all the American continent. Some scientific works indicate that factors such as diet, sexual dimorphism, ontogeny, mutations and distribution may influence the composition of the venoms and consequently the poisoning. The present work aims to characterize the enzymatic and immunological properties of the venoms of some species and subspecies of Crotalus ssp. (C. atrox, C. scutulatus scutulatus, C. viridis viridis, C. vegrandis, C. durissus cascavella, C. collilineatus and C. d. terrificus). The results indicated few variability among the electrophoretic profiles of the venoms, however the differences were in the relative concentration of the proteins. The proteomic analysis identified serinopeptidases, metallopeptidases and phospholipases A2, which were the most abundant components of the venoms. In addition, zymography assays indicate that the all the venoms showed proteolytic activity, furthermore, the North American venoms, presented more hydrolysis in all zimograms. The caseinolytic activity was less intense compared with other substrates. Regarding the gelatinolytic activity of the samples, inhibition of the enzymatic activity of some components could be observed using EDTA, mainly in the C. atrox and C. vegrandis venoms. Partial or total inhibition was observed of the serinopeptidases activity of the crotalic gelatinases. Among the hyaluronidases, variations between crotalic venoms, in relation to the enzymes mass and degradation intensity were identified. In addition, when incubated at different pHs, the hyaluronidase profile presented different patterns in the activity. In the quantitative enzymatic assays (azocaseinolytic phospholipasic, peptidasic) the North American venoms displayed higher activity in relation to the South American venoms. In the Western Blotting assays, the samples reacted with antibodies present in the Brazilian anti-crotalic and bothropic sera, indicating cross-reactive antigenicity between the homologous and heterologous samples. Besides that, there was immunoreactivity between the anti-jararrhagin serum and some components of all North American crotalic venoms.

As cascavéis são classificadas como grupo monofilético contendo dois gêneros descritos ao grupo: Crotalus ssp. e Sistrurus ssp., os quais surgiram no México a aproximadamente 20 milhões de anos, colonizando então, praticamente todo o continente americano. Fatores como dieta, dimorfismo sexual, ontogenia, mutações e distribuição geográfica podem influenciar na composição dos venenos e consequentemente no envenenamento. O presente trabalho tem como objetivo caracterizar o perfil proteico, bem como as propriedades enzimáticas e imunológicas dos venenos de algumas espécies e subespécies de Crotalus ssp. (C. atrox, C. scutulatus scutulatus, C. viridis viridis, C. vegrandis, C. durissus cascavella, C. d. collilineatus e C. d. terrificus). Os resultados indicaram pouca variabilidade entre os perfis eletroforéticos dos venenos, contudo as diferenças foram na concentração relativa das proteínas. A análises proteômica identificou alguns componentes dos venenos e serinopeptidases, metalopeptidases e fosfolipases A2 foram as mais abundantes. Além disso, por zimografia, observou-se que todos os venenos analisados apresentaram atividade proteolítica e que os venenos norte-americanos em todos os zimogramas foram mais hidrolíticos. Em caseína, a atividade enzimática dos venenos foi menos intensa comparado aos outros substratos. Em relação às gelatinases das amostras estudadas, pôde ser observado inibição da atividade enzimática induzida por alguns componentes utilizando EDTA, principalmente nos venenos de C. atrox e C. vegrandis. Em relação à inibição das serinopeptidases, foi observado que todas as gelatinases dos venenos crotálicos apresentaram inibição total ou parcial da atividade hidrolítica. Houve variabilidade entre as hialuronidases encontradas dos venenos crotálicos, tanto em relação à massa das enzimas e intensidade da degradação, quanto em diferentes pHs. Nos ensaios enzimáticos quantitativos (azocaseinolítico fosfolipásico e peptidásico) os venenos Norte Americanos demonstraram conter mais proteases em relação aos venenos Sul Americanos. Por Western Blotting, as amostras reagiram com os anticorpos presentes nos soros anti-crotálico e anti-botrópico, apresentando reatividade antigênica cruzada entre as amostras homólogas e heterólogas. Além disso, houve imunoreatividade entre o soro anti-jararagina e alguns componentes de todos os venenos crotálicos norte-americanos. / The rattlesnakes are classified as a monophyletic group containing two genera referring to the group: Crotalus ssp. and Sistrurus ssp., which arose in Mexico 20 millions of years ago, colonizing then, practically all the American continent. Some scientific works indicate that factors such as diet, sexual dimorphism, ontogeny, mutations and distribution may influence the composition of the venoms and consequently the poisoning. The present work aims to characterize the enzymatic and immunological properties of the venoms of some species and subspecies of Crotalus ssp. (C. atrox, C. scutulatus scutulatus, C. viridis viridis, C. vegrandis, C. durissus cascavella, C. collilineatus and C. d. terrificus). The results indicated few variability among the electrophoretic profiles of the venoms, however the differences were in the relative concentration of the proteins. The proteomic analysis identified serinopeptidases, metallopeptidases and phospholipases A2, which were the most abundant components of the venoms. In addition, zymography assays indicate that the all the venoms showed proteolytic activity, furthermore, the North American venoms, presented more hydrolysis in all zimograms. The caseinolytic activity was less intense compared with other substrates. Regarding the gelatinolytic activity of the samples, inhibition of the enzymatic activity of some components could be observed using EDTA, mainly in the C. atrox and C. vegrandis venoms. Partial or total inhibition was observed of the serinopeptidases activity of the crotalic gelatinases. Among the hyaluronidases, variations between crotalic venoms, in relation to the enzymes mass and degradation intensity were identified. In addition, when incubated at different pHs, the hyaluronidase profile presented different patterns in the activity. In the quantitative enzymatic assays (azocaseinolytic phospholipasic, peptidasic) the North American venoms displayed higher activity in relation to the South American venoms. In the Western Blotting assays, the samples reacted with antibodies present in the Brazilian anti-crotalic and bothropic sera, indicating cross-reactive antigenicity between the homologous and heterologous samples. Besides that, there was immunoreactivity between the anti-jararrhagin serum and some components of all North American crotalic venoms.

No description available.

A doença de Chagas, uma infecção parasitária amplamente distribuída na América Latina, é um problema grave de saúde pública com consequências devastadoras em termos de morbidade e mortalidade humana. O arsenal terapêutico contra a doença é bastante limitado e insuficiente em todos os aspectos clínicos. Visando o desenvolvimento de novos agentes antichagásicos, várias proteínas do parasita têm sido exploradas como alvos terapêuticos. Neste contexto, a enzima cruzaína, uma cisteíno protease envolvida nos estágios de desenvolvimento e diferenciação do Trypanosoma cruzi, foi selecionada para os nossos estudos, visando a identificação de inibidores através do uso do método de planejamento de fármacos baseado na estrutura do receptor (SBDD, do inglês, structure-based drug design). Esta metodologia engloba uma diversidade de estratégias, empregando estruturas cristalográficas de proteínas alvo, disponíveis usualmente no Protein Data Bank (PDB). Entre as técnicas modernas utilizadas no SBDD, destaca-se a triagem virtual baseada na estrutura do receptor (SBVS, do inglês, structure-based virtual screening), que possibilita a seleção de novos candidatos a ligantes de proteínas alvo, a partir de grandes bases de dados de compostos. No presente trabalho de dissertação, a seleção de 19 estruturas da enzima cruzaína, em complexo com ligantes, permitiu a aplicação de métodos de SBDD. Um conjunto com cerca de 3,4 milhões de compostos, com característica líder-similar (do inglês, lead-like), e outro conjunto com aproximadamente 450.000 compostos, com característica fragmento-similar (do inglês, fragment-like), foram coletados da base de dados ZINC. O programa DOCK 3.5.54 foi empregado na triagem virtual das bases de dados utilizando-se a estrutura cristalográfica PDB ID: 3KKU. Um subconjunto com 35.000 moléculas foi selecionado para estudos posteriores com os programas GOLD e Surflex. As 500 melhores moléculas selecionadas por cada um dos programas foram analisadas visualmente considerando-se diversas características estruturais dos subsítios da enzima cruzaína e dos ligantes (e.g., complementaridade molecular, flexibilidade, lipofilia do subsítio S2, presença de doadores e aceptores de hidrogênio entre os subsítios S2 e S1). Desta forma, um conjunto final de 18 compostos foi priorizado para os ensaios bioquímicos frente a enzima cruzaína. Destes 18 compostos, 6 apresentaram atividade inibitória frente a cruzaína, com destaque para os 2 mais promissores, com valores de IC50 (concentração de inibidor necessária para reduzir em 50% a atividade enzimática) de 20 µM e 580 nM. O inibidor mais potente da série foi selecionado da base fragmento-similar e apresentou um valor de eficiência do ligante (EL) de 0,53 kcal/mol/átomo, considerado significativo para otimização em química medicinal. A integração de técnicas computacionais e experimentais permitiu a descoberta de ligantes com inovação estrutural, abrindo novas perspectivas para o planejamento de inibidores mais potentes e seletivos da enzima cruzaína de T. cruzi. / Chagas disease, a parasitic infection widely distributed in Latin America, is a serious public health problem with devastating consequences in terms of human morbidity and mortality. The therapeutic arsenal against the disease is very limited and insufficient in all clinical aspects. This has led to a new paradigm for the discovery of new agents that act on specific enzymes or metabolic pathways. The enzyme cruzain, a cysteine protease essential for the survival of the parasite Trypanosoma cruzi, has been selected in this work as an attractive target for the development of new inhibitors through the use of structure-based drug design (SBDD). This approach brings together a diversity of strategies, which employs crystal structures of target proteins, usually available in the Protein Data Bank (PDB). Structure-based virtual screening (SBVS), one of the most important techniques used in SBDD, allows the selection of new ligands of target proteins from large libraries of compounds. In this work, 19 crystal structures of the cruzain enzyme, in complex with ligands, allowed the application of SBDD methods. A data set of about 3.4 million compounds, with lead-like characteristics, and a second data set, with approximately 450,000 compounds, with fragment-like characteristics, were collected from the ZINC data base. The docking program DOCK 3.5.54 was employed in the virtual screening of the data sets using the crystal structure PDB ID: 3KKU. A subset of 35,000 compounds was selected for further studies with the programs GOLD and Surflex. The 500 top ranked molecules for each of the programs were visually inspected considering a number of structural characteristics of the subsites of the cruzain enzyme, as well as of the ligands (e.g., molecular complementarity, flexibility, the hydrophobic S2 subsite, and the presence of hydrogen donors and acceptors between the subsites S2 and S1). Thus, a final subset of 18 compounds was prioritized for the biochemical assays against the cruzain enzyme. Six out of 18 compounds exhibited enzyme inhibition, with the most two promising inhibitors having IC50 values (IC50 refers to the concentration of compound required for 50% inhibition of cruzain) of 20 µM e 580 nM. The most potent inhibitor of the series was selected from the fragment-like data set and showed a ligand efficiency of 0,53 kcal/mol/atom, which is considered significant in drug design. The integration of computational and experimental approaches allowed the discovery of compounds with innovative structures, providing new perspectives for the design of inhibitors of T.cruzi cruzain having increased potency and selectivity.

Im Bereich der medizinischen Diagnostik spielen DNA-Chips eine immer wichtigere Rolle. Dabei werden Glas- oder Silikon-Oberflächen mit Tausenden von einzelsträngigen DNA-Fragmenten, sog. Sonden, bestückt, die mit den passenden DNA-Fragmenten in der zugefügten Patientenprobe verschmelzen. Die Auswertung solcher Messungen liefert die Diagnose für Krankheiten wie z.B. Krebs, Alzheimer oder für den Nachweis pathogener Erreger. Durch fortschreitende Miniaturisierung dieser Meßsysteme können bis zu 40.000 Genfragmente des Menschen in einer einzigen Messung analysiert werden. Neben den DNA-Fragmenten können Bio-Chips auch für andere biologische Komponenten wie Antikörper und Proteine eingesetzt werden, wobei bei letzteren neben der Bindung auch die Aktivität ein wichtiger Diagnoseparamter ist. <br /> <br /> Am Fraunhofer-Institut für medizinische Technik und am Lehrstuhl für Analytische Biochemie der Universität Potsdam wurden im Rahmen einer Doktorarbeit Methoden entwickelt, die es ermöglichen auf nukleinsäuremodifizierten Sensoroberflächen die Aktivität von Proteinen zu messen. Es wurden Nukleinsäuren auf Oberflächen optischer Sensoren verankert. Diese fungierten als Rezeptor für die Proteine sowie auch als Substrat für Restriktionsenzyme, die Nukleinsäuren schneiden und Polymerasen, die Nukleinsäuren synthetisieren und verlängern können.<br /> <br /> Seine Anwendung fand diese Messmethode in der Messung der Aktivität des Proteins Telomerase, das in 90% aller Tumore erhöhte Aktivität gegenüber gesunden Zellen aufweist. Die Vorteile dieses neuen Assays gegenüber älteren Methoden liegt im Verzicht auf radioaktiv-markierten Komponenten und einer deutlich verkürzten Analysezeit. Die Arbeit schliesst mit einem funktionsfähigen Nachweis der Telomeraseaktivität im Zellextrakt von gesunden und kranken Zellen. Der direkte Einfluß von Hemmstoffen auf die Aktivität konnte sichtbar gemacht werden, und steht daher bei der Entwicklung neuer Tumor-Diagnostika und Therapeutika zur Verfügung. / In the field of medical diagnostic the importance of DNA chips is growing continuously. On silica surfaces hundreds of single stranded DNA fragments are immobilised which finally detect the complementary sequences in samples of patients by hybridisation. These methods enable the detection of serious diseases as cancer, Alzheimer's disease or infection by pathogens. Biomolecules as nucleic acids, antibodies and proteins can be used as receptors on the solid surfaces whereas in case of proteins not only the binding but also the activity are of high interest for medical diagnosis.<br /> <br /> In this thesis a biosensoric approach has been developed to determine the activity of nucleic-acid modifying enzymes. Optical sensors as, e.g., the grating coupler, were used to monitor the association and dissociation of unlabeled compounds on the sensor surface in real time, by virtue of evanescent-field. Furthermore sensors based on total internal reflection fluorescence measured the activity of restriction enzymes and polymerases. The general approach included the immobilisation of oligonucleotides which acted as the receptor for the enzymes as well as the substrate for the enzymatic reaction. Enzymes as EcoRI and Klenow were used to establish a model system to measure the activity of DNA-modifying enzymes on optical surfaces. As most nucleic acid detection systems use amplification steps such as polymerase chain reaction (PCR) to increase the amount of the probe the new optical systems facilitated the analysis of the enzymatic activity by measuring the DNA-synthesis or restriction directly.<br /> <br /> These systems were finally used to detect the activity of the telomerase, an enzymatic marker for the cancerous development of cells. In 90% of cancer cells the activity of telomerase is higher than in normal cells. Additionally the increase of the telomerase activity in cells induced by carcinogenic substances was detected. Furthermore no purification steps of the samples were required as all measurements were performed with crude cell extract.<br /> <br /> Also the effect of inhibitors of the telomerase could be shown in real time measurements underlining the potential of this assay for further developments of new cancer therapeutics.

A doença de Chagas, uma infecção parasitária amplamente distribuída na América Latina, é um problema grave de saúde pública com consequências devastadoras em termos de morbidade e mortalidade humana. O arsenal terapêutico contra a doença é bastante limitado e insuficiente em todos os aspectos clínicos. Visando o desenvolvimento de novos agentes antichagásicos, várias proteínas do parasita têm sido exploradas como alvos terapêuticos. Neste contexto, a enzima cruzaína, uma cisteíno protease envolvida nos estágios de desenvolvimento e diferenciação do Trypanosoma cruzi, foi selecionada para os nossos estudos, visando a identificação de inibidores através do uso do método de planejamento de fármacos baseado na estrutura do receptor (SBDD, do inglês, structure-based drug design). Esta metodologia engloba uma diversidade de estratégias, empregando estruturas cristalográficas de proteínas alvo, disponíveis usualmente no Protein Data Bank (PDB). Entre as técnicas modernas utilizadas no SBDD, destaca-se a triagem virtual baseada na estrutura do receptor (SBVS, do inglês, structure-based virtual screening), que possibilita a seleção de novos candidatos a ligantes de proteínas alvo, a partir de grandes bases de dados de compostos. No presente trabalho de dissertação, a seleção de 19 estruturas da enzima cruzaína, em complexo com ligantes, permitiu a aplicação de métodos de SBDD. Um conjunto com cerca de 3,4 milhões de compostos, com característica líder-similar (do inglês, lead-like), e outro conjunto com aproximadamente 450.000 compostos, com característica fragmento-similar (do inglês, fragment-like), foram coletados da base de dados ZINC. O programa DOCK 3.5.54 foi empregado na triagem virtual das bases de dados utilizando-se a estrutura cristalográfica PDB ID: 3KKU. Um subconjunto com 35.000 moléculas foi selecionado para estudos posteriores com os programas GOLD e Surflex. As 500 melhores moléculas selecionadas por cada um dos programas foram analisadas visualmente considerando-se diversas características estruturais dos subsítios da enzima cruzaína e dos ligantes (e.g., complementaridade molecular, flexibilidade, lipofilia do subsítio S2, presença de doadores e aceptores de hidrogênio entre os subsítios S2 e S1). Desta forma, um conjunto final de 18 compostos foi priorizado para os ensaios bioquímicos frente a enzima cruzaína. Destes 18 compostos, 6 apresentaram atividade inibitória frente a cruzaína, com destaque para os 2 mais promissores, com valores de IC50 (concentração de inibidor necessária para reduzir em 50% a atividade enzimática) de 20 &micro;M e 580 nM. O inibidor mais potente da série foi selecionado da base fragmento-similar e apresentou um valor de eficiência do ligante (EL) de 0,53 kcal/mol/átomo, considerado significativo para otimização em química medicinal. A integração de técnicas computacionais e experimentais permitiu a descoberta de ligantes com inovação estrutural, abrindo novas perspectivas para o planejamento de inibidores mais potentes e seletivos da enzima cruzaína de T. cruzi. / Chagas disease, a parasitic infection widely distributed in Latin America, is a serious public health problem with devastating consequences in terms of human morbidity and mortality. The therapeutic arsenal against the disease is very limited and insufficient in all clinical aspects. This has led to a new paradigm for the discovery of new agents that act on specific enzymes or metabolic pathways. The enzyme cruzain, a cysteine protease essential for the survival of the parasite Trypanosoma cruzi, has been selected in this work as an attractive target for the development of new inhibitors through the use of structure-based drug design (SBDD). This approach brings together a diversity of strategies, which employs crystal structures of target proteins, usually available in the Protein Data Bank (PDB). Structure-based virtual screening (SBVS), one of the most important techniques used in SBDD, allows the selection of new ligands of target proteins from large libraries of compounds. In this work, 19 crystal structures of the cruzain enzyme, in complex with ligands, allowed the application of SBDD methods. A data set of about 3.4 million compounds, with lead-like characteristics, and a second data set, with approximately 450,000 compounds, with fragment-like characteristics, were collected from the ZINC data base. The docking program DOCK 3.5.54 was employed in the virtual screening of the data sets using the crystal structure PDB ID: 3KKU. A subset of 35,000 compounds was selected for further studies with the programs GOLD and Surflex. The 500 top ranked molecules for each of the programs were visually inspected considering a number of structural characteristics of the subsites of the cruzain enzyme, as well as of the ligands (e.g., molecular complementarity, flexibility, the hydrophobic S2 subsite, and the presence of hydrogen donors and acceptors between the subsites S2 and S1). Thus, a final subset of 18 compounds was prioritized for the biochemical assays against the cruzain enzyme. Six out of 18 compounds exhibited enzyme inhibition, with the most two promising inhibitors having IC50 values (IC50 refers to the concentration of compound required for 50% inhibition of cruzain) of 20 &micro;M e 580 nM. The most potent inhibitor of the series was selected from the fragment-like data set and showed a ligand efficiency of 0,53 kcal/mol/atom, which is considered significant in drug design. The integration of computational and experimental approaches allowed the discovery of compounds with innovative structures, providing new perspectives for the design of inhibitors of T.cruzi cruzain having increased potency and selectivity.

No description available.

Glykerat hemoglobin, HbA1c, är en indikation på genomsnittligt glukosvärde. HbA1c används vid diagnostisering av diabetes men också uppföljning av diagnostiserade diabetiker. Uppföljningen visar hur väl diabetiker förhåller sig till kost men också medicinering. Informationen av patientens HbA1c värde spelar en stor roll i vidare behandlingar. Analysmetoden HbA1c är inte helt standardiserad vilket har medfört att flera analysmetoder utvecklats för HbA1c. Syftet med denna studie var att undersöka om en enzymatisk, Direct enzymatic HbA1c eller immunologisk analysmetod, Hemoglobin A1c kan lösa problemet med hemoglobin-variationer vid analys av HbA1c som idag analyseras med HPLC som rutin på klinisk kemi-laboratorium i Västerås. Genomförandet gjordes på två instrument, TOSOH G7 och AU 680. TOSOH:s värden (HPLC) användes vid jämförelse av de två analysmetoderna på AU680. Förberedelse och behandling, såsom hemolysering, skedde innan proven sattes i instrumentet AU680. Resultatet (n=134) visade att analysmetoden Hemoglobin A1c förhöll sig väl till HPLC analysmetod (R 2=0,98) jämfört med vad analysmetoden Direct enzymatic HbA1c gjorde (R2=0,86). Likartade resultat kunde observeras för Hemoglobin A1c (R2=0,98) och Direct enzymatic Hba1c (R2=0,95) då bara patientprov med hemoglobin-varianter analyserades (n=10). Mann-Whitney’s U-test vid analys av hemoglobin varianter med Hemoglobin A1c visade en tendens till signifikant skillnad gentemot HPLC analysen (p=0,051; n=34). Fel reagens erhölls från reagenstillverkaren gällande Direct enzymatic, Detta kan förklara det erhållna resultatet och kräver fler analyser med korrekta reagens. Hemoglobin A1c bör även undersökas vidare med mer omfattande provmaterial för möjlig standardisering i rutin hos KKTM i Västerås. / Glycated hemoglobin, HbA1c, is an indication of average long-term glucose. HbA1c is used as a diagnostic method for diabetes but also as a follow-up for diagnosed diabetics. Follow-ups shows how well a diabetic relates to diet but also medication. The information of the patient's HbA1c value plays an important factor in further treatments. The analysis method for HbA1c is not standardized, which has resulted in several analysis methods developed for HbA1c. The purposes of this study were to investigate whether an enzymatic, Direct enzymatic HbA1c or immunological assay method, Hemoglobin A1c, can solve the problem of hemoglobin variations in the analysis of HbA1c, which is currently analyzed by HPLC as a routine at the clinical chemistry laboratory in Västerås The implementation was performed on two instruments, TOSOH G7 and AU 680. The values from TOSOH (HPLC) were used for comparison of the two analysis methods applied on the AU680. Preparation and treatment, such as hemolysis, occurred before putting the samples into the AU680 instrument. The result showed that the Hemoglobin A1c assay method was well-matched with HPLC assay (R2 = 0.98) in comparison to that of the Direct enzymatic HbA1c assay method (R2 = 0.86). Similar results could be observed for Hemoglobin A1c (R2 = 0.98) and Direct Enzyme HbA1c (R2 = 0.95) when only samples from patients with hemoglobin variants were analyzed (n=10). Solely analysis of hemoglobin variants with Hemoglobin A1c showed a boundary case for a significant difference compared to HPLC analysis (P = 0.051; n=134). Incorrect reagents were obtained from the reagent manufacturer in the case of Direct enzymatic. This can explain the results obtained. Hemoglobin A1c should also be investigated with more extensive test materials for possible standardization in the routine of KKTM in Västerås.

<p>Истраживање има фокус на хуманим ензимима из суперфамилије алдо-кето редуктаза, које имају велики метаболички значај за хомеостатско функционисање организма. Неки од чланова подфамилије 1С алдо-кето редуктаза (AKR1C) имају улогу у развоју одређених патолошких стања, као што су леукемија, тумори простате,<br />дојке и ендометријума, као и у смањивању ефекта хемотерапија. До сада није регистрован лек који директно утиче на протеине ове групе и самим тим је акценат на изналажењу специфичних лиганада (супстрата, инхибитора), који би могли да имају фармаколошку примену, али и на утврђивању везе између структуре и функције испитиваних лиганада према ензиму. Теза је имала фокус на протеину<br />AKR1C3. У овој дисертацији је представљена оптимизација ензимског есеја и испитивање потенцијалних лиганада и њиховог ефекта на ензимску активност одређених хуманих изоформи протеина из подфамилије AKR1C. Тестирана су синтетисанa стероиднa jeдињења, комерцијална једињења и биљни екстракти. Стероидни лиганди (<strong>AKR-1, -2, -3, -7, -9, -19</strong> и <strong>-22</strong>) који су показали добре инхибиторне карактеристике су детаљније описани одређеним добијеним<br />кинетичким параметрима и затим су кокристализовани са протеином и<br />кофакторм. Од 7 различитих комплекса протеина са најбољиминхибитором, за два комплекса су добијене дифракције са инхибитором и решене кристалне структуре са лигандом у везном месту и врло добром резолуцијом, <strong>AKR-7: 1.7 &Aring;, AKR -19: 1.6 &Aring;.</strong> Ови резултати представљају прве протеинске кристале чију су структуру решили истраживачи из Србије, а у научном смислу и одличну основу за даљи дизајн и тестирање једињења и кокристализације.</p> / <p>Istraživanje ima fokus na humanim enzimima iz superfamilije aldo-keto reduktaza, koje imaju veliki metabolički značaj za homeostatsko funkcionisanje organizma. Neki od članova podfamilije 1S aldo-keto reduktaza (AKR1C) imaju ulogu u razvoju određenih patoloških stanja, kao što su leukemija, tumori prostate,<br />dojke i endometrijuma, kao i u smanjivanju efekta hemoterapija. Do sada nije registrovan lek koji direktno utiče na proteine ove grupe i samim tim je akcenat na iznalaženju specifičnih liganada (supstrata, inhibitora), koji bi mogli da imaju farmakološku primenu, ali i na utvrđivanju veze između strukture i funkcije ispitivanih liganada prema enzimu. Teza je imala fokus na proteinu<br />AKR1C3. U ovoj disertaciji je predstavljena optimizacija enzimskog eseja i ispitivanje potencijalnih liganada i njihovog efekta na enzimsku aktivnost određenih humanih izoformi proteina iz podfamilije AKR1C. Testirana su sintetisana steroidna jedinjenja, komercijalna jedinjenja i biljni ekstrakti. Steroidni ligandi (<strong>AKR-1, -2, -3, -7, -9, -19</strong> i <strong>-22</strong>) koji su pokazali dobre inhibitorne karakteristike su detaljnije opisani određenim dobijenim<br />kinetičkim parametrima i zatim su kokristalizovani sa proteinom i<br />kofaktorm. Od 7 različitih kompleksa proteina sa najboljiminhibitorom, za dva kompleksa su dobijene difrakcije sa inhibitorom i rešene kristalne strukture sa ligandom u veznom mestu i vrlo dobrom rezolucijom, <strong>AKR-7: 1.7 &Aring;, AKR -19: 1.6 &Aring;.</strong> Ovi rezultati predstavljaju prve proteinske kristale čiju su strukturu rešili istraživači iz Srbije, a u naučnom smislu i odličnu osnovu za dalji dizajn i testiranje jedinjenja i kokristalizacije.</p> / <p>This research focuses on human enzymes of the aldo-keto reductase superfamily, whose functions have a significant metabolic impact on organism homeostasis. Some members of the 1C aldo-keto reductase (AKR1C) subfamily play role in the development of specific pathological conditions, such as leukaemia, prostate cancer, breast cancer and endometrial cancer, as well as reducing the effectivness of chemotherapy. However, currently there are no approved and registered drugs that directly affect proteins from this subfamily. Therefore our main aim was to screen for specific ligands (substrates, inhibitors) with potential pharmacological applications, and to establish structure-activity relationships for these ligands and enzymes. This thesis mainly focuses on isoform AKR1C3. In this dissertation, optimization of an enzymatic assay and testing of potential&nbsp; ligands and their effects on the enzymatic<br />activity of specific human isoforms of proteins from subfamily AKR1C are presented. Tested ligands include synthetic steroidal compounds, commercial compounds and plant extracts. Steroid compounds, <strong>AKR-1, -2, -3, -7, -9, -19</strong> and -<strong>22</strong>, were found to be&nbsp;&nbsp; good inhibitors of AKR1C3, and further kinetic studies were conducted. Finally, cocrystalization of protein AKR1C3 with cofactor and these inhibitors was accomplished. From 7 different complexes of protein with inhibitors, two structures were solved to very high resolution, <strong>AKR-7: 1.7 &Aring;, AKR -19: 1.6 &Aring;</strong>. These results represent the first protein crystal structures solved by researchers from Serbia, and&nbsp; results provide an excellent basis for further design and testing of new inhibitors.</p>