Plasmodium falciparum e Toxoplasma gondii são protozoários parasitas pertencentes ao filo Apicomplexa. Apirases são enzimas metabolizadoras de nucleotídeos extracelulares. Nesta tese mostramos pela primeira vez a presença de um membro desta família de enzimas em P. falciparum, o qual foi capaz de degradar ATP extracelular. Análises por RT-qPCR revelaram a expressão da apirase durante todo o ciclo intraeritrocítico. A adição de inibidores desta classe de enzimas foi capaz de prejudicar o desenvolvimento dos parasitas e a invasão de novas hemácias pelos merozoitos, sugerindo assim um papel da apirase nestes processos. A via de sinalização por Ca2+ é universal e vital para todas as células. Para melhor entender a fisiologia celular de P. falciparum construímos uma nova linhagem de parasitas transgênicos, PfGCaMP3, que nos tornam capazes de monitorar a dinâmica de Ca2+ sem o uso de protocolos invasivos de marcação. De modo semelhante utilizamos uma nova linhagem de T. gondii expressando de forma estável o indicador de Ca2+ GCaMP3 para estudar o papel deste íon na saída da célula. T. gondii possui o Ca2+ necessário para promover este processo, entretanto Ca2+ extracelular age como um fator intensificador neste passo essencial do ciclo lítico. / Plasmodium falciparum and Toxoplasma gondii are protozoan parasites that belong to phylum Apicomplexa. Apirases are metabolizing enzymes of extracellular nucleotides. In this work we show for the first time the presence of an apyrase in P. falciparum, which was able to degrade extracellular ATP. RTqPCR analysis revealed the expression of apyrase throughout the intraerythrocytic cycle. Addition of apyrase inhibitors was able to impair the development of the parasites and the invasion of new erythrocytes by merozoites, thus suggesting a role of apyrase in these processes. Calcium signaling is universal and vital to all cells. To better understand the cellular physiology of P. falciparum we construct a new strain of transgenic parasites, PfGCaMP3, which enable us to monitor the Ca2+ dynamics without using invasive protocols. Similarly we use a new strain of T. gondii that stably express the Ca2+ indicator GCaMP3 to study the role Ca2+ in parasite egress. T. gondii has the Ca2+ required to promote this process, however extracellular Ca2+ acts as an enhancer factor in this crucial step of the lytic cycle.

Detecting mutations and genomic variations is fundamental indiagnosis, isolating disease genes, association studies,functional genomics and pharmacogenomics. The objective hasbeen to use and further develop a variety of tools andtechnologies to analyze these genetic alterations andvariations. The p53 tumor suppressor gene and short arm of chromosome 9have been used as genetic markers to investigate fundamentalquestions concerning early events preceding non-melanoma skincancers, clonal progression and timing of different mutationsand deletions. Conventional gel based DNA sequencing andfragment analysis of microsatellite markers were utilized forthis purpose. In addition, a sequence-specific PCR-mediatedartifact is discussed. Pyrosequencing, a bioluminometric technique based onsequencing-by-synthesis, has been utilized to determinemutation ratios in the p53 gene. In addition, in the case ofmultiple mutations, pyrosequencing was adopted to determineallelic distribution of mutations without the use of cloningprocedures. Exons 5 to 8 of the p53 gene were also sequenced bythis method. The possibility of typing single base variations bypyrosequencing has been evaluated. Two different nucleotidedispensation orders were investigated and data were comparedwith the predicted pattern for each alternative of the variableposition. Analysis of loss of heterozygosity was possible byutilizing single nucleotide polymorphisms. A modified allele-specific extension strategy for genotypingof single nucleotide polymorphisms has been developed. Throughthe use of a real-time bioluminometric assay, it has beendemonstrated that reaction kinetics for a mismatchedprimer-template is slower than the matched configuration,butthe end-point signals are comparable. By introduction ofapyrase, the problems associated with mismatch extensions havebeen circumvented and accurate data has been obtained. Keywords:fragment analysis, microsatellite, loss ofheterozygosity, DNA sequencing, pyrosequencing, cancer,mutation, variation, single nucleotide polymorphism,allele-specific extension, bioluminescence, apyrase. / QC 20100415

<p>In this thesis techniques are presented that aim to determine individual DNA signatures by controlled synthesis of nucleic acid multimers. Allele-specific extension reactions with an improved specificity were applied for several genomic purposes. Since DNA polymerases extend some mismatched 3’-end primers, an improved specificity is a concern. This has been possible by exploiting the faster extension of matched primers and applying the enzymes apyrase or Proteinase K. The findings were applied to methods for resequencing and viral and single nucleotide polymorphism (SNP) genotyping.</p><p>P53 mutation is the most frequent event in human cancers. Here, a model system for resequencing of 15 bps in p53 based on apyrase-mediated allele-specific extension (AMASE) is described, investigated and evaluated (Paper I). A microarray format with fluorescence detection was used. On each array, four oligonucleotides were printed for each base to resequence. Target PCR products were hybridized and an AMASE-reaction performed in situ to distinguish which of the printed oligonucleotides matched the target. The results showed that without the inclusion of apyrase, the resulting sequence was unreadable. The results open the possibilities for developing large-scale resequencing tools.</p><p>The presence of certain types of human papillomaviruses (HPV) transforms normal cells into cervical cancer cells. Thus, HPV type determination is clinically important. Also, multiple HPV infections are common but difficult to distinguish. Therefore, a genotyping platform based on competitive hybridization and AMASE is described, used on clinical sample material and evaluated by comparison to Sanger DNA sequencing (Papers II and III). A flexible tag-microarray was used for detection and the two levels of discrimination gave a high level of specificity. Easy identification of multiple infections was possible which provides new opportunities to investigate the importance of multiply infected samples.</p><p>To achieve highly multiplexed allele-specific extension reactions, large numbers of primers will be employed and lead to spurious hybridizations. Papers IV to VI focus on an alternative approach to control oligomerization by using protease mediated allele-specific extension (PrASE). In order to maintain stringency at higher temperatures, Proteinase K, was used instead of apyrase, leading to DNA polymerase degradation and preventing unspecific extensions. An automated assay with tag-array detection for SNP genotyping was established. First PrASE was introduced and characterized (Paper IV), then used for genotyping of 10 SNPs in 442 samples (Paper V). A 99.8 % concordance to pyrosequencing was found. PrASE is a flexible tool for association studies and the results indicate an improved assay conversion rate as compared to plain allele-specific extension.</p><p>The highly polymorphic melanocortin-1 receptor gene (MC1R) is involved in melanogenesis. Twenty-one MC1R variants were genotyped with PrASE since variants in the gene have been associated to an increased risk of developing melanoma. A pilot study was performed to establish the assay (Paper VI) and subsequently a larger study was executed to investigate allele frequencies in the Swedish population (Paper VII). The case and control groups consisted of 1001 and 721 samples respectively. A two to sevenfold increased risk of developing melanoma was observed for carriers of variants.</p>

In this thesis techniques are presented that aim to determine individual DNA signatures by controlled synthesis of nucleic acid multimers. Allele-specific extension reactions with an improved specificity were applied for several genomic purposes. Since DNA polymerases extend some mismatched 3’-end primers, an improved specificity is a concern. This has been possible by exploiting the faster extension of matched primers and applying the enzymes apyrase or Proteinase K. The findings were applied to methods for resequencing and viral and single nucleotide polymorphism (SNP) genotyping. P53 mutation is the most frequent event in human cancers. Here, a model system for resequencing of 15 bps in p53 based on apyrase-mediated allele-specific extension (AMASE) is described, investigated and evaluated (Paper I). A microarray format with fluorescence detection was used. On each array, four oligonucleotides were printed for each base to resequence. Target PCR products were hybridized and an AMASE-reaction performed in situ to distinguish which of the printed oligonucleotides matched the target. The results showed that without the inclusion of apyrase, the resulting sequence was unreadable. The results open the possibilities for developing large-scale resequencing tools. The presence of certain types of human papillomaviruses (HPV) transforms normal cells into cervical cancer cells. Thus, HPV type determination is clinically important. Also, multiple HPV infections are common but difficult to distinguish. Therefore, a genotyping platform based on competitive hybridization and AMASE is described, used on clinical sample material and evaluated by comparison to Sanger DNA sequencing (Papers II and III). A flexible tag-microarray was used for detection and the two levels of discrimination gave a high level of specificity. Easy identification of multiple infections was possible which provides new opportunities to investigate the importance of multiply infected samples. To achieve highly multiplexed allele-specific extension reactions, large numbers of primers will be employed and lead to spurious hybridizations. Papers IV to VI focus on an alternative approach to control oligomerization by using protease mediated allele-specific extension (PrASE). In order to maintain stringency at higher temperatures, Proteinase K, was used instead of apyrase, leading to DNA polymerase degradation and preventing unspecific extensions. An automated assay with tag-array detection for SNP genotyping was established. First PrASE was introduced and characterized (Paper IV), then used for genotyping of 10 SNPs in 442 samples (Paper V). A 99.8 % concordance to pyrosequencing was found. PrASE is a flexible tool for association studies and the results indicate an improved assay conversion rate as compared to plain allele-specific extension. The highly polymorphic melanocortin-1 receptor gene (MC1R) is involved in melanogenesis. Twenty-one MC1R variants were genotyped with PrASE since variants in the gene have been associated to an increased risk of developing melanoma. A pilot study was performed to establish the assay (Paper VI) and subsequently a larger study was executed to investigate allele frequencies in the Swedish population (Paper VII). The case and control groups consisted of 1001 and 721 samples respectively. A two to sevenfold increased risk of developing melanoma was observed for carriers of variants. / QC 20101028

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-08-17T12:32:21Z No. of bitstreams: 1 micheliaantoniadonascimentogusmao.pdf: 2641235 bytes, checksum: 154c338e19fb47129f176bdceb0cf92e (MD5) / Rejected by Adriana Oliveira (adriana.oliveira@ufjf.edu.br), reason: on 2017-08-24T11:26:04Z (GMT) / Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-08-24T15:14:14Z No. of bitstreams: 0 / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-30T14:21:34Z (GMT) No. of bitstreams: 0 / Made available in DSpace on 2017-08-30T14:21:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-02-17 / A esquistossomose é uma doença tropical negligenciada que acomete milhões de pessoas no mundo. A falta de vacina para essa doença helmíntica impulsiona a busca por moléculas antigênicas oriundas dos parasitos do gênero Schistosoma. Dentre as moléculas consideradas como promissoras, se encontra a ATP difosfohidrolase de Schistosoma mansoni. O parasito possui duas isoformas de desta enzima que são denominadas SmATPDases e são expressas em todos as fases do ciclo de vida deste helminto. A apirase de batata é uma proteína vegetal pertencente à mesma família de enzimas do parasito, compartilha epitopos com as isoformas de SmATPDases. Dessa forma, o objetivo deste trabalho foi realizar a caracterização imunológica da apirase de batata em modelo de infecção murina experimental com base nessa homologia protéica. A proteína vegetal foi inoculada em camundongos C57BL/6 que posteriormente foram infectados com cercárias de S. mansoni. A proteína vegetal induziu níveis significativos de anticorpos IgG e IgG1 após a imunização, os quais se mantiveram elevados na análise 60 dias após a infecção. A cultura de esplenócitos dos animais imunizados respondeu ao estímulo com a proteína vegetal, induzindo a produção de níveis significativos de IFN-γ, IL-10 e IL-5, mas não de IL-13. O processo de imunização não conferiu proteção, sendo incapaz de promover a redução da carga parasitária. O efeito imunomodulador da imunização foi observado através de uma redução da área afetada pela reação granulomatosa hepática nos animais imunizados. Em relação à ligação de anticorpos IgE, a apirase de batata mostrou reatividade significativa com estes anticorpos presentes em amostras do plasma de pacientes com esquistossomose. Em ensaios de Western blotting, os anticorpos IgE foram capazes de reconhecer três proteínas presentes no homogeneizado de vermes adultos do helminto, com 91, 63 e 55 kDa. Foi possível inferir que todas as bandas reveladas correspondem a isoformas de SmAPTDases com distintos processamentos pós-tradução, tais como glicosilação e proteólise. Comprovando esta reatividade, foi possível verificar que a imunização dos animais (C57BL/6) com a proteína vegetal foi capaz de induzir a produção significativa de anticorpos IgE detectáveis em técnicas de ELISA e Western blotting. Como a apirase de batata possui identidade com a NTPDase 1 (CD39) de mamíferos sugerimos que os pacientes possuam anticorpos naturais contra NTPDases envolvidos na manutenção da homeostase do organismo. O conjunto de dados obtidos mostra que a apirase de batata possui um perfil imunoregulatório na esquistossomose, podendo contribuir para a redução da morbidade, induzindo a produção de anticorpos IgE, tidos como protetores na esquistossomose. / Schistosomiasis is a neglected tropical disease that affects millions of people around the world. The lack of vaccine for this helminth disease prompts a search for antigenic molecules from the parasites of the genus Schistosoma. Among the molecules considered promising are an ATP diphosphohydrolase from Schistosoma mansoni. The parasite has two enzyme isoforms which are termed SmATPDases and are expressed in all as life cycle phases of this helminth. The potato apyrase is a plant protein belonging to the same family of parasite enzymes, shared epitopes with SmATPDase isoforms. Thus, the objective of this work was to perform an immunological characterization of potato apyrase in a model of experimental murine infection based on this protein homology. Plant protein was inoculated into C57BL / 6 mice that were infected with S. mansoni cercariae. Plant protein induced significant levels of IgG and IgG1 antibodies after immunization, which remained high for analysis 60 days after infection. The solenocytes culture of the immunized animals responded to the stimulus with a plant protein, inducing a significant production of IFN-γ, IL-10 and IL-5, but not IL-13. The immunization process did not provide protection, being unable to promote a reduction of the parasitic load. The immunomodulatory effect of immunization was observed through a reduction of the area affected by the hepatic granulomatous reaction in the immunized animals. Concerning the binding of IgE antibodies, potato apyrase showed significant reactivity with these antibodies present in plasma samples from patients with schistosomiasis. In Western blotting assays, IgE antibodies were able to recognize three non-homogenized languages of adult helminth worms at 91, 63 and 55 kDa. It was possible to infer that all the bands revealed correspond to SmAPTDase isoforms with various post-translational processing, such as glycosylation and proteolysis. (C57BL / 6) with a plant protein capable of inducing a significant production of IgE antibodies detected in ELISA and Western blotting techniques. As potato apyrase has an identity with a mammalian NTPDase 1 (CD39), we suggest that patients have an antibody against the NTPDases involved in maintaining the body's homeostasis. The obtained data show that the potato harvest has an immunoregulatory profile in schistosomiasis, and may contribute to the reduction of morbidity, inducing a production of IgE antibodies, considered as protectors in schistosomiasis.

Apyrasen (NTPDasen) sind Nukleosidtri- und diphosphat spaltende Enzyme. Bisher konnten Apyrasen in allen untersuchten Pro- und Eukaryonten nachgewiesen werden. Im Gegensatz zu tierischen Organismen, in denen Apyrasen gut untersucht sind und eine Rolle in der Nukleotid-vermittelten Signaltransduktion spielen, ist in Pflanzen weit weniger bekannt. In dem Modellorganismus A. thaliana wurden bisher zwei Apyrasen – AtAPY1 und AtAPY2 – als funktionell beschrieben. Durch Knockoutstudien konnte gezeigt werden, dass beide Apyrasen redundant sind. Der Doppelknockout der AtAPY1 und AtAPY2 ist im Gegensatz zum Einzelknockout einer Apyrase letal. Auf Grund von Vorarbeiten wurde die AtAPY1 extrazellulär im Apoplasten vermutet, wo sie eine Rolle im ATP-Signalweg spielen könnte. In der vorliegenden Arbeit sollte die Apyrase 1 (AtAPY1) biochemisch charakterisiert und subzellulär lokalisiert werden. Die Aufklärung der biochemischen Eigenschaften und der subzellullären Lokalisation der AtAPY1 würde entscheidend mithelfen, die Funktion der Apyrasen in Pflanzen aufzuklären. Für die biochemische Charakterisierung wie der Bestimmung des pH-Optimums und des Substratspektrums der AtAPY1 war die Reinigung einer aktiven AtAPY1 notwendig. Da eine Überexpression einer aktiven AtAPY1 in E. coli nicht möglich war, wurde zur biochemischen Charakterisierung die AtAPY1 mit verschiedenen Systemen in vitro translatiert. Bei Verwendung von Retikulozytenextrakten konnte die AtAPY1 in vitro translatiert werden, zeigte aber in den Aktivitätstests keine Aktivität. Auf Grund ihrer enzymatischen Aktivität und Struktur scheint die AtAPY1 inhibierend auf verschiedene Expressionssysteme zu wirken, was die Gewinnung von aktiver AtAPY1 stark limitiert. In einem weiteren Ansatz wurde die AtAPY1-GFP nativ aus transgenen A. thaliana mittels anti-GFP markierter Matrix gereinigen. Die Reinigung der AtAPY1-GFP aus dem Gesamtproteinextrakt war erfolgreich und die immobilisierte AtAPY1-GFP zeigte eine Apyraseaktivität. Eine anschließende Elution des Proteins von der Matrix war allerdings zu stringent und führte zum vollständigen Aktivitätsverlust. Für die subzelluläre Lokalisation wurden Apyraseeinzelknockouts in Vorarbeiten mit zwei unabhängigen Konstrukten transformiert: zum einen wurde die Atapy1 C-terminal mit dem SNAP-Tag fusioniert und unter ihrem nativen Promotorbereich exprimiert, zum anderen erfolgte eine Überexpression der AtAPY1-GFP unter dem konstitutiven CaMV 35S-Promotor. Die komplementierten Pflanzen zeigten keine phänotypischen Unterschiede im Vergleich zum Wildtyp. Durch Immunfluoreszenz und in vivo Mikroskopie konnte die AtAPY1 in vesikelartigen Strukturen, jedoch nicht in der Plasmamembran oder extrazellulären Matrix nachgewiesen werden. Um die detektierten Strukturen einem Organell zuzuordnen, wurden Co-Lokalisationsstudien durchgeführt. Für Co-Lokalisation wurden die AtAPY1-GFP Pflanzen mit Markerproteinen transformiert oder mit den entsprechenden transgenen Pflanzen gekreuzt. Zum Nachweis der AtAPY1-GFP im sekretorischen Weg oder endozytotischen Vesikeln wurden transgene AtAPY1-GFP Pflanzen mit RabE1d-YFP transformiert, was jedoch keine Co-Lokalisation zeigte. Anschließend erfolgten Kreuzungen mit transgenen Pflanzen, die die Golgi-Markerproteine Membrin 12, Syntaxin of plants 32 oder Golgi transport protein 1-Homolog exprimierten. Mit allen drei Kreuzungen konnte eine Co-Lokalisation der AtAPY1-GFP mit dem entsprechenden Markerprotein im Golgi gezeigt werden. Durch eine zusätzliche Behandlung der AtAPY1-GFP Pflanzen mit dem Membranfarbstoff FM4-64, welcher das trans-Golgi-Netzwerk aber nicht den Golgi-Apparat anfärbt und dem fungiziden Toxin Brefeldin A, welches zur Bildung von BFA-Kompartimenten durch die Fusion des trans-Golgi-Netzwerks mit Endosomen und Teilen des trans-Golgi-Apparates führt, konnte die AtAPY1-GFP dem Golgi-Apparat zugewiesen werden. Weiterhin wurde untersucht, ob die AtAPY1 löslich oder membrangebunden im Golgi-Apparat vorliegt. Um zwischen löslichen, peripheren und integralen Membranproteinen zu unterscheiden, wurde das mikrosomale Pellet mit verschiedenen Detergenzien und Salzen behandelt. Hohe Salz- (2 M NaCl) und alkalische Bedingungen (0,2 M Na2CO3) führten zur Ablösung peripherer Proteine von der Membran. Harnstoff (4 M) und das anionische Detergenz SDS (0,2 %) führten zur Denaturierung von Proteinen und zum Nachweis integraler Proteine. Es konnte gezeigt werden, dass die AtAPY1-GFP ein integrales Membranprotein ist, da sie ausschließlich in den mit SDS und Harnstoff behandelten Fraktionen im Überstand mittels Western Blot nachgewiesen werden konnte. Die genaue Funktion der AtAPY1 im Golgi-Apparat ist noch ungeklärt, da der Fokus der bisherigen Apyraseforschung von einer Lokalisation der AtAPY1 in der Plasmamembran ausging und frühere Ergebnisse in neuem Kontext diskutiert werden müssen.

Apyrasen (NTPDasen) sind Nukleosidtri- und diphosphat spaltende Enzyme. Bisher konnten Apyrasen in allen untersuchten Pro- und Eukaryonten nachgewiesen werden. Im Gegensatz zu tierischen Organismen, in denen Apyrasen gut untersucht sind und eine Rolle in der Nukleotid-vermittelten Signaltransduktion spielen, ist in Pflanzen weit weniger bekannt. In dem Modellorganismus A. thaliana wurden bisher zwei Apyrasen – AtAPY1 und AtAPY2 – als funktionell beschrieben. Durch Knockoutstudien konnte gezeigt werden, dass beide Apyrasen redundant sind. Der Doppelknockout der AtAPY1 und AtAPY2 ist im Gegensatz zum Einzelknockout einer Apyrase letal. Auf Grund von Vorarbeiten wurde die AtAPY1 extrazellulär im Apoplasten vermutet, wo sie eine Rolle im ATP-Signalweg spielen könnte. In der vorliegenden Arbeit sollte die Apyrase 1 (AtAPY1) biochemisch charakterisiert und subzellulär lokalisiert werden. Die Aufklärung der biochemischen Eigenschaften und der subzellullären Lokalisation der AtAPY1 würde entscheidend mithelfen, die Funktion der Apyrasen in Pflanzen aufzuklären. Für die biochemische Charakterisierung wie der Bestimmung des pH-Optimums und des Substratspektrums der AtAPY1 war die Reinigung einer aktiven AtAPY1 notwendig. Da eine Überexpression einer aktiven AtAPY1 in E. coli nicht möglich war, wurde zur biochemischen Charakterisierung die AtAPY1 mit verschiedenen Systemen in vitro translatiert. Bei Verwendung von Retikulozytenextrakten konnte die AtAPY1 in vitro translatiert werden, zeigte aber in den Aktivitätstests keine Aktivität. Auf Grund ihrer enzymatischen Aktivität und Struktur scheint die AtAPY1 inhibierend auf verschiedene Expressionssysteme zu wirken, was die Gewinnung von aktiver AtAPY1 stark limitiert. In einem weiteren Ansatz wurde die AtAPY1-GFP nativ aus transgenen A. thaliana mittels anti-GFP markierter Matrix gereinigen. Die Reinigung der AtAPY1-GFP aus dem Gesamtproteinextrakt war erfolgreich und die immobilisierte AtAPY1-GFP zeigte eine Apyraseaktivität. Eine anschließende Elution des Proteins von der Matrix war allerdings zu stringent und führte zum vollständigen Aktivitätsverlust. Für die subzelluläre Lokalisation wurden Apyraseeinzelknockouts in Vorarbeiten mit zwei unabhängigen Konstrukten transformiert: zum einen wurde die Atapy1 C-terminal mit dem SNAP-Tag fusioniert und unter ihrem nativen Promotorbereich exprimiert, zum anderen erfolgte eine Überexpression der AtAPY1-GFP unter dem konstitutiven CaMV 35S-Promotor. Die komplementierten Pflanzen zeigten keine phänotypischen Unterschiede im Vergleich zum Wildtyp. Durch Immunfluoreszenz und in vivo Mikroskopie konnte die AtAPY1 in vesikelartigen Strukturen, jedoch nicht in der Plasmamembran oder extrazellulären Matrix nachgewiesen werden. Um die detektierten Strukturen einem Organell zuzuordnen, wurden Co-Lokalisationsstudien durchgeführt. Für Co-Lokalisation wurden die AtAPY1-GFP Pflanzen mit Markerproteinen transformiert oder mit den entsprechenden transgenen Pflanzen gekreuzt. Zum Nachweis der AtAPY1-GFP im sekretorischen Weg oder endozytotischen Vesikeln wurden transgene AtAPY1-GFP Pflanzen mit RabE1d-YFP transformiert, was jedoch keine Co-Lokalisation zeigte. Anschließend erfolgten Kreuzungen mit transgenen Pflanzen, die die Golgi-Markerproteine Membrin 12, Syntaxin of plants 32 oder Golgi transport protein 1-Homolog exprimierten. Mit allen drei Kreuzungen konnte eine Co-Lokalisation der AtAPY1-GFP mit dem entsprechenden Markerprotein im Golgi gezeigt werden. Durch eine zusätzliche Behandlung der AtAPY1-GFP Pflanzen mit dem Membranfarbstoff FM4-64, welcher das trans-Golgi-Netzwerk aber nicht den Golgi-Apparat anfärbt und dem fungiziden Toxin Brefeldin A, welches zur Bildung von BFA-Kompartimenten durch die Fusion des trans-Golgi-Netzwerks mit Endosomen und Teilen des trans-Golgi-Apparates führt, konnte die AtAPY1-GFP dem Golgi-Apparat zugewiesen werden. Weiterhin wurde untersucht, ob die AtAPY1 löslich oder membrangebunden im Golgi-Apparat vorliegt. Um zwischen löslichen, peripheren und integralen Membranproteinen zu unterscheiden, wurde das mikrosomale Pellet mit verschiedenen Detergenzien und Salzen behandelt. Hohe Salz- (2 M NaCl) und alkalische Bedingungen (0,2 M Na2CO3) führten zur Ablösung peripherer Proteine von der Membran. Harnstoff (4 M) und das anionische Detergenz SDS (0,2 %) führten zur Denaturierung von Proteinen und zum Nachweis integraler Proteine. Es konnte gezeigt werden, dass die AtAPY1-GFP ein integrales Membranprotein ist, da sie ausschließlich in den mit SDS und Harnstoff behandelten Fraktionen im Überstand mittels Western Blot nachgewiesen werden konnte. Die genaue Funktion der AtAPY1 im Golgi-Apparat ist noch ungeklärt, da der Fokus der bisherigen Apyraseforschung von einer Lokalisation der AtAPY1 in der Plasmamembran ausging und frühere Ergebnisse in neuem Kontext diskutiert werden müssen.:ABKÜRZUNGEN 7 1. EINLEITUNG 9 1.1. APYRASEN 9 1.2. APYRASEN IN TIEREN 11 1.2.1. ROLLE DER NTPDASEN BEI DER THROMBOZYTENAGGREGATION 11 1.3. APYRASEN IN PFLANZEN 12 1.3.1. ROLLE EXTRAZELLULÄRER APYRASEN 13 1.3.2. GOLGI LOKALISIERTE APYRASEN 15 1.3.3. KENNTNISSTAND ÜBER APYRASEN IN A. THALIANA 16 1.4. VORARBEITEN 20 1.5. ZIELSTELLUNG 21 2. MATERIAL 22 2.1. GERÄTE 22 2.2. CHEMIKALIEN 23 2.3. HÄUFIG GENUTZTE PUFFER 24 2.4. BESONDERE VERBRAUCHSMATERIALIEN 24 2.5. KITS/STANDARDS 25 2.6. ENZYME 25 2.7. VEKTOREN 26 2.8. ZELLLINIEN 26 2.9. OLIGONUKLEOTIDE 27 2.10. ANTIKÖRPER (AK) 28 2.11. ANTIBIOTIKA/HERBIZIDE 28 2.12. VERWENDETE PFLANZENLINIEN 29 2.13. SCHLÜSSELNUMMERN (ACCESSION CODES) 29 2.14. SPEZIELLE SOFTWARE 30 3. METHODEN 31 3.1. ALLGEMEINE METHODEN 31 3.1.1. PFLANZENKULTIVIERUNG 31 3.1.1.1. Arabidopsis thaliana 31 3.1.1.2. Nicotiana benthamiana 31 3.1.2. KREUZEN VON A. THALIANA 31 3.1.3. TRANSFORMATION 31 3.1.3.1. Bakterien 31 3.1.3.2. Arabidopsis thaliana 32 3.2. MOLEKULARBIOLGISCHE METHODEN 33 3.2.1. PLASMIDPRÄPARATION 33 3.2.2. RNA-EXTRAKTION 33 3.2.3. REVERSE TRANSKRIPTION 33 3.2.4. NACHWEIS DER INTEGRITÄT VON RNA UND CDNA 34 3.2.5. DNA-EXTRAKTION AUS PFLANZEN 34 3.2.6. POLYMERASE-KETTENREAKTION (PCR) 35 3.2.7. AGAROSE-GELELEKTROPHORESE VON DNA 35 3.2.8. REINIGUNG VON DNA-FRAGMENTEN AUS AGAROSEGELEN 36 3.2.9. DNA-FÄLLUNG 36 3.2.10. KLONIERUNG DER ATAPY1-HIS UND SNAP-HIS IN E. COLI 36 3.3.1. KOMPLEMENTATION VON APYRASE DOPPELKNOCKOUT MUTANTEN 37 3.3.2. IMMUNFLUORESZENZ 38 3.3.2.1. Probenpräparation 38 3.3.2.2. Konfokale Mikroskopie 39 3.3.3. IN VIVO MIKROSKOPIE VON ATAPY1-GFP EXPRIMIERENDEN KEIMLINGEN 39 3.3.3.1. FM4-64 und Brefeldin A – Behandlung 39 3.3.3.2. Co-Lokalisation mit RabE1d, MEMB12, GOT1P-Homolog, SYP32 40 3.3.4. PROTOPLASTENISOLATION 40 3.3.5. PH-WECHSEL IM APOPLASTEN VON ATAPY1-GFP-KEIMLINGEN 41 3.4. PROTEIN-BIOCHEMISCHE METHODEN 41 3.4.1. PROTEINISOLATION 41 3.4.2. SOLUBILISIERUNG VON MEMBRANPROTEINEN 41 3.4.3. PROTEINBESTIMMUNG 42 3.4.4. SDS-PAGE 42 3.4.5. WESTERN BLOT 43 3.4.6. COOMASSIE-FÄRBUNG 43 3.4.7. KOLLOIDALE COOMASSIE-FÄRBUNG 44 3.4.8. PONCEAU-S-FÄRBUNG 44 3.4.9. IMMUNDETEKTION 44 3.4.10. PROTEINREINIGUNG MITTELS NI-NTA-SÄULENCHROMATOGRAPHIE 45 3.4.11. ISOLATION UND REINIGUNG DER ATAPY1-GFP AUS A. THALIANA 46 3.4.12. IN-VITRO TRANSLATION (IVT) ATAPY1-GFP UND ATAPY1-SNAP 46 3.4.13. QUANTIFIZIERUNG DES ATAPY1-SNAP-PROTEINS (IVT) 47 3.4.14. AKTIVITÄTSMESSUNG DER ATAPY1 48 3.4.14.1. Eisensulfat-Test 48 3.4.14.2. Malachitgrün-Test 49 4. ERGEBNISSE 50 4.1. SUBZELLULÄRE LOKALISATION DER ATAPY1 50 4.1.1. KOMPLEMENTATION DES LETALEN ATAPY1 UND ATAPY2 DOPPELKNOCKOUTS 50 4.1.2. DIE ATAPY1 IST IM GOLGI-APPARAT LOKALISIERT 55 4.1.2.1. Indirekte Immunfluoreszenz von AtAPY1-SNAP in Vesikeln 55 4.1.2.2. AtAPY1-GFP lokalisiert in Vesikeln 56 4.1.2.3. Keine Lokalisation der AtAPY1 in Plasmamembran und Apoplast 58 4.1.3. CO-LOKALISATIONSSTUDIEN DER ATAPY1-GFP 59 4.1.3.1. Keine Co-Lokalisation in sekretorischen Vesikeln 60 4.1.3.2. AtAPY1 ist Brefeldin A-sensitiv 61 4.1.3.3. AtAPY1 co-lokalisiert nicht mit FM4-64 64 4.1.3.4. Co-Lokalisation mit den Golgimarkerproteinen MEMB12, GOT1P-Homolog und SYP32 65 4.1.4. ATAPY1 IST EIN INTEGRALES MEMBRANPROTEIN 67 4.2. BIOCHEMISCHE CHARAKTERISIERUNG DER ATAPY1 69 4.2.1. EXPRESSION DER ATAPY1 IN E. COLI 69 4.2.2. IN VITRO TRANSLATION DER ATAPY1 71 4.2.3. REINIGUNG DER ATAPY1 AUS A. THALIANA 74 4.2.4. AKTIVITÄTSBESTIMMUNG DER ATAPY1 75 5. DISKUSSION 77 5.1. BEDEUTUNG DER LOKALISATION DER ATAPY1 FÜR DIE APYRASEFORSCHUNG 77 5.2. FUNKTION DER ATAPY1 IM GOLGI-APPARAT 78 5.2.1. AKKUMULATION VON STÄRKEGRANULA IN CHLOROPLASTEN 79 5.2.2. ROLLE DER APYRASEN BEI DER ZELLDIFFERENZIERUNG 81 6. AUSBLICK 86 7. ZUSAMMENFASSUNG 88 8. ABBILDUNGS- UND TABELLENVERZEICHNIS 90 9. LITERATURVERZEICHNIS 92 10. ANHANG 106

Lectin nucleotide phosphohydrolases (LNPs) are proteins which possess both apyrase catalytic activity (E.C. 3.6.1.5) and specific carbohydrate binding properties, and these are linked. To investigate the structural and functional properties for these proteins, two putative soluble plant LNPs, 4WC and 7WC (from white clover), and a putative soluble plant apyrase 6RG (from ryegrass) were chosen. Rabbit polyclonal antibodies for each plant apyrase were generated using highly purified, overexpressed recombinant 4WC or 7WC. In the case of 6RG, the C-terminal half of the protein constituted the best antigen for generating polyclonal antibodies. These antibodies showed high specificity and sensitivity. Active, recombinant 4WC and 6RG were overexpressed and purified using the baculoviral insect cell expression system (4WCbac-sup and 6RG:Hisbac), while 7WC (7WCcoli) was produced from E. coli inclusion bodies and subsequently refolded to give active enzyme. In course of overexpression, recombinant 4WC was localised in both the cellular fraction (4WCbac) and in the media supernatant (4WCbac-sup), while recombinant 6RG:Hisbac was only found in the cellular fraction (6RG:Hisbac) indicating that it was not secreted during insect cell growth. Secretion of 4WCbac was found to be dependent on N-glycosylation at N313 but not at N85 and elimination of one or both of these sites appeared to have little influence on apyrase activity. In addition, both 4WCbac and 6RG:Hisbac from the cellular fraction were fully functional. These results were compared with similar work performed on the animal ecto-apyrases which have different specific N-glycosylation sites required for secretion and activity. The 4WCbac-sup, 7WCcoli and 6RG:Hisbac proteins all showed apyrase activity, that is they catalysed the hydrolysis of nucleotide tri- and/or di-phosphates to their corresponding nucleotide monophosphates, and released inorganic phosphate in a divalent cation-dependent manner. However, the proteins exhibited different activities, substrate specificities, pH profiles and influence of inhibitors: 4WCbac-sup had a preference for NDPs with a pH optimum ≥9.5; 7WCcoli had a modest preference for NTPs with a pH optimum at 8.5; 6RG:Hisbac was almost exclusively an NTPase with a pH optimum at 6.5. Contrary to predictions based on phylogeny the proteins all bound to sulphated disaccharides and their catalytic activities were influenced both positively and negatively by the binding of specific chitosans. The data indicates that all three soluble plant apyrases investigated here were LNPs, in contrast to predictions from the literature. In order to pinpoint the regions responsible for determining substrate specificity and chitosan binding, chimeras were made using the N- and C-terminal halves of 4WC and 6RG. This resulted in fully functional reciprocal chimeras. Comparison of the apyrase activity for parents and chimeras, substrate specificity, optimal pH, influence of inhibitors on activity and effects of chitosans indicated that the C-terminus was responsible for determining substrate specificity. However, the influence of specific chitosans on the chimeras appeared to be dependent on both the N- and C-terminal portions of the proteins. In addition, chimeras were found to bind to the same sulphated disaccharides as the parent proteins. Preliminary crystal screening experiments were performed with highly purified preparations of 7WCcoli and 6RG:Hisbac. Under specific conditions 7WCcoli was found to form cube-like crystalline arrangements while 6RG:Hisbac formed hexagonal-like crystalline structures. A potential model for carbohydrate binding by LNPs is proposed and the possible biological roles of plant LNPs are discussed.

Nucleic acid technology has assumed an essential role invarious areas ofin vitrodiagnostics ranging from infectious diseasediagnosis to human genetics. An important requirement of suchmolecular methods is that they achieve high sensitivity andspecificity with a fast turnaround time in a cost-effectivemanner. To this end, in this thesis we have focused on thedevelopment of sensitive nucleic acid strategies thatfacilitate automation and high-throughput analysis. The success of nucleic acid diagnostics in the clinicalsetting depends heavily on the method used for purification ofthe nucleic acid target from biological samples. Here we havefocused on developing strategies for hybridisation capture ofsuch templates. Using biosensor technology we observed that thehybridisation efficiency could be improved using contiguousoligonucleotide probes which acted co-operatively. Byimmobilising one of the probes and annealing the second probein solution, we achieved a marked increase in target capturedue to a base stacking effect between nicked oligonucleotidesand/or due to the opening up of secondary structure. Suchco-operatively interacting modular probes were then combinedwith bio-magnetic bead technology to develop a capture systemfor the extraction of hepatitis C RNA from serum. Viral capturewith such co-operatively interacting probes extracted 2-foldmore target as capture with only a single probe achieving asimilar sensitivity to the conventional extraction protocol. Ananalogous strategy was designed to enrich for sequencingproducts prior to gel electrophoresis removing sequencingreagents and template DNA which interfere with the separationand detection of sequencing ladders, especially in the case ofcapillary gel electrophoresis. This protocol facilitates highthroughput clean-up of cycle sequencing reactions resulting inaccurate sequence data at a low cost, which is a pre-requisitefor large-scale genome sequencing products. Currently, a large effort is directed towards differentialsequencing to identify mutations or polymorphisms both in theclinical laboratory and in medical genetics. Inexpensive, highthroughput methods are therefore required to rapidly screen atarget nucleic acid for sequence based changes. In the clinicalsetting, sequence analysis of human immunodeficiency virus(HIV-1) is used to determine the presence of drug resistancemutations. Here we describe a bioluminometric pyrosequencingapproach to rapidly screen for the presence of drug resistancemutations in the protease gene of HIV-1. This sequencingstrategy can analyse the protease gene of HIV-1 from eightpatients in less than an hour and such non-gel based approachesshould be useful in the future in a clinical setting for rapid,robust mutation detection. Microarray technology facilitates large-scalemutation/polymorphism detection and here we developed amicroarray based single nucleotide polymorphism (SNP)genotyping strategy based on apyrase mediated allele specificextension (AMASE). AMASE exploits the fact that mismatchedprimers exhibit slower reaction kinetics than perfectly matchedprimers by including a nucleotide degrading enzyme (apyrase)which results in degradation of the nucleotides before themismatched primer can be extended. We have successfully typed200 genotypes (14% were incorrect without apyrase) by AMASEwhich cluster into three distinct groups representing the threepossible genotypes. In the future, AMASE on DNA microarraysshould facilitate association studies where an accuracy&gt;99%is required. <b>Keywords:</b>nucleic acid capture, modular probes,biosensor, bio-magnetic separation, hepatitis C, sequencing,pyrosequencing, mutation detection, HIV-1, drug resistance,SNP, allele-specific extension, apyrase, genotyping.

The ability to determine nucleic acid sequences is one ofthe most important platforms for the detailed study ofbiological systems. Pyrosequencing technology is a relativelynovel DNA sequencing technique with multifaceted uniquecharacteristics, adjustable to different strategies, formatsand instrumentations. The aims of this thesis were to improvethe chemistry of the Pyrosequencing technique for increasedread-length, enhance the general sequence quality and improvethe sequencing performance for challenging templates. Improvedchemistry would enable Pyrosequencing technique to be used fornumerous applications with inherent advantages in accuracy,flexibility and parallel processing. Pyrosequencing technology, at its advent, was restricted tosequencing short stretches of DNA. The major limiting factorwas presence of an isomer of dATPaS, a substitute for thenatural dATP, which inhibited enzyme activity in thePyrosequencing chemistry. By removing this non-functionalnucleotide, we were able to achieve DNA read-lengths of up toone hundred bases, which has been a substantial accomplishmentfor performance of different applications. Furthermore, the useof a new polymerase, called Sequenase, has enabled sequencingof homopolymeric T-regions, which are challenging for thetraditional Klenow polymerase. Sequenase has markedly madepossible sequencing of such templates with synchronizedextension. The improved read-length and chemistry has enabledadditional applications, which were not possible previously.DNA sequencing is the gold standard method for microbial andvial typing. We have utilized Pyrosequencing technology foraccurate typing ofhuman papillomaviruses, and bacterial andfungal identification with promising results. Furthermore, DNA sequencing technologies are not capable oftyping of a sample harboring a multitude of species/types orunspecific amplification products. We have addressed theproblem of multiple infections/variants present in a clinicalsample by a new versatile method. The multiple sequencingprimer method is suited for detection and typing of samplesharboring different clinically important types/species(multiple infections) and unspecific amplifications, whicheliminates the need for nested PCR, stringent PCR conditionsand cloning. Furthermore, the method has proved to be usefulfor samples containing subdominant types/species, and sampleswith low PCR yield, which avoids reperforming unsuccessfulPCRs. We also introduce the sequence pattern recognition whenthere is a plurality of genotypes in the sample, whichfacilitates typing of more than one target DNA in the sample.Moreover, target specific sequencing primers could be easilytailored and adapted according to the desired applications orclinical settings based on regional prevalence ofmicroorganisms and viruses. Pyrosequencing technology has also been used forclone-checking by using preprogrammed nucleotide additionorder, EST sequencing and SNP analysis, yielding accurate andreliable results. <b>Keywords:</b>apyrase, bacterial identification, dATPaS, ESTsequencing, fungal identification, human papillomavirus (HPV),microbial and viral typing, multiple sequencing primer method,Pyrosequencing technology, Sequenase, single-strandedDNA-binding protein (SSB), SNP analysis