<p>Nucleic acid technology has assumed an essential role invarious areas of<i>in vitro</i>diagnostics 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.</p><p>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.</p><p>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.</p><p>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>99%is required.</p><p><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.</p>

<p>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.</p><p>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.</p><p>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.</p><p>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.</p><p>Pyrosequencing technology has also been used forclone-checking by using preprogrammed nucleotide additionorder, EST sequencing and SNP analysis, yielding accurate andreliable results.</p><p><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</p>

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.

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.

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.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-11T13:29:19Z No. of bitstreams: 1 micheliaantoniadonascimentogusmao.pdf: 2029497 bytes, checksum: 4ab5e6f5d64e0b6010c2ea583d1ed036 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-06-20T18:43:57Z (GMT) No. of bitstreams: 1 micheliaantoniadonascimentogusmao.pdf: 2029497 bytes, checksum: 4ab5e6f5d64e0b6010c2ea583d1ed036 (MD5) / Made available in DSpace on 2016-06-20T18:43:57Z (GMT). No. of bitstreams: 1 micheliaantoniadonascimentogusmao.pdf: 2029497 bytes, checksum: 4ab5e6f5d64e0b6010c2ea583d1ed036 (MD5) Previous issue date: 2013-03-06 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A esquistossomose é uma doença que acomete cerca de 200 milhões de pessoas no mundo causando grande morbidade. Devido à falta de vacina para essa doença, muitos antígenos são pesquisados. Um domínio antigênico B (r156-195) da ATPDase 2 de Schistosoma mansoni foi previamente descrito, e dois peptídeos sintéticos (SmB1LJ, r155-176; SmB2LJ, r175-194) derivados deste domínio foram obtidos com o objetivo de avaliar a imunogenicidade dessa proteía. O inóculo de cada peptídeo em camundongos BALB/c induziu atividade imunoestimulatória significativa, elevando os níveis de anticorpos IgG1 e IgG2, como detectado por ELISA. Os anticorpos policlonais anti-SmB1LJ e anti-SmB2LJ, quando imobilizados em Proteína A-Sepharose, imunoprecipitaram aproximadamente 42-96% da atividade ADPásica da preparação de verme adulto. Por “Western blots” do complexo imunoprecipitado, três bandas de 76, 63 e 55 kDa foram identificadas pelos dois soros imunes, confirmando a identidade da ATPDase 2, e sugerindo que esta proteína está sujeita a vários processamentos pós-tradução, tais como glicosilação e proteólise, resultando finalmente em uma proteína secretada de 55 kDa. Adicionalmente, anticorpos policlonais anti-SmB2LJ inibiram 62% e 37% das atividades ATPásica e ADPásica, respectivamente, enquanto anticorpos policlonais anti-SmB1LJ inibiram ADPase (92%), mas não atividade ATPásica, sugerindo que o domínio B da ATPDase 2 do S. mansoni está envolvido na regulação catalítica da enzima, sendo um novo alvo para o desenho de inibidores. Imunomarcação com anticorpos policlonais anti-SmB2LJ de cortes de fígado de camundongo infectado revelaram reações positivas na superfície externa do miracídio no ovo de S. mansoni. Fluorescência intensa foi detectada na região entre o miracídio e o lado interno da casca do ovo, localizada no envelope de von Lichtenberg, a qual é uma região de produção de componentes imunogênicos. Fluorescência foi também detectada no lado de fora da casca do ovo, aprisionada pelos microespinhos de superfície, confirmando que esta proteína, como no verme adulto, é também secretada de ovos de S. mansoni. Nenhuma reatividade foi observada nos tecidos granulomatosos circundantes. Amostras de plasmas de camundongos Swiss infectados com S. mansoni foram testadas por ELISA usando SmB1LJ or SmB2LJ como antígeno. Reatividade significativamente maior de anticorpos IgG, IgG1 ou IgG2a foi detectada com SmB1LJ, enquanto somente anticorpo IgG1 foi altamente reativo com SmB2LJ. A reatividade de amostras de plasma (diluídas 1:200) de camundongos Swiss previamente inoculados com apirase de batata ou r-potDomínio B, um polipeptídio com cauda de hexahistidina derivado do domínio B (r78-117) da apirase de batata, e homólogo ao domínio B da ATPDase 2, foi também testada por ELISA usando os peptídeos como antígenos. Anticorpos policlonais IgG e IgG1 anti-apirase de batata reagiram significativamente com SmB1LJ, enquanto somente IgG1 reagiu com SmB2LJ. Anticorpos policlonais anti-r-potDomínio B não reagiram significativamente com SmB2LJ. Comparado aos controles, amostras de plasma (diluídas 1:200) destes animais pré-imunizados com apirase de batata ou r-potDomínio B e desafiados com S. mansoni não reagem significativamente com os peptídeos. Os resultados confirmaram que o domínio B da ATPDase 2 está potencialmente envolvido na resposta imune do hospedeiro, e os peptídeos SmB1LJ e SmB2LJ podem ser usados em estudos da esquistossomose. / Schistosomiasis is a disease that affects approximately 200 million people worldwide causing great morbidity. Due to lack of vaccine for this disease, many antigens are searched. An antigenic domain B (r156-195) from the Schistosoma mansoni ATPDase 2 isoform was previously described, and two synthetic peptides (SmB1LJ, r155-176; SmB2LJ, r175-194) belonging to this domain were obtained. Inoculation of each peptide in healthy BALB/c mice induced significant immunostimulatory activity, increasing the IgG1 and IgG2a antibody levels, as detected by ELISA. The polyclonal anti-SmB1LJ and anti-SmB2LJ antibodies, when immobilized on Protein A-Sepharose, immunoprecipitated approximately 42-96% of the ADPase activity from the adult worm preparation. By Western blots of immunoprecipitated complex, three bands of 76, 63 and 55 kDa were identified by the two immune sera, confirming the ATPDase 2 identity, and suggesting that this protein is subject to several posttranslational processing, such as glycosylation and proteolysis, resulting finally in a secreted protein of 55 kDa. Additionally, polyclonal anti-SmB2LJ antibodies inhibited 62% and 37% of the ATPase and ADPase activities, respectively, whereas polyclonal anti-SmB1LJ antibodies inhibited ADPase (92%), but not ATPase activity, suggesting that the domain B from the S. mansoni ATPDase 2 is involved in enzyme catalytic regulation, being a new target for inhibitor design. Immunolabelled cryostat sections of infected mouse liver by the anti-SmB2LJ polyclonal antibodies revealed positive reactions on the external surface from the miracidium in the S. mansoni egg. Intense fluorescence was detected in the region between the miracidium and the inner side of the egg-shell, located in von Lichtenberg’s envelope, which is a region of production of immunogenic components. Fluorescence was also detected in the outer side of the egg-shell and entrapped by the surface microspines, confirming that this protein, like in the adult worm, is also secreted from the S. mansoni eggs. No reactivity was observed in the surrounding granumomatous tissues, which are rich en inflammatory cells. Serum samples from S. mansoni-infected Swiss mice were tested by ELISA using SmB1LJ or SmB2LJ as coating antigen. Significantly higher IgG, IgG1 or IgG2a antibody reactivity was detected against SmB1LJ, whereas only IgG1 antibody was highly reactive against SmB2LJ. The reactivity of plasma samples (diluted 1:200) from Swiss mice previously inoculated with potato apyrase or r-potDomain B, a 6xHis tag polypeptide belonging to the domain B (r78-117) from the potato apyrase, homologue to the domain B from the ATPDase 2, was also tested by ELISA using the peptides as coating antigens. Polyclonal IgG and IgG1 anti-potato apyrase antibodies significantly reacted with SmB1LJ, whereas only IgG1 reacted with SmB2LJ. Polyclonal anti-r-potDomain B antibodies did not significantly react with SmB2LJ. Compared to controls, plasma samples (diluted 1:200) from these animals pre-immunized with potato apyrase or r-potDomain B and challenged with S. mansoni did not react significantly with peptides. The results confirmed that the domain B from the ATPDase 2 is potentially involved in the host immune response, and peptides SmB1LJ and SmB2LJ can be used in schistosomiasis studies.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-06-22T11:07:10Z No. of bitstreams: 1 michelledelimadetoni.pdf: 1224744 bytes, checksum: a3734f99b34853e12e12fb5964be66e5 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-07T19:20:05Z (GMT) No. of bitstreams: 1 michelledelimadetoni.pdf: 1224744 bytes, checksum: a3734f99b34853e12e12fb5964be66e5 (MD5) / Made available in DSpace on 2017-08-07T19:20:05Z (GMT). No. of bitstreams: 1 michelledelimadetoni.pdf: 1224744 bytes, checksum: a3734f99b34853e12e12fb5964be66e5 (MD5) Previous issue date: 2009-03-06 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Calliandra brevipes apresenta uma galha globosa (GG) e uma fusiforme (GF), induzidas por Tanaostigmodes mecanga e T. ringueleti, respectivamente. As variações sazonais no conteúdo fenólico de ambas as galhas, de caule e folha não galhados (CS e FS, respectivamente) foram analisadas durante 12 meses. O perfil de variação do teor fenólico nos tecidos galhados e não galhados de C. brevipes foi similar ao longo do período analisado, apresentando-se fortemente influenciado pelas variações na precipitação pluviométrica, ocorrendo maior acúmulo fenólico quando o vegetal estava sob estresse hídrico. Os conteúdos fenólicos de ambas as galhas foram similares e, no período de drásticas mudanças na precipitação pluviométrica, estes sofreram alterações significativamente menores do que as sofridas pelos tecidos não galhados, sugerindo que as duas espécies de Tanaostigmodes modulam o conteúdo fenólico no tecido galhado de maneira similar. Assim, notou-se que as alterações no conteúdo fenólico podem estar primariamente associadas às mudanças pluviométricas, constituindo uma defesa direta contra o estresse hídrico. Na triagem de metabólitos secundários, flavonóides, cumarinas e taninos foram detectados em tecidos galhados e não galhados de C. brevipes. Saponinas foram encontradas somente em amostras de tecidos não galhado, mostrando que as galhas apresentam menor toxicidade para o herbívoro. Triterpenóides foram encontrados em GG e esteróides em GF, sugerindo que cada galhador explora diferencialmente o metabolismo do hospedeiro. Adicionalmente, o conteúdo de flavonóides e a atividade antioxidante também estavam diferenciados em ambas as galhas, sendo maiores na galha fusiforme. Na análise cromatográfica e densitométrica de carboidratos, GG e GF apresentaram conteúdos de sacarose, glicose e frutose maiores do que os encontrados em tecidos não galhados, 1,5-3 vezes e 2 vezes, respectivamente. A análise de saponinas e carboidratos evidenciou que ambas as galhas apresentam menor toxicidade e constituem uma fonte mais nutritiva para o herbívoro do que o tecido não galhado. O fracionamento e a análise densitométrica de proteínas, detectou polipeptídeos comuns a ambas as galhas (80, 69, 61, 52, 32 kDa) provavelmente essenciais para a organização básica desta neoformação, bem como específicos a cada uma (galha globosa: 97, 75, 34 kDa; galha fusiforme: 40, 33 kDa) que podem estar associadas aos distintos morfotipos e suas vias metabólicas. Em conjunto, a análise de proteínas, flavonóides e a atividade antioxidante mostram que cada galhador manipula o hospedeiro de maneira peculiar, mesmo quando duas espécies galhadoras filogeneticamente tão próximas parasitam um hospedeiro comum. Quanto à análise fosfohidrolítica, CS hidrolisou igualmente ATP, ADP, UDP ou GDP. Na GG, o UDP, GDP, e ADP foram hidrolisados aproximadamente 1,5-3 vezes mais do que no CS, já o AMP foi 10 vezes mais hidrolisado na GG. Em conjunto, estes dados confirmam que este tecido exacerba os mecanismos capazes de hidrolisar nucleotídeos, possivelmente devido sua maior atividade metabólica. Atividades ADPásica e UDPásica de CS e GG foram 10-32% estimuladas por 5 mM de Ca2+, e inibidas pela adição de 5 mM EDTA ou EGTA (88 a 100%), confirmando a dependência destas atividades à cátions bivalentes. Uma pequena inibição por ortovanadato foi detectada sugerindo a presença de P-ATPases, enquanto azida sódica, DCCD e bafilomicina não inibiram significativamente a atividade ATPásica. Uma apirase de 75 kDa foi identificada em CS e GG de C. brevipes, e provavelmente contribuiu com a atividade fosfohidrolítica observada. Foi constatada a presença de apirase e de atividade ADPásica nas células nutritivas que delineiam a câmara larvar por microscopia de imunofluorescência e citoquímica, respectivamente. A alta atividade apirásica na galha globosa de C. brevipes pode estar associada à biossíntese de carboidratos, recuperação de nucleosídeos e/ou proliferação celular, contribuindo assim, para a nutrição e sobrevivência do galhador. / Calliandra brevipes presents globose (GG) and fusiform galls (GF) induced by Tanaostigmodes mecanga and T. ringueleti, respectively. The seasonal changes in phenolic content of both galls, and in non-galled stem and leaf were analyzed over one year. The phenolic variation profile of galled or non-galled tissues of C. brevipes was similar during the analyzed period, but it is strongly influenced by the variations in the pluviometric levels increasing during hydric stress. The phenolic levels were in similar amounts between the two galls and, in the period of drastic changes in pluviometric levels, the alterations were significantly higher in non-galled tissues when compared to the galled tissues, suggesting that the two distinct species of Tanaostigmodes manipulate the phenolic level in a similar way. Therefore, the alterations in phenolic contents could be primarily associated to the pluviometric changes, constituting a direct defense against the hydric stress. The phytochemical screening showed the presence of flavonoids, coumarins and tannins in galled and non-galled samples. Saponins were found only in non-galled samples, suggesting that the galled tissues are less toxic to the herbivory. Triterpenoids were detected in GG, but not in GF, suggesting that each inducer explore in a different way the plant metabolism. In addition, flavonoids content and antioxidant activity were differentiated in both galls, being significantly higher in fusiform gall. Chromatographic and densitometric analyses showed that GG and GF present sucrose, glucose and fructose contents 1.5-3 and 2 times higher, respectively than those found in non galled tissue. The secondary metabolites and carbohydrate analyses evidenced that both galls tissues are less toxic and constitute a better nutrient source for the herbivory than the non-galled tissues. Protein fractionation and densitometric analysis showed common polypeptides to both galls (80, 69, 61, 52, 32 kDa) possibly essentials for their basic neoformations, and specific polypeptides in either globose (globose: 97, 75, 34 kDa) or fusiform gall (40, 33 kDa), which could be associated to the different gall morphotypes and their metabolic pathways. Together, the protein, flavonoid and antioxidant analyses showed that each inducer, although closer phylogenetically, manipulate the host by specific pathways in the same plant. Phosphohydrolitic analysis showed that CS equally hydrolyzed either ATP, ADP, UDP or GDP. In GG, the UDP, GDP, and ADP were hydrolyzed approximately 1.5-3 folds higher than that found in CS. AMP hydrolyzes was 10 folds higher in GG confirming that this tissue increase their mechanisms capable of hydrolyzing nucleotides. ADPase and UDPase activities from CS and GG were 10-32% stimulated by 5 mM calcium, and inhibited by the addition of 5 mM EDTA or EGTA (88 to 100%), thus confirming the divalent cation dependence. Little ATPase inhibition by orthovanadate was detected suggesting the presence of a P-type ATPase, while sodium azide, dicyclohexylcarbodiimide, and bafilomycin did not affect significantly the ATPase activity. A band of 75 kDa was recognized by polyclonal anti-potato apyrase antibodies in CS or GG, confirming the presence of an apyrase in Calliandra brevipes. By immunofluorescence microscopy, using anti-potato apyrase antibodies, and by cytochemical techniques, using ADP as substrate, the apyrase was located on the external surface and inside of the nutritive cells that compose the central chamber. The high apyrase activity in globose gall of C. brevipes could be participating in carbohydrate biosynthesis, in the salvage pathway of nucleosides and/or cell proliferation, for the feeding and survival of the insect.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-08-31T15:40:53Z No. of bitstreams: 0 / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-09-01T11:35:40Z (GMT) No. of bitstreams: 0 / Made available in DSpace on 2017-09-01T11:35:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2013 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Por alinhamento das sequências de aminoácidos da apirase de S. tuberosum e isoformas de outras espécies de plantas, alta identidade (46% a 94%) e similaridade (64% a 96%) foram identificadas entre elas, incluindo espécies diferentes de Fabaceae, e estreita relação estrutural foi encontrada entre os domínios B destas proteínas. Anticorpos policlonais anti-apirase de batata S. tuberosum inibiram 65-80% da atividade catalítica desta proteína. Soros imunes policlonais produzidos contra os peptídeos sintéticos homólogos potB1LJ, LbB1LJ e SmB1LJ ou potB2LJ, LbB2LJ e SmB2LJ, derivados de domínios B de isoformas de NTPDases, inibiram significativamente (32-44%) a atividade da apirase de batata. Estes anticorpos podem ser ferramentas moleculares auxiliares para o estudo das isoformas de apirase. A atividade apirásica de tecidos de folha (NL) ou caule (NS) normais e de galha globosa (GG) de Calliandra brevipes BENTH (Fabaceae: Mimosoidae) foi testada em meio de reação pH 7,5 e na presença de C12E9, e a hidrólise de ADP, UDP ou GDP por GG foi 1,8 a 2,4 vezes maiores que tecidos não galhados. Anticorpos policlonais anti-apirase de batata inibiram significativamente (70-90%) a hidrólise de ATP, ADP, GDP ou UDP por NS. Em GG, similarmente, a atividade ATPásica, GDPásica, ADPásica ou UDPásica foi significativamente inibida (40-66%). Para o isolamento e identificação de apirase de tecidos normais e galha globosa de Calliandra brevipes, amostra de tecido de NS ou NL não galhado, ou de GG previamente homogeneizada em Triton X-100 mais deoxicolato de sódio foi fracionada em gel não desnaturante contendo os mesmos detergentes. Usando ADP como o substrato, a atividade catalítica da enzima permitiu o aparecimento de uma única banda de precipitado de fosfato de cálcio mostrando idêntica mobilidade eletroforética nas amostras NS, NL e GG, sendo mais intensa nesta última, sugerindo que a apirase é superexpressa neste tecido. Nesta banda de maior mobilidade, distintos polipeptídeos de 52 a 75 kDa foram reconhecidos pela imunoreatividade cruzada de anticorpos anti-apirase de batata, sugerindo a existência de formas ativas da mesma proteína com diferentes cargas líquidas, possivelmente como um resultado de diferentes níveis de glicosilação. Além disso, uma banda de aproximadamente 52 kDa, de menor mobilidade e atividade, foi também identificada em todos os tecidos, possivelmente a apirase em sua forma completamente deglicosilada. Usando técnicas histoquímicas e microscopia óptica, cortes micrométricos corados por lugol ou azul de astra-safranina evidenciaram a câmara pulpal amplamente circundada por tecido nutritivo contendo amido em sua porção distal, e parênquima cortical delimitado por anéis esclerenquimáticos lignificados. Por técnicas histoquímicas, a atividade ADPásica ou ATPásica foi encontrada como um depósito eletrodenso granular de fosfato de chumbo distribuído na superfície externa, e nos tecidos nutritivos da galha globosa. Por técnicas imunohistoquímicas, usando Microscópio Confocal de Varredura a Laser e anticorpos anti-apirase de batata e anti-peptídeos sintéticos, a apirase de galha globosa foi encontrada nos mesmos sítios. Hipóteses funcionais para esta proteína estão sob investigação. Além disso, o tempo de germinação de semente de C. brevipes foi determinado, e no quinto dia após a instalação do teste, a primeira folha foi aberta, identificando tecidos de crescimento rápido apropriados para a investigação da regulação da hidrólise de ATP por apirase na proliferação celular. / By alignment of amino acid sequences of the S. tuberosum apyrase and isoforms from other plant species, high identity (46 to 94%) and similarity (64 to 96%) were identified between them, including distinct species of Fabaceae, and closer structural relationship was found between the domains B from these proteins. Polyclonal antibodies anti- S. tuberosum potato apyrase inhibited 65-80% of the catalytic activity of this protein. In addition, polyclonal immune sera produced against homologue synthetic peptides potB1LJ, LbB1LJ and SmB1LJ or potB2LJ, LbB2LJ and SmB2LJ, derivatives from B domains of NTPDases isoforms, significantly inhibited (32 to 44%) potato apyrase activity. These antibodies could be useful molecular tools for studies of the apyrase isoforms. The apyrase activity from the non-galled stem (NS), non-galled leaf (NL), or globose gall (GG) tissue from Calliandra brevipes BENTH (Fabaceae: Mimosoidae) was tested in reaction medium pH 7.4 and in the presence of C12E9, the ADP, UDP or GDP hydrolysis by GG was 1.8 to 2.4 fold higher than non-galled tissues. Polyclonal antibodies anti-potato apyrase significantly inhibited (70 to 90%) the ATP, ADP, GDP or UDP hydrolysis by NS. In GG, similarly, the ATPase, GDPase, ADPase or UDPase activity was also significantly inhibited (40-66%). For isolation and identification of apyrase from normal tissues and globose gall from the Calliandra brevipes, sample of NS, NL or GG tissue was previously homogenised in Triton X-100 plus sodium deoxycholate, and fractionated in non-denaturing gel containing the same detergents. Using ADP as the substrate, the enzyme catalytic activity gave rise to the appearance of a single calcium phosphate precipitate band displaying identical electrophoretic mobilities in NS, NL, and GG samples, being more intense in this last one suggesting that apyrase is over-expressed in this tissue. In this band of higher mobility, a number of distinct polypeptides ranging from 52 to 75 kDa were recognized by cross-immunoreactivity with polyclonal anti-potato apyrase antibodies, suggesting the existence of active forms of the same protein with different net charges, possibly as a result of distinct glycosylation levels. In addition, an immunoreactive band of approximately 52 kDa, of lower mobility and activity, was also identified in all tissues, possibly apyrase in completely deglycosylated form. By histochemical techniques and light microscopy, micrometric sections stained with lugol or Astra Blue-Saphranine evidenced the chamber pulpal widely surrounded by nutritive tissue containing starch in its distal portion, and cortical parenchime delimited by rings sclereides lignified. By histochemical techniques, the ADPase or ATPase activity was found as a granular dense lead phosphate deposit distributed at the external surface, and inside of the nutritive cells of the globose gall. By immunohistochemical techniques, using confocal laser scanning microscopy and antibodies anti-potato apyrase and anti-synthetic peptides, the gall globose apyrase was found at same sites. In addition, the time of laboratory germination of C. brevipes seed was determined, and on the fifth day after test installation, the first leaf opened, identifying rapid growing tissues suitable for further investigations on the participation of both apyrase and regulation of ATP hydrolysis in cell proliferation.

Disturbances of host integrity have the potential to evoke activation of innate immunologic and hemostatic protection mechanisms in blood. Irrespective of whether the activating stimulus is typically immunogenic or thrombotic, it will generally affect both the complement system and platelets to a certain degree. The theme of this thesis is complement and platelet activity, which is intersected in all five included papers. The initial aim was to study the responses and mechanisms of the complement cascade in relation to platelet activation. The secondary aim was to use an applied approach to regulate platelets and complement on model biomaterial and cell surfaces.    Complement activation was found in the fluid phase in response to platelet activation in whole blood. The mechanism was traced to platelet release of stored chondroitin sulfate-A (CS-A) and classical pathway activation via C1q. C3 was detected at the platelet surface, though its binding was independent of complement activation. The inhibitors factor H and C4-binding protein (C4BP) were detected on activated platelets, and their binding was partly dependent on surface-exposed CS-A. Collectively, these results showed that platelet activation induces inflammatory complement activation in the fluid phase. CS-A was shown to be a central molecule in the complement-modulatory functions of platelets by its interaction with C1q, C4BP, and factor H. Platelet activation and surface adherence were successfully attenuated by conjugating an ADP-degrading apyrase on a model biomaterial. Only minor complement regulation was seen, and was therefore targeted specifically on surfaces and cells by co-immobilizing a factor H-binding peptide together with the apyrase. This combined approach led to a synchronized inhibition of both platelet and complement activation at the interface of biomaterials/xenogeneic cells and blood. In conclusion, here presents a novel crosstalk-mechanism for activation of complement when triggering platelets, which highlights the importance of regulating both complement and platelets to lower inflammatory events. In addition, a strategy to enhance the biocompatibility of biomaterials and cells by simultaneously targeting ADP-dependent platelet activation and the alternative complement C3-convertase is proposed.