published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Theoretical studies focusing on the nature of landscapes that correlate molecular sequences to molecular function have mainly been carried out in silico due to the vast amounts of data that are needed for analysis. In vitro selections of aptamers are a good model system to study theoretical questions at a experimental level. With the introduction of robotic platforms that conduct in vitro selections, it is now capable of producing significant amounts of data in a short time, making theoretical modeling with real experimental data attainable. I will be using a Biomek 2000 Laboratory Automation Workstation to carry out multiple in vitro nucleic acid selections in parallel. I will explore the sequence space to examine whether existing in vitro selection systems are optimal at isolating the best winning species. New methods will be introduced that will allow for the selection of identical targets with identical pools free of cross contamination on the open robotic system. This will open the doors to further conduct selections against other identical or highly similar targets, such as complex cellular targets. Finally, I will investigate the methods to improve the effectiveness at isolating aptamers against the highly complex lung cancer cell lines. These targets are highly challenging for isolating specific aptamers because of the great diversity of biomarkers found among them. Moreover, their highly morphological similarity of the cultured cells makes selections for specific aptamers very difficult. I explore the different methods that will allow for the generation of aptamers that can distinguish between non-small cell lung cancer and small cell lung cancer, and between non-small cell lung cancer and normal lung cells. Fine-tuning of this process is essential at transferring this process to automated platforms for large-scale generation of biosensors against tumor biomarkers.

The loss of muscle mass leading to cachexia is rarely identified in early lung cancer. Fasting blood and muscle biopsy were collected in 59 non-small cell lung cancer (NSCLC) and 16 non-cancer patients, at the beginning of thoracic surgery. Serum C-reactive protein (CRP), and IL-6 were higher in NSCLC. In weight-losing NSCLC, food intake and serum albumin were lower, CRP, and TNF-alpha were higher. Although the expression of genes of the ubiquitin-proteasome system was not different, ubiquitinated-protein levels were lower and negatively correlated with ph-FOX01 in weight-losing patients. This would suggest lower muscle proteolytic rates in the early stages of NSCLC. Ph-FOXO1 also related to the degree of weight loss and stage of NSCLC. These data suggest that in early stages of the disease, weight and muscle loss could be mainly due to reduced food intake, rather than accelerated proteolysis, which reinforces the potential for successful dietary interventions to prevent or delay the onset of cachexia.

The Jaagsiekte Sheep Retrovirus (JSRV), an exogenous type B/D-retrovirus with about 10-15 endogenous counterparts in all normal sheep genomes, causes Jaagsiekte (JS) or ovine pulmonary adenocarcinoma (OPA), a contagious lung cancer of sheep. This sheep lung cancer has been identified as the best natural out-bred model that can be used to study human epithelial tumours. The close similarity between the pathology of the sheep disease and Human Bronchiolo-alveolar carcinoma are highly suggestive that the human disease could have a similar aetiology and mechanism to the sheep disease. However, in the case of sheep at the time of the study there was a need for an assay that could be used to screen for infected sheep. The isolation, cloning and subsequent sequencing of the first full-length exogenous and endogenous forms of JSRV contributed greatly towards JSRV research. Until recently the diagnosis of OPA was based mostly on clinical presentation with confirmation by micro and macro examination of the affected lungs by expert pathologists. In the absence of a specific humoral response no serology-based tests were available to diagnose the disease early in live animals. Control and management of the disease was primarily by regular flock inspections and prompt culling of the suspected cases. The objective of this research project was therefore to assess and investigate the serological and molecular diagnosis of JSRV. In an attempt to develop a serology based assay three proteins were identified as candidate diagnostic antigens, the group specific antigen JSRV p26, the transmembrane and the orf-X proteins. Genes coding for all three proteins were isolated, cloned and expressed. The JSRV p26 was sufficiently purified and its potential as a diagnostic antigen was evaluated in both a Western blot and ELISA. Our studies confirmed that there were no circulating antibodies to the JSRV capsid protein. Evidence suggested that the immune response was localised to the lungs. Lung lavage samples were therefore collected from infected and normal sheep and analysed for the presence of JSRV p26 antibodies using an in-house JSp26 peroxidase conjugate in an antigen capture assay. This assay lacked sensitivity but the results indicated that there was a specific localised immune response to JSRV in the lungs of OPA affected sheep. This was confirmed with an in-house antigen capture assay that we developed. JS antigen was detected in the lung and nasal fluid of affected sheep, but not in equivalent samples from normal sheep. Three molecular assays were investigated for their sensitivity and specificity, the LTR-gag PCR, U3/LTR hemi-nested PCR and the PCR that covered the V1/V2 region. The U3/LTR hemi-nested assay was 2 logs more sensitive than the LTR-gag PCR. However, it detected the endogenous JSRV5.9A1 loci at higher concentrations. This was overcome by designing a more specific primer P3M for the first step of the U3/LTR hemi-nested PCR and the use of the AmpliTaq Gold DNA polymerase. This assay proved to be both sensitive and specific enough to screen for the infectious exogenous JSRV in peripheral blood samples from individual sheep. It is now possible to use this assay to selectively eradicate the disease from a flock through a selective culling programme. Furthermore, the assay could be made quantitative by the inclusion of concentration standards. / Thesis (M.Med.)-University of KwaZulu-Natal, 2005.

Hybrid plasmonic-photonic structures were introduced as novel platforms for on-chip biochemical sensing and spectroscopy. By appropriate coupling of photonic and plasmonic modes, a hybrid architecture was realized that can benefit from the advantages of integrated photonics such as the low propagation loss, ultra-high Q modes, and robustness, as well as the advantages of nanoplasmonics such as extreme light localization, large sensitivities, and ultra-high field enhancements to bring about unique performance advantages for efficient on-chip sensing. These structures are highly sensitive and can effectively interact with the target biological and chemical molecules. It was shown that interrogation of single plasmonic nanoparticles is possible using a hybrid waveguide and microresonator-based structure, in which light is efficiently coupled from photonic structures to the integrated plasmonic structures. The design, implementation, and experimental demonstration of hybrid plasmonic-photonic structures for lab-on-chip biochemical sensing applications were discussed. The design goal was to achieve novel, robust, highly efficient, and high-throughput devices for on-chip sensing. The sensing scenarios of interest were label-free refractive index sensing and SERS. Nanofabrication processes were developed to realize the hybrid plasmonic-photonic structures. Silicon nitride was used as the material platform to realize the integrated photonic structure, and gold was used to realize plasmonic nanostructures. Special optical characterization setups were designed and implemented to test the performance of these nanoplasmonic and nanophotonic structures. The integration of the hybrid plasmonic-photonic structures with microfluidics was also optimized and demonstrated. The hybrid plasmonic-photonic-fluidic structures were used to detect different analytes at different concentrations. A complete course of research from design, fabrication, and characterization to demonstration of sensing applications was conducted to realize nanoplasmonic and integrated photonic structures. The novel structures developed in this research can open up new potentials for biochemical sensors with advanced on-chip functionalities and enhanced performances.

Two areas of the HTLV-I genome were targeted for an in-house molecular diagnostic test, namely the pol and env regions. The pol primers proved the most sensitive (100%)and specific (100%). Amplification using the env primer pair was not reproducible, and was not pursued further. The AmpliSensor assay (Acugen Systems, Lowell, MA) was also tested. The assay was very specific, but not as sensitive as our in-house PCR. To investigate the predominant HTLV-I subtype in the region, a 1535 by env gene was isolated from peripheral blood obtained from five local HTLV-I seropositive patients. Four of the patients presented with HAM/TSP, and the fifth presented with a skin disease. Nucleotide sequencing of the amplified products revealed the local strains to be very conserved, differing by 0.1% to 0.9% among themselves. No apparent difference was noted for the two clinical manifestations. Phylogenetic analysis was performed using repesentative strains from around the world. The local strains clearly fell within the cosmopolitan subtype. The local strains were most closely related to the North American strains suggesting an unexpected link between the two countries. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1998.

Aspergillus fumigatus is a foodborne and airborne human pathogen which produces mycotoxins such as gliotoxin, helvolic acid, fumigallin, and fumigaclavin. The most common disease caused by A. fumigatus is aspergillosis, which is often fatal, especially among AIDS, cancer, and organ-transplant patients. In this study, random cDNA cloning was performed from a cDNA library of A. fumigatus (IMI 385708) constructed on &amp / #955 / ZAP Express. Some of these clones were selected according to their insert sizes and were further subjected to sequence analysis. The sequences were then analyzed by a BLAST search (NCBI Genome Database) to determine the possible functions of these genes. Two of the clones were identified as the primase and 60S ribosomal protein L1-b genes. These genes and a third gene corresponding to the antigenic cell wall galactomannoprotein gene of A. fumigatus were used for the design of three distinct primer pairs. For the primer design, a software was written to differentiate nonconserved regions by multiple sequence alignment. Designed primers were employed in PCR experiments against genomic DNAs of different Aspergillus species. Unique bands were obtained only against A. fumigatus genomic DNA. It was concluded that this PCR-based detection method can be further developed for the rapid detection of A. fumigatus spores from air and food samples.

Orientador: Helio Langoni / Resumo: Mycoplasma spp., tem distribuição mundial e é um patógeno relevante em medicina veterinária. As mastites causadas por Mycoplasma spp. mais frequentes em grandes rebanhos leiteiros, porém este patógeno é subestimado no Brasil, onde se têm poucos relatos como agente causador de mastite, o que se deve possivelmente a quantidade reduzida de laboratórios que inclui a análise de M. bovis em sua rotina. Devido a necessidade de meios seletivos e de condições especiais para o seu isolamento. Diante disso um dos objetivos do presente estudo foi pesquisar a participação de Mycoplasma bovis na etiologia das mastites clínicas em amostras de leite de vacas de propriedades leiteiras de sete estados do Brasil, totalizando 561 amostras de leite, que foram cultivas em meio Hayflick adicionado de acetato de tálio a 0,01%, incubadas em ambiente de microaerofilia com 5% de CO2. Foram também submetidas a reação em cadeia da polimerase (PCR) para detecção de Mycoplasma spp. e Mycoplasma bovis. Pesquisouse também a microbiota aeróbica envolvida nas mastites, cultivando-se as mesmas amostras de leite em meios de ágar sangue bovino 5% e ágar MacConkey resultando 225 amostras positivas. Obtiveram-se 11 (1,96%) amostras positivas para Mycoplasma spp., no exame microbiológico, enquanto na detecção molecular obteve-se um total de 17 (3,03%) amostras positivas para Mycoplasma bovis. Pode-se concluir com os resultados obtidos pela presença e dispersão do Mycoplasma bovis nos rebanhos leiteiros avaliados, ap... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Introdução: A rede lisossômica é um complexo de vias metabólicas que influenciam processos como degradação de organelas danificadas ou senescentes, processamento de antígenos, reutilização de aminoácidos essenciais e, em última instância, um sistema de fundamental importância para a fisiologia celular normal. Nesse contexto, os lisossomos possuem uma relevância muito grande, uma vez que é nessa organela que ocorre a destruição das moléculas envolvidas em todos os processos citados acima. Entre as unidades operacionais nos lisossomos estão as hidrolases lisossômicas, que são mais de 50 enzimas com capacidade, em ambiente ácido, de realizar a quebra de substratos específicos. A GlcNAc-fosfotransferase é um complexo hexamérico (J2K2L2) residente na porção cis do complexo de Golgi que realiza a adição de resíduos de manose-6- fosfato nas cadeias de oligossacarídeos das hidrolases lisossômicas. Com ação subsequente, a enzima descobridora realiza a remoção da manose, expõe os resíduos de fosfato e possibilita que as hidrolases sejam reconhecidas pelos receptores de manose-6- fosfato e direcionadas aos compartimentos lisossomais. As subunidades J e K da GlcNAc-fosfotransferase são codificadas pelo gene GNPTAB, localizado no cromossomo 12, constituído de 21 éxons, e a subunidade L pelo gene GNPTG, localizado no cromossomo 16, constituído de 11 éxons. Alterações patogênicas em GNPTAB podem causar as doenças Mucolipidose II ou III alfa/beta e alterações em GNPTG causam a doença Mucolipidose III gama. O defeito genético leva à atividade residual, ou nula, da enzima que acaba por gerar o extravasamento das hidrolases lisossômicas ao meio extracelular e o acúmulo de substratos nos lisossomos. Objetivos: (1) caracterizar as alterações patogênicas em GNPTAB em um grupo de pacientes brasileiros não relacionados com Mucolipidose II ou III alfa/beta, e (2) definir um protocolo de pesquisa molecular para os pacientes brasileiros. Metodologia: É um estudo transversal, com amostragem por conveniência, e inclui pacientes com diagnóstico clínico e bioquímico de Mucolipidose II ou III. Foi extraído DNA genômico dos pacientes a partir de sangue obtido por punção venosa periférica. O gene GNPTAB foi sequenciado através da técnica de Sanger. As alterações do tipo troca de sentido foram analisadas pelos programas de Bioinformática Polyphen2, Sift e Consurf, e as preditas como patogênicas foram pesquisadas em alelos controles brasileiros e analisadas por estudos funcionais, quando possível. A geração de construtos das alterações p.Ser385Leu e c.3503_3504delTC foi realizada por mutagênese sítio-dirigida e a atividade residual destes foi avaliada 24 horas após expressão em células HEK. Para a análise financeira, valores atuais de equipamentos, reagente de biologia molecular e materiais plásticos foram utilizados para estimar o custo de uma extração de DNA, reação de PCR, purificação com PEG8000 e sequenciamento. Resultados: Foram incluídos 13 pacientes (ML II= 8; ML III= 5) e, adicionalmente, de uma mãe de paciente com diagnóstico clínico e bioquímico de ML II. A análise molecular identificou seis alterações patogênicas novas, as c.831delT, c.1763insA, c.1927delAATT, p.Ser385Leu, p.(Asp76Gly) e p.Try1111*. A análise de bioinformática das alterações do tipo troca de sentido as caracterizaram como prejudiciais para a função da proteína e os resíduos 76 e 385 como estrutural e funcional, respectivamente, além de ambos como altamente conservados entre as espécies. A análise funcional dos mutantes p.Ser385Leu e c.3503_3504delTC identificaram atividades residuais de 1,5% e nula, respectivamente. Também foram identificadas outras seis alterações patogênicas previamente descritas. A alteração c.3503_3504delTC foi a que apresentou a maior frequência (40%, n= 10/25 alelos), seguido pela p.Ile403The (12%, n=3/25 alelos). Quanto às relações genótipo-fenótipo, sete pacientes com ML II possuem genótipos combinados de alterações do tipo mudança de fase de leitura e sem sentido, enquanto que os cincos pacientes com ML III alfa/beta apresentam pelo menos uma alteração do tipo troca de sentido, o que evidencia a relação entre alterações que impactam a funcionalidade da proteína e fenótipos mais graves. A análise retrospectiva definiu o protocolo 1.0 que finalizaria o diagnóstico com um custo médio de R$ 338,45, em uma amostra de 25 pacientes. A análise prospectiva do protocolo 2.0, sobre a mesma amostra de pacientes, indicou que o mesmo finalizaria o diagnóstico com o custo médio de R$ 299,80, uma economia de 25%. Discussão/Conclusão: As novas alterações patogênicas descritas nesse trabalho confirmam a alta heterogeneidade alélica do gene GNPTAB. A análise funcional da alteração p.Ser385Leu confirma sua patogenicidade, que está de acordo com o fenótipo ML II do paciente, e evidencia a necessidade de mais estudos a fim de constatar o motivo desse resíduo ser importante para a proteína. A síntese dos protocolos demonstrou ser uma estratégia interessante e economicamente importante, uma vez que diminui os gastos envolvidos para finalizar o diagnóstico molecular. / Introduction: The lysosomal network is a complex of metabolic ways that influence processes like damaged or senescent organelles degradation, antigen processing, essential amino acid reutilization, and, in the last instance, it is important for normal cell physiology. In this context, lysosomes have a great relevance since it is in this organelle that the destruction of the molecules involved in all the processes mentioned above occurs. The operational units in the lysosomes are the lysosomal hydrolases that are more than 50 enzymes with capacity, in an acid environment, to breakdown specific substrates. GlcNAc-phosphotransferase is a hexameric complex (J2K2L2) located in the cis portion of the Golgi complex that performs the addition of mannose-6-phosphate residues in oligosaccharides chains on lysosomal hydrolases. In a subsequent way, the uncovering enzyme removes the mannose residues, exposes the phosphate residues, and enables the recognition of hydrolases by mannose-6-phosphate receptors. The J and K subunits are codified by GNPTAB gene, which is located in chromosome 12 and consists of 21 exons, and the L subunit, encoded by GNPTG gene, that is located in chromosome 16 and consists of 11 exons. The consequences of pathogenic alterations in GNPTAB are Mucolipidosis II or III alpha/beta diseases and alterations in the GNPTG are Mucolipidosis III gamma disease. The genetic defect leads to residual or absent activity of enzyme which ultimately generates an overflow of lysosomal hydrolases to the extracellular environment and accumulation of substrates in lysosomes. Objectives: (1) to characterize, by sequencing of the GNPTAB gene, the pathogenic alterations in a group of unrelated Brazilian patients with Mucolipidosis II or III alpha/beta, and (2) to define a molecular research protocol for Brazilian patients. Methodology: It is a crosssectional study with convenience sampling, and it includes patients with biochemical and clinical diagnosis of Mucolipidosis II or III. The DNA was amplified by PCR technique and sequencing by Sanger technique. All patients in the present study had all exons amplified. The missense alterations were analyzed by Polyphen2, Sift, and ConSurf softwares, and the alterations predicted as pathogenic were studied through research in Brazilian control alleles. The p.Ser385Leu and c.3503_3504delTC were evaluated by site-direct mutagenesis and the residual activity was evaluated 24 hours after expression in HEK cells, through radioactive assays. For cost-price analysis, current values for equipment, molecular biology reagents, and plastic materials were utilized to estimate the cost of DNA extraction, PCR reaction, PEG8000 purification, and sequencing. Results: Of the 13 patients, 8 were clinically diagnosed with Mucolipidosis II and 5 with Mucolipidosis III alpha/beta and, additionally, a mother of one patient with biochemical and clinical diagnosis of Mucolipidosis II was also analyzed. The DNA analysis identified six novel pathogenic alterations in GNPTAB: c.831delT, c.1763insA, c.1927delAATT, p.Ser385Leu, p.(Asp76Gly), and p.Tyr1111*. The bioinformatics analysis of missense alterations were characterized as damaging for protein function, and residues 76 and 385 as structural and functional, respectively, and both as highly conserved among the species. The functional analysis of mutants p.Ser385Leu and c.3503_3504delTC showed the residual activity of GlcNAcphosphotransferase of 1.5% and 0%, respectively. Six others pathogenic alterations previously described were also identified. The alteration c.3503_3504delTC showed the highest frequency (40%, n=10/25 alleles) followed by p.Ile403The (12%, n=3/25 alleles). The retrospective analysis defined the 1.0 protocol that finalized the molecular diagnosis at the cost of R$ 338,45 per sample, in a group of 25 patients. The prospective analysis of 2.0 protocol, in the same patients, indicated that it would finalize diagnosis at the cost of R$ 299,80 per sample, a saving of 25%. Discussion/Conclusion: The novel pathogenic alterations described confirm the high allelic heterogeneity of GNPTAB gene. In the genotype-phenotype relationship, 7 patients with Mucolipidosis II have combined genotype of frameshift or nonsense alterations, or both, and 5 Mucolipidosis III alpha/beta patients have at least one missense alteration, that shows the correlation between alterations that cause impact on the protein function and severe phenotype. The functional analysis of alteration p.Ser385Leu confirms its pathogenicity and makes evident the need of more studies in order to determine the reason this residue is so important for protein function. Protocol synthesis proves to be an interesting and economically important strategy, once it decreases costs to conclude the molecular diagnosis.

Orientador: Rosangela Zacarias Machado / Banca: Cristiane Divan Baldani / Banca: Carlos Termignoni / Banca: Marcos Rogério André / Banca: Cristina Harumi Adania / Resumo: Agentes das Ordens Rickettsiales, Haemosporida e Rhizobiales são causadores de enfermidades que acometem várias espécies animais, podendo representar um risco à saúde dos mesmos, inclusive de seres humanos. Aves carnívoras podem infestar-se e infectar-se com muitas espécies de parasitas, incluindose carrapatos e hemoparasitas, sendo os hábitos de predação fatores de favorecimento à transmissão e translocação de diversos agentes etiológicos. O objetivo deste estudo foi pesquisar, por meio de métodos indiretos (Reação de Imunofluorescência Indireta - RIFI) e diretos (esfregaços sanguíneos, PCR em Tempo Real e PCR Convencional) os agentes das Famílias Anaplasmataceae (Ehrlichia, Anaplasma, Neorickettsia), Bartonellaceae (Bartonella spp.) e Ordem Haemosporida (Plasmodium, Haemoproteus e Leucocytozoon) em ectoparasitas e amostras de sangue de aves carnívoras, realizando-se um estudo filogenético dos agentes encontrados. Para tanto, procedeu-se à colheita de 121 amostras de sangue total e 88 amostras de soro de aves pertencentes às Ordens Accipitriformes, Falconiformes, Strigiformes e Cathartiformes, capturadas nos estados de São Paulo e Rio de Janeiro. Em relação aos ectoparasitas, foram recolhidas três larvas e uma ninfa ingurgitada de carrapatos do gênero Amblyomma de um gavião carijó (Rupornis magnirostris) no Estado de São Paulo. Na RIFI para E. chaffeensis e A. phagocytophilum, 03 (3,41%) e 05 (5,68%) amostras apresentaram sororreatividade, respectivamente. Todas as amostras testadas na RIFI para E. canis mostraram-se negativas. Na avaliação de esfregaços sanguíneos corados pelo Giemsa, não foram encontradas estruturas compatíveis com hemoparasitas. Em relação a PCR em Tempo Real (qPCR), não foram encontrados animais PCR positivos para Ehrlichia chaffeensis (gene vlpt) e Anaplasma phagocytophilum (gene msp-2). Na qPCR Multiplex para Ehrlichia spp., Anaplasma spp. (gene groE) e... / Abstract: Rickettsiales, Haemosporida and Rhizobiales agents cause diseases that affect various animal species, including humans. Carnivorous birds become infested and infected with many species of parasites, including ticks and hemoparasites, and the predation habits favoring transmission and translocation of various etiological agents. The aim of this study was to investigate, using indirect methods (Immunofluorescence - IFAT) and direct methods (blood smears, Real-Time PCR and Conventional PCR) agents of Families Anaplasmataceae (Ehrlichia, Anaplasma, Neorickettsia), Bartonellaceae (Bartonella spp.) and Haemosporida Order (Plasmodium, Haemoproteus and Leucocytozoon) in ectoparasites and blood samples carnivorous birds, performing a phylogenetic study of these agents. Therefore, we collected 121 blood samples and 88 serum samples from birds belonging to the Accipitriformes, Falconiformes, Strigiformes and Cathartiformes Orders, captured in the states of São Paulo and Rio de Janeiro. Regarding the ectoparasites, we collected three larvae and one engorged nymphal Amblyomma ticks of a Rupornis magnirostris in São Paulo. At IFAT for E. chaffeensis and A. phagocytophilum, 03 (3.41%) and 05 (5.68%) samples showed seroreactivity, respectively. Blood smears stained by Giemsa were analyzed and hemoparasites were not found. Regarding the Real-Time PCR (qPCR), in multiplex qPCR for Ehrlichia spp., Anaplasma spp. (groE gene) and Bartonella spp. (nuoG gene) observed 12 samples positive for Anaplasma spp. (9.91%). In conventional PCR-based 16SrRNA gene for Anaplasmataceae and cytochrome B gene for hemosporidia, eight birds were PCR positive for Ehrlichia spp (6.61%, five in PCR for E. chaffeensis and three in PCR for E. canis), three for Haemoproteus spp (2.48%) and Plasmodium spp (0.83%). In phylogenetic analysis, four samples of Ehrlichia spp. positioned on the same branch Ehrlichia identified in wild animals in Brazil and the US human isolate. The ... / Doutor