Thesis advisor: Gregory R. Chiklis / Thesis advisor: Kathleen Dunn / In molecular diagnostics, the polymerase chain reaction (PCR) is used to amplify small amounts of nucleic acids found in patient samples, allowing for detection of diseases within hours of infection. This early detection allows medical professionals to diagnose and treat patients with greater success. It is crucial that internal controls, such as NATtrol™-treated microorganisms, are used in these PCR assays to avoid false-negative results and ensure accurate diagnosis of patients. NATtrol™ treatment renders microorganisms non-infectious while leaving them fully intact with their complete RNA or DNA genomes. Therefore, NATtrol™-treated microorganisms can be used in PCR as full-process internal controls that are spiked into patient samples and co-extracted and co-amplified within the sample. If the spiked NATtrol™ control returns expected results on the test, then the patient sample result can also be trusted. Here, we performed studies to validate the use of NATtrol™-treated MS2 virus as a universal full-process internal molecular control. In these studies, a quantitative, real-time, reverse-transcription PCR (qRT-PCR) assay was performed on the Roche LightCycler 480 instrument. Studies included working range validation, limit of detection, within-run precision, between-run precision, real-time stability, freeze-thaw (transport) stability, and open-vial (use-life) stability. All studies demonstrated the precision and stability of the MS2 NATtrol™ molecular control. / Thesis (BS) — Boston College, 2014. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Biology Honors Program. / Discipline: Biology.

No Brasil, casos de doença exantemática em bovinos e humanos têm sido relatados em diversas regiões, cujo agente causal é o virus vaccinia pertencente ao gênero Orthopoxirus da família Poxviridae. Classicamente, a varíola bovina é causada pelo Cowpoxvirus, entretanto, outros Poxvirus, como o vírus vaccinia podem desenvolver sintomatologia clínica semelhante. Além de comprometer a cadeia produtiva de leite, uma vez que dificulta a ordenha, predispõe a mastite e descarte do produto, é uma zoonose e atualmente está incluída no diagnóstico de doença vesicular. A origem desses casos bem como a epidemiologia da doença ainda é pouco conhecida. Assim, objetivou-se no presente estudo 1) Avaliar a soroprevalência de Orthopoxirus em rebanhos bovinos do circuito sete do Estado de São Paulo que compreendem as regiões do Vale do Paraíba e Mogi das Cruzes e associar os possíveis fatores de risco envolvidos na transmissão da doença; 2) Detectar e caracterizar a presença de Orthopoxirus em amostras suspeitas de doença vesicular, no período de 2007 a 2009, provenientes de diversas regiões do Brasil utilizando técnicas convencionais e moleculares: microscopia eletrônica, isolamento viral, PCR e RFLP; 3) Sequenciamento e análise filogenética das amostras positivas para o vírus vaccinia. Para o inquérito soroepidemiológico foram analisadas 76 propriedades pertencentes ao circuito 7 que compreendem as regiões do Vale do Paraíba e Mogi das Cruzes, estado de São Paulo, selecionadas aleatoriamente, totalizando 619 animais, estratificados em fêmeas acima de dois anos. A soroprevalência de Orthopoxirus no Vale do Paraíba foi de 32,3% (200/619) pela virusneutralização. Associação positiva foi encontrada para presença de animais silvestres. No diagnóstico virológico foram analisadas 227 amostras de epitélio, negativas para outras doenças vesiculares. Encontrou-se frequencia de 63,4% (144/227) positivas para o Orthopoxirus em praticamente todas as regiões do país. A análise por RFLP revelou perfil de padrão para o vírus vaccinia. O sequenciamento dos isolados confirmou que o vírus vaccinia é a estirpe circulante e está agrupado no grupo I de isolados de vaccinia brasileiros. / In Brazil, cases of rash illness in cattle and humans have been reported in several regions whose causal agent is the vaccinia virus belonging to the genus Orthopoxirus (OPV) family Poxviridae. Classically, cowpox is caused by Cowpoxvirus, however, other poxviruses such as vaccinia virus may develop same clinical signs. In addition to compromising the production chain of milk, as it hampers milking, predisposes to mastitis and discard the product, also it is a zoonosis and is currently included in the differential diagnosis of vesicular disease. The origin of these cases as well as the epidemiology of the disease is still unknown. Thus, the aim of the present study 1) assess the serum prevalence of OPV in cattle herds circuit seven Vale do Paraíba and associate the possible risk factors involved in disease transmission, 2) identify and characterize the presence of OPV in samples suspected vesicular disease in the period 2007-2009, from various regions of Brazil using conventional techniques and molecular electron microscopy, virus isolation, PCR and RFLP; 3) Sequencing and phylogenetic analysis of the samples positive for OPV. For epidemiological survey were analyzed 76 properties in the region of Vale do Paraíba, São Paulo, randomly selected, totaling 619 animals, stratified in females more than two years. The serum prevalence of OPV in the Paraíba Valley was 32.3% (200/619) by virus neutralization. Positive association was found for presence of wild animals. The virological diagnosis were analyzed 227 samples of epithelium, negative for other vesicular diseases. Met frequency of 63.4% (144/227) positive for OPV in practically all regions of the country. RFLP analysis revealed default profile for the vaccinia virus. The sequencing of the isolates confirmed that vaccinia virus strain is circulating and is clustered in group I isolates of Brazilians vaccinia.

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No. of bitstreams: 2 Tese - Marc Alexandre Duarte Gigonzac - 2016.pdf: 12763622 bytes, checksum: 3479eadda35402525c2387337a3a0d69 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-02-02 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / X-Fragile Syndrome (FXS) is the leading cause of inherited intellectual disability in the world and the second of genetic etiology, with an estimated prevalence of 1/4000 men and 1/8000 women. The most common molecular mechanism in SXF is due to changes in the expression of the FMR1 gene, located in Xq27.3, due to CGG trinucleotide expansions in the promoter region and subsequent methylation of the gene. In spite of presenting consistent clinical findings, they are not exclusive, and the existence of carriers of alteration in the FMR1 gene without apparent clinical manifestations makes it impossible to diagnose SXF based only on the evaluation. In the present study, a methodological proposal for the molecular diagnosis of X-Fragile Syndrome was developed from the methylation-specific triple amplification of the promoter region of the FMR1 gene combined with capillary electrophoresis. Thirty-four patients with clinical indication of SXF were referred to a laboratory of the public health network. After extraction and quantification of the DNA, the samples were amplified in an optimized protocol and the products submitted to 36cm capillary electrophoresis to verify the amount of CGG repeats and the degree of DNA methylation of each sample. Pre-mutation (3%) and six complete mutations (18%) were detected, all of which revealed a high degree of methylation. Considering the clinical signs commonly presented, the patients were also analyzed for the occurrence of Autism Spectrum Disorder (ASD), which shadowing and overlapping the SXF, verifying that 100% of the individuals with complete mutation presented the phenotype. Thus, it was possible to observe small behavioral differences in the patients analyzed, indicating a lighter clinical picture regarding aspects of social interaction and stereotypies. Thus, the new methodological proposal allows to effectively determine the CGG trinucleotide expansions in FMR1 allowing an assertive diagnosis of SXF for the families of patients attended in the public health network in Goiás. / A Síndrome do X-Frágil (SXF) é a principal causa de deficiência intelectual herdável no mundo e a segunda de etiologia genética, com uma prevalência estimada de 1/4000 homens e 1/8000 mulheres. O mecanismo molecular mais comum na SXF é decorrente de alterações na expressão do gene FMR1, localizado em Xq27.3, devido a expansões trinucleotídicas CGG na região promotora e subsequente metilação do gene. Apesar de apresentar achados clínicos consistentes, os mesmos não são exclusivos, e a existência de portadores de alteração no gene FMR1 sem manifestações clínicas aparentes impossibilitam o diagnóstico da SXF baseado apenas no exame físico. No presente estudo foi desenvolvido uma proposta metodológica para o diagnóstico molecular da Síndrome do X-Frágil a partir da amplificação tripla metilação-específica da região promotora do gene FMR1 combinada a eletroforese em capilar. Foram utilizados 34 pacientes com indicação clínica de SXF encaminhados para um laboratório da rede pública de saúde. Após extração e quantificação do DNA, as amostras foram amplificadas em protocolo otimizado e os produtos submetidos a eletroforese em capilar de 36cm para verificar a quantidade de repetições CGG e o grau de metilação do DNA de cada amostra. Foram detectadas uma pré-mutação (3%) e seis mutações completas (18%), sendo que todas estas últimas revelaram um alto grau de metilação. Considerando os sinais clínicos comumente apresentados, os pacientes foram também analisados para a ocorrência do Transtorno do Espectro do Autismo (TEA), que sombreia e se sobrepõe à SXF, verificando que 100% dos indivíduos com mutação completa apresentaram o fenótipo. Foi possível observar pequenas diferenças comportamentais nos pacientes analisados, indicando um quadro clínico mais leve quanto aos aspectos da interação social e das estereotipias. Sendo assim, a nova proposta metodológica permite determinar de forma eficaz as expansões trinucleotídicas CGG no FMR1 permitindo um diagnóstico assertivo da SXF para as famílias de pacientes atendidos na rede pública de saúde em Goiás.

Wheat bulb fly (WBF) (Delia coarctata, Fallén, Diptera: Anthomyiidae) is a pest of commercial importance in cereal crops. Control is dependent on organophosphates some of which are restricted in the UK, while current oviposition monitoring techniques are labour intensive and subjective. Eggs are not laid in association with a host-plant, therefore, prompt location of a suitable host is critical to the survival of the newly hatched larvae. Wheat bulb fly larvae have been shown to exhibit a positive chemotactic response to wheat and other host-plant seedlings and their root exudates. The objective of this study was to improve the control and population monitoring methodology associated with WBF, by investigating the ecology and specifically the chemical ecology of the WBF. Bioassays were used to investigate the behavioural response of WBF to known chemical constituents of host-plant exudates. Four secondary metabolites were found to be attractive while CO2 was found to alter the behaviour of larvae. Wheat bulb fly oviposition was assessed in field situations to describe egg laying spatially and through time. Geostatistical and ecological techniques were used to observe the spatial dependence and dispersion of oviposition and construct contour maps or scale-sized dot graphs of oviposition density. The traditional single line transect sampling pattern was compared against a more intensive sampling regime. Oviposition monitoring was conducted over a three year period to ascertain the time of peak egg density of this fly. A molecular based diagnostic test to assess WBF egg populations for damage forecasting was developed. A real time polymerase chain reaction (PCR) protocol was produced to estimate field populations of WBF eggs through the quantification of eggs from field samples. In addition endpoint PCR was used to identify the presence or absence of eggs from samples. This study gives the potential to advance current control methodology by providing the basis for the development of a lure and kill or confusion/disruption strategy, while offering a more accurate sampling system and a molecular diagnostic test, for improvement of the management of WBF.

It is well known that cells in tissues display a large heterogeneity in gene expression due to differences in cell lineage origin and variation in the local environment at different sites in the tissue, a heterogeneity that is difficult to study by analyzing bulk RNA extracts from tissue. Recently, genome-wide transcriptome analysis technologies have enabled the analysis of this variation with single-cell resolution. In order to link the heterogeneity observed at molecular level with the morphological context of tissues, new methods are needed which achieve an additional level of information, such as spatial resolution. In this thesis I describe the development and application of padlock probes and rolling circle amplification (RCA) as molecular tools for spatially-resolved transcriptome analysis. Padlock probes allow in situ detection of individual mRNA molecules with single nucleotide resolution, visualizing the molecular information directly in the cell and tissue context. Detection of clinically relevant point mutations in tumor samples is achieved by using padlock probes in situ, allowing visualization of intra-tumor heterogeneity. To resolve more complex gene expression patterns, we developed in situ sequencing of RCA products combining padlock probes and next-generation sequencing methods. We demonstrated the use of this new method by, for the first time, sequencing short stretches of transcript molecules directly in cells and tissue. By using in situ sequencing as read-out for multiplexed padlock probe assays, we measured the expression of tens of genes in hundreds of thousands of cells, including point mutations, fusions transcripts and gene expression level. These molecular tools can complement genome-wide transcriptome analyses adding spatial resolution to the molecular information. This level of resolution is important for the understanding of many biological processes and potentially relevant for the clinical management of cancer patients. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

No Brasil, casos de doença exantemática em bovinos e humanos têm sido relatados em diversas regiões, cujo agente causal é o virus vaccinia pertencente ao gênero Orthopoxirus da família Poxviridae. Classicamente, a varíola bovina é causada pelo Cowpoxvirus, entretanto, outros Poxvirus, como o vírus vaccinia podem desenvolver sintomatologia clínica semelhante. Além de comprometer a cadeia produtiva de leite, uma vez que dificulta a ordenha, predispõe a mastite e descarte do produto, é uma zoonose e atualmente está incluída no diagnóstico de doença vesicular. A origem desses casos bem como a epidemiologia da doença ainda é pouco conhecida. Assim, objetivou-se no presente estudo 1) Avaliar a soroprevalência de Orthopoxirus em rebanhos bovinos do circuito sete do Estado de São Paulo que compreendem as regiões do Vale do Paraíba e Mogi das Cruzes e associar os possíveis fatores de risco envolvidos na transmissão da doença; 2) Detectar e caracterizar a presença de Orthopoxirus em amostras suspeitas de doença vesicular, no período de 2007 a 2009, provenientes de diversas regiões do Brasil utilizando técnicas convencionais e moleculares: microscopia eletrônica, isolamento viral, PCR e RFLP; 3) Sequenciamento e análise filogenética das amostras positivas para o vírus vaccinia. Para o inquérito soroepidemiológico foram analisadas 76 propriedades pertencentes ao circuito 7 que compreendem as regiões do Vale do Paraíba e Mogi das Cruzes, estado de São Paulo, selecionadas aleatoriamente, totalizando 619 animais, estratificados em fêmeas acima de dois anos. A soroprevalência de Orthopoxirus no Vale do Paraíba foi de 32,3% (200/619) pela virusneutralização. Associação positiva foi encontrada para presença de animais silvestres. No diagnóstico virológico foram analisadas 227 amostras de epitélio, negativas para outras doenças vesiculares. Encontrou-se frequencia de 63,4% (144/227) positivas para o Orthopoxirus em praticamente todas as regiões do país. A análise por RFLP revelou perfil de padrão para o vírus vaccinia. O sequenciamento dos isolados confirmou que o vírus vaccinia é a estirpe circulante e está agrupado no grupo I de isolados de vaccinia brasileiros. / In Brazil, cases of rash illness in cattle and humans have been reported in several regions whose causal agent is the vaccinia virus belonging to the genus Orthopoxirus (OPV) family Poxviridae. Classically, cowpox is caused by Cowpoxvirus, however, other poxviruses such as vaccinia virus may develop same clinical signs. In addition to compromising the production chain of milk, as it hampers milking, predisposes to mastitis and discard the product, also it is a zoonosis and is currently included in the differential diagnosis of vesicular disease. The origin of these cases as well as the epidemiology of the disease is still unknown. Thus, the aim of the present study 1) assess the serum prevalence of OPV in cattle herds circuit seven Vale do Paraíba and associate the possible risk factors involved in disease transmission, 2) identify and characterize the presence of OPV in samples suspected vesicular disease in the period 2007-2009, from various regions of Brazil using conventional techniques and molecular electron microscopy, virus isolation, PCR and RFLP; 3) Sequencing and phylogenetic analysis of the samples positive for OPV. For epidemiological survey were analyzed 76 properties in the region of Vale do Paraíba, São Paulo, randomly selected, totaling 619 animals, stratified in females more than two years. The serum prevalence of OPV in the Paraíba Valley was 32.3% (200/619) by virus neutralization. Positive association was found for presence of wild animals. The virological diagnosis were analyzed 227 samples of epithelium, negative for other vesicular diseases. Met frequency of 63.4% (144/227) positive for OPV in practically all regions of the country. RFLP analysis revealed default profile for the vaccinia virus. The sequencing of the isolates confirmed that vaccinia virus strain is circulating and is clustered in group I isolates of Brazilians vaccinia.

Rapid, point-of-care infectious disease diagnostics have the potential to dramatically improve health care provision in low-income world regions. However, the development of technologies such as electrochemical DNA biosensors is hindered by slow turnaround times from design to working prototype. In order to facilitate biosensor development, a rapid prototyping method has been applied to the fabrication of wrinkled nano-/micro-structured electrodes in this work. An electrocatalytic DNA hybridization detection scheme is optimized for use with the wrinkled electrodes by adjusting the concentrations of redox agents FiCN and RuHex. Characterization of the electrodes by electrochemical and fluorescence-based methods showed tunability of important detection-related parameters – namely, the density of DNA probe molecules and the hybridization-induced electrocatalytic signal change – by altering parameters of deposition time, molar fraction of DNA probes relative to diluent molecules, and thickness of the wrinkled gold film. / Thesis / Master of Applied Science (MASc)

The 16S, 23S, and 5S ribosomal RNA (rRNA) genes are highly conserved sequences in bacteria. For this reason, rRNA genes are often used for phylogenetic classification. On the other hand, the regions between the structural sequences, known as intergenic spacer regions (ITS), are under less evolutionary pressure to be conserved. Because they are not as highly conserved, they can be used to differentiate strains of the same bacterial specie. The purpose of this study was to evaluate the 16S-23S ITS of Flavobacterium columnare, an important pathogen of cultured fish, by comparing the 16S-23S ITS sequences from 70 isolates. We developed two PCR assays that amplify overlapping regions of one large previously identified ITS. The primers targeted the 16S sequence and isoleucine tRNA encoding sequences and the 23S sequence and alanine tRNA encoding sequences. The PCR products were cloned and sequenced. We also targeted I-CeuI restriction fragments from the ATCC type strain that were separated by pulse field gel electrophoresis and analyzed the 16S-23S ITS regions. We found that the genome of this species harbors at least 6 intergenic spacer regions that are very similar and contain the same tRNA encoding sequences. This suggests that earlier studies that used the ITS for distinguishing between strains of Flavobacterium columnare may be comparing sequences from different structural RNA operons and thus have misleading data.

There is an urgent need for detection of viral respiratory pathogens to identify the causal agent(s) involved and to prevent the spread of related diseases. The first part of this dissertation focuses on development, optimization and validation of Real-time reverse transcription polymerase chain reaction (rRT-PCR) assays for the detection of several common equine viral pathogens: equine arteritis virus (EAV), equine influenza virus and equine rhinitis viruses A and B. Emphasis of the second part of this dissertation is on studying the role of EAV envelope proteins in virus attachment and entry. Using an infectious cDNA clone of EAV and reverse genetics, a panel of chimeric viruses was generated by swapping the N-terminal ectodomains and full-lengths of the two major envelope proteins (GP5 and M) from porcine reproductive and respiratory syndrome virus (PRRSV). The recombinant viruses expressing the N-terminal ectodomain of PRRSV GP5 or M or both (GP5ecto, Mecto, and GP5&Mecto, respectively) in an EAV backbone were viable and genetically stable. Compared to the parental virus, these three chimeric viruses produced lower titers and smaller plaque sizes indicating that they have a crippled phenotype. Interestingly, the three chimeric viruses could only infect EAV susceptible cell lines but not the PRRSV susceptible cell line. Therefore, the exchange of GP5 and/or M protein N-terminal ectodomains from PRRSV did not alter the cellular tropism of the chimeric viruses. We also investigated the role of one of the minor envelope proteins (E) of EAV in virus attachment and entry. The results showed that EAV infection of equine endothelial cells is heparin-dependent and the Cterminus of the E protein contains a putative heparin-binding domain. We generated a panel of arginine to glycine mutations in the conserved region of both the full-length EAV infectious cDNA clone and individual E protein expression vectors. The triple mutation R52,60,65G construct grew significantly slower and produced much smaller plaques. The double mutant R52,60G completely blocked the interaction between E protein and heparin. Taken together, these data indicated that E protein interacts with heparin to facilitate virus attachment and plays a major role in EAV infection.

UA Open Access Publishing Fund / Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decision-making. Our novel methodology, droplet-on-thermocouple silhouette real-time polymerase chain reaction (DOTS qPCR), uses interfacial effects for droplet actuation, inhibition relief, and amplification sensing. DOTS qPCR has sample-to-answer times as short as 3 min 30 s. In infective endocarditis diagnosis, DOTS qPCR demonstrates reproducibility, differentiation of antibiotic susceptibility, subpicogram limit of detection, and thermocycling speeds of up to 28 s/cycle in the presence of tissue contaminants. Langmuir and Gibbs adsorption isotherms are used to describe the decreasing interfacial tension upon amplification. Moreover, a log-linear relationship with low threshold cycles is presented for real-time quantification by imaging the droplet-on-thermocouple silhouette with a smartphone. DOTS qPCR resolves several limitations of commercially available real-time PCR systems, which rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection, we anticipate extending this technology to biological research applications such as single cell, single nucleus, and single DNA molecule analyses. Our work is the first demonstrated use of interfacial effects for sensing reaction progress, and it will enable point-of-care molecular diagnosis of infections.