Doctor of Philosophy / Department of Physics / Thomas W. Phillips / The emergence of heritable high-level resistance to the fumigant gas phosphine in stored-product insects is of grave concern to many grain growing countries around the world. The research reported in this dissertation was designed to determine the presence of phosphine resistance in 34 field collected populations of Rhyzopertha dominica (F.) from the United States and Canada, the potential to control resistant R. dominica populations utilizing high dose and longer exposure time strategies, and to develop a rapid assay for phosphine resistance detection. Using a discriminatory dose assay called the FAO number 16 method, adult R. dominica were sampled and subjected to a phosphine dose of 20 ppm for a fumigation exposure period of 20 h to distinguish a susceptible R. dominica adult by death from a resistant beetle that survives the treatment. Results from the study showed that 32 out of the 34 geographic populations surveyed had beetles resistant to phosphine, and the frequency of resistance varied from 97% in a population from Parlier, California to 0% in beetles from both Carnduff, Saskatchewan and Starbuck, Manitoba. A 20-hour dose response assay was used to characterize the level of resistance by calculating the resistance ratio factors using beetles from a laboratory susceptible strain and those from five of the populations sampled. This resistance ratio (RR) was based on the ratio of LC50 (estimate for the concentration to kill 50% of a test group) in the sampled population to the LC50 for the susceptible strain. The highest RR for the five resistant populations was nearly 596-fold in beetles from Belle Glade, Florida, which represented the “strong” resistance phenotype, whereas the lowest RR in that group was 9-fold in Wamego, Kansas, representing the “weak” resistance phenotype. Manipulation of concentration and exposure periods can be utilized to manage strongly resistant R. dominica populations. The effect of several phosphine concentrations and fumigation exposure periods were assessed on progeny of mixed life stage colonies of the strongly resistant R. dominica. A 48 hours dose response assay was carried out on these two strongly resistant populations to re-characterize their levels of resistance. Results from this assay showed that a phosphine dose of 730-870 ppm could control all resistant adult R. dominica. Additionally, phosphine concentrations ranging from 400-800 ppm phosphine for 96 hours completely killed mixed life stage colonies of strongly resistant lesser grain borers from the two populations studied. Lastly, fumigations done beyond 4 days at phosphine concentrations between 450-700 ppm controlled all phosphine resistant populations of lesser grain identified from our previous work. Phosphine applied at high concentrations is known to elicit a knockdown effect that can vary between susceptible and resistance grain insects. Using 18 of the 34 R. dominica populations this study sought to determine among three knockdown time (KT) techniques which method had potential to be utilized in an effective rapid assay for phosphine resistance in R. dominica. Adult R. dominica were exposed to a high concentration of phosphine (3000 ppm) to assess the time to knockdown 50%, 100% of a group of ten insects and that of single insects from the 18 geographically distinct populations vis a vis the resistance frequencies using the FAO method. KT100 quick test was better than the KT50 and Ktsingle, because bioassays were able to clearly distinguish among susceptible, weak and strong resistant individuals. Time for KT100 from susceptible populations did not exceed 30 minutes, while resistant populations had KT100 times above 30 min, with strong-resistant populations times longer than 100 min.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Dana Vanlandingham / Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus maintained among swine and avian species. In infected pigs, replication of JEV leads to the onset of viremia and the development of neurological and reproductive disease in young and naïve pregnant animals. The high-titer viremia levels associated with JEV infection in pigs, whilst important to the enzootic transmission cycle responsible for viral maintenance, also have human health implications within the zoonotic cycle. Sensitive and specific veterinary diagnostic methods capable of readily detecting JEV infection are critical components of JEV surveillance programs in the Asian Pacific region. In this study, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assays were evaluated for use in veterinary diagnosis of JEV. Our hypotheses for this research project were that RT-qPCR assays with fewer oligonucleotide mismatches between the primers and probes of the assays and JEV genomes will be more sensitive for the diagnosis of JEV infection and that oral shedding of JEV in swine would allow for detection of viral RNA using oral fluids. The sensitivity and specificity of three RT-qPCR assays for the detection of JEV were determined using tissue culture fluids of five representative JEV strains belonging to four endemic genotypes. The first assay (assay #1), targeting the highly conserved NS5 gene and 3UTR regions, provided optimum detection for the current predominant genotype, GI-b. All three assays were highly specific for JEV when tested against other selected flaviviruses in the JEV serocomplex. A rope-based collection method allowed for the simplified collection of oral fluids from three-week-old piglets challenged with endemic JEV strain JE-91. These fluids were then evaluated using RT-qPCR assays for the presence of viral RNA. The results suggest that the shedding of JEV in oral fluids can be readily detected and that non-invasive oral fluid collection can serve as a novel sampling method for the diagnosis and surveillance of JEV in swine.

Made available in DSpace on 2014-06-11T19:32:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-16Bitstream added on 2014-06-13T20:03:10Z : No. of bitstreams: 1 vieira_dcm_dr_arafcf.pdf: 1317618 bytes, checksum: ac08bac3ba14ebc0cb17729af3a88219 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / A cefuroxima sódica (CAS 56238-63-2) é uma cefalosporina de segunda geração, indicada nas infecções provocadas por micro-organismos Gram-positivos e Gram-negativos. Apesar deste fármaco ser altamente estudado e pesquisado no que concerne à atividade antimicrobiana, farmacocinética e farmacodinâmica, há poucos estudos na literatura em relação ao desenvolvimento de metodologia analítica para esta cefalosporina. Desta forma, pesquisas envolvendo métodos analíticos são de fundamental importância e altamente relevantes para otimizar sua análise na indústria farmacêutica e garantir a qualidade do produto já comercializado. Neste trabalho foram desenvolvidas e validadas técnicas de análise para cefuroxima sódica forma farmacêutica injetável: (i) espectrofotometria na região do UV a 280,0 nm com faixa de concentração 5,0 a 14,0 μg/mL, utilizando água como solvente, com exatidão de 100,82% e teor de 99,49%; (ii) doseamento microbiológico, método de difusão em ágar na faixa de concentração de 30,0 a 120,0 μg/mL, utilizando Micrococcus luteus ATCC 9341, com exatidão de 100,77% e teor de 99,96%; (iii) doseamento microbiológico, método turbidimétrico na faixa de concentração de 30,0 a 120,0 μg/mL, utilizando Micrococcus luteus ATCC 9341, com exatidão 100,21% e teor de 99,97%; (iv) espectrofotometria na região do visível a 510,0 nm, com concentração de 100,0 a 300,0 μg/mL utilizando o-fenantrolina como ligante, com exatidão de 99,98% e teor de 99,82%; (v) espectrofotometria na região do IV, com exatidão de 99,83% e teor de 100,25%; (vi) acidimetria, com exatidão de 100,32% e teor de 100,51%; (vii) volumetria em meio nãoaquoso, com exatidão de 100,40% e teor de 100,86%; (viii) iodometria, com exatidão de 99,97% e teor de 100,27%; (ix) cromatografia líquida de alta eficiência com detector UV a 280,0 nm, utilizando metanol:água (70:30) como fase... / Cefuroxime sodium (CAS 56238-63-2) is a second generation cephalosporin indicated in the infections caused by Gram-positive and Gram-negative microorganisms. Although this drug highly to be studied and to be searched with respect to the antimicobial pharmacokinetics and pharmacodynamics activity, there are few studies in relation to the development of analytical methodology for this cephalosporin in literature. In such a way, research involving highly excellent analytical methods is very important to optimize its analysis in the pharmaceutical industry and to guarantee the commercialized product quality. In this work, analytical methods for determination of cefuroxime injectable were validated: (i) UV spectrophotometry at 280 nm with concentration range of 5.0 a 14.0 μg/mL using water as solvent, with accuracy of 100.8% and quantitation of 99.49%; (ii) microbiological assay, agar diffusion method and (iii) turbidimetric method at concentration range 30.0 to 120.0 μg/mL, using Micrococcus luteus ATCC 9341 as indicator microorganism, accuracy 100.77%, quantitation of 99.96% and accuracy of 100.21% and quantitation 99.97%, respectively; (iv) visible spectrophotometric method at 510.0 nm with concentration range of 100.0 at 300.0 μg/mL, using o-phenantrolin as reagent, with accuracy of 99.98% and quantitation of 99.82%; (v) IR spectrophotometry, accuracy of 99.83% and quantitation of 100.25%; (vi) acidimetry, accuracy of 100.32% and quantitation of 100.51%; (vii) volumetry ina a non aqueous, accuracy of 100.40% and quantitation of 100.86%; (viii) iodometry accuracy of 99.97% and quantitation of 100.27%; (ix) HPLC method with UV detector at 280.0 nm using methanol and water (70:30), v/v) as mobile phase and concentration range of 10.0 a 15.0 μg/mL, flow of 0.8 ml/min, accuracy of 100.10% and quantitation of 99.84% and mean retention time of 1.8 minutes. Preliminary study of sodium cefuroxime...(Complete abstract click electronic access below)

Master of Science / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / African swine fever is a hemorrhagic disease of domestic pigs caused by African swine fever virus (ASFV), a double-stranded DNA virus and the only member of the family Asfarviridae. The structure of this multilayer virion contains more than 34 proteins including the protein p72 which is the major capsid protein. A single conformational neutralizing epitope has been identified on p72, but information on the other antigenic regions (epitopes) is lacking. The objective of this study was to identify p72 epitopes using polyclonal swine sera and a panel of monoclonal antibodies with the ultimate goal being the development of a blocking ELISA assay for the detection of anti-ASFV antibodies. The segment of the p72 protein from amino acids 1 to 345 was divided into five overlapping fragments which were then commercially synthesized. These fragments were cloned into the pHUE expression vector and transformed into Escherichia coli competent cells. The recombinant proteins were expressed in vitro, purified, and used as antigens in indirect ELISAs and western blots to test monoclonal antibodies and polyclonal swine sera. The monoclonal antibodies were produced against the p72 protein based on the ASFV Georgia/07 strain. The polyclonal sera were obtained from pigs immunized with a defective alphavirus replicon particle, RP-sHA-p72, expressing a recombinant protein composed of the extracellular domain of the ASFV HA protein together with the whole p72 protein. The polyclonal sera reacted to p72 in two distinct regions: between amino acids 1 and 83 and between amino acids 250 and 280. The anti-p72 reactive monoclonal antibodies reacted with p72 in three regions: between amino acids 100 and 171, amino acids 180 and 250, and amino acids 280 and 345. Fine mapping with oligopeptides allowed for the identification of six different linear epitopes. Among the monoclonal antibodies selected for blocking assay development, two have been shown to be promising candidates for further evaluation using sera from ASFV-infected pigs.

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Resumo: A cefuroxima sódica (CAS 56238-63-2) é uma cefalosporina de segunda geração, indicada nas infecções provocadas por micro-organismos Gram-positivos e Gram-negativos. Apesar deste fármaco ser altamente estudado e pesquisado no que concerne à atividade antimicrobiana, farmacocinética e farmacodinâmica, há poucos estudos na literatura em relação ao desenvolvimento de metodologia analítica para esta cefalosporina. Desta forma, pesquisas envolvendo métodos analíticos são de fundamental importância e altamente relevantes para otimizar sua análise na indústria farmacêutica e garantir a qualidade do produto já comercializado. Neste trabalho foram desenvolvidas e validadas técnicas de análise para cefuroxima sódica forma farmacêutica injetável: (i) espectrofotometria na região do UV a 280,0 nm com faixa de concentração 5,0 a 14,0 μg/mL, utilizando água como solvente, com exatidão de 100,82% e teor de 99,49%; (ii) doseamento microbiológico, método de difusão em ágar na faixa de concentração de 30,0 a 120,0 μg/mL, utilizando Micrococcus luteus ATCC 9341, com exatidão de 100,77% e teor de 99,96%; (iii) doseamento microbiológico, método turbidimétrico na faixa de concentração de 30,0 a 120,0 μg/mL, utilizando Micrococcus luteus ATCC 9341, com exatidão 100,21% e teor de 99,97%; (iv) espectrofotometria na região do visível a 510,0 nm, com concentração de 100,0 a 300,0 μg/mL utilizando o-fenantrolina como ligante, com exatidão de 99,98% e teor de 99,82%; (v) espectrofotometria na região do IV, com exatidão de 99,83% e teor de 100,25%; (vi) acidimetria, com exatidão de 100,32% e teor de 100,51%; (vii) volumetria em meio nãoaquoso, com exatidão de 100,40% e teor de 100,86%; (viii) iodometria, com exatidão de 99,97% e teor de 100,27%; (ix) cromatografia líquida de alta eficiência com detector UV a 280,0 nm, utilizando metanol:água (70:30) como fase... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Cefuroxime sodium (CAS 56238-63-2) is a second generation cephalosporin indicated in the infections caused by Gram-positive and Gram-negative microorganisms. Although this drug highly to be studied and to be searched with respect to the antimicobial pharmacokinetics and pharmacodynamics activity, there are few studies in relation to the development of analytical methodology for this cephalosporin in literature. In such a way, research involving highly excellent analytical methods is very important to optimize its analysis in the pharmaceutical industry and to guarantee the commercialized product quality. In this work, analytical methods for determination of cefuroxime injectable were validated: (i) UV spectrophotometry at 280 nm with concentration range of 5.0 a 14.0 μg/mL using water as solvent, with accuracy of 100.8% and quantitation of 99.49%; (ii) microbiological assay, agar diffusion method and (iii) turbidimetric method at concentration range 30.0 to 120.0 μg/mL, using Micrococcus luteus ATCC 9341 as indicator microorganism, accuracy 100.77%, quantitation of 99.96% and accuracy of 100.21% and quantitation 99.97%, respectively; (iv) visible spectrophotometric method at 510.0 nm with concentration range of 100.0 at 300.0 μg/mL, using o-phenantrolin as reagent, with accuracy of 99.98% and quantitation of 99.82%; (v) IR spectrophotometry, accuracy of 99.83% and quantitation of 100.25%; (vi) acidimetry, accuracy of 100.32% and quantitation of 100.51%; (vii) volumetry ina a non aqueous, accuracy of 100.40% and quantitation of 100.86%; (viii) iodometry accuracy of 99.97% and quantitation of 100.27%; (ix) HPLC method with UV detector at 280.0 nm using methanol and water (70:30), v/v) as mobile phase and concentration range of 10.0 a 15.0 μg/mL, flow of 0.8 ml/min, accuracy of 100.10% and quantitation of 99.84% and mean retention time of 1.8 minutes. Preliminary study of sodium cefuroxime...(Complete abstract click electronic access below) / Orientador: Hérida Regina Nunes Salgado / Coorientador: Magali Benjamim de Araújo / Banca: Maria Virginia Costa Scarpa / Banca: Taís Maria Bauab / Banca: Pierina Sueli Bonato / Banca: Maria José Vieira Fonseca / Doutor

Orientador: Wilma Aparecida Starke Buzetti / Banca: Edson Guilherme Vieira / Banca: Willian Marinho Dourado Coelho / Resumo: A leishmaniose visceral (LV), doença causada pelo protozoário Leishmania infantum e transmitida pelo flebotomíneo Lutzomyia longipalpis, é considerada um problema de saúde pública no Brasil. O objetivo deste trabalho foi realizar um estudo epidemiológico sobre a ocorrência da LV em cães e gatos e de flebotomíneos em dois abrigos de animais, durante o período de um ano, no município de Ilha Solteira, SP. Amostras de material biológico foram coletadas de 192 cães e de 197 gatos, para realização de exames sorológicos por meio do ensaio imunoenzimático indireto (ELISA), da reação de imunofluorescência indireta (RIFI) para ambas espécies animais e da reação intradérmica de Montenegro para cães. Dos cães examinados, 17,2% (33/192) estavam sorologicamente positivos para LV pelo método ELISA, 19,8% (38/192) pela RIFI e 45,4% (15/33) pela reação intradérmica. Para cães, a concordância entre as técnicas sorológicas foi classificada de razoável a boa, mas quando comparadas à reação de Montenegro foi considerada ruim. No inquérito felino das 197 amostras, a soroprevalência foi de 32,4% (64/197) e 31,9% (63/197) no ELISA e RIFI, respectivamente. Embora a maioria dos cães e dos gatos entregue nos abrigos já estivesse infectada, seis cães e cinco gatos infectaram-se no interior desses abrigos, após algum tempo de permanência nesses recintos. Um total de 131 flebotomíneos foram capturados por meio das armadilhas luminosas, colocadas no interior dos dois abrigos durante o período de dois anos. Os fatores de riscos associados à LV foram determinados estatisticamente pela análise univariada, com valor significativo (p ≤ 0,05), onde o porte pequeno dos cães, o uso de coleira de deltametrina e o diagnóstico precoce foram variáveis determinantes que influenciaram a positividade dos animais, ou seja, as duas últimas variáveis determinaram significativamente na diminuição de casos positivos da doença nos... / Abstract: Visceral leishmaniasis (VL), a disease caused by Leishmania infantum and transmitted by the sand fly Lutzomyia longipalpis, is considered a public health problem in Brazil. The study aimed to carry out an epidemiological study on the occurrence of VL in dogs and cats, and sand flies during the period of one year in two animal shelters from Ilha Solteira, SP. A total of 197 and 192 biological material samples were collected, respectively from cats and dogs for serological survey by an indirect enzyme-linked immunosorbent assay (ELISA) and an indirect fluorescent antibody test (IFAT) for both species of animals, and the intradermal reaction of Montenegro (only for dogs). During the study with dogs, 17,2% (33/192) were serologically positive for LV by ELISA, 19,8% (38/192) by IFAT and 45,4% (15/33) by the intradermal method. The correlation analysis between the serological techniques was ranged from reasonable to good for dogs, but was bad when compared to the Montenegro reaction. The feline seroprevalence was 32,4% (64/197) and 31,9% (63/197) for ELISA and IFAT, respectively. Although the most of dogs and cats in shelters were infected, six dogs and five cats infected inside the shelters. A total of 131 sand flies were captured by the light traps in both shelters, during two years. The risk factors, statistically determined by univariate analysis (p ≤ 0.05), demonstrated that the small size of the dogs, the use of deltamethrin collar and the precocious diagnosis, significantly influenced the animals positivity for VL. It was concluded that these animal shelters were vulnerable to this parasitic infection because of the presence of sand flies and positive animals for LV / Mestre

Plants have been utilized as a source of medicines since ancient times. They contain a vast range of secondary metabolites which play important roles in different diseases. The Scope of this study is to define the function of secondary metabolites from Andrographis paniculata (Kalmegh) and Silybum marianum (Milk Thistle) against DNA damage, which initiates many diseases. Sequential extraction of both plant materials was performed with different polarity solvents. Qualitative analysis was performed with Gas (GC) and High Performance Liquid Chromatography (HPLC). Primary extracts screening studies were performed against cytotoxicity, antioxidant and soluble collagen assays (Sircol dye). Further bioactivity was confirmed using the single-cell gel electrophoresis (Comet assay) to estimate levels of DNA damage. Fourier Transform Infrared analysis of bioactive extracts was performed to identify the functional groups present in them . Subsequently, bioactive extracts were further separated into acid, base, phenol and neutral fractions. These fractions and bioactive extracts were screened with the free radical assays to identify the scavenging activity. Chemical mapping of the bioactive fraction was performed with High Performance Liquid Chromatography (HPLC). Preparative-HPLC was performed to separate the compounds which were present in the bioactive fractions. MTT assay, Hydroxyl radical and nitric oxide radical scavenging activity assays were performed to screen the fractions and DNA protective activity of the bioactive fractions was confirmed with single-cell gel electrophoresis. These bioactive fractions were de-replicated with hyphenated techniques like LCMS and LCMS/MS to identify the molecular weight of the compounds and Quadrupole time-of-flight mass spectrometry was performed to identify the accurate mass of the compounds. Sequential extraction separates the non-polar and polar compounds present in the plant material. Qualitative analysis confirmed the presence of fatty acids in the non-polar extracts of both plants using GC and the presence of standard constituents in the polar extracts of both plants using HPLC. It also helps in chemical mapping of the extracts. Acetone, Methanol1 and Methanol2 extracts from either plant are non-cytotoxic. The high antioxidant activity is observed in methanol extracts from Andrographis paniculata and in acetone/methanol2 extracts from Silybum marianum. Extracts that protect against UVA and UVB damage also increased soluble collagen production in Human Dermal Fibroblast (HDF) in culture. Primary Screening helped to select six extracts out of twelve extracts for further analysis. Comet assay confirmed DNA protective activity in Methanol1 extract of Milk thistle and Acetone, Methanol2 extracts from Kalmegh. These three extracts were further fractionated into 38 fractions out of which three fractions that are F1, F13 and F31 fractions confirm the DNA protection activity. De-replication of the bioactive fractions was performed with LC-ESI-MS/MS which confirm twenty one compounds and accurate mass of fifteen compounds was determined using Q-tof mass spectrometry.

abstract: Cancer claims hundreds of thousands of lives every year in US alone. Finding ways for early detection of cancer onset is crucial for better management and treatment of cancer. Thus, biomarkers especially protein biomarkers, being the functional units which reflect dynamic physiological changes, need to be discovered. Though important, there are only a few approved protein cancer biomarkers till date. To accelerate this process, fast, comprehensive and affordable assays are required which can be applied to large population studies. For this, these assays should be able to comprehensively characterize and explore the molecular diversity of nominally "single" proteins across populations. This information is usually unavailable with commonly used immunoassays such as ELISA (enzyme linked immunosorbent assay) which either ignore protein microheterogeneity, or are confounded by it. To this end, mass spectrometric immuno assays (MSIA) for three different human plasma proteins have been developed. These proteins viz. IGF-1, hemopexin and tetranectin have been found in reported literature to show correlations with many diseases along with several carcinomas. Developed assays were used to extract entire proteins from plasma samples and subsequently analyzed on mass spectrometric platforms. Matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometric techniques where used due to their availability and suitability for the analysis. This resulted in visibility of different structural forms of these proteins showing their structural micro-heterogeneity which is invisible to commonly used immunoassays. These assays are fast, comprehensive and can be applied in large sample studies to analyze proteins for biomarker discovery. / Dissertation/Thesis / M.S. Biochemistry 2012