Biomarker validation remains one of the most important constraints to development of new clinical diagnostic assays. To address this challenge, an immuno-mass spectrometric assay known as SISCAPA has been developed for quantitation of protein biomarkers in human blood. The SISCAPA assay overcomes the sensitivity barrier facing most mass spectrometric approaches by utilizing high affinity antibodies for enrichment of specific surrogate peptide analytes from complex mixtures such as trypsin-digested human plasma. However, several technological barriers remain before the SISCAPA technology gains widespread use for biomarker validation. Improvements are required in areas such as selection of high affinity anti-peptide antibodies, peptide detection sensitivity and increasing sample throughput to allow biomarker validation on large sample sets. The work presented in this dissertation describes the development of new methods for antibody selection and for high-throughput application of SISCAPA technology to biomarker measurement in human plasma. Specifically, two technological developments are described: 1) an assay called MiSCREEN was developed, which allows high-throughput screening of anti-peptide antibodies, enabling selection of high affinity reagents for de novo SISCAPA assays and 2) a liquid chromatography (LC)-free SISCAPA assay was developed that enables quantitation of surrogate peptides using both MALDI-TOF and RapidFire/MS platforms. Taken together, these technological advances provide a meaningful solution to the biomarker validation dilemma and allow a unified system for biomarker qualification, verification, validation and development of clinical assays for diagnosis and monitoring of a variety of diseases. To demonstrate the utility of the unified SISCAPA system for biomarker measurement, an assay was developed for protein C inhibitor (PCI) as a marker for prediction of biochemical recurrence in prostate cancer patients. The PCI-specific analyte was shown to predict biochemical recurrence of prostate cancer after radiation/hormone treatment. Early stage detection of recurrence was achieved, when compared to the ‘gold standard’ marker for prostate cancer, prostate specific antigen (PSA). Two-dimensional gel electrophoresis studies on PCI, revealed unique protein spots in a serum sample from a biochemically recurrent patient. Studying such alterations at the protein level may enable understanding of the molecular mechanisms by which PCI is involved in prostate cancer progression. / Graduate

Several obstacles to widespread use of bacteriocins in food have been identified, including lack of specific, rapid quantitation methods, and little data on their efficacy in food systems. The first objective of this study was to develop a specific, rapid quantitation method for bacteriocins that did not rely on bioassays and their associated limitations. Phage display was chosen to reduce reliance on continued use of animals and produce antibodies to the bacteriocins leucocin A, piscicolin 126, and brochocin-A. Although the antibody libraries generated by phage display were not successful for antibody production, a strong immune response to leucocin A and piscicolin 126 was observed in mice. The second objective of the study was to determine the efficacy of brochocin-C against Clostridium botulinum in a model system and in a vacuum-packaged, chopped and formed pork product stored at refrigeration temperature. Group I Cl. botulinum was not controlled by brochocin-C, and was found to inactivate brochocin-C and several class IIa bacteriocins by proteolysis. Cell counts revealed that Group II Cl. botulinum was controlled by brochocin-C in a model meat system, but was not controlled in the chopped and formed pork product. Powdered smoke and NaCl in the pork product had a synergistic interaction against Group II Cl. botulinum, as shown by minimum inhibitory concentration testing. The choice of media for isolation of Cl. botulinum from the chopped and formed pork product was important, as the presence of background microflora isolated from the meat was found to impact growth of Group II Cl. botulinum on plating media. In the presence of the background microflora, which were identified by 16S rDNA sequencing as carnobacteria and staphylococci, inclusion of phosphate in the plating medium was found to allow growth of Cl. botulinum. Other nutrients such as magnesium, sulphur, or increased protein sources added to the medium had no effect on growth of Cl. botulinum. Two of the background microflora strains, Carnobacterium maltaromaticum MH3 and Staphylococcus pasteuri EIV-21, inhibited Cl. botulinum, while one strain, C. maltaromaticum MH2, stimulated growth of Cl. botulinum. / Food Science and Technology

Doctor of Philosophy / Department of Statistics / Christopher Vahl / Veterinary drugs are used extensively for disease prevention and treatment in food producing animals. The residues of these drugs and their metabolites can pose risks for human health. Therefore, a withdrawal time is established to ensure consumer safety so that tissue, milk or eggs from treated animals cannot be harvested for human consumption until enough time has elapsed for the residue levels to decrease to safe concentrations. Part of the process to establish a withdrawal time involves a linear regression to model drug residue depletion over time. This regression model is used to calculate a one-sided, upper tolerance limit for the amount of drug residue remaining in target tissue as a function of time. The withdrawal period is then determined by finding the smallest time so that the upper tolerance limit falls below the maximum residue limit. Observations with measured residue levels at or below the limit of quantitation (LOQ) of the analytical method present a special challenge in the estimation of the tolerance limit. Because values observed below the LOQ are thought to be unreliable, they add in an additional source of uncertainty and, if dealt with improperly or ignored, can introduce bias in the estimation of the withdrawal time. The U.S. Food and Drug Administration (FDA) suggests excluding such data while the European Medicine Agency (EMA) recommends replacing observations below the LOQ with a fixed number, specifically half the value of the LOQ. However, observations below LOQ are technically left censored and these methods are do not effectively address this fact. As an alternative, a regression method accounting for left-censoring is proposed and implemented in order to adequately model residue depletion over time. Furthermore, a method based on generalized (or fiducial) inference is developed to compute a tolerance limit with results from the proposed regression method. A simulation study is then conducted to compare the proposed withdrawal time calculation procedure to the current FDA and EMA approaches. Finally, the proposed procedures are applied to real experimental data.

Ebastine is a second generation antihistaminic drug used to treat allergic rhinitis and urticaria. It is available in the Brazilian market as tablets and syrup. There are no official methods for ebastine analysis in pharmaceutical formulations. In the present work, methods for quantification and dissolution evaluation of the drug in tablets were developed and validated. Spectrophotometric and liquid chromatographic (LC) methods were used for drug determination. In the spectrophotometric method ebastine can be quantified at 258 nm, using acetonitrile and 0.01M HCl as diluents. The LC analysis were performed on a C18 column maintained at ambient temperature. The mobile phase composed of acetonitrile:phosphoric acid 0.1% pH 3.0 (55:45, v/v), was run at a flow rate of 1.2 mL/min with UV detection at 254 nm. The methods showed good linearity (r>0.999), precision (RSD<2%), and accuracy. The proposed methods were applied for the analysis of pharmaceutical products, showing significant correlation (P>0.05) of the results. The optimization of dissolution test conditions for in vitro quality control of ebastine in tablets was evaluated. The use of 900 mL of 0.01M HCl at 37.0 ± 0.5°C, paddle as apparatus at 75 rpm and 60 minutes of test provided satisfactory results for tested product. The LC method was validated to evaluate dissolution testing and showed to be specific, linear, precise and accurate. The sample tablets containing ebastine were subjected to accelerated stability study under conditions of controlled temperature and relative humidity (40 °C ± 2 °C e 75% ± 5 %, respectively) for six months and then evaluated by LC and mass spectrometry. / A ebastina é um fármaco anti-histamínico de segunda geração utilizado para tratamento de rinite alérgica e urticária. No mercado brasileiro encontra-se disponível na forma de comprimidos e solução oral. Não há monografias descritas em farmacopéias para análise de ebastina em formulações farmacêuticas. No presente trabalho, métodos para quantificação e avaliação da dissolução do fármaco em comprimidos foram desenvolvidos e validados. Os métodos usados para a quantificação do fármaco foram a espectrofotometria no ultravioleta e a cromatografia líquida de alta eficiência (CL). No método espectrofotométrico a ebastina pode ser quantificada em 258 nm, utilizando acetonitrila e HCl 0,01M como diluentes. As análises por CL foram realizadas em coluna C18 mantida a temperatura ambiente. A fase móvel, composta de acetonitrila: ácido fosfórico 0,1% pH 3,0 (55:45, v/v), foi eluída isocraticamente com vazão de 1,2 mL/min com detecção no ultravioleta em 254 nm. Os métodos mostraram boa linearidade (r>0,999), precisão (DPR<2%) e exatidão. Os métodos propostos foram aplicados na análise de produtos farmacêuticos, demonstrando correlação significativa dos resultados (p>0,05). A otimização das condições para o teste de dissolução in vitro para ebastina comprimidos foi avaliada. As condições que forneceram resultados satisfatórios para os produtos testados foram 900 mL de ácido clorídrico 0,01M a 37,0 ± 0,5 ºC, aparato pá, 75 rpm e 60 minutos de teste. O método por CL foi validado para quantificação das amostras do teste de dissolução e mostrou ser específico, linear, preciso e exato. Amostras de comprimidos contendo ebastina foram submetidas a estudo de estabilidade acelerada sob condições controladas de temperatura e umidade relativa (40 °C ± 2 °C e 75% ± 5%, respectivamente) por seis meses e posteriormente avaliadas por cromatografia líquida e espectrometria de massa.

abstract: Biological fluids, in particular blood plasma, provide a vital source of information on the state of human health. While specific detection of biomarker species can aid in disease diagnostics, the complexity of plasma makes analysis challenging. Despite the challenge of complex sample analysis, biomarker quantification has become a primary interest in biomedical analysis. Due to the extremely specific interaction between antibody and analyte, immunoassays are attractive for the analysis of these samples and have gained popularity since their initial introduction several decades ago. Current limitations to diagnostics through blood testing include long incubation times, interference from non-specific binding, and the requirement for specialized instrumentation and personnel. Optimizing the features of immunoassay for diagnostic testing and biomarker quantification would enable early and accurate detection of disease and afford rapid intervention, potentially improving patient outcomes. Improving the limit of quantitation for immunoassay has been the primary goal of many diverse experimental platforms. While the ability to accurately quantify low abundance species in a complex biological sample is of the utmost importance in diagnostic testing, models illustrating experimental limitations have relied on mathematical fittings, which cannot be directly related to finite analytical limits or fundamental relationships. By creating models based on the law of mass action, it is demonstrated that fundamental limitations are imposed by molecular shot noise, creating a finite statistical limitation to quantitative abilities. Regardless of sample volume, 131 molecules are necessary for quantitation to take place with acceptable levels of uncertainty. Understanding the fundamental limitations of the technique can aid in the design of immunoassay platforms, and assess progress toward the development of optimal diagnostic testing. A sandwich-type immunoassay was developed and tested on three separate human protein targets: myoglobin, heart-type fatty acid binding protein, and cardiac troponin I, achieving superior limits of quantitation approaching ultimate limitations. Furthermore, this approach is compatible with upstream sample separation methods, enabling the isolation of target molecules from a complex biological sample. Isolation of target species prior to analysis allows for the multiplex detection of biomarker panels in a microscale device, making the full optimization of immunoassay techniques possible for clinical diagnostics. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2015

Os sistemas agroindustriais são estruturas complexas que são influenciadas pelas transformações econômicas, políticas e tecnológicas da sociedade. Assim, as organizações públicas e privadas que estão inseridas nesses sistemas precisam de informações setoriais que as permitam identificar cenários futuros e ajustar seus recursos internos às novas situações geradas pelo ambiente externo. Uma das alternativas para dispor dessa informação é a aplicação de ferramentas de projeção por meio da simulação de cenários. Este trabalho, nesse sentido, tem como foco principal a simulação de cenários para o setor sucroenergético brasileiro a partir do método de mapeamento e quantificação de sistemas agroindustriais. Para atingi- lo, foi aplicada uma pesquisa exploratória e qualitativa, cuja execução foi divida em três fases. A primeira considerou o desenho do sistema agroindustrial da cana-de-açúcar no Brasil, identificando os seus principais setores e variáveis participantes; a segunda compreendeu a quantificação do sistema, estimando, a partir de relações entre as variáveis, os fluxos comerciais dos elos de insumos agrícolas, produção de matéria-prima, insumos industriais e processamento industrial, no ano fiscal de 2008; e, finalmente, na terceira fase foi feita uma simulação de cenários para o período 2011-2015 com base em projeções de mercado e uso de ferramenta eletrônica. A coleta de dados foi realizada por meio de dados primários e secundários, levantados em entrevistas com executivos, pesquisadores, instituições governamentais e organizações setoriais dos diferentes elos do sistema agroindustrial, além de publicações como anuários estatísticos, relatórios, prognósticos, entre outros, elaborados por instituições governamentais e não governamentais. Já o processamento e simulação de cenários foram feitos com a utilização de um software de planilha eletrônica. Como resultados desta pesquisa, portanto, foram apresentadas as etapas necessárias para desenhar, quantificar e simular cenários para o setor sucroenergético, assim como os valores obtidos nos cálculos, que permitirá, em pesquisas futuras, explorar o aperfeiçoamento do modelo de simulação por meio da inserção de novas variáveis e da atualização dos valores atribuídos a elas. / Agroindustrial systems are complex structures that are subject to economic, political and technological changes within society. Therefore, the organization held within these systems must frequently adjust their specific resources to the situations created by the environment. One of the alternatives to identify opportunities and threats to the systems is the application of tools for exploring the future through scenario analysis. Thus, this research focuses on simulating scenarios for the Brazilian Sugarcane Agroindustrial System through the application of the Method for Mapping and Quantifying Agroindustrial Systems. In order to achieve this objective, an exploratory and qualitative research has been made in three phases. The first phase has considered the structure of the Sugarcane Agroindustrial System in Bra zil, identifying their key sectors and variables; the second one has addressed the quantification of the system, estimating the trade flows between farm input suppliers, sugarcane producers, industrial input supplier and sugarcane mills in 2008; and finally, in the third phase a scenario simulation has been done for the period of 2011-2015 based on market projections with the use of an electronic tool. Both primary and secondary data have been used. Primary data have been collected thought interviews with executives, researchers, governmental institutions and industrial organizations representatives, while secondary data have been gathered from publications such as statistical reports and prognostics from private and governmental institutions. The data processing and the scenario simulations have been done by using an electronic spreadsheet software. The results of the research show the necessary stages for drawing the systems structure, quantifying the trade flows between its links and simulating scenarios for the Sugarcane Agroindustrial System. They also show the values obtained from the calculations, which allows the further improvement of the simulation model in future research by updating the values given to the variables as well as by inserting new var iables.

A specific method has been developed for the extraction and measurement of staphylococcal nuclease in cheese in which Staphylococcus aureus has grown. Ten grams of cheese sample were homogenized with ninety milliliters of pH ten buffer for three minutes. Ammonium sulfate fractionation was used and a forty to eighty percent fraction was collected and concentrated using ultrafilters. The nuclease activity was determined using a toluidine blue deoxyribonucleic acid agar slide method and a spectophotometric method. The DNA agar slide method was used to compare staphylococcal growth with nuclease production in cheese under varying conditions. When Staphylococcus aureus plate counts indicated populations of three to four thousand per milliliter, it was possible to detect nuclease in the cheese sample. A method has also been developed to detect Staphylococcus aureus colonies using DNase agar and toluidine blue, utilizing the heat stability of Staphylococcus aureus nuclease.

No / Mannitol is a polymorphic pharmaceutical excipient, which commonly exists in three forms: alpha, beta and delta. Each polymorph has a needle-like morphology, which can give preferred orientation effects when analysed by X-ray powder diffractometry (XRPD) thus providing difficulties for quantitative XRPD assessments. The occurrence of preferred orientation may be demonstrated by sample rotation and the consequent effects on X-ray data can be minimised by reducing the particle size. Using two particle size ranges (<125 and 125¿500 ¿m), binary mixtures of beta and delta mannitol were prepared and the delta component was quantified. Samples were assayed in either a static or rotating sampling accessory. Rotation and reducing the particle size range to <125 ¿m halved the limits of detection and quantitation to 1 and 3.6%, respectively. Numerous potential sources of assay errors were investigated; sample packing and mixing errors contributed the greatest source of variation. However, the rotation of samples for both particle size ranges reduced the majority of assay errors examined. This study shows that coupling sample rotation with a particle size reduction minimises preferred orientation effects on assay accuracy, allowing discrimination of two very similar polymorphs at around the 1% level.

In the last two decades, protein drugs have enjoyed a rapid growth and achieved a tremendous success in treating human diseases. However, the presence of physiological barriers greatly impedes the efficient delivery of such unconventional large molecule drugs, and therefore limits their clinical utility. An elegant way to address this challenge takes advantage of certain endogenous transporter proteins, such as human transferrin (Tf), whose ability to traverse physiological barriers has been extensively exploited. However, methods to investigate Tf-based drug delivery remained insufficient and unsatisfactory until recent development of quantitative mass spectrometry (MS). Hereby, MS-based methods have been developed and validated for quantitation of exogenous Tf in biological fluids. Particularly, different O18-labeling based approaches have been evaluated, modified and developed in this work, in order to achieve the most reliable quantitation. Alternatively, a novel approach based on indium labeling and inductively coupled plasma mass spectrometry (ICP-MS) detection has been developed for sensitive quantitation of Tf in biological fluids. The second aspect of this dissertation work focuses on the application of MS-based methods for characterization of protein drugs at different levels, ranging from protein identification, covalent structure, conformation, and interaction with physiological partners. Particularly, an O18-labeling assisted approach has been developed to identification of protein deamidation products. This new approach can readily distinguish between the two deamidated isomers. Also, an LC-MS based method has been developed for ranking the susceptibility of protein disulfide bonds to reduction, which could be applied to several disulfide bond-related analyses. Finally, a recently designed growth hormone transferrin fusion protein was studied using MS-based methods, and the molecular basis for its successful oral delivery was revealed.

Fast, sensitive, quantitative, and low-cost analysis of environmental pollutants is highly valuable for environmental monitoring. Due to its single-molecule sensitivity and fingerprint specificity, surface-enhanced Raman spectroscopy (SERS) has been widely employed for heavy metal, organic compound, and pathogen detection. However, SERS quantitation is challenging because 1) analytes do not stay in the strongest enhancing region ("hot spots") and 2) SERS reproducibility is poor. In this dissertation, gold nanoparticle/bacterial cellulose (AuNP/BC) substrates were developed to improve SERS sensitivity by increasing hot spot density within the laser excitation volume. Environmentally relevant organic amines were fixed at "hot spots" by lowering solution pH below the analyte pKa and thus enabling SERS quantitation. In addition, a new SERS internal standard was developed based upon the electromagnetic enhancement mechanism that relates Rayleigh (elastic) and Raman (in-elastic) scattering. Rayleigh scattering arising from the amplified spontaneous emission of the excitation laser was employed as a normalization factor to minimize the inherent SERS signal variation caused by the heterogeneous distribution of "hot spots" across a SERS substrate. This highly novel technique, hot spot-normalized SERS (HSNSERS), was subsequently applied to evaluate the efficiency of SERS substrates, provide in situ monitoring of ligand exchange kinetics on the AuNP surface, and to reveal the relationship between the pKa of aromatic amines and their affinity to citrate-coated AuNPs (cit-AuNPs). Finally, colloidally stable stable pH nanoprobes were synthesized using co-solvent mediated AuNP aggregation and subsequent coating of poly(ethylene) glycol (PEG). These nanoprobes were applied for pH detection in cancer cells and in phosphate buffered aerosol droplets. The latter experiments suggest that stable pH gradients exist in aerosol droplets. / PHD