Orientador: Reinaldo Marchetto / Coorientador: Edson Crusca Junior / Banca: Saulo Santesso Garrido / Banca: José Luiz de Souza Lopes / Resumo: O peptídeo antimicrobiano Pantinina-3 (P3) possui 13 resíduos e é derivado do veneno do escorpião africano Pandinus imperator. Ele apresenta alta atividade antimicrobiana contra bactérias Gram-positivas, principalmente Enterococcus resistente à vancomicina (VRE-S13) in vitro. Entretanto, este peptídeo também possui atividade hemolítica. Neste contexto, foram desenhados e sintetizados cinco análogos de P3 a partir de substituições pontuais de resíduos específicos por um resíduo de lisina, os quais foram denominados L2K, I5K, N7K, I9K e L13K. O objetivo da criação destes análogos foi estudar a relação de estrutura e atividade do peptídeo P3, a fim de investigar o efeito da modificação de suas características biofísicas em sua atividade biológica. Para isso, os peptídeos foram sintetizados por meio da técnica de síntese em fase sólida (SPFS), purificados por cromatografia líquida de alta eficiência em coluna de fase reversa (CLAE-FR) e identificados por espectrometria de massas (SM). Os peptídeos purificados foram testados quanto à sua atividade biológica e foi verificado que o análogo L2K apresentou atividade somente para a bactéria Gram-negativa testada, o análogo N7K mostrou ser mais ativo que P3 tanto para Gram-positivas quanto para Gram-negativas e os outros análogos não apresentaram atividade antimicrobiana. Foi avaliada também a atividade hemolítica dos peptídeos, e foi verificado que o único análogo que apresentou atividade contra eritrócitos foi N7K. A hidrofobicidade d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The antimicrobial peptide Pantinin-3 (P3) has 13 residues and is derived from the venom of the African scorpion Pandinus imperator. It has high antimicrobial activity against Gram-positive bacteria, mainly vancomycin-resistant Enterococcus (VRE-S13) in vitro. However, this peptide also has hemolytic activity. In this context, five P3 analogs were designed and synthesized from point substitutions of specific residues by a lysine residue, which were named L2K, I5K, N7K, I9K and L13K. The objective of the creation of these analogues was to study the structure and activity relationship of the P3 peptide in order to investigate the effect of the modification of its biophysical characteristics on its biological activity. For this, the peptides were synthesized using the solid-phase peptide synthesis technique (SPPS), purified by high performance liquid chromatography on a reverse phase column (HPLC-RF) and identified by mass spectrometry (MS). The purified peptides were tested for their biological activity and it was found that the L2K analog showed activity only for the Gram-negative bacteria tested, the N7K analog showed to be more active than P3 for both Gram-positive and Gram-negative and the others did not present antimicrobial activity. The hemolytic activity of the peptides was also evaluated, and it was verified that the only analog that showed activity against erythrocytes was N7K. The hydrophobicity of the peptides was analyzed by the retention time obtained by HPLC-RF an... (Complete abstract click electronic access below) / Mestre

Malaria is a serious disease that can lead to fatal consequences if not treated. It is mainly spread via Plasmodium falciparum, a parasite carried by mosquitoes as host organisms. As a potential way of treating malaria, research is being done on possible inhibitors of calmodulin (CaM) in the parasite. CaM is a highly conserved protein found in all eukaryotes, and is important in many essential biochemical reactions. The potential inhibitor analysed in this study is calmidazolium (CZM). This study aims to characterise structure and stability differences between the C-lobes of human and P. falciparum CaM, while analysing the effect of the presence of CZM.  Previous studies have proven that CZM acts as an inhibitor to human CaM by binding to the C-lobe, with a dissociation constant in the nano molar range. In other studies, thermal stability measurements have shown that the secondary structure of P. falciparum CaM is more stable than that of human CaM.  In this study, the stability measurements showed that for the ANS binding site and around tyrosines, the C-lobe of human CaM was more stable than the C-lobe of P. falciparum CaM, knowledge which was previously unknown. When studying the entire secondary structure, the C-lobe of P. falciparum CaM was found to be more stable, which is in agreement with previous studies for the secondary structure of the complete CaM variants. For binding, the dissociation constants for both the C-lobe of human CaM and for the C-lobe of P. falciparum CaM were proven to be at a lower range than micro molar, most likely in the nano molar range. This is in agreement with earlier findings regarding the entire human CaM. Furthermore, CaM and CZM were proven to have their absorbance at the same wavelengths. Finally, several amino acid differences between the C-lobes of human and P. falciparum CaM were found that could play a role in binding and stability. One specific amino acid that was suggested to contribute to the stabilisation of the C-lobe of P. falciparum CaM was isoleucine. In the C-lobe of human CaM, these isoleucines were exchanged to threonine and arginine. Another amino acid difference that could potentially play a key role was the valine versus isoleucine, where valine might contribute to the stabilisation of the ANS binding site of the C-lobe of human CaM. To perform this study, the methods fluorescence spectroscopy, UV spectroscopy and circular dichroism were used, as well as several bioinformatic tools.  Overall, both stability and structure analyses have helped determine several differences between the two CaM variants, opening up possibilities to find an inhibitor that targets only the CaM of P. falciparum. CZM still remains as an interesting potential inhibitor, and can hopefully be a part of future research in malaria treatment.

Nitrogen dioxide (NO₂) is a pollutant of interest for study both because of its controlling role in the oxidant capacity of the atmosphere and the health risks it poses. Concerns about the health effects of NO₂ and its role in forming deleterious atmospheric species have made it desirable to have low-cost, sensitive ambient measurements of NO₂. A continuous-wave laser-diode laser-induced fluorescence (LIF) system for NO₂ was developed here which operates at ambient pressure, thereby eliminating the need for an expensive pumping system. The current prototype system has achieved sensitivity several orders of magnitude beyond previous efforts at ambient pressure (limit of detection of 2 ppb, 60 s averaging time). Ambient measurements of NO₂ were made in Portland, Oregon using both the standard NO₂ chemiluminescence method and the LIF instrument and showed good agreement (r² = 0.92). In addition, investigations into surface mediated chemistry involving oxides of nitrogen (namely, NO₂) have stimulated new inquiry into potential heterogeneous sources of NO₂ as well as challenged the stability of permanent sinks for NO₂. The possibility that surface mediated chemistry plays a significant role in NOy chemistry in urban air has for the past few decades received considerable attention. The AP-LIF NO₂ instrument is uniquely suited to measure surface chemistry under near ambient conditions. The so called 'renoxification' reaction of gaseous NO with surface bound HNO₃ yielding NO₂ (2HNO₃(surface) + NO--> 3NO₂ +H₂O(surface)) was suggested as a potentially important source of NO₂ which also degraded the stability of nitric acid as a sink of active oxides of nitrogen. Yet, there is disagreement in the literature as to the importance of this reaction. The disagreement stems from differing measurements of the rate for the renoxification reaction. Because there are differences in experimental setups no one research group has studied the renoxification reaction under ambient conditions, i.e., at moderate concentrations of NOy and in a static cell held at 1 atm. In this work, the production of NO₂ was measured using a novel AP-LIF. This setup made it possible to measure the rate of production of NO₂ due to the heterogeneous reaction of NO with HNO₃ under ambient conditions. Under these conditions it was found that renoxification due to gas-phase NO on surface HNO₃ is not a significant source of NO₂. However, this study did show the importance of water vapor in the renoxification of surface HNO₃.

Warfarin is a well-known anticoagulant drug that is used to prevent cardiovascular disease and blood coagulation such as thrombosis. In this study, the main aim was to investigate the photo physical characteristics of warfarin in the different molecular environments provided by sodium dodecyl sulfate (SDS) micelles by using ultraviolet absorption and fluorescence emission spectroscopic techniques. Warfarin and a structural analogue not existing in solution as a cyclic hemiketal, phenprocoumon, were mixed with different concentrations of SDS and spectral changes for these warfarin and phenprocoumon were recorded. Interestingly, results demonstrated, based on an evident increase in the absorption intensity at 273 nm and an evident blue shift in the fluorescence emission spectrum after the addition of an increasing concentration of SDS, that primarily the cyclic hemiketal isomer of warfarin was found to be solvated by SDS micelles at an apparent recorded critical micelle concentration of ~8mM.  Altogether these observations suggest that warfarin may be used as a molecular probe to explore the polarities of complex colloidal mixtures. Moreover, the possibility of using micelles for controlling the isomeric state of warfarin is interesting and can potentially be used for better controlling dosage of warfarin thereby reducing side effects.

The continuing need for greater energy security and energy independence has motivated researchers to develop new energy technologies for better energy resource management and efficient energy usage. The focus of this dissertation is the development of optical (spectroscopic) sensing methodologies for various fuels, and energy applications. A fiber-optic NIR sensing methodology was developed for predicting water content in bio-oil. The feasibility of using the designed near infrared (NIR) system for estimating water content in bio-oil was tested by applying multivariate analysis to NIR spectral data. The calibration results demonstrated that the spectral information can successfully predict the bio-oil water content (from 16% to 36%). The effect of ultraviolet (UV) light on the chemical stability of bio-oil was studied by employing laser-induced fluorescence (LIF) spectroscopy. To simulate the UV light exposure, a laser in the UV region (325 nm) was employed for bio-oil excitation. The LIF, as a signature of chemical change, was recorded from bio-oil. From this study, it was concluded that phenols present in the bio-oil show chemical instability, when exposed to UV light. A laser-induced breakdown spectroscopy (LIBS)-based optical sensor was designed, developed, and tested for detection of four important trace impurities in rocket fuel (hydrogen). The sensor can simultaneously measure the concentrations of nitrogen, argon, oxygen, and helium in hydrogen from storage tanks and supply lines. The sensor had estimated lower detection limits of 80 ppm for nitrogen, 97 ppm for argon, 10 ppm for oxygen, and 25 ppm for helium. A chemiluminescence-based spectroscopic diagnostics were performed to measure equivalence ratios in methane-air premixed flames. A partial least-squares regression (PLS-R)-based multivariate sensing methodology was investigated. It was found that the equivalence ratios predicted with the PLS-R-based multivariate calibration model matched with the experimentally measured equivalence ratios within 7 %. A comparative study was performed for equivalence ratios measurement in atmospheric premixed methane-air flames with ungated LIBS and chemiluminescence spectroscopy. It was reported that LIBS-based calibration, which carries spectroscopic information from a “point-like-volume,” provides better predictions of equivalence ratios compared to chemiluminescence-based calibration, which is essentially a “line-of-sight” measurement.

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This research uses Regression analysis of fluorescence spectroscopy results to correlate with Biochemical Oxygen Demand (BOD5). Fluorescence spectroscopy was applied to samples taken from seven sample sites in the Provo and Orem waste water treatment plants found in Utah County. A total of 161 samples were collected for this research. 23 samples each were taken from four sites in the Provo waste water treatment plant namely Provo head works, aeration basin, primary filter settlement basin and the Provo effluent basin. The Orem head works, the clarifier and the Orem effluent basin were the three sample sites in the Orem waste water treatment plant where 23 samples each were collected to carry out the analysis. The fluorescent characteristics of the samples were determined using fluorescence spectrometry. These intensities were correlated with standard five day Biochemical Oxygen Demand (BOD5) values which were used as a measure of the amount of biodegradable organic material present. Chemical oxygen demand (COD) data were also taken from these treatment plants for correlation purposes. Three different correlation analyses were made which were the correlation of fluorescence spectroscopy excitation-emission matrix (EEM) against (1) individual sites BOD and COD values (2) Provo only and Orem only BOD and COD values (3) combined Provo and Orem BOD and COD values. The correlation of Individual site EEMs against BOD and COD values produced the best results. There was a higher correlation of EEM with BOD data than COD data. The R-squared for the combined Provo and Orem BOD data was 0.756 and that for COD was 0.729. Very high R-squared was obtained for Provo Influent data and Orem Influent data which were 0.955 and 0.946 respectively. This method can be used by wastewater stakeholders in deriving quick results in determining potential pollution events within a shorter time frame. This research demonstrates that there is a correlation between EEM and BOD/COD.

Secretory phospholipase A2 hydrolyzes phospholipids at a lipid-water interface, resulting in pro-inflammatory products being released from cell membranes. Healthy cells are resistant to cleavage by this enzyme, but apoptotic cells become susceptible to its activity. Only bilayers with certain characteristics are able to be hydrolyzed. Most recently, studies in this lab have emphasized the idea that the biophysical state of the bilayer (in terms of lipid order, spacing, and fluidity) is relevant in determining the probability of one phospholipid escaping the membrane to be hydrolyzed. Prior to this study, it had been shown that apoptotic cells undergo biophysical alterations that weaken inter-lipid interactions early in apoptosis. The purpose of this dissertation was to examine these changes in more detail, define them more clearly on the molecular level, and suggest possible mechanisms responsible for their occurrence. First, the role of increased membrane permeability in susceptibility to the phospholipase was investigated. S49 cells were treated with ionomycin or apoptotic agents and assayed for merocyanine 540 staining of the membrane and membrane permeability to a vital dye. Human group X and snake venom isoforms were active towards all treated cells, but human groups V and IIa only hydrolyzed cells that were moderately permeable to the vital dye. Different isoforms must then be sensitive to different membrane properties. Second, the role of membrane oxidation in cell membrane vulnerability to the phospholipase (specifically human group IIa) was tested. The temporal onset of lipid peroxidation was assayed during apoptosis. This correlated with the onset of susceptibility to the IIa isoform. Direct oxidizers were then used to verify this result in isolation from other apoptotic membrane changes. Third, biophysical alterations during thapsigargin-induced apoptosis were examined using TMA-DPH and Patman. Data from these probes in artificial bilayers undergoing phase transitions were used to quantify the decrease in interlipid interactions and predict a 50 -- 100-fold increase in the probability of phospholipid protrusions. Patman equilibration kinetics also revealed more molecular detail about the biophysical changes related to susceptibility. Finally, temperature- and ionomyin-induced alterations in membrane properties were compared. Both increased fluidity, but only ionomycin caused susceptibility. Patman equilibration kinetic analysis could distinguish responsible membrane properties. Actin fragmentation during apoptosis or calcium loading is proposed as the mechanism.

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