Voltage-gated K+ channels associate with multiple regulatory proteins to form complexes with diverse gating properties and pharmacological sensitivities. Small molecules which activate or inhibit channel function are valuable tools for dissecting the assembly and function of these macromolecular complexes. My thesis focuses on the discovery and use of small molecules to probe the structure and function of the KCNQ family of voltage-gated K+ channels. One protein that obligatorily assembles with KCNQ channels to mediate proper assembly, trafficking, and gating is the calcium sensor, calmodulin. Although resolution of the crystal structures of calmodulin associated with isolated peptide fragments from other ion channels has provided some insight into how calmodulin interacts with and modulates KCNQ channels, structural information for calmodulin bound to a fully folded ion channel in the membrane is unknown. In Chapter II, I developed an intracellular tethered blocker approach to determine the location of calmodulin binding with respect to the KCNQ ion-conducting pathway. Using distance restraints from a panel of these intracellular tethered blockers we then generated models of the KCNQ-calmodulin complex. Our model places calmodulin close to the gate of KCNQ channels, providing structural insight into how CaM is able to communicate changes in intracellular calcium levels to KCNQ channel complexes. In addition to pore blockers, chemical modification of ion channels has been used to probe ion channel function. During my initial attempt to chemically activate KCNQ channels, I discovered that some boronates modulate KCNQ complexes. In Chapter III, the activating derivative, phenylboronic acid, is characterized. Characterization of activation by phenylboronic acid showed that it targeted the ion conduction pathway of KCNQ channels with some specificity over other voltage-gated K+ channels. The commercial availability of thousands of boronic acid derivatives provides a large class of compounds with which to systematically dissect the mechanisms of KCNQ gating and may lead to the discovery of a potent activator of KCNQ complexes for the treatment of channelopathies. All of the electrophysiological studies presented in this thesis were conducted in Xenopus oocytes. Unexpectedly, during the studies described above, the quality of our Xenopus oocytes declined. The afflicted oocytes developed black foci on their membranes, had negligible electric resting potentials, and poor viability. Culturing the compromised oocytes determined that they were infected with multi-drug resistant Stenotrophomonas maltophilia, Pseudomonas fluorescens and Pseudomonas putida. Antibiotic testing showed that all three species of bacteria were susceptible to amikacin and ciprofloxacin, which when included in the oocyte storage media prevented the appearance of black foci and resulted in oocytes that were usable for electrophysiological recordings. This study provides a solution to a common issue that plagues many electrophysiologists who use Xenopus oocytes. Taken together, these findings provide new insights into activation of KCNQ channel complexes and provide new tools to study the structure-function relationship of voltage-gated K+ channels.

Author did not provide abstract.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-09-08T13:48:31Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE_DANILO FONTES_PPGCF_UFPE.pdf: 6750693 bytes, checksum: ec0d551dfa4fb27855a7caf940d95aab (MD5) / Made available in DSpace on 2016-09-08T13:48:31Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE_DANILO FONTES_PPGCF_UFPE.pdf: 6750693 bytes, checksum: ec0d551dfa4fb27855a7caf940d95aab (MD5) Previous issue date: 2016-03-18 / FACEPE / Os hidróxidos duplos lamelares (HDL), também conhecidos como compostos tipo hidrotalcita, são materiais capazes de incorporar espécies biologicamente ativas, negativamente carregadas, na sua região interlamelar, de modo a neutralizar as cargas po-sitivas das lamelas, através do mecanismo de troca iônica. Além deste mecanismo, os HDL possuem alta capacidade de adsorção de materiais não iônicos e carregados positivamente, através de interações eletrostáticas e ligações de hidrogênio na sua vasta área superficial. Os HDL possuem ocorrência natural e também podem ser sintetizados em laboratório por rotas simples e de baixo custo, que permitem o isolamento de sólidos de alta pureza. O presente trabalho tem por objetivo a síntese e caracterização de HDL (CaAl-HDL e MgAl-Cl-HDL), e sua aplicação junto aos fármacos antiretrovirais Efavirenz (EFZ) e Zidovudina (AZT), e do anti-chagásico Benznidazol (BNZ). Os materiais obtidos foram caracterizados pelas técnicas de difração de raios-X (DRX), termogravimetria (TG), calorimetria exploratória diferencial (DSC), análise térmica diferencial (DTA), espectroscopia no infravermelho (IV), microscopia eletrônica de varredura (MEV), microscopia de luz polarizada e análise elementar de metais e de carbono, hidrogênio e nitrogênio (CHN). Foi possível observar que em sistemas com HDL de cálcio e alumínio (CaAl-HDL), obtido pelo método de evaporação do solvente, contendo até 30% de EFZ e até 20% do BNZ, o fármaco tornou-se uma molécula com características amorfas, perdendo seu caráter cristalino. Este fenômeno pôde ser comprovado através da ausência de planos cristalinos do fármaco no DRX, e também do seu ponto de fusão no DSC. Nos testes de liberação, estes sistemas obtiveram destaque no incremento de solubilidade, sendo a proporção HDL-EFZ 30% a mais promissora, com aumento de 558% do EFZ solúvel em relação ao fármaco isolado; e o sistema HDL-BNZ 20%, proporcionando 702% de aumento na solubilidade do fármaco. Desta maneira comprovou-se que associação entre o CaAl-HDL e o BNZ e EFZ (fármacos de baixa solubilidade) confere incremento de solubilidade, promovendo uma maior taxa de dissolução nos estudos in vitro. A solubilidade aquosa de um fármaco constitui requisito prévio à absorção e obtenção de resposta clínica, para a maioria dos medicamentos administrados por via oral. No sistema contendo MgAl-Cl-HDL e AZT, obtido pelo método de síntese por co-precipitação a pH constante, foi possível notar que, o fármaco tornou-se amorfo através da interação com a superfície do HDL, como evidenciado através do DRX, TG, IV e MEV. No estudo de liberação, foi possível obter um perfil de liberação prolongada do AZT, de 90% de fármaco em 24 horas de estudo. Os sistemas CaAL-HDL:EFZ e MgAl-Cl-HDL:AZT tornaram-se menos tóxicos quando comparados a seus respectivos fármacos isolados, enquanto o sistema CaAL-HDL:BNZ, não alterou a toxicidade do BNZ, quando testados em linhagem de macrófagos humanos. / Layered double hydroxides (LDH), also known as hydrotalcite compounds, are materials able to incorporate biologically active species, negatively charged, into the interlayer region, to neutralize the positive charges of the lamellae through the ion exchange mechanism. Besides this mechanism, LDH have a high adsorption capacity for positively charged non-ionic materials, through electrostatic interactions and hydrogen bonds on its vast surface area. LDH are found in nature and can also be synthesized by simple and low-cost routes, that allow for the isolation of high purity solids. This paper presents the synthesis and characterization of the LDH (CaAl-LDH e MgAl-Cl-LDH), its applications with the antiretroviral drugs efavirenz and zidovudine, and the anti-chagas drug Benznidazol. The materials were characterized by the techniques of x-ray diffraction (XRD), thermogravimetry (TG), differential scanning calorimetry (DSC), differential thermal analysis (DTA), infrared spectroscopy (IR), scanning electron microscopy (SEM), polarized light microscopy, and elemental analysis of metals and carbon, hydrogen and nitrogen (CHN). It was observed that in systems with CaAl-HDL, obtained by the solvent evaporation method, containing up to 30% of EFZ and 20% of BNZ, the drug became a molecule with amorphous characteristics, losing its crystalline character. This phenomenon could be demonstrated by the absence of the drug crystal planes in the XRD analysis, and also by its melting point in the DSC analysis. In the release experiments, these systems stood out because they promoted an increase in solubility, with LDH-EFZ 30% being the most promising, with an increase in the EFV solubility of 558% compared to the drug itself; and the LDH-BNZ 20% system providing a 702% increase in solubility. Thus, the association between CaAl-LDH and BNZ and EFZ (low solubility drugs) was proven to increase the solubility of these drugs, promoting a higher dissolution rate in in vitro studies. The aqueous solubility of a drug is a prerequisite for obtaining absorption and clinical response for most drugs administered orally. In the system containing MgAl-Cl-LDH and AZT, obtained by the method of co-precipitation at constant pH, it was noticeable that, after the synthesis, the drug became amorphous due to the interaction with the surface of the LDH, as evidenced by the XRD, TG, SEM, and IR analyses. In the release study, it was possible to obtain a profile of the prolonged release of AZT, 90% of the drug in a 24-hour experiment. The cell viability experiment showed that the CaAl-LDH:EFZ and MgAl-Cl-HDL:AZT systems became less toxic than the isolated drugs and the CaAl-HDL:BNZ system did not alter the toxicity of the drug itself, when tested in human macrophage lineage.

A dentina esclerosada é um substrato comumente encontrado em pacientes idosos. No entanto, existem poucos estudos comparativos entre dentina humana e bovina esclerosadas. O objetivo deste estudo foi comparar os componentes inorgânicos e orgânicos da dentina saudável e esclerosada humana e bovina, através de cinco parâmetros: nanodureza, módulo de elasticidade, análise quantitativa da concentração de Cálcio (Ca) e Fósforo (P), densidade tubular e morfologia do colágeno. Trinta dentes humanos e 30 bovinos foram distribuídos em 4 grupos experimentais (n=15 por grupo): dentina humana saudável (DHS), esclerosada humana (DEH), bovina saudável (DBS), e bovina esclerosada (DBE). Os dentes saudáveis foram preparados na mesma altura e inclinação dos dentes esclerosados expondo níveis similares da dentina e obtendo fragmentos com 2mm de espessura. Foram realizadas 3 medições por espécime em 3 áreas pré determinadas de dentina intertubular com a utilização do Nanoindentador (carga de 500N por 5 s). Cinco espécimes de cada grupo foram preparados para Microscopia Eletrônica de Varredura (MEV). Com o auxílio da Energia Dispersiva por Raios-X EDX foram obtidos os valores (em percentagem) das concentrações de Ca e P e calculada a relação Ca:P. A contagem dos túbulos por área foi realizada em todas as eletromicrografias. Após descalcificação e preparo, o restante dos espécimes foi analisado em Microscopia Eletrônica de Transmissão (MET). DBS obteve maiores valores de nanodureza comparada à DBE e DHS. DHE sem diferenças com DHS e DBE (p=0,0008). DBS exibiu maiores valores de módulo de elasticidade somente comparada à DHS (p=0,000). A análise estatística não demonstrou diferenças estatisticamente significantes (p=0,71) entre as concentrações de Ca e P. Quanto à densidade tubular (número de túbulos/mm²), os grupos saudáveis foram maiores que os esclerosados e os humanos maiores que os bovinos. As fibras colágenas da DBS mostraram-se mais compactadas e mais desorganizadas que as demais. Pôde se concluir que apenas as concentrações de Ca e P foram similares e que as dentinas esclerosadas humana e bovina mostraram similaridade. Esta pesquisa teve suporte da Fapesp sob o número 2008/10290-8. / The sclerotic dentin has been commonly found in elderly patients. However, there are scarce reports in the literature comparing on the use of human and bovine sclerotic dentins. The objective of this study was to compare inorganic and organic components of healthy and sclerotic dentins from human and bovine. Five parameters were analyzed: nanohardness, elastic modulus, quantitative analysis of Calcium (Ca) and Phosphorous (P) concentrations, tubular density and ultrastructural morphology. Thirty human teeth plus 30 bovine teeth were distributed in 4 experimental groups (n=15 per group): human healthy dentin (HHD), human sclerotic dentin (HSD), bovine healthy dentin (BHD) and bovine sclerotic dentin (BSD). Healthy teeth were cut in the same level and inclination of the sclerotic superficial dentins. The nanohardness and elastic modulus (GPa) of three pre determined areas of each exposed dentin was measured using a nanoindenter (500N for 5s). Five samples of each group were prepared for scanning electron microscopy (SEM) examination. Energy Dispersive X-ray (EDX) was used for obtaining the Ca/P ratio. The tubular density was obtained by counting the tubules in scanning electron micrographs taken in the same magnification and work distance. Data were statistically analyzed by ANOVA complemented by the Tukeys test (p0.05). The ultrastructure of the dentins was observed in specimens processed for transmission electron microscopy (TEM). BHD exhibited significant higher nanohardness than BSD and HHD. HSD nanohardness was similar to those of HHD and BSD (p=0,0008). BHD exhibited significant higher elastic modulus than HHD (p=0,000). The Ca:P ratios were similar amongst all groups (p=0.71). The tubular densities were higher in the healthy dentins than in the sclerotic for both human and bovine. The human dentins presented higher tubular densities than bovine dentins (p=0.000). The intertubular dentin of BHD showed short collagen fibers distributed in a condensed fashion; whereas the other dentins exhibited well-organized long bundles of collagen fibers. It was concluded that sclerotic dentins of human and bovine share most morphological and structural characteristics. This research was supported by Fapesp grants number 2008/10290-8.

Orientador: Marcelo Giannini / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-18T10:52:55Z (GMT). No. of bitstreams: 1 Aguiar_ThaianeRodrigues_D.pdf: 24572936 bytes, checksum: 8141f58fcbce6835d1091f52127552a7 (MD5) Previous issue date: 2011 / Resumo: O emprego dos cimentos resinosos na fixação de peças protéticas têm sido avaliado em muitos estudos, entretanto, trabalhos que simulem os desafios presentes na cavidade bucal e o comportamento a longo prazo dos cimentos resinosos autoadesivos ainda são escassos na literatura. Diante disso, o presente estudo teve como objetivo: 1) verificar a composição inorgânica das partículas de carga e caracterizá-las quanto ao tipo e morfologia; 2) analisar a ultramorfologia da interface de união dentina-cimento resinoso através da microscopia confocal por varredura a laser e da microscopia eletrônica de varredura (MEV); 3) avaliar o efeito do armazenamento por 1 ano ou da ciclagem mecânica (50.000 ciclos) na resistência de união dentina-cimento resinoso. Quatro cimentos resinosos foram avaliados: dois autoadesivos (RelyX Unicem, 3M ESPE e Clearfil SA Luting, Kuraray Medical Inc.) e dois convencionais (RelyX ARC/Adper Scotchbond Multi-Purpose Plus, 3M ESPE e Clearfil Esthetic Cement/Clearfil DC Bond, Kuraray Medical Inc.). Para analisar os principais componentes inorgânicos e avaliar as características das partículas de carga, o conteúdo orgânico dos cimentos resinosos foi removido por solventes orgânicos e as amostras analisadas em MEV/EDX (Espectroscopia de energia dispersiva por raios-X). Terceiros molares humanos foram utilizados na avaliação da ultramorfologia da interface dentina-cimento resinoso e no ensaio de resistência de união à dentina. Na análise da ultramorfologia da área de união, corantes (fluoresceína e rodamina) foram previamente adicionados aos materiais estudados para possibilitar a obtenção das imagens. Para o ensaio de resistência de união, discos de resina composta indireta foram confeccionados visando simular as restaurações indiretas. Após a cimentação, os dentes restaurados foram submetidos a três tratamentos e ao ensaio de microtração: 1- espécimes testados após 24 h (grupo controle); 2- ciclagem mecânica e em seguida testados; 3- armazenados em saliva artificial por 1 ano na forma de "palitos" e testados. Os dados obtidos a partir do ensaio de resistência de união foram analisados estatisticamente (ANOVA e teste Tukey, p<0,05). Nos outros dois estudos, a análise descritiva dos dados obtidos a partir das imagens microscópicas foi realizada. Na avaliação MEV/EDX notou-se elevado conteúdo de silício em todos os materiais. O cimento RelyX ARC apresentou partículas de carga esféricas e irregulares, enquanto os outros materiais demonstraram partículas com formato irregular. Nas fotomicrografias, a hibridização dentinária somente foi observada para os sistemas de cimentação convencional. No ensaio de resistência de união, os tratamentos não influenciaram a resistência de união dos cimentos resinosos à dentina. Os cimentos autoadesivos apresentaram médias de resistência de união significantemente superiores aos chamados convencionais. Embora os materiais avaliados apresentem partículas de carga com distintas composições inorgânicas e interagem com a dentina através de diferentes mecanismos, os tratamentos de envelhecimentos propostos não reduziram a resistência de união à dentina, quando comparados ao grupo controle. Os cimentos autoadesivos não formam camada híbrida na dentina intertubular, nem tags de resina nos túbulos dentinários, entretanto, apresentam partículas de carga com maior tamanho e demonstraram os maiores valores médios de resistência de união à dentina entre os cimentos estudados / Abstract: Resin luting materials to fixed prosthetic restorations have been evaluated in many studies, however, little is known about self-adhesive resin cements, mainly involving long-term durability and when these materials have been submitted to challenges of oral environment. Thus, the purpose of this study was: 1- to verify the filler particles components and to characterize them according to type and morphology; 2- to analyze the ultramorphology of the dentin-resin interfaces using confocal laser scanning microscopy and scanning electron microscopy (SEM) and 3- to evaluate the effect of the storage for 1 year or the mechanical cycling (50.000 cycles) on bond strength of resin cements to dentin. Four resin cements were used in this study: two self-adhesive resin cements (RelyX Unicem, 3M ESPE and Clearfil SA Luting, Kuraray Medical Inc.) and two conventional resin cements (RelyX ARC/Adper Scotchbond Multi- Purpose Plus, 3M ESPE and Clearfil Esthetic Cement/Clearfil DC Bond, Kuraray Medical Inc.). For indentifying the main inorganic components and to evaluate the filler particles characteristics, the organic phase of resin cements was removed using organic solvents and the sample was analyzed by SEM/EDX (Energy-dispersive X-ray spectroscopy). Human third molars were used for micromorphology analysis of resin-dentin interfaces and microtensile bond strength test. For micromorphology analyses of resin-dentin interfaces, two fluorescent dyes (fluorescein and rhodamine B) were incorporated into the materials previously. For microtensile bond strength, pre-polymerized resin discs were prepared to simulate indirect restorations. After bonding procedures, the restored teeth were submitted to three treatments: 1- control group (tested 24 h after specimen preparation); 2- tested after mechanical cycling; 3- beams were storage for one year in artificial saliva and tested. Bond strength data were statistically analyzed by two-way ANOVA and Tukey test (?<0.05). Qualitative analysis was performed for two other studies using microscopic images. For SEM/EDX measurements, high amount of silicon was identified for all cements. The resin cement RelyX ARC showed spherical and irregular particles, while other cements presented irregular shape fillers. Hybrid layer formation was observed only for conventional resin cements in micromorphology analyses. For microtensile test, the treatments did not reduce the bond strength between dentin-resin cement. Self-adhesive resin cements provide significantly higher bond strength than those conventional materials. Despite the resin cements showed filler particles with differences in the inorganic composition and different bonding mechanisms, the mechanical cycling and storage for one year did not affect the bond strength to dentin when it was compared to control group. The self-adhesive resin cements showed no hybrid layer formation in intertubular dentin and nor resin tags in the dentinal tubules were detected. However, these materials showed the biggest filler particles and higher bond strength to dentin than conventional resin cements studied / Doutorado / Dentística / Doutor em Clínica Odontológica

A central concept in the physiology of neurosecretion is that a rise in cytosolic [Ca2+] in the vicinity of plasmalemmal Ca2+ channels due to Ca2+ influx, elicits exocytosis. This dissertation examines the effect on both spontaneous and elicited exocytosis of a rise in focal cytosolic [Ca2+] in the vicinity of ryanodine receptors (RYRs) due to release from internal stores in the form of Ca2+ syntillas. Ca2+ syntillas are focal cytosolic transients mediated by RYRs, which we first found in hypothalamic magnocellular neuronal terminals. (Scintilla, Latin for spark, found in nerve terminals, normally synaptic structures.) We have also observed Ca2+ syntillas in mouse adrenal chromaffin cells (ACCs). Here the effect of Ca2+syntillas on exocytosis is examined in ACCs, which are widely used as model cells for the study of neurosecretion. Elicited exocytosis employs two sources of Ca2+, one due to influx from the cell exterior through voltage-gated Ca2+ channels (VGCCs) and another due to release from intracellular stores. To eliminate complications arising from Ca2+ influx, the first part of this dissertation examines spontaneous exocytosis where influx is not activated. We report that decreasing syntillas leads to an increase in spontaneous exocytosis measured amperometrically. Two independent lines of experimentation each lead to this conclusion. In one case release from stores was blocked by ryanodine; in another, stores were partially emptied using thapsigargin plus caffeine after which syntillas were decreased. We conclude that Ca2+syntillas act to inhibit spontaneous exocytosis, and we propose a simple model to account quantitatively for this action of syntillas. The second part of this dissertation examines the role of syntillas in elicited exocytosis whereby Ca2+ influx is activated by physiologically relevant levels of stimulation. Catecholamine and neuropeptide release from ACCs into the circulation is controlled by the sympathetic division of the Autonomic Nervous System. To ensure proper homeostasis tightly controlled exocytic mechanisms must exist both in resting conditions, where minimal output is desirable and under stress, where maximal, but not total release is necessary. It is thought that sympathetic discharge accomplishes this task by regulating the frequency of Ca2+ influx through VGCCs, which serves as a direct trigger for exocytosis. But our studies on spontaneous release in ACCs revealed the presence of Ca2+ syntillas, which had the opposite effect of inhibiting release. Therefore, assuming Ca2+-induced Ca2+ release (CICR) via RYRs due to Ca2+ influx through VGCCs, we are confronted with a contradiction. Sympathetic discharge should increase syntilla frequency and that in turn should decreaseexocytosis, a paradox. A simple “explanation” might be that the increase in syntillas would act as a brake to prevent an overly great exocytic release. But upon investigation of this question a different finding emerged. We examined the role of syntillas under varying levels of physiologic stimulation in ACCs using simulated action potentials (sAPs) designed to mimic native input at frequencies associated with stress, 15 Hz, and the basal sympathetic tone, 0.5 Hz. Surprisingly, we found that sAPs delivered at 15 Hz or 0.5 Hz were able to completely abolish Ca2+ syntillas within a time frame of two minutes. This was not expected. Further, a single sAP is all that was necessary to initiate suppression of syntillas. Syntillas remained inhibited after 0.5 Hz stimulation but were only temporarily suppressed (for 2 minutes) by 15 Hz stimulation, where global [Ca2+]i was raised to 1 – 2 μM. Thus we propose that CICR, if present in these cells, is overridden by other processes. Hence it appears that inhibition of syntillas by action potentials in ACCs is due to a new process which is the opposite of CICR. This process needs to be investigated, and that will be one of the very next steps in the future. Finally we conclude that syntilla suppression by action potentials is part of the mechanism for elicited exocytosis, resolving the paradox. In the last chapter speculation is discussed into the mechanisms by which physiologic input in the form of an action potential can inhibit Ca2+ syntillas and furthermore, how the Ca2+ syntilla can inhibit exocytic output.

The utility of a model organism for studying biological processes is closely tied to its amenability to genome manipulation. Although tools for targeted genome engineering in mice have been available since 1987, most organisms including zebrafish have lacked efficient reverse genetic tools, which has stymied their broad implementation as a model system to study biological processes. The development of zinc finger nucleases (ZFNs) that can create double-strand breaks at desired sites in a genome has provided a universal platform for targeted genome modification. ZFNs are artificial restriction endonucleases that comprise of an array of 3- to 6-C2H2-zinc finger DNA-binding domains fused with the dimeric cleavage domain of the type IIs endonuclease FokI. C2H2-zinc fingers are the most common, naturally occurring DNA-binding domain, and their specificity can be engineered to recognize a variety of DNA sequences providing a strategy for targeting the appended nuclease domain to desired sites in a genome. The utility of ZFNs for gene editing relies on their activity and precision in vivo both of which depend on the generation of ZFPs that bind desired target sites high specificity and affinity. Although various methods are available that allow construction of ZFPs with novel specificities, ZFNs assembled using existing approaches often display negligible in vivo activity, presumably resulting from ZFPs with either low affinity or suboptimal specificity. A root cause of this deficiency is the presence of interfering interactions at the finger-finger interface upon assembly of multiple fingers. In this study we have employed bacterial-one-hybrid (B1H)-based selections to identify two-finger zinc finger units (2F-modules) containing optimized interface residues that can be combined with published finger archives to rapidly yield ZFNs that can target more than 95% of the zebrafish and human protein-coding genes while maintaining a success rate higher than that of ZFNs constructed using available methods. In addition to genome engineering in model organisms, this advancement in ZFN design will aid in the development of ZFN-based therapeutics. In the process of creating this archive, we have undertaken a broader study of zinc finger specificity to better understand fundamental aspects of DNA recognition. In the process we have created the largest protein-DNA interaction dataset for zinc fingers to be described that will facilitate the development of better predictive models of recognition. Ultimately, these predictive models would enable the rational design of synthetic zinc finger proteins for targeted gene regulation or genomic modification, and the prediction of genomic binding sites for naturally occurring zinc finger proteins for the construction of more accurate gene regulatory networks.

The hydrophobic effect and hydrogen bonding interactions have long been considered to be the dominant forces in protein folding. However, the contribution of hydrogen bonds to stabilizing proteins has been difficult to clarify. As the intramolecular hydrogen bonds are formed in place of hydrogen bonds with solvent during folding, measures of stability fail to give a significant change in free energy. Previous studies on hydrogen bonding interactions have shown that they are only marginally important. Three long-range side chain-main chain hydrogen bonds have been found in the alpha subunit of tryptophan synthase (αTS), a (βα)8TIM barrel protein. These long-range noncovalent interactions connect either the N-terminus of one β-strand with the C-terminus of the succeeding and anti-parallel α-helix (F19-D46 and I97-D124) or the N-terminus of an α-helix with the C-terminus of the succeeding β-strand (A103-D130). By analogy, these interactions are designated as βα- or αβ-hairpin clamps. Surprisingly, the removal of any one of these clamp interactions, by replacement of the aspartic acid with alanine, results in significantly decreased thermodynamic stability for the native state and a substantial loss of secondary structure. When compared to several other side chain-side chain and short-range side chain-main chain interactions in αTS, these hairpin clamps clearly play a unique role in the structure and stability of αTS. The generality of these observations for βα-hairpin clamps in TIM barrel proteins was tested by experimental analysis of the clamps in a pair of homologous indole-3-glycerol phosphate synthase (IGPS) TIM barrels of low sequence identity. The results suggest that only the subset of conserved βα-hairpin clamps with hydrogen bond length less than 2.80 Å make substantive contributions to stability and/or structure. Those clamps with longer hydrogen bonds make modest contributions to stability and structure, similar to other types of side chain-main chain or side chain-side chain hydrogen bonds. The role of these clamps in defining the structures of the super-family of TIM barrel proteins was examined by a survey of 71 TIM barrel proteins from the structural database. Conserved features of βα-hairpin clamps are consistent with a 4-fold symmetry, with a predominance of main chain amide hydrogen bond donors near the N-terminus of the odd-number β-strands and side chain hydrogen bond acceptors in the loops between the subsequent α-helices and even-numbered β-strands. In this configuration, the clamps provide an N-terminal cap to odd-number β- strands in the β-barrel. Taken together, these findings suggest that βα-hairpin clamps are a vestigial signature of the fundamental βαβ building block for the (βα)8 motif and an integral part of the basic TIM barrel architecture. The relative paucity of βα-hairpin clamps remaining in TIM barrel structures and their variable contributions to stability imply that other determinants for structure and stability of the barrel have evolved to render a subset of the clamp interactions redundant. Distinct sequence preferences for the partners in the βα-hairpin clamps and the neighboring segments may be useful in enhancing algorithms for structure prediction and for engineering stability in TIM barrel proteins.

Iron oxide nanoparticles have received sustained interest for biomedical applications as synthetic approaches are continually developed for precise control of nanoparticle properties. This thesis presents an investigation of parameters in the benzyl alcohol synthesis of iron oxide nanoparticles. A modified seed growth method was designed for obtaining optimal nanoparticle properties for magnetic fluid hyperthermia. With a one or two addition process, iron oxide nanoparticles were produced with crystallite sizes ranging from 5-20 nm using only benzyl alcohol and iron precursor. The effects of reaction environment, temperature, concentration, and modified seed growth parameters were investigated to obtain precise control over properties affecting radiofrequency heat generation. The reaction A2-24(205)_B2-24(205) produced monodispersed (PDI=0.265) nanoparticles with a crystallite size of 19.5±1.06 nm and the highest radiofrequency heating rate of 4.48 (°C/min)/mg (SAR=1,175.56 W/g, ILP=3.1127 nHm2/kg) for the reactions investigated. The benzyl alcohol modified seed growth method offers great potential for synthesizing iron oxide nanoparticles for radiofrequency hyperthermia.

É crescente a demanda por materiais que atendam as exigências para construção de barreiras selantes (liners) em obras para disposição de resíduos. A utilização de solos tropicais para esta finalidade só não é maior por falta de conhecimento sobre muitas de suas características técnicas, principalmente a migração de contaminantes. Tentativas de melhoria das qualidades de solos supostamente inadequados também são importantes. Esta tese apresenta estudos sobre a migração dos íons \'CD POT.2+\', \'K POT.+\', \'CL POT.1-\' e \'F POT.-\' em latossolos residuais das formações Botucatu e Serra Geral e três diferentes misturas destes solos, com ênfase nos processos de sorção e difusão molecular. O potencial de retenção dos íons é avaliado através da construção de 33 isotermas de sorção resultantes de ensaios de equilíbrio em lote. Seus parâmetros são obtidos da adequação dos modelos linear, Freundlich e Langmuir as isotermas. O potencial de transporte dos íons é avaliado através de ensaios de difusão em colunas estanques, onde curvas teóricas produzidas no programa POLLUTE são ajustadas aos dados de concentração para a estimativa dos parâmetros de difusão. Os resultados dos estudos de retenção mostram que o \'CL POT.-\' experimenta sorção sob certas condições, o \'K POT.+\' e \'F POT.-\' possuem taxas semelhantes de retenção, e que a sorção do \'CD POT.2+\' se mostrou altamente dependente da composição da solução contaminante. Os ensaios de difusão resultaram em vários valores para o coeficiente de difusão efetiva (D*), mostrando que o fluxo difusivo dos íons diminui para solos mais argilosos. / There is a growing demand for materials that can be used in lining systems for waste disposal facilities. The use of tropical soils for this purpose is restricted due to the lack of technical knowledge about this type of material. Attempts to improve the quality of supposed unsuitable soils play also an important role. This thesis presents studies concerning the migration of the ions \'CD POT.2+\', \'K POT.+\', \'CL POT.-\' and \'F POT.-\' in latosoils from the Botucatu and Serra Geral formations and three different mixtures of these soils, with emphasis on sorption and molecular diffusion. Batch equilibrium tests were conduced to examine the retention capacity of the soils, and the sorption parameters are estimated by fitting the linear, Freundlich and Langmuir models to the experimental isotherm plots. The potential for these soils to transport ions is evaluated by diffusion tests, where theoretical curves produced in the POLLUTE code are used to estimate the effective diffusion coefficients (D*). The results show that the \'CL POT.-\' is sorbed under specific conditions and, therefore, it can not always be used as a conservative ion (i.e. nonreactive ion), \'K POT.+\' and \'F POT.-\' are sorbed in similar amounts, and that the \'CD POT.2+\' sorption is highly dependent on the contaminant solution composition. Several D* values are reported, showing that the diffusion rates are decreased for soils with more clay.