Effects of TI-299423 on Neuronal Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors (nAChRs) are pentameric, ligand-gated, cation channels found throughout the central and peripheral nervous system, whose endogenous ligand is acetylcholine, but which can also be acted upon by nicotine. The subunit compositions of nAChR determine their physiological and pharmacological properties, with different subunits expressed in different combinations or areas throughout the brain. The behavioral and physiological effects of nicotine are elicited by its agonistic and desensitizing actions selectively on neuronal nAChRs. The midbrain is of particular interest due to its population of nAChRs expressed on dopaminergic neurons, which are important for reward and reinforcement, and possibly contribute to nicotine dependence. The α6-subunit is found on dopaminergic neurons but very few other regions of the brain, making it an interesting drug target. We assayed a novel nicotinic agonist, called TI-299423 or TC299, for its possible selectivity for α6-containing nAChRs. Our goal was to isolate the role of α6-containing nAChRs in nicotine reward and reinforcement, and provide insight into the search for more effective smoking cessation compounds. This was done using a variety of in vitro and behavioral assays, aimed dually at understanding TI-299423’s exact mechanism of action and its downstream effects. Additionally, we looked at the effects of another compound, menthol, on nicotine reward. Understanding how reward is generated in the cholinergic system and how that is modulated by other compounds contributes to a better understand of our complex neural circuitry and provides insight for the future development of therapeutics.

Structure-Function Studies of Nicotinic Acetylcholine Receptors Using Selective Agonists and Positive Allosteric Modulators

This dissertation primarily describes chemical-scale studies of nicotinic acetylcholine receptors (nAChRs) in order to better understand ligand-receptor selectivity and allosteric modulation influences during receptor activation. Electrophysiology coupled with canonical and non-canonical amino acids mutagenesis is used to probe subtle changes in receptor function.

The first half of this dissertation focuses on differential agonist selectivity of α4β2-containing nAChRs. The α4β2 nAChR can assemble in alternative stoichiometries as well as assemble with other accessory subunits. Chapter 2 identifies key structural residues that dictate binding and activation of three stoichiometry-dependent α4β2 receptor ligands: sazetidine-A, cytisine, and NS9283. These do not follow previously suggested hydrogen-bonding patterns of selectivity. Instead, three residues on the complementary subunit strongly influence binding ability of a ligand and receptor activation. Chapter 3 involves isolation of a α5α4β2 receptor-enriched population to test for a potential alternative agonist binding location at the α5 α4 interface. Results strongly suggest that agonist occupation of this site is not necessary for receptor activation and that the α5 subunit only incorporates at the accessory subunit location.

The second half of this dissertation seeks to identify residue interactions with positive allosteric modulators (PAMs) of the α7 nAChR. Chapter 4 focuses on methods development to study loss of potentiation of Type I PAMs, which indicate residues vital to propagation of PAM effects and/or binding. Chapter 5 investigates α7 receptor modulation by a Type II PAM (PNU 120596). These results show that PNU 120596 does not alter the agonist binding site, thus is relegated to influencing only the gating component of activation. From this, we were able to map a potential network of residues from the agonist binding site to the proposed PNU 120596 binding site that are essential for receptor potentiation.

DNA-Mediated Charge Transport Devices for Protein Detection

Detection of biologically relevant targets, including small molecules, proteins, DNA, and RNA, is vital for fundamental research as well as clinical diagnostics. Sensors with biological elements provide a natural foundation for such devices because of the inherent recognition capabilities of biomolecules. Electrochemical DNA platforms are simple, sensitive, and do not require complex target labeling or expensive instrumentation. Sensitivity and specificity are added to DNA electrochemical platforms when the physical properties of DNA are harnessed. The inherent structure of DNA, with its stacked core of aromatic bases, enables DNA to act as a wire via DNA-mediated charge transport (DNA CT). DNA CT is not only robust over long molecular distances of at least 34 nm, but is also especially sensitive to anything that perturbs proper base stacking, including DNA mismatches, lesions, or DNA-binding proteins that distort the π-stack. Electrochemical sensors based on DNA CT have previously been used for single-nucleotide polymorphism detection, hybridization assays, and DNA-binding protein detection. Here, improvements to (i) the structure of DNA monolayers and (ii) the signal amplification with DNA CT platforms for improved sensitivity and detection are described.

First, improvements to the control over DNA monolayer formation are reported through the incorporation of copper-free click chemistry into DNA monolayer assembly. As opposed to conventional film formation involving the self-assembly of thiolated DNA, copper-free click chemistry enables DNA to be tethered to a pre-formed mixed alkylthiol monolayer. The total amount of DNA in the final film is directly related to the amount of azide in the underlying alkylthiol monolayer. DNA monolayers formed with this technique are significantly more homogeneous and lower density, with a larger amount of individual helices exposed to the analyte solution. With these improved monolayers, significantly more sensitive detection of the transcription factor TATA binding protein (TBP) is achieved.

Using low-density DNA monolayers, two-electrode DNA arrays were designed and fabricated to enable the placement of multiple DNA sequences onto a single underlying electrode. To pattern DNA onto the primary electrode surface of these arrays, a copper precatalyst for click chemistry was electrochemically activated at the secondary electrode. The location of the secondary electrode relative to the primary electrode enabled the patterning of up to four sequences of DNA onto a single electrode surface. As opposed to conventional electrochemical readout from the primary, DNA-modified electrode, a secondary microelectrode, coupled with electrocatalytic signal amplification, enables more sensitive detection with spatial resolution on the DNA array electrode surface. Using this two-electrode platform, arrays have been formed that facilitate differentiation between well-matched and mismatched sequences, detection of transcription factors, and sequence-selective DNA hybridization, all with the incorporation of internal controls.

For effective clinical detection, the two working electrode platform was multiplexed to contain two complementary arrays, each with fifteen electrodes. This platform, coupled with low density DNA monolayers and electrocatalysis with readout from a secondary electrode, enabled even more sensitive detection from especially small volumes (4 μL per well). This multiplexed platform has enabled the simultaneous detection of two transcription factors, TBP and CopG, with surface dissociation constants comparable to their solution dissociation constants.

With the sensitivity and selectivity obtained from the multiplexed, two working electrode array, an electrochemical signal-on assay for activity of the human methyltransferase DNMT1 was incorporated. DNMT1 is the most abundant human methyltransferase, and its aberrant methylation has been linked to the development of cancer. However, current methods to monitor methyltransferase activity are either ineffective with crude samples or are impractical to develop for clinical applications due to a reliance on radioactivity. Electrochemical detection of methyltransferase activity, in contrast, circumvents these issues. The signal-on detection assay translates methylation events into electrochemical signals via a methylation-specific restriction enzyme. Using the two working electrode platform combined with this assay, DNMT1 activity from tumor and healthy adjacent tissue lysate were evaluated. Our electrochemical measurements revealed significant differences in methyltransferase activity between tumor tissue and healthy adjacent tissue.

As differential activity was observed between colorectal tumor tissue and healthy adjacent tissue, ten tumor sets were subsequently analyzed for DNMT1 activity both electrochemically and by tritium incorporation. These results were compared to expression levels of DNMT1, measured by qPCR, and total DNMT1 protein content, measured by Western blot. The only trend detected was that hyperactivity was observed in the tumor samples as compared to the healthy adjacent tissue when measured electrochemically. These advances in DNA CT-based platforms have propelled this class of sensors from the purely academic realm into the realm of clinically relevant detection.

Biological Activity of Rhodium Metalloinsertors and the Design of Bifunctional Conjugates

The Barton laboratory has established that octahedral rhodium complexes bearing the sterically expansive 5,6-chrysene diimine ligand can target thermodynamically destabilized sites, such as base pair mismatches, in DNA with high affinity and selectivity. These complexes approach DNA from the minor groove, ejecting the mismatched base pairs from the duplex in a binding mode termed metalloinsertion. In recent years, we have shown that these metalloinsertor complexes also exhibit cytotoxicity preferentially in cancer cells that are deficient in the mismatch repair (MMR) machinery.

Here, we establish that a sensitive structure-activity relationship exists for rhodium metalloinsertors. We studied the relationship between the chemical structures of metalloinsertors and their effect on biological activity for ten complexes with similar DNA binding affinities, but wide variation in their lipophilicity. Drastic differences were observed in the selectivities of the complexes for MMR-deficient cells. Compounds with hydrophilic ligands were highly selective, exhibiting preferential cytotoxicity in MMR-deficient cells at low concentrations and short incubation periods, whereas complexes with lipophilic ligands displayed poor cell-selectivity. It was discovered that all of the complexes localized to the nucleus in concentrations sufficient for mismatch binding; however, highly lipophilic complexes also exhibited high mitochondrial uptake. Significantly, these results support the notion that mitochondrial DNA is not the desired target for our metalloinsertor complexes; instead, selectivity stems from targeting mismatches in genomic DNA.

We have also explored the potential for metalloinsertors to be developed into more complex structures with multiple functionalities that could either enhance their overall potency or impart mismatch selectivity onto other therapeutic cargo. We have constructed a family of bifunctional metalloinsertor conjugates incorporating cis-platinum, each unique in its chemical structure, DNA binding interactions, and biological activity. The study of these complexes in MMR-deficient cells has established that the cell-selective biological activity of rhodium metalloinsertors proceeds through a critical cellular pathway leading to necrosis.

We further explored the underlying mechanisms surrounding the biological response to mismatch recognition by metalloinsertors in the genome. Immunofluorescence assays of MMR-deficient and MMR-proficient cells revealed that a critical biomarker for DNA damage, phosphorylation of histone H2AX (γH2AX) rapidly accumulates in response to metalloinsertor treatment, signifying the induction of double strand breaks in the genome. Significantly, we have discovered that our metalloinsertor complexes selectively inhibit transcription in MMR-deficient cells, which may be a crucial checkpoint in the eventual breakdown of the cell via necrosis. Additionally, preliminary in vivo studies have revealed the capability of these compounds to traverse the complex environments of multicellular organisms and accumulate in MMR-deficient tumors. Our ever-increasing understanding of metalloinsertors, as well as the development of new generations of complexes both monofunctional and bifunctional, enables their continued progress into the clinic as promising new chemotherapeutic agents.

Conversão do metanol em olefinas catalisada por zeólitas com diferentes características ácidas e estruturais

A reação de transformação de metanol em olefinas leves foi investigada sobre as peneiras moleculares HZSM-5, HFER, SAPO-34 e HMCM-22. A caracterização físico-química das amostras foi realizada através das técnicas de FRX, DRX, fisissorção de nitrogênio, MEV, espectrometria no IV com adsorção de piridina e TPD de NH3. O desempenho catalítico das mesmas foi comparado em condições de isoconversão inicial de 755%. Verificou-se que as características ácidas e estruturais exerceram forte influência sobre o desempenho catalítico quanto à atividade, estabilidade e seletividade aos produtos da reação. A amostra mais estável foi a HZSM-5 que apresentou maior densidade de sítios fortes e uma estrutura porosa que permite uma circulação tridimensional das moléculas. Já a menos estável, SAPO-34, apresentou a menor concentração de sítios ácidos fortes dentre os materiais estudados e uma estrutura com cavidades com aberturas estreitas (4Å) que oferecem restrições ao acesso dos reagentes aos sítios ácidos do catalisador. Quanto à seletividade a olefinas, a primeira foi mais seletiva a propeno e a segunda, a eteno. A ferrierita não se mostrou seletiva às olefinas leves tendo apresentado, no entanto, comportamento promissor quanto a formação de DME a partir do metanol. Já a HMCM-22 foi seletiva às olefinas leves e aos hidrocarbonetos com 4, 5 e 6 ou mais átomos de carbono. A influência da temperatura no desempenho catalítico foi investigada variando-se a temperatura de reação (300, 400 e 500C). Verificou-se que para a HZSM-5 e HMCM-22, perda da atividade catalítica foi intensificada a partir de 400C. Quanto à seletividade a olefinas leves, apenas a SAPO-34 não se mostrou sensível a variações na temperatura, efeito este que foi nitidamente observado nos outros três catalisadores: um aumento na temperatura promoveu um aumento na seletividade a olefinas leves no caso da HZSM-5 e da HMCM-22 e queda nesse valor para a HFER

The design of multimesh, multidepth gillnet fleets for use in the Lake Victoria Fisheries Research Project

Multimesh, multidepth gillnet fleets are useful in assessing fish stock abundance, size distribution and depth distribution. Using data collected on net mesh selectivity for Nile perch, Lates niloticus (L.), in the Kenyan waters of Lake Victoria, suitable mesh sizes for gillnet fleets for use in the Lake Victoria Fisheries Research Project were determined. The modal selection length for Nile perch in the mesh sized used in the earlier experiment were determined, as was the size range vulnerable to capture. Initial trials suggest 60% of the Nile perch swim within 5 m of the bottom. Setting and hauling of the nets is simple and quick, allowing the nets to be used at the same time as other sampling programmes.

记录结构对体光栅各向异性衍射选择特性研究

以静态体光栅各向异性衍射理论分析模型为基础，将与体光栅厚度有关的记录光强调制度引入到各向异性耦合波方程中．利用耦合波理论分析了光折变晶体中体光栅的各向异性衍射性能，研究了在不同初始记录光强比的条件下，记录光入射角的改变对光折变体光栅各向异性布喇格衍射性能的影响。结果表明：记录光入射角和记录光初始光强比均对体光栅的各向异性衍射具有选择性；与此相比，记录光入射角和记录光初始光强比对于体光栅各向同性衍射不具备选择性。

Transferência de renda e capital portador de juros: uma insidiosa captura

Esta dissertação tem como objeto de estudo a relação entre capital portador de juros e programas de transferência de renda na atualidade. O objetivo pretendido é a análise da dinâmica de organização desta forma de capital e seus desdobramentos na contemporaneidade, bem como os impactos e determinações impostas às políticas sociais em tempos de financeirização do capital. Partimos da hipótese de que os programas de transferência de renda contribuem para o processo de contra-reforma das políticas sociais, tanto pela focalização preconizada como pela priorização orçamentária, em detrimento dos demais programas e projetos da assistência social, além de permitirem a remuneração do capital portador de juros por meio dos recursos que destinam às instituições bancárias. A pesquisa realizada utilizou-se de análise documental e teórica com base na tradição marxista, buscando analisar criticamente as configurações das políticas sociais na atualidade e o papel das transferências de renda. Buscamos ainda analisar o orçamento público no âmbito federal referente à Seguridade Social e, em particular, aos programas de transferência de renda no âmbito da Assistência Social. Escolhemos estudar o orçamento da Renda Mensal Vitalícia, o Benefício de Prestação Continuada e o Programa Bolsa-Família, entre os anos de 2006 e 2009, analisando os recursos que estes destinam às instituições bancárias que operacionalizam o repasse dos benefícios. O resultado da pesquisa demonstrou que os programas de transferência de renda são estratégias de contra-reformar as políticas sociais ao incentivar o processo de assistencialização da Seguridade Social visto que apresentam um aumento contínuo e substancial no âmbito orçamentário e ao operarem com a seletividade do atendimento aos mais pobres na contramão dos princípios fundamentais presentes no texto constitucional. São ainda mecanismos de alimentação do capital portador de juros ao repassarem consideráveis somas de recursos para as instituições bancárias e inserirem a classe trabalhadora pobre no mundo das finanças.

Stock assessment of Lates niloticus (L.), Oreochromis niloticus (L.) and Rastrineobola argentea (Pellegrin) using fisheries-dependent data from Tanzania waters of Lake Victoria

Three commercially important fish species, Lates niloticus (L.), Rastrineobola argentea (Pellegrin) and Oreochromis niloticus (L.) that are fished by artisanal fishermen of Lake Victoria, Tanzania part, were studied in Kagera, Mwanza and Mara beaches from October 1997 to July 1999. Catches, effort, exploitation and stock structure were investigated. Beaches for sampling were selected based on importance for landing the above named fish species. The number of boats found on beach that day, the number that lay idle and their means of propulsion were recorded. As many boats as possible were sampled for gear type and gear size. The catches were sorted into species and measured. Variation in the species and size composition of landings was observed between regions, between months and between gears used. The implications of the findings to management are discussed.

Anisotropic diffraction of stratified volume holographic grating in photorefractive media

We have studied the anisotropic diffraction properties of the stratified volume holographic gratings recorded in photorefractive media using the anisotropic coupled wave theory. It is shown that the diffraction efficiency of such system exhibit the uniform periodic Bragg selectivity properties. In addition the dependence of the stratified volume holographic optical elements (SVHOEs) diffraction properties on the buffer-layer thickness, grating-layer thickness, number of modulation layers, and total thickness of system are discussed in detail. (c) 2005 Elsevier GrnbH. All rights reserved.

Desenvolvimento de membranas de poli(éter imida) sultonada para a permeacão de gases

A busca por membranas com propriedades adequadas a separação de gases em escala industrial tem levado a modificação e sIntese de polImeros de engenharia, com objetivo de obter membranas com propriedades adequadas. Uma das modificaçoes que tem se apresentado promissora é a inserção de grupos sulfônicos em polImeros comerciais. Espera-se que o polImero sulfonado apresente um aumento na permeação de gases polares, em relação a gases apolares, devido a sua estrutura mais polar e flexIvel. Neste contexto, o objetivo do presente trabalho é a sIntese e caracterização de membranas de poli(éter imida) sulfonada para a permeação de gases. Um planejamento experimental foi desenvolvido, em diferentes condiçoes reacionais de temperatura, tempo e excesso de um dos reagentes (ácido acético), para a sIntese de poli(éter imida) sulfonada (SPEI). Através deste planejamento, constatou-se que as variáveis que mais influenciam o grau de sulfonação são a temperatura e o tempo. O polImero com o maior grau de sulfonação, determinado por capacidade de troca iônica (IEC= 92 mEq H+/g), foi utilizado para o preparo da membrana de SPEI, obtida pela técnica de inversão de fase por evaporação do solvente, utilizando-se clorofórmio como solvente. Este filme foi caracterizado a partir das seguintes análises: espectroscopia de infravermelho (FTIR), calorimetria diferencial de varredura (DSC), análise termogravimétrica (TGA) e microscopia eletrônica de varredura (MEV), a fim de avaliar a influência da inserção do grupo sulfônico na matriz polimérica. O espectro de infravermelho de SPEI apresentou bandas relacionadas as vibraçoes assimétricas em 1240 cm-1 (ligação O=S=O), ligação simétrica em 1171 cm-1 (O=S=O) e ligação S-O entre 1010-1024 cm-1. Isto indica a presença de grupos sulfônicos. A análise de DSC foi realizada entre 150-250C. Nesta faixa, não foram observadas alteraçoes na temperatura de transição vItrea (Tg) do polImero modificado (217C). Acredita-se que a decomposição do grupo sulfona aconteça antes da temperatura atingir o Tg do polImero. Esta suposição é confirmada na análise de TGA. As imagens de MEV mostraram que foram obtidos filmes livres de poros e defeitos. A membrana da SPEI foi utilizada no ensaio de permeaçao dos gases 02, N2 e C02, a fim de determinar a permeabilidade e seletividade da membrana. As permeabilidades encontradas para o gas oxigênio foram de 0,76 barrer para a PEI e 0,46 barrer para a SPEI. A seletividade do dióxido de carbono em relaçao ao oxigênio aumentou de 3,5, na membrana de PEI, para 4,83, na membrana de SPEI. Em relaçao ao nitrogênio, as permeabilidades medidas foram 0,064 barrer e 0,043 barrer, para a PEI e para a SPEI, respectivamente, enquanto a seletividade em relaçao ao C02 aumentou de 41,1 para 55,5. Estes resultados indicam que o efeito de sorçao predominou devido ao aumento das interaçöes moleculares, reduzindo assim o volume livre, o que tornou a membrana sulfonada mais compacta, com permeabilidade menor e maior seletividade. Estes resultados corroboram com a premissa de que a sulfonaçao é um processo promissor para o desenvolvimento de membranas mais eficientes.

MDT三维工具集在齿轮造型与分析中的应用

通过MDT三维工具集方便地实现了直齿轮的造型，重点说明了用基于特征造型的方法在MDT环境下实现斜齿轮造型的详细过程，并用MDT的有限元分析功能对齿轮的受力善进行了分析。整个过程在MDT的集成环境下完成，对齿轮的工程设计具有较高的实用价值。

Analysis of an ultrashort pulsed finite beam diffracted by volume gratings

We obtain analytical solutions of the coupled wave equations that describe the Bragg diffraction of ultrashort pulsed finite beams by a thick planar grating, using two-dimensional coupled wave theory. The diffraction properties for the case of an ultrashort pulsed finite beam with Gaussian profiles in both the time and spatial domains are investigated. The spectral bandwidth of the diffracted beam, the Bragg selectivity bandwidth and the diffraction efficiency of the volume grating are influenced by the geometry parameter and the input bandwidth. Therefore extra attention should be paid to designing optical elements based on volume gratings for use with ultrashort pulsed waves in applications of pulse shaping and processing.

Do salvacionismo à segregação: a experiência dos Centros Integrados de Educação Pública no Rio de Janeiro

Este trabalho versa sobre a relação entre a implantação de um projeto de educação pública e a sua receptividade social. Resgata as concepções que deram origem ao programa de implantação das escolas de tempo integral no estado do Rio de Janeiro e como hoje elas são vistas por seus usuários. Discute os resultados inesperados que teve o projeto educacional salvador (não só da educação como também das populações empobrecidas do estado do Rio de Janeiro). Pretendendo ser inclusivo, dando ao pobre acesso a benefícios que não tinha, produziu mais segregação, repetindo a seletividade que a escola pública brasileira apresenta.

Diffraction properties of ultrashort pulsed beams with arbitrary temporal profiles studied with a volume holographic grating

Using modified two- dimensional coupled- wave theory, the diffraction properties of ultrashort pulsed beams with arbitrary temporal profiles are studied with a volume holographic grating. Analytical expressions for the profiles of the transmitted and diffracted beams are obtained. It is shown that the Bragg selectivity bandwidth of the volume grating can be influenced by the geometry parameter. Numerical results are illustrated for three different temporal profiles. For different temporal profiles, the ratios of the diffraction bandwidths to input bandwidths are discussed.