Role of G protein coupled inwardly rectifier K+ channels (GIRK) on the neurobiology depression

353 págs.

光学蛋白质芯片关键技术研究及生物医学应用

蛋白质芯片，又称蛋白质微阵列，是继基因芯片之后又一对生物医学研究乃至人类健康具有重大应用价值的生物芯片。目前，蛋白质芯片的发展还处于初级阶段，还没有真正走出基因芯片的阴影，在蛋白质芯片的制作和检测中借用了很多基因芯片技术。许多在基因芯片上没有解决的问题，如低丰度信号的检测、不同的点样质量带来的干扰和基于标记方法的检测技术问题，也同样在蛋白质芯片上出现，并表现得更为突出，主要原因在于蛋白质分子与DNA分子之间的巨大差异。基于椭偏光学成像技术的无标记光学蛋白质芯片技术的提出正是为了解决目前蛋白质芯片发展中存在的一些问题。椭偏光学成像技术是近来发展起来的一种新型的光学检测技术，目前国际上尚未发展出适合于其检测特点的蛋白质芯片技术。本文研究的无标记光学蛋白质芯片是多学科高度交叉的生物技术，它主要包含五方面的内容：（1）芯片设计；（2）配基装配；（3）芯片反应器；（4）芯片信号采样和处理；（5）芯片数据库。本文的研究工作集中在前三部分内容，创新性主要表现在以下几方面：研制了微流道蛋白质芯片系统；建立了高通量蛋白质芯片制备方法；发展了多种芯片表面改性和配基固定方法；开展了无标记光学蛋白质芯片在生物医学领域的应用。本文研制了微流道蛋白质芯片系统，建立了化学格式化法高通量蛋白质芯片制备方法。通过这两种方法制备的蛋白质芯片能够满足椭偏光学成像技术定量检测的要求，而且这两种蛋白质芯片与本实验室早期发展的生物活性探针和多元蛋白质芯片结合在一起形成了较为完整的无标记光学蛋白质芯片系列，使之不但能够简单方便地进行低通量蛋白质检测，而且也具有了高通量蛋白质分析的能力。本文建立的微流道蛋白质芯片系统把微流控芯片和微阵列芯片二者的优势结合在一起，以微型流动控制见长的微流控芯片被设计成微阵列芯片的微型点样仪与微型高效率反应器，而以并行分析见长的微阵列芯片成为微流道系统的专用传感器件，并且实现了在同一微型分析系统中进行蛋白质芯片的制备与检测。微流道蛋白质芯片系统改变了阵列式生物芯片整体反应模式，使得芯片的使用更加灵活方便。通过微流道蛋白质芯片系统进行蛋白质芯片制备与检测，显著降低了试剂和样品的消耗，缩短了检测时间，把检测灵敏度提高到了纳克／毫升量级。能够多次重复使用的微流道蛋白质芯片系统，使得蛋白质芯片的使用成本大幅度降低。本文针对椭偏光学成像技术的检测特点、不同的芯片设计和配基发展了多种表面改性及配基固定技术，实现了配基分子共价连接、抗体分子定向固定、混合硅烷膜层对硅基底的表面改性以及混合烷硫醇SAM对金基底的表面改性。这些技术的使用明显提高了配基分子在蛋白质芯片表面上的稳定性，较好地保持了配基分子的生物活性，从而大幅度提高了无标记光学蛋白质芯片的检测灵敏度。本文在上述关键技术发展的基础上，还成功地开展了无标记蛋白质芯片在生物医学领域的应用。实现了在一块蛋白质芯片上进行乙肝五项指标同时检测；通过蛋白质芯片对病毒一噬菌体进行了直接检测；乙肝表面抗原检测和乳腺癌标志物定量检测已经能够达到临床免疫检测的水平；还通过无标记蛋白质芯片技术同时研究了多对生物分子之间的相互作用，并通过模型化分析获得了相互作用动力学常数。

Asymmetric organocatalytic cascade reactions

302 p. : gráf.

Structural requirements for catalysis: studies on RTEM-1 β-lactamase

β-lactamases are a group of enzymes that confer resistance to penam and cephem antibiotics by hydrolysis of the β-lactam ring, thereby inactivating the antibiotic. Crystallographic and computer modeling studies of RTEM-1 β-lactamase have indicated that Asp 132, a strictly conserved residue among the class A β-lactamases, appears to be involved in substrate binding, catalysis, or both. To study the contribution of residue 132 to β-lactamase function, site saturation mutagenesis was used to generate mutants coding for all 20 amino acids at position 132. Phenotypic screening of all mutants indicated that position 132 is very sensitive to amino acid changes, with only N132C, N132D, N132E, and N132Q showing any appreciable activity. Kinetic analysis of three of these mutants showed increases in K_M, along with substantial decreases in k_(cat). Efforts to trap a stable acyl-enzyme intermediate were unsuccessfuL These results indicate that residue 132 is involved in substrate binding, as well as catalysis, and supports the involvement of this residue in acylation as suggested by Strynadka et al.

Crystallographic and computer modeling studies of RTEM-1 β-lactamase have indicated that Lys 73 and Glu 166, two strictly conserved residues among the class A β-lactamases, appear to be involved in substrate binding, catalysis, or both. To study the contribution of these residues to β-lactamase function, site saturation mutagenesis was used to generate mutants coding for all 20 amino acids at positions 73 and 166. Then all 400 possible combinations of mutants were created by combinatorial mutagenesis. The colonies harboring the mutants were screened for growth in the presence of ampicillin. The competent colonys' DNA were sequenced, and kinetic parameters investigated. It was found that lysine is essential at position 73, and that position 166 only tolerated fairly conservative changes (Aspartic acid, Histidine, and Tyrosine). These functional mutants exhibited decreased kcat's, but K_M was close to wild-type levels. The results of the combinatorial mutagenesis experiments indicate that Lysis absolutely required for activity at position 73; no mutation at residue 166 can compensate for loss of the long side chain amine. The active mutants found--K73K/E166D, K73KIE166H, and K73KIE166Y were studied by kinetic analysis. These results reaffirmed the function of residue 166 as important in catalysis, specifically deacylation.

The identity of the residue responsible for enhancing the active site serine (Ser 70) in RTEM-1 β-lactamase has been disputed for some time. Recently, analysis of a crystal structure of RTEM-1 β-lactamase with covalently bound intermediate was published, and it was suggested that Lys 73, a strictly conserved residue among the class A β-lactamases, was acting as a general base, activating Ser 70. For this to be possible, the pK_a of Lys 73 would have to be depressed significantly. In an attempt to assay the pK_a of Lys 73, the mutation K73C was made. This mutant protein can be reacted with 2-bromoethylamine, and activity is restored to near wild type levels. ^(15)N-2-bromoethylamine hydrobromide and ^(13)C-2-bromoethylamine hydrobromide were synthesized. Reacting these compounds with the K73C mutant gives stable isotopic enrichment at residue 73 in the form of aminoethylcysteine, a lysine homologue. The pK_a of an amine can be determined by NMR titration, following the change in chemical shift of either the ^(15)N-amine nuclei or adjacent Be nuclei as pH is changed. Unfortunately, low protein solubility, along with probable label scrambling in the Be experiment, did not permit direct observation of either the ^(15)N or ^(13)C signals. Indirect detection experiments were used to observe the protons bonded directly to the ^(13)C atoms. Two NMR signals were seen, and their chemical shift change with pH variation was noted. The peak which was determined to correspond to the aminoethylcysteine residue shifted from 3.2 ppm down to 2.8 ppm over a pH range of 6.6 to 12.5. The pK_a of the amine at position 73 was determined to be ~10. This indicates that residue 73 does not function as a general base in the acylation step of the reaction. However the experimental measurement takes place in the absence of substrate. Since the enzyme undergoes conformational changes upon substrate binding, the measured pK_a of the free enzyme may not correspond to the pK_a of the enzyme substrate complex.

SAMUDRA Report No. 56, July 2010

(PDF contains 56 pages)

Carbon-carbon bond forming reactions from bis(carbene)-platinum(II) complexes and olefin polymerization and oligomerization using group 4 post-metallocene complexes

A long-standing challenge in transition metal catalysis is selective C–C bond coupling of simple feedstocks, such as carbon monoxide, ethylene or propylene, to yield value-added products. This work describes efforts toward selective C–C bond formation using early- and late-transition metals, which may have important implications for the production of fuels and plastics, as well as many other commodity chemicals.

The industrial Fischer-Tropsch (F-T) process converts synthesis gas (syngas, a mixture of CO + H₂) into a complex mixture of hydrocarbons and oxygenates. Well-defined homogeneous catalysts for F-T may provide greater product selectivity for fuel-range liquid hydrocarbons compared to traditional heterogeneous catalysts. The first part of this work involved the preparation of late-transition metal complexes for use in syngas conversion. We investigated C–C bond forming reactions via carbene coupling using bis(carbene)platinum(II) compounds, which are models for putative metal–carbene intermediates in F-T chemistry. It was found that C–C bond formation could be induced by either (1) chemical reduction of or (2) exogenous phosphine coordination to the platinum(II) starting complexes. These two mild methods afforded different products, constitutional isomers, suggesting that at least two different mechanisms are possible for C–C bond formation from carbene intermediates. These results are encouraging for the development of a multicomponent homogeneous catalysis system for the generation of higher hydrocarbons.

A second avenue of research focused on the design and synthesis of post-metallocene catalysts for olefin polymerization. The polymerization chemistry of a new class of group 4 complexes supported by asymmetric anilide(pyridine)phenolate (NNO) pincer ligands was explored. Unlike typical early transition metal polymerization catalysts, NNO-ligated catalysts produce nearly regiorandom polypropylene, with as many as 30-40 mol % of insertions being 2,1-inserted (versus 1,2-inserted), compared to <1 mol % in most metallocene systems. A survey of model Ti polymerization catalysts suggests that catalyst modification pathways that could affect regioselectivity, such as C–H activation of the anilide ring, cleavage of the amine R-group, or monomer insertion into metal–ligand bonds are unlikely. A parallel investigation of a Ti–amido(pyridine)phenolate polymerization catalyst, which features a five- rather than a six-membered Ti–N chelate ring, but maintained a dianionic NNO motif, revealed that simply maintaining this motif was not enough to produce regioirregular polypropylene; in fact, these experiments seem to indicate that only an intact anilide(pyridine)phenolate ligated-complex will lead to regioirregular polypropylene. As yet, the underlying causes for the unique regioselectivity of anilide(pyridine)phenolate polymerization catalysts remains unknown. Further exploration of NNO-ligated polymerization catalysts could lead to the controlled synthesis of new types of polymer architectures.

Finally, we investigated the reactivity of a known Ti–phenoxy(imine) (Ti-FI) catalyst that has been shown to be very active for ethylene homotrimerization in an effort to upgrade simple feedstocks to liquid hydrocarbon fuels through co-oligomerization of heavy and light olefins. We demonstrated that the Ti-FI catalyst can homo-oligomerize 1-hexene to C₁₂ and C₁₈ alkenes through olefin dimerization and trimerization, respectively. Future work will include kinetic studies to determine monomer selectivity by investigating the relative rates of insertion of light olefins (e.g., ethylene) vs. higher α-olefins, as well as a more detailed mechanistic study of olefin trimerization. Our ultimate goal is to exploit this catalyst in a multi-catalyst system for conversion of simple alkenes into hydrocarbon fuels.

Bimetallic olefin polymerization catalysis : mechanisms and applications of proximal effects

This dissertation covers progress with bimetallic polymerization catalysts. The complexes we have designed were aimed at expanding the capabilities of homogeneous polymerization catalysts by taking advantage of multimetallic effects. Such effects were examined in group 4 and group 10 bimetallic complexes; proximity and steric repulsion were determined to be major factors in the effects observed.

Chapters 2 and 3 introduce the rigid p-terphenyl dinucleating framework utilized in most of this thesis. The permethylation of the central arene allows for the separation of syn and anti atropisomers of the terphenyl compounds. Kinetic studies were carried out to examine the isomerization of the dinucleating bis(salicylaldimine) ligand precursors. Metallation of the syn and anti bis(salicylaldimine)s using Ni(Me)2(tmeda) and excess pyridine afforded dinickel bisphenoxyiminato complexes with a methyl and a pyridyl ligand on each nickel. The syn and anti atropisomers of the dinickel complexes were structurally characterized and utilized in ethylene and ethylene/α-olefin polymerizations. Monometallic analogues were also synthesized and tested for polymerization activity. Ethylene polymerizations were performed in the presence of primary, secondary, and tertiary amines – additives that generally deactivate nickel polymerization catalysts. Inhibition of this deactivation was observed with the syn atropisomer of the bimetallic species, but not with the anti or monometallic analogues. A mechanism was proposed wherein steric repulsion of the substituents on proximal nickel centers disfavors simultaneous ligation of base to both of the metal centers. The bimetallic effect has been explored with respect to size and binding ability of the added base.

Chapter 4 presents the optimization of the bisphenoxyimine ligand synthesis and synthesis of syn and anti m-terphenyl analogues. Metallation with NiClMe(PMe₃)₂ yielded phosphine-ligated dinickel complexes, which have been structurally characterized. Ethylene/1-hexene copolymerizations in the presence of amines using Ni(COD)₂ as a phosphine scavenger showed significantly improved activity relative to the pyridine-ligated analogues. Incorporation of amino olefins in copolymerizations with ethylene was accomplished, and a mechanism was proposed based on proximal effects. Copolymerization trials with a variety of amino olefins and ethylene/1-hexene/amino olefin terpolymerizations were completed.

Early transition metal complexes based on the rigid p-terphenyl framework were designed with a variety of donor sets (Chapter 5 and Appendix B). Chapter 5 details the use of syn dizirconium di[amine bis(phenolate)] complexes for isoselective 1-hexene and propylene homopolymerizations. Ligand variation and monometallic complexes were studied to determine the origin of tacticity control. A mechanistic proposal was presented based on the symmetry at zirconium and the steric effects of the proximal metal center. Appendix B covers additional studies of bimetallic early transition metal complexes based on the p-terphenyl. Dititanium, dizirconium, and asymmetric complexes with bisphenoxyiminato ligands and derivatives thereof were targeted. Progress toward the synthesis of these complexes is described along with preliminary polymerization data. 1-hexene/diene copolymerizations and attempted polymerizations in the presence of ethers and esters with the syn dizirconium di[amine bis(phenolate)] complexes demonstrate the potential for further applications of this system in catalysis.

Appendix A includes work toward palladium catalysts for insertion polymerization of polar monomers. These complexes were based on dioxime and diimine frameworks with the intent of binding Lewis acidic metals at the oxime oxygens, at pendant phenolic donors, or at pendant aminediol moieties. The synthesis and structural characterization of a number of palladium and Lewis acid complexes is presented. Due to the instability of the desired species, efforts toward isolation of the desired complexes proved unsuccessful, though preliminary ethylene/methyl acrylate copolymerizations using in situ activation of the palladium species were attempted.

Designing conformational control of human tissue transglutaminase

Understanding the mechanisms of enzymes is crucial for our understanding of their role in biology and for designing methods to perturb or harness their activities for medical treatments, industrial processes, or biological engineering. One aspect of enzymes that makes them difficult to fully understand is that they are in constant motion, and these motions and the conformations adopted throughout these transitions often play a role in their function.

Traditionally, it has been difficult to isolate a protein in a particular conformation to determine what role each form plays in the reaction or biology of that enzyme. A new technology, computational protein design, makes the isolation of various conformations possible, and therefore is an extremely powerful tool in enabling a fuller understanding of the role a protein conformation plays in various biological processes.

One such protein that undergoes large structural shifts during different activities is human type II transglutaminase (TG2). TG2 is an enzyme that exists in two dramatically different conformational states: (1) an open, extended form, which is adopted upon the binding of calcium, and (2) a closed, compact form, which is adopted upon the binding of GTP or GDP. TG2 possess two separate active sites, each with a radically different activity. This open, calcium-bound form of TG2 is believed to act as a transglutaminse, where it catalyzes the formation of an isopeptide bond between the sidechain of a peptide-bound glutamine and a primary amine. The closed, GTP-bound conformation is believed to act as a GTPase. TG2 is also implicated in a variety of biological and pathological processes.

To better understand the effects of TG2’s conformations on its activities and pathological processes, we set out to design variants of TG2 isolated in either the closed or open conformations. We were able to design open-locked and closed-biased TG2 variants, and use these designs to unseat the current understanding of the activities and their concurrent conformations of TG2 and explore each conformation’s role in celiac disease models. This work also enabled us to help explain older confusing results in regards to this enzyme and its activities. The new model for TG2 activity has immense implications for our understanding of its functional capabilities in various environments, and for our ability to understand which conformations need to be inhibited in the design of new drugs for diseases in which TG2’s activities are believed to elicit pathological effects.

SAMUDRA Report No. 57, November 2010

Mackerel mayhem: On the ongoing dispute in northern Europe over the boom in mackerel stocks. Small and mighty: The Banjul civil society declaration on sustainable livelihoods in African fisheries. Building partnerships: The case of Red Sea fisheries management shows how fishers’ rights can be strengthened. The write stuff: The website of Comité Local des Pêches Le Guilvinec celebrates its second anniversary. Frankenfish salmon: The United States is close to approving genetically engineered salmon. Trawl brawl: Indian and Sri Lankan fishermen have worked to co-exist in the Palk Bay. Tsunami recovery: On the traditional tenure system of the fishing community of Juan Fernández. New goals from Nagoya: The Nagoya meeting of the Convention on Biological Diversity made some progress. Beyond Bangkok: The civil society workshop in Costa Rica focused on small-scale fishers in Latin America. Securing small-scale fisheries. Recommendations adopted at the San José FAO workshop dealt with small-scale fisheries. (PDF contains 56 pages)

Exciplexes in fluorescence quenching of aromatic hydrocarbons by tertiary aliphatic amines

Strong quenching of the fluorescence of aromatic hydrocarbons by tertiary aliphatic amines has been observed in solution at room temperature. Accompanying the fluorescence quenching of aromatic hydrocarbons, an anomalous emission is observed. This new emission is very broad, structureless and red-shifted from the original hydrocarbon fluorescence.

Kinetic studies indicate that this anomalous emission is due to an exciplex formed by an aromatic hydrocarbon molecule in its lowest excited singlet state with an amine molecule. The fluorescence quenching of the aromatic hydrocarbons is due to the depopulation of excited hydrocarbon molecules by the formation of exciplexes, with subsequent de-excitation of exciplexes by either radiative or non-radiative processes.

Analysis of rate constants shows the electron-transfer nature of the exciplex. Through the study of the effects on the frequencies of exciplex emissions of substituents on the hydrocarbons, it is concluded that partial electron transfer from the amine molecule to the aromatic hydrocarbon molecule in its lowest excited singlet state occurs in the formation of exciplex. Solvent effects on the exciplex emission frequencies further demonstrate the polar nature of the exciplex.

A model based on this electron-transfer nature of exciplex is proposed and proves satisfactory in interpreting the exciplex emission phenomenon in the fluorescence quenching of aromatic hydrocarbons by tertiary aliphatic amines.

Catalisadores para hidroisomerização de n-hexadecano

No processo de hidrocraqueamento para a produção de lubrificantes ocorre a formação de uma corrente rica em compostos parafínicos que possuem alto ponto de fluidez, apesar dos mesmos apresentarem excelentes desempenhos em termos de estabilidade térmica e oxidativa. A transformação das n-parafinas obtidas nestas correntes em isoparafinas e compostos naftênicos, os quais possuem menores pontos de fluidez, se faz necessária a fim de enquadrar esta propriedade. Uma das rotas catalíticas mais importantes neste sentido é a hidroisodesparafinação ou HIDW (hydroisodewaxing) que consiste na conversão de n-parafinas nas respectivas isoparafinas, onde são empregados catalisadores bifuncionais zeolíticos com a ocorrência de seletividade de forma. No caso dos catalisadores industriais, se faz necessária a dispersão da fase metálica e da zeólita em uma matriz amorfa para viabilizar sua conformação e melhorar a resistência mecânica do catalisador final. Neste cenário, o objetivo deste trabalho foi preparar e analisar o desempenho de uma série de catalisadores à base de zeólita beta inseridos numa matriz de alumina, variando-se o teor de zeólita e o tipo de precursor de Pt utilizado. Os catalisadores foram avaliados na reação de hidroisomerização de um composto modelo, no caso, n-hexadecano. Os testes realizados para avaliação da atividade e seletividade foram conduzidos em um reator de fluxo contínuo em alta pressão e fase líquida em unidade de laboratório. Os catalisadores foram testados em condições operacionais que proporcionassem uma ampla faixa de conversões do n-C16. Verificou-se que as atividades dos catalisadores foram proporcionais ao teor de zeólita no catalisador, indicando que a função ácida, neste catalisador bifuncional, é a etapa limitante do processo. Quanto à natureza do precursor de Pt, o catalisador preparado com ácido cloroplatínico foi sensivelmente mais ativo que os preparados com o complexo aminplatina. No entanto, para todos os catalisadores, a distribuição de produtos em função da conversão foi similar, independente do teor de zeólita e da natureza do precursor de platina. Foi também determinado o ponto de fluidez de uma série de produtos de reação, obtendo-se valores entre 17,5 C (n-hexadecano) e - 41 C (produto com 98% de conversão). Obteve-se uma boa correlação entre o ponto de fluidez e a composição dos produtos, considerando-se a presença de isômeros mono, di e tri-substituídos e compostos de menor peso molecular que C16

Effect of environmental conditions on the distribution of Pacific mackerel (Scomber japonicus) larvae in the California Current system

We modeled the probability of capturing Pacif ic mackerel (Scomber japonicus) larvae as a function of environmental variables for the Southern California Bight (SCB) most years from 1951 through 2008 and Mexican waters offshore of Baja California from 1951 through 1984. The model exhibited acceptable fit, as indicated by the area under a receiver-operating-characteristic curve of 0.80 but was inconsistent with the zero catches that occurred frequently in the 2000s. Two types of spawners overlapped spatially within the survey area: those that exhibited peak spawning during April in the SCB at about 15.5°C and a smaller group that exhibited peak spawning in August near Punta Eugenia, Mexico, at 20°C or greater. The SCB generally had greater zooplankton than Mexican waters but less appropriate (lower) geostrophic f lows. Mexican waters generally exhibited greater predicted habitat quality than the SCB in cold years. Predicted quality of the habitat in the SCB was greater from the 1980s to 2008 than in the earlier years of the survey primarily because temperatures and geostrophic flows were more appropriate for larvae. However, stock size the previous year had a larger effect on predictions than any environmental variable, indicating that larval Pacific mackerel did not fully occupy the suitable habitat during most years.

Uma leitura dos textos epistolares da Nancy Huston

Este trabalho tem como objetivo discutir e analisar os dois textos epistolares de Nancy Huston: À lAmour comme à la guerre (1984) e Lettres parisiennes (1986). Os dois livros são escritos em momentos diferentes da vida da autora e possuem temáticas diferenciadas. Os dois volumes se constituem como troca de cartas com dois correspondentes: Sam Kinser e Leïla Sebbar, respectivamente. A análise presente nesta dissertação se divide em três momentos. No primeiro, examinamos a evolução da carta ao longo do tempo. No segundo, destacamos alguns temas tratados no corpus, buscando estabelecer relações com outros textos da autora e evidenciar o espaço que ocupam na obra de Nancy Huston. No terceiro momento, estudamos as (in)definições de gênero literário e as características da autobiografia e do ensaio, cotejando-as com os textos epistolares do corpus a fim de analisarmos em que medida essas cartas se configuram como autobiografia e ensaio. O percurso de pesquisa busca, pois, localizar os textos epistolares na produção crítica e literária de Nancy Huston

Effect of towing speed on retention of zooplankton in bongo nets

Long-term time series of zooplankton data provide invaluable information about the fluctuations of species abundance and the stability of marine community structure. These data have demonstrated that environmental variability have a profound effect on zooplankton communities across the Atlantic basin (Beaugrand et al., 2002; Frank et al., 2005; Pershing et al., 2005). The value of these time series increases as they lengthen, but so does the likelihood of changes in sampling or processing methods. Sam-pling zooplankton with nylon nets is highly selective and biased because of mesh selectivity, net avoidance, and damage to fragile organisms. One sampling parameter that must be standardized and closely monitored is the speed of the net through the water column. Tow speed should be as fast as possible to minimize net avoid-ance by the organisms, but not so fast as to damage soft bodied zooplankters or extrude them through the mesh (Tranter et al., 1968; Anderson and Warren, 1991).