Quantitative, Time-Resolved Proteomic Analysis Using Bio-Orthogonal Non-Canonical Amino Acid Tagging

Bio-orthogonal non-canonical amino acid tagging (BONCAT) is an analytical method that allows the selective analysis of the subset of newly synthesized cellular proteins produced in response to a biological stimulus. In BONCAT, cells are treated with the non-canonical amino acid L-azidohomoalanine (Aha), which is utilized in protein synthesis in place of methionine by wild-type translational machinery. Nascent, Aha-labeled proteins are selectively ligated to affinity tags for enrichment and subsequently identified via mass spectrometry. The work presented in this thesis exhibits advancements in and applications of the BONCAT technology that establishes it as an effective tool for analyzing proteome dynamics with time-resolved precision.

Chapter 1 introduces the BONCAT method and serves as an outline for the thesis as a whole. I discuss motivations behind the methodological advancements in Chapter 2 and the biological applications in Chapters 2 and 3.

Chapter 2 presents methodological developments that make BONCAT a proteomic tool capable of, in addition to identifying newly synthesized proteins, accurately quantifying rates of protein synthesis. I demonstrate that this quantitative BONCAT approach can measure proteome-wide patterns of protein synthesis at time scales inaccessible to alternative techniques.

In Chapter 3, I use BONCAT to study the biological function of the small RNA regulator CyaR in Escherichia coli. I correctly identify previously known CyaR targets, and validate several new CyaR targets, expanding the functional roles of the sRNA regulator.

In Chapter 4, I use BONCAT to measure the proteomic profile of the quorum sensing bacterium Vibrio harveyi during the time-dependent transition from individual- to group-behaviors. My analysis reveals new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.

Overall, this work describes how to use BONCAT to perform quantitative, time-resolved proteomic analysis and demonstrates that these measurements can be used to study a broad range of biological processes.

Quantitative data on the feeding of Cyclops strenuus Fischer and Cyclops viridis Jurine. [Translation from: Trudy saratov.Otd.vses.nauchno-issled.Inst.ozer.rech.ryb.Khoz. 1, 163-176, 1951.]

Cyclopids, exactly in the same way as daphnids, significant component in the nutrition of plankton-f and the young of the majority of fishes. It is established that the food spectrum of cyclopids is extremely broad: daphnids, planarians, Copepodite stages of copepods (cannibalism), rotifers, protists, bacteria, phytoplankton and so on. It is clear that the problem of studying these or other components of feeding in the general food spectrum can be definitely resolved only after obtaining exact quantitative data on the feeding of cyclopids. This article attempts to fill the gap in the study of the quantitative side of the feeding of cyclopias; in it is investigated the size of the 24-hour ration of cyclopids feeding on protists, the dependence of the ration on some factors of the external medium, and the difference of 24-hour consumption per unit weight of tody with two species of cyclopids (Cyclops strenuus and Cyclops viridis).

Quantitative measurement of domain inversion in RuO2 : LiNbO3 crystal by digital holographic interferometry

We quantitatively study the domain inversion in a RuO2:LiNbO3 crystal wafer by the digital holographic interferometry. The crystal wafer is placed into one arm of a Mach-Zehnder-type interferometer to record a series of holograms. Making use of the angular spectrum backward propagation algorithm, we reconstruct the optical wave field in the crystal plane. The extracted phase difference from the reconstructed optical wave field is a well linear function of the applied external voltage. We deduce that the linear electro-optic coefficient of the detected RuO2:LiNbO3 crystal sample is 9.1x10(-12) m/V. An unexpected phase contrast at the antiparallel domain wall is observed and the influence of the applied external voltage on it is studied in detail. Also the built-in internal field is quantitatively measured as 0.72 kV/mm. (c) 2006 American Institute of Physics.

Percepções de formandos do ensino médio acerca das contribuições da escola e da classe social de origem sobre suas chances de ascensão educacional: o que os alunos pensam, querem e como interpretam suas realidades

O presente estudo traz como lócus central de interesse apreender as percepções que alunos formandos do ensino médio de uma escola estadual do município do Rio de Janeiro, possuem sobre suas chances de terem ascensão educacional, via ingresso no ensino superior, focalizando, em especial, como percebem as contribuições da escola e da classe social a que pertencem nesse processo. O estudo foi realizado apenas durante o turno da manhã, utilizando-se de aporte teórico-metodológico ancorado nas pesquisas sobre os efeitos das escolas, campo de estudo que se iniciou fora do Brasil, e que recentemente tem experimentado mais entrada no campo educacional brasileiro. Esta pesquisa congregou o uso das metodologias quantitativa e qualitativa, com aplicação de questionários estruturados a todos os alunos formandos, bem como buscou conhecer mais das expectativas destes estudantes, através de entrevistas semi-estruturadas realizadas com dois alunos de cada turma. Estudos apontam que, ainda que os fatores extra-escolares, especialmente aqueles relacionados às condições socioeconômicas dos indivíduos, exerçam forte influência sobre o desempenho escolar dos alunos, sobre suas trajetórias escolares, e, posteriormente, sobre suas trajetórias profissionais, outros trabalhos vêm sugerindo que há mais elementos que precisam ser introduzidos na análise, elementos estes que dizem respeito a aspectos relacionados à própria escola, que se convencionou chamar no Brasil como efeitos das escolas, ou simplesmente, efeito-escola. Acredita-se, segundo esta perspectiva de análise, que as escolas podem vir a afetar as trajetórias dos alunos, assim como suas expectativas de vida e suas motivações, embaralhando a influência dos fatores socioeconômicos, que, ainda que presentes, não seriam os únicos condicionantes. Estas pesquisas revelam que, depois de controladas as variáveis concernentes às condições socioeconômicas dos indivíduos, os dados sugeririam que as escolas exerceriam influência sobre o desempenho dos alunos, em maior ou menor grau, com maior ou menor êxito, mostrando que as escolas podem fazer diferença, e que não são apenas os fatores extra-escolares os únicos determinantes para o sucesso e insucesso escolar dos indivíduos.

Rapid quantitative detection of Yersinia pestis by lateral-flow immunoassay and up-converting phosphor technology-based biosensor

Up-converting phosphor technology (UPT)-based lateral-flow immunoassay has been developed for quantitative detection of Yersinia pestis rapidly and specifically. In this assay, 400 nm up-converting phosphor particles were used as the reporter. A sandwich immumoassay was employed by using a polyclonal antibody against F1 antigen of Y. pestis immobilized on the nitrocellulose membrane and the same antibody conjugated to the UPT particles. The signal detection of the strips was performed by the UPT-based biosensor that could provide a 980 nm IR laser to excite the phosphor particles, then collect the visible luminescence emitted by the UPT particles and finally convert it to the voltage as a signal. V-T and V-c stand for the multiplied voltage units for the test and the control line, respectively, and the ratio V-T/V-C is directly proportional to the number of Y pestis in a sample. We observed a good linearity between the ratio and log CFU/ml of Y pestis above the detection limit, which was approximately 10(4) CFU/mI. The precision of the intra- and inter-assay was below 15% (coefficient of variation, CV). Cross-reactivity with related Gram-negative enteric bacteria was not found. The UPT-LF immunoassay system presented here takes less than 30 min to perform from the sample treatment to the data analysis. The current paper includes only preliminary data concerning the biomedical aspects of the assay, but is more concentrated on the technical details of establishing a rapid manual assay using a state-of-the-art label chemistry. (c) 2006 Elsevier B.V. All rights reserved.

Data Gathering Bias: Trait Vulnerability to Psychotic Symptoms?

Background Jumping to conclusions (JTC) is associated with psychotic disorder and psychotic symptoms. If JTC represents a trait, the rate should be (i) increased in people with elevated levels of psychosis proneness such as individuals diagnosed with borderline personality disorder (BPD), and (ii) show a degree of stability over time. Methods The JTC rate was examined in 3 groups: patients with first episode psychosis (FEP), BPD patients and controls, using the Beads Task. PANSS, SIS-R and CAPE scales were used to assess positive psychotic symptoms. Four WAIS III subtests were used to assess IQ. Results A total of 61 FEP, 26 BPD and 150 controls were evaluated. 29 FEP were revaluated after one year. 44% of FEP (OR = 8.4, 95% CI: 3.9-17.9) displayed a JTC reasoning bias versus 19% of BPD (OR = 2.5, 95% CI: 0.8-7.8) and 9% of controls. JTC was not associated with level of psychotic symptoms or specifically delusionality across the different groups. Differences between FEP and controls were independent of sex, educational level, cannabis use and IQ. After one year, 47.8% of FEP with JTC at baseline again displayed JTC. Conclusions JTC in part reflects trait vulnerability to develop disorders with expression of psychotic symptoms.

Étude quantitative de l'upwelling sur le plateau continental ivorien

A wind-driven upwelling occurs on the continental shelf of Ivory Coast during the northern summer months; by studying the average conditions in the wind field, it has been found that in steady state the vertical speed upwards does not exceed 70 cm per day. The vertical flow per km super(2) is estimated in 46 m super(3)/s for a channel 50 m depth and in 92m super(3)/s for a channel 300 m depth. This study does not include the inclination of isopycnes in geostrophic adjustment with the variations of the Guinea current.

Qualitative and quantitative aspects of the food of Ethmalosa fimbriata (Bowdich) in the Ebrié lagoon (Côte d'Ivoire)

The food of Ethmalosa fimbriata in the central part of the Ebrié lagoon, where the salinity is low, consists on limnic phytoplankton. In the region near Abidjan, which is more strongly influenced by coastal water, it consists of marine phyto- and zooplankton. The daily ration of a 12.5 cm fork-length fish is estimated to be between 2 and 3 % of its body weight.