We perform a spectroscopic analysis of asphaltene in solution and in crude oil with the goal of designing an optical probe of asphaltene precipitation inside high-pressure cells. Quantitative analysis of steady-state spectroscopic data is employed to identify fluorescence and Raman contributions to the observed signals. Time-resolved fluorescence spectroscopy indicates that fluorescence lifetime can be used as a spectroscopic probe of asphaltene in crude oil. Quantitative confocal laser-scanning microscopy studies of asphaltene in n-heptane are used to calculate particle-size distributions as a function of time, both at the sample surface and asphaltene interior. The resulting precipitation kinetics is well described by stochastic numerical simulations of diffusion-limited aggregation. Based on these results, we present the design and construction of an apparatus to optically probe the in situ precipitation of asphaltene suitable for studies inside high pressure cells. Design considerations include the use of a spatial light modulator for aberration correction in microscopy measurements, together with the design of epi-fluorescence spectrometer, both fiber-based and for remote sensing fluorescence spectroscopy.

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Perturbations in the native structure, often caused by stressing cellular conditions, not only impair protein function but also lead to the formation of aggregates, which can accumulate in the cell leading to harmful effects. Some organisms, such as plants, express the molecular chaperone HSP100 (homologous to HSP104 from yeast), which has the remarkable capacity to disaggregate and reactivate proteins. Recently, studies with animal cells, which lack a canonical HSP100, have identified the involvement of a distinct system composed of HSP70/HSP40 that needs the assistance of HSP110 to efficiently perform protein breakdown. As sessile plants experience stressful conditions more severe than those experienced by animals, we asked whether a plant HSP110 could also play a role in collaborating with HSP70/HSP40 in a system that increases the efficiency of disaggregation. Thus, the gene for a putative HSP110 from the cereal Sorghum bicolor was cloned and the protein, named SbHSP110, purified. For comparison purposes, human HsHSP110 (HSPH1/HSP105) was also purified and investigated in parallel. First, a combination of spectroscopic and hydrodynamic techniques was used for the characterization of the conformation and stability of recombinant SbHSP110, which was produced folded. Second, small-angle X-ray scattering and combined predictors of protein structure indicated that SbHSP110 and HsHSP110 have similar conformations. Then, the chaperone activities, which included protection against aggregation, refolding, and reactivation, were investigated, showing that SbHSP110 and HsHSP110 have similar functional activities. Altogether, the results add to the structure/function relationship study of HSP110s and support the hypothesis that plants have multiple strategies to act upon the reactivation of protein aggregates.

As proteínas são as macromoléculas mais importantes nas células, pois desempenham diversas funções biológicas nos organismos. Para que as proteínas desempenhem suas funções elas precisam adotar uma estrutura tridimensional estável. Há proteínas capazes de auxiliar na formação da estrutura nativa, conhecidas como chaperonas moleculares, as quais constituem uma família de proteínas que auxiliam na formação da estrutura tridimensional de polipeptídeos clientes, previnem o enovelamento incorreto e a agregação, sendo, portanto, essenciais na manutenção da homeostase proteica. Em animais, identificaram a participação da Hsp110 no sistema da Hsp70, em um sistema de desagregação. A Hsp110 foi analisada por diferentes técnicas como dicroísmo circular e fluorescência, para elucidar sua estrutura e obter informações que ajudem a compreender seu mecanismo funcional. O Sorgo é muito importante para a agricultura sendo o quinto cereal mais produzido no mundo.

Clonagem, expressão, purificação e caracterização da proteína Hsp110 de sorgo visando a compreensão do sistema de desagregação de proteínas em plantas CAMPINAS 2018 JULIANA CROTTI FRANCO Clonagem, expressão, purificação e caracterização da proteína Hsp110 de sorgo visando a compreensão do sistema de desagregação de proteínas em plantas