Fluorescence-based techniques are widely used in bioscience, offering a high sensitivity and versatility. In this work, fluorescence electronic energy migration/ transfer is applied to measure intramolecular distances in two types of systems and under various conditions. The main part of the thesis utilizes the process of donor-acceptor energy transfer to probe distances within the ribosomal protein S16. Proteins are essential to all organisms. Therefore, it is of great interest to study protein structure and function in order to understand and prevent protein malfunction. Moreover, it is also important to try to study the proteins in an environment which resembles its natural habitat. Here two protein homologs were investigated; S16Thermo and S16Meso, isolated from a hyperthemophilic bacterium and a mesophilic bacterium, respectively. It was concluded that the chemically induced unfolded state ensemble of S16Thermo is more compact than the corresponding ensemble of S16Meso. This unfolded state compaction may be one reason for the increased thermal stability of S16Thermo as compared to S16Meso. The unfolded state of S16 was also studied under highly crowded conditions, mimicking the environment found in cells. It appears that a high degree of crowding, induced by 200 mg/mL dextran 20, forces the unfolded state ensemble of S16Thermo to become even more compact. Further, intramolecular distances in the folded state of five S16 mutants were investigated upon increasing amounts of dextran 20. We found that the probed distances in S16Thermo are unaffected by increasing degree of crowding. However, S16Meso shows decreasing intramolecular distances for all three studied variants, up to 100 mg/mL dextran. At higher concentrations, the change in distance becomes anisotropic. This suggests that marginally stable proteins like s16Meso may respond to macromolecular crowding by fine-tuning its structure. More stable proteins like S16Thermo however, show no structural change upon increasing degree of crowding. We also investigated the possibility of local specific interactions between the protein and crowding agent, by means of fluorescence quenching experiments. Upon increasing amounts of a tyrosine labelled dextran, a diverse pattern of fluorescence quantum yield and lifetime suggests that specific, local protein-crowder interactions may occur. In a second studied system, electronic energy migration between two donor-groups, separated by a rigid steroid, was studied by two-photon excitation depolarization experiments. Data were analysed by using recent advances, based on the extended Förster theory, which yield a reasonable value of the distance between the two interacting donor-groups. To the best of our knowledge, this is the first quantitative analysis of energy migration data, obtained from two-photon excited fluorescence.

No description available.

No description available.

Understanding the mechanisms of virus capsid assembly has been an important research objective over the past few decades. Determining critical points along the pathways by which virus capsids form could prove extremely beneficial in producing more stable DNA vectors or pinpointing targets for antiviral therapy. The inability of current experimental technology to address this objective has resulted in a need for alternative approaches. Theoretical and computational studies offer an unprecedented opportunity for detailed examination of capsid assembly. The Schwartz Lab has previously developed a discrete event stochastic simulator to model virus assembly based upon local rules detailing the geometry and interaction kinetics of individual capsid subunits. Applying numerical optimization methods to learn kinetic rate parameters that fit simulation output to in vitro static light scattering data has been a successful avenue to understand the details of virus assembly systems; however, information describing in vitro assembly processes does not necessarily translate to real virus assembly pathways in vivo. There are a number of important distinctions between experimental and realistic assembly environments that must be addressed to produce an accurate model. This thesis will describe work expanding upon previous parameter estimation algorithms for more complex data over three model icosahedral virus systems: human papillomavirus (HPV), hepatitis B virus (HBV) and cowpea chlorotic mottle virus (CCMV). Then it will consider two important modifications to assembly environment to more accurately reflect in vivo conditions: macromolecular crowding and the presence of nucleic acid about which viruses may assemble. The results of this work led to a number of surprising revelations about the variability in potential assembly rates and mechanisms discovered and insight into how assembly mechanisms are affected by changes in concentration, fluctuations in kinetic rates and adjustments to the assembly environment.

Proteins function as worker molecules in the cell and their natural environment is crowded. How they fold in a cell-like environment and how they recognize their interacting partners in such conditions, are questions that underlie the work of this thesis. Two distinct subjects were investigated using a combination of biochemical- and biophysical methods. First, the unfolding/dissociation of a heptameric protein (cpn10) in the presence of the crowding agent Ficoll 70. Ficoll 70 was used to mimic the crowded environment in the cell and it has been used previously to study macromolecular crowding effects, or excluded volume effects, in protein folding studies. Second, the conformational changes upon interaction between the Mediator subunit Med25 and the transcription factor Dreb2a from Arabidopsis thaliana. Mediator is a transcriptional co-regulator complex which is conserved from yeast to humans. The molecular mechanisms of its action are however not entirely understood. It has been proposed that the Mediator complex conveys regulatory signals from promoter-bound transcription factors (activators/repressors) to the RNA polymerase II machinery through conformational rearrangements. The results from the folding study showed that cpn10 was stabilized in the presence of Ficoll 70 during thermal- and chemical induced unfolding (GuHCl). The thermal transition midpoint increased by 4°C, and the chemical midpoint by 0.5 M GuHCl as compared to buffer conditions. Also the heptamer-monomer dissociation was affected in the presence of Ficoll 70, the transition midpoint was lower in Ficoll 70 (3.1 μM) compared to in buffer (8.1 μM) thus indicating tighter binding in crowded conditions. The coupled unfolding/dissociation free energy for the heptamer increased by about 36 kJ/mol in Ficoll. Altogether, the results revealed that the stability effect on cpn10 due to macromolecular crowding was larger in the individual monomers (33%) than at the monomer-monomer interfaces (8%). The results from the interaction study indicated conformational changes upon interaction between the A. thaliana Med25 ACtivator Interaction Domain (ACID) and Dreb2a. Structural changes were probed to originate from unstructured Dreb2a and not from the Med25-ACID. Human Med25-ACID was also found to interact with the plant-specific Dreb2a, even though the ACIDs from human and A. thaliana share low sequence homology. Moreover, the human Med25-interacting transcription factor VP16 was found to interact with A. thaliana Med25. Finally, NMR, ITC and pull-down experiments showed that the unrelated transcription factors Dreb2a and VP16 interact with overlapping regions in the ACIDs of A. thaliana and human Med25. The results presented in this thesis contribute to previous reports in two different aspects. Firstly, they lend support to the findings that the intracellular environment affects the biophysical properties of proteins. It will therefore be important to continue comparing results between in vitro and cell-like conditions to measure the magnitude of such effects and to improve the understanding of protein folding and thereby misfolding of proteins in cells. Better knowledge of protein misfolding mechanisms is critical since they are associated to several neurodegenerative diseases such as Alzheimer’s and Parkinson's. Secondly, our results substantiate the notion that transcription factors are able to bind multiple targets and that they gain structure upon binding. They also show that subunits of the conserved Mediator complex, despite low sequence homologies, retain a conserved structure and function when comparing evolutionary diverged species.

Universiẗat, Diss.--Göttingen, 2004.

Thesis (Ph.D.)--Uniformed Services University of the Health Sciences, 2006 / Typescript (photocopy)

abstract: Crowding and satisfaction remain widely studied concepts among those seeking to understand quality visitor experiences. One area of interest in this study is how the order of crowding and satisfaction items on a survey affects their measurement levels. An additional area of interest is the influence of personality traits on experience-use-history, crowding, and satisfaction. This study used two versions of a survey: A) crowding measured prior to satisfaction and B) satisfaction measured prior to crowding, to explore the influence of item order on crowding and satisfaction levels. Additionally, the study explored the influence of personality traits (extraversion and neuroticism) and experience use history (EUH) on crowding and satisfaction. EUH was included as a variable of interest given previous empirical evidence of its influence on crowding and satisfaction. Data were obtained from an onsite self-administered questionnaire distributed to day use visitors at a 16,000 acre desert landscape municipal park in Arizona. A total of 619 completed questionnaires (equally distributed between the two survey versions) were obtained. The resulting response rate was 80%. One-way ANOVA's indicated significant differences in crowding and satisfaction levels with both crowding and satisfaction levels being higher for survey version B. Path analysis was used to test the influence of personality traits and EUH on crowding and satisfaction. Two models, one for each version of the survey were developed using AMOS 5. The first model was tested using data in which crowding was measured prior to satisfaction. The second model relied on data in which satisfaction was measured prior to crowding. Results indicated that personality traits influenced crowding and satisfaction. Specifically, in the first model, significant relationships were observed between neuroticism and crowding, neuroticism and EUH, EUH and crowding, and between crowding and satisfaction. In the second model, significant relationships were observed between extraversion and crowding, extraversion and satisfaction, and between EUH and satisfaction. Findings suggest crowding and satisfaction item order have a potential to influence their measurement. Additionally, results indicate that personality traits potentially influence visitor experience evaluation. Implications of these findings are discussed. / Dissertation/Thesis / M.S. Recreation and Tourism Studies 2011

O presente trabalho teve por objetivo ajustar um processo para fabricação de chapas de partículas de bagaço de cana-de-açúcar e resina poliuretana bi componente à base de óleo de mamona, avaliar tecnicamente as propriedades físico-mecânicas, micro estrutural e de durabilidade desse material e validar seu uso como componente de painel portante para uso como fechamento lateral de instalações zootécnicas para bovino. A qualidade das chapas foi avaliada com base nas prescrições dos documentos normativos NBR 14810 (2006) e ANSI A208.1 (1999). Foram fabricados chapas com partículas com dimensão de até 5 e 8 mm, a fim de identificar a dimensão adequada dessa matéria-prima, para a aplicação das chapas na composição do painel portante. Posteriormente, foi desenvolvido e fabricado um protótipo de painel portante em madeira de reflorestamento e chapas de partículas de bagaço de cana-de-açúcar, que teve seu desempenho estrutural avaliado por meio de ensaio de impacto de corpo-mole. Os resultados obtidos indicam que as chapas com partículas de dimensão de até 8 mm apresentaram valores de propriedades físico-mecânicas que atendem as recomendações mínimas dos documentos normativos para uso estrutural. O painel portante apresentou desempenho estrutural adequado para resistir às cargas de impacto nas condições de uso, sendo possível indicá-lo com alternativa para fechamento lateral em centro de manejo para bovinos. / The objective of this study was to adjust a process for the manufacture of particleboards of sugarcane bagasse and bi-component polyurethane resin a base of castor oil, toevaluate its physic-mechanical properties, microstructure and durability of this material and to validate its use as component of self-bearing panel for use as a lateral closure of zootechnics installations for cattle. The particleboard quality was evaluated based on the requirements of normative documents NBR 14810 (2006) and ANSI A208.1 (1999). Particleboards were manufactured with particles size up to 8 and 5 mm, to identify the appropriate size of the raw material forthe application in particleboards for composition of the self-bearing panel. Then, a prototype ofself-bearing panel in reforestation wood and sugarcane particleboards it was developed and manufactured, which had its structural performance evaluated by soft body impact test. The results indicate that particleboards with particles size up to 8 mm showed values of physical-mechanical properties that met the minimum recommendations of the normative documents for structural use. The self-bearing panel showed adequate structural performance to withstand impact loads under the conditions of use and it can be designate asan alternative to closing side of cattle handling facilities.

Reducing ED wait times is a top health care priority for the Ontario government and hospitals in Ontario are incentivised to meet provincial ED wait time targets. In this study, we considered the costs and benefits associated with increasing the number of acute-care beds to reduce the time an admitted patient spends boarding in the ED. A shorter hospital LOS has often been cited as a potential benefit associated with shorter ED wait times. We derived a multivariable Cox regression model to examine this association. We found no significant association between ED boarding times and the time to discharge. Using a Markov model, we estimated an increased annual operating cost of $2.1m to meet the prescribed wait time targets. We concluded that increasing acute-care beds to reduce ED wait times would require significant funding from hospitals and would have no effect on total length of stay of hospitalized patients.