No description available.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 98-107).

Critical to a cell's survival is its ability to deal with stress by making an appropriate response. This response often takes place in the cytoplasm, which is everything contained within the cell's plasma membrane that is not the nucleus. The cytoplasm is a dynamic environment and its ability to reorganize is essential to the cell's function. This dissertation presents a novel, previously undiscovered state of cytoplasm organization for the model system Schizosaccharomyces pombe, also known as fission yeast. Typically the fission yeast cytoplasm is a fluid-like environment in which endogenous lipid granules subject to thermal fluctuations, move freely as they explore their local surroundings through diffusion. When the cell is in a nutrient depleted environment it is exposed to the stress of advanced glucose starvation. As a result, we find that the cytoplasm undergoes drastic reorganization reminiscent of a phase transition; it is now a solid-like environment in which there is no visible motion. Lipid granules throughout the cell appear to be completely immobilized and are unable to move through the cytoplasm, despite the application of force through optical tweezers. We term this cytoplasmic state the cell frozen state. The cell frozen state is a physiological state, one that the cell can recover from with the addition of fresh nutrients. It is characterized by an anomalous diffusion exponent of [alpha] = 0.23 ± 0.01, which is a significant reduction from the anomalous diffusion exponent [alpha] = 0.66 ± 0.01 found for exponentially growing cells in which there is visible motion. To account for the cell wide immobilization of lipid granules, we hypothesize the formation of a polymer network all through the cytoplasm, and identify septins 1-3 as the most likely filament formers. In addition, we find there is an increase in the number of vacuoles in the cytoplasm during starvation, and propose a vacuole-septin model to describe the cytoplasm reorganization for the cell frozen state. / text

Forces are increasingly recognized as major regulators of cell physiology and function, and the mechanical properties of cells are essential to the mechanisms by which cells sense forces, transmit them to the cell interior or to other cells, and transduce them into chemical signals that impact a spectrum of cellular responses. Furthermore, cells can sense their extracellular environment and regulate their own mechanics and biology. Due to limitation of methodology, the cortical property of cells has been extensively characterized; however, the mechanics and dynamics of cytoplasm which consists all key cellular organelles, remains poorly understood. Moreover, a basic understanding of cell mechanics, such as which parameters correlates with cell stiffness and therefore impact cell biology is unknown. In this thesis, we firstly present a thorough investigation of the mechanical and dynamic properties of the cytoplasm, including direct measurement of cytoplasmic material property using optical tweezers, and visualization of intracellular dynamics by tracer particles. By combining these two measurements we obtain a directly characterization of the cytoplasmic forces; we further apply this method to study cancer cells and cells without vimentin intermediate filament, and find that cancer cells have significantly stronger intracellular forces, which vimentin intermediate filament does not have effect on the force generation. Secondly, we present our result on the role of cell volume in cell mechanics and cell biology. We show that the volume of a cell changes upon the property of the extracellular environment; the change in cell volume directly induces change in the mechanical property of both cytoplasm and cell cortex. We further show that the change in cell volume is due to intracellular water influx/efflux, and this has significant impact on cell biology, such as stem cell differentiation. Finally, we present a direct characterization of the equation of state of living cells by measuring cell volume under increasing osmotic pressure. We show that a living cell, under osmotic compression, behaves as Van der Waals gas with a hard sphere excluded volume; the minimum volume of cells is determined by cellular proteins, which the equation of state of living cells is dominated by intracellular ions. / Engineering and Applied Sciences

Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. "August 2008" Includes bibliographical references.

No description available.

Os venenos de serpentes contém uma mistura complexa de toxinas (proteínas e enzimas) que destroem os processos bioquímicos ou fisiológicos da presa. A crotamina é um dos principais componentes do veneno da cascavel Sul Americana Crotalus durissus terrificus. Essa proteína pertence à família dos polipeptídeos miotóxicos de baixo peso molecular (SBMPs). Uma importante atividade da crotamina é a habilidade de penetrar rapidamente em células proliferamente ativas, podendo ser usada como nanocarreadora, levando DNA plasmidial e drogas para dentro de células tumorais. Outra atividade interessante desta toxina é a indução de morte celular dose dependente em células cancerígenas proliferamente ativas sem prejudicar células normais, demonstrando promissora atividade anticâncer. A crotamina apresenta forte atividade antifúngica e moderada atividade contra procariotos, age bloqueando canais de potássio dependente de voltagem, sendo seletiva para os canais Kv1.1, Kv1.2 e Kv1.3, e induz paralisia dos membros posteriores de camundongo. Dada a importância das atividades da crotamina, a toxina foi usada para produzir na forma recombinante. De forma geral, as toxinas animais são peptídeos secretados para o lúmen da glândula de veneno e são ricos em pontes dissulfeto, comumente expressas em sistemas procarióticos na forma de corpúsculos de inclusão ou em sistemas de expressão periplasmática. Foram construídos três genes sintéticos, um para a expressão em citoplasma e dois para a expressão em periplasma bacteriano utilizando o vetor pRSET-A (Invitrogen ®). Para a expressão de crotamina em citoplasma bacteriano, a região do DNA que codifica a região do peptídeo maduro foi clonada em fusão com uma cauda simples de 6 x His separada por uma seqüência codificante do sítio de clivagem de enterokinase em substituição à proteína de fusão do vetor comercial pRSETA. Para a expressão em periplasma a região codificante da crotamina madura foi clonada sob o controle da seqüência sinal da OmpA de E. coli. Todos os códons raros da crotamina madura foram otimizados de acordo com os códons preferenciais de E. coli. Em uma das construções para a expressão em periplasma, além da crotamina madura, o peptídeo sinal de OmpA também teve seus códons raros otimizados. Os plasmídeos desenhados para a expressão da crotamina madura em citoplasma e periplasma foram transformados em bactéria competente BL21(DE3) e BL21-AI e a indução da proteína foi feita por IPTG e arabinose, respectivamente. A expressão foi analisada por SDS-PAGE e Western Blotting, mostrando que foi possível expressar as três construções em BL21-AI. / Snake venoms contain a complex cocktail of toxins (proteins and enzymes) which disrupt physiological or biochemical processes of the pray. Crotamine is one of the major components present in the venom of the South American rattlesnake Crotalus durissus terrificus. It belongs to the small basic myotoxins peptide SBMPs. An important activity of crotamine is the ability to rapidly penetrate proliferative cells, acting as a nanocarrier, carrying plasmid DNA and drugs into tumor cells. Another interesting activity of this toxin is the ability to promote cell death of actively proliferating cancer cells in a dose dependent manner. Crotamine shows strong antifungical activity and modest activities against prokaryotes, it acts by blocking voltage-dependent potassium channels being selective for channels Kv1.1, Kv1.2 e Kv1.3 and inducing paralysis of the hind limbs of mice. Given the importance of the activities of crotamine, the toxin was chosen for production in the recombinant form. Generally, animal toxins are peptides secreted into the lumen of the venom gland and are rich in disulfide bridges. When expressed in prokaryotic system, animal toxins are commonly found in the form of inclusion bodies. They can also be expressed in periplasmic sytem. Three synthetic genes were constructed, one for expression in the cytoplasm and two for expression in the bacterial periplasm using pRSET-A (Invitrogen ®) system. For cytoplasm expression, the DNA region encoding the mature peptide was cloned in fusion with a 6 x His tag separated by a site. For expression in the periplasm, the mature crotamine coding region was cloned in fusion with E. coli OmpA signal sequence. All crotamine and OmpA signal peptide rare codons were optimized according to E. coli preferred codon usage. The plasmids designed for crotamine expression in periplasm and cytoplasm have been transformed in strains BL21(DE3) and BL21-AI were used as hosts, and the induction of recombinant protein was done by IPTG and arabinose, respectively. The induced culture products were analyzed by SDSPAGE and Western Blot, showing that it was possible to express the three constructs in BL21-AI.

Xenopus embryos begin development by undergoing a series of extremely rapid cell divisions that occur without growth, gap phases, or cell cycle checkpoints. This cell cycle program, which allows the fertilized egg to rapidly subdivide its contents into many separate cells, is made possible by the extraordinary ability of these embryos to replicate DNA quickly. After a dozen such divisions, the time required to complete S phase and complete the cell cycle increases sharply amidst other embryonic changes during the midblastula transition (MBT). Successful completion of the MBT is essential for viability, but the mechanism responsible for actuating these changes remains unknown. Previous work has shown that the onset of the MBT is dependent upon the embryo reaching a critical nucleus to cytoplasm (N/C) ratio, but it is unclear how this controls cell cycle lengthening. Here, we use Xenopus egg extracts to investigate the mechanism responsible for S phase lengthening at the MBT. As in embryos, high N/C extracts exhibit lengthened S phases, and this is due to both reduced utilization of origins of replication and reduced replication fork progression. Although recent work has suggested that developmental activation of the ATR/Chk1 pathway may provide the stimulus for cell cycle remodeling at the MBT, we find that this pathway is not activated more efficiently at high N/C ratio. Rather, the Chk1 phosphorylation observed at high N/C is simply the aggregated, basal checkpoint activity associated with normal replication in a large number of nuclei. Instead, we provide evidence that the reduced replication rates at high N/C ratio are the result of the depletion of maternal factors by the increased number of nuclei, and these factors are involved in both the initiation of replication and replication fork progression. We provide evidence that protein phosphatase 2A (PP2A) activity is the limiting factor for origin firing in high N/C extracts. Likewise, partial depletion of PP2A is sufficient to prevent the high levels of origin firing observed in low N/C extracts. These results suggest a mechanism by which PP2A levels control the rate of origin firing in Xenopus egg extracts and in Xenopus embryos at the MBT.

We have employed a differential detector combined with dielectrophoretic (DEP) translation in a microfluidic channel to monitor dielectric response of single cells and particularly to track phenomenon related to apoptosis. Two different cell lines were studied; Chinese hamster ovary cells (CHO) and MRC5 cells. Dielectric response was quantified by a factor called Force Index. Force Index was studied statistically to identify apoptotic subpopulations. Another direction of this work was to monitor changes in the cytoplasm conductivity following inhibition of mitochondrial ATP production by Oligomycin. To make the DEP response mostly sensitive to the cytoplasm conductivity, medium conductivity and DEP frequency were adjusted such that Clausius Mossotti factor and hence DEP response become less sensitive to cell radius. Chinese hamster ovary cells were used in this work and the impact of different concentrations of Oligomycin has been studied. We show that following exposure to Oligomycin at 8 μg/ml, cytoplasm conductivity drops. The majority of the changes takes place within one hour of exposure to the drug. Furthermore, double shell models has been used to estimate cytoplasm conductivity in a medium with conductivity of 0.42 S/m and the drop in the cytoplasm conductivity following treatment with Oligomycin was estimated to be ≈ 0.16 S/m. The magnitude of the decrease in the cytoplasm conductivity is evidence that Glycolysis is active as an energy production pathway within the cell. This approach can be used to quantify Glycolysis versus mitochondria ATP production which has an application in Warburg effect in cancer cells and monitoring bioprocesses.

In 2007, U.S. Congress created the "Energy Independence and Security Act" with primary goals focused on increasing the knowledge in production of renewable fuels, increasing the percentages of renewable fuels in the transportation sector and decreasing the emissions of greenhouse gases from fossil fuel sources. To achieve these goals, many species have been pointed as sources of feedstock for the biofuel industry. Photoperiod sensitive (PS) biomass sorghum for the lignocellusosic based conversion is one. In this study, three main objectives were addressed regarding the relative performance for biomass yield and biomass composition of PS biomass sorghum. First, genetic and environmental variation effects on the biomass yield and biomass composition, and usefulness of pre-classification of genotypes by biomass lignin content were evaluated. On the set of genotypes and locations tested, the environmental effect had the largest influence on the biomass composition, yield and its components. Although smaller, the genetic variation effect was significant for most of the traits, some traits had significant genotype by environment GXE interaction. The pre-classification of genotypes according to lignin content proved to be an efficient system of separating genotypes as groups, but failed to be efficient in separating on the entries bases. Assessment of growth patterns for biomass yield and composition, characterized photoperiod sensitive sorghum as capable of producing a harvestable crop as soon as 4 months, but variations in the concentration of constituents and moisture percentage, pointed to a harvest window that can be extended up to the 7th month after planting. Genetic variation was observed in this trail for most agronomic and composition traits, but a strong environmental effect was also observed. Lastly, the influence of three diverse cytoplasm male sterility (CMS) systems in biomass sorghum hybrids was assessed. The presence of A1, A2 or A3 CMS in the hybrids tested in this study had no influence on the biomass yield performance or in the biomass composition. Therefore, any of the CMS systems can be used in the production of biomass sorghum hybrid seed. Also, in this trial the environmental effects were significant and strong for most traits evaluated.