The study presented herein investigated a potential low-energy method to separate and concentrate glucose from a lignocellulosic hydrolysate. The motivation for this method was twofold: 1) to provide the fermentation microorganism an optimal glucose concentration and 2) to supply a fermentation media free of inhibitory compounds. Two sizes of porous silica particles (with 7 nm and 2.3 nm pores) were synthesized and their ability to adsorb glucose from solution confirmed. Next, the ability of two different microorganisms, Saccharomyces cerevisiae and Streptococcus bovis, to utilize sugars adsorbed to the porous silica particles was investigated. Both the 7 nm and 2.3 nm pore-size particles were capable of adsorbing glucose from solution. Fermentations with glucose provided adsorbed to nanoparticles were compared to fermentations with glucose dissolved in media. The success of the fermentation was assessed by using high performance liquid chromatography to compare the concentration of fermentation products at harvest time. Results indicated that although the fermentation with soluble glucose produced significantly more end-products, S. bovis demonstrated some ability to metabolize the glucose adsorbed to the 7 nm pore silica particles. No evidence was detected that S. cerevisiae could metabolize glucose adsorbed to silica particles of either pore size.

Gene regulation mediated by transcription factors (TFs) is essential for all organisms. The functionality of TFs can largely be described by the fraction of time they occupy their regulatory binding sites on the chromosome. DNA-binding proteins have been shown to find their targets through facilitated diffusion in vitro. In its simplest form this means that the protein combines a random 3D search in the cytoplasm with 1D sliding along DNA. This has been proposed to speed up target location. It is difficult to mimic the in vivo conditions for gene regulation in biochemistry experiments; i.e. the ionic strength, chromosomal structure, and the presence of other DNA-binding macromolecules.    In this thesis single molecule imaging assays for live cell measurements were developed to study the kinetics of the Escherichia coli transcription factor LacI. The low copy number LacI, in fusion with a fluorescent protein (Venus) is detected as a localized near-diffraction limited spot when being DNA-bound for longer than the exposure time. An allosteric inducer is used to control binding and release. Using this method we can measure the time it takes for LacI to bind to different operator sequences. We then extend the assay and show that LacI slides in to and out from the operator site, and that it is obstructed by another DNA-binding protein positioned next to its target. We present a new model where LacI redundantly passes over the operator many times before binding.    By combining experiments with molecular dynamics simulations we can characterize the details of non-specific DNA-binding. In particular, we validate long-standing assumptions that the non-specific association is diffusion-controlled. In addition it is seen that the non-specifically bound protein diffuses along DNA in a helical path.    Using microfluidics we design a chase assay to measure in vivo dissociation rates for the LacI-Venus dimer. Based on the comparison of these rates with association rates and equilibrium binding data we suggest that there might be a short time following TF dissociation when transcription initiation is silenced. This implies that the fraction of time the operator is occupied is not enough to describe the regulatory range of the promoter.

<p>  </p> <p>Plants synthesize and release a variety of volatile organic compounds (VOCs) that are important for their reproduction, defense, and communication. These low-molecular-weight, lipophilic molecules also serve as practical products in industries such as food additives, fragrances, colorants, nutraceuticals, and pharmaceuticals. In addition, they have agricultural applications such as sustainable methods for pest control. Therefore, identifying the biological mechanisms involved in volatile emission could help researchers develop new ways to control the timing and release of volatiles, defend against pests, and engineer the production of these valuable chemicals.</p> <p>While progress has been made in understanding plant volatile biosynthesis, their release from the cell remains incomplete. For plant VOCs to be emitted into the environment, they must move from their site of biosynthesis through the cytosol, transverse the plasma membrane, hydrophilic cell wall, and sometimes cuticle to exit the cell. It was previously shown by mathematical modeling that to achieve observed emission rates solely by diffusion, VOCs would accumulate in the cellular membranes to levels that are likely detrimental to the membrane integrity and function. Hence, it was proposed that there are biological mechanisms involved to lower VOC concentrations in membranes. In this work, we focus on the aqueous cell wall, the thickest layer among the three subcellular barriers that should act as a barrier for the diffusion of VOCs. We hypothesize that the transport of VOCs across the cell wall is facilitated by lipid transfer proteins (LTPs) which enhance the solubility of hydrophobic volatiles in the aqueous environment, prevent their back partition into the plasma membrane after entering the cell wall, and hence enhance their net diffusion. To investigate if the presence of LTPs has influence on the total VOC efflux, we use reverse-genetic, biochemical, and mathematical modeling approaches. Out of three highly expressed LTPs identified in the petunia petal, only downregulation of <em>PhLTP3</em> expression led to a decrease in VOC emission in the corresponding transgenic plants. A facilitated diffusion model was built to quantify the VOC flux difference with the presence of LTPs in the cell wall. Modeling of the steady state system revealed the facilitation of VOC flux by LTPs is greatest when the VOC concentration gradient across the cell wall is shallow, which is a physiologically relevant condition. In addition, there exists an optimal protein dissociation constant value for maximal facilitated flux, indicating the balance between the binding and the unloading of VOC is critical. With the in vitro displacement assay, the binding constants of candidate PhLTPs with VOCs were obtained and were all found to be in the µM range, which is close to our model predicted optimal value. The results revealed that LTPs, specifically PhLTP3, play a role in the export of VOCs from the plasma membrane, across the cell wall, to the cuticle.</p> <p>In our earlier mathematical model, the emission of VOCs from the petunia flowers was modeled assuming negligible mass transfer resistance on the surface of the cuticle because of their high volatility. However, the resistance imposed by the surface boundary layer was not considered. To examine if surface convection influences VOC emission, a model system which utilized a model cuticular wax film containing 2-phenylethanol (2-PE) was built to imitate the VOC emission from plant cuticle. The convection mass transfer coefficient of 2-PE emission from a model cuticular wax film was obtained by experimental data fitting and calculated from the correlation that involves Sherwood number. The obtained values that were smaller than unity indicates that the surface boundary layer imposes a higher mass transfer resistance than the model cuticle for the emission of 2-PE in the range of wind velocities investigated. The examination of petunia flowers under air flow showed increases in total emission but no significant differences in total internal pools, which indicates an increase in biosynthesis. The emission changes of individual compounds were different and does not clearly correlate to any molecular properties of the compounds.  </p>

O transporte de &#945;-metil glicosídio ( &#945;-MG) em Saccharomyces cerevisiae foi recentemente reportado como transporte ativo, do tipo simporte de &$945;-MG com H+ mediado pela permease Agt1p. A cepa AP77-11B (cepa selecionada em nosso laboratório) 14C-&#945;-MG pelo mecanismo descrito como difusão facilitada porque não existe co-transporte de H+ durante o transporte de &#945;-MG. Os genes HXT1-HXT17 pertencem à família dos transportadores de hexoses em Saccharomyces cerevisiae. Então, nós decidimos investigar a possibilidade que o transporte de &#945;-MG poderia ser mediado pelos transportadores de hexoses. Nós demonstramos que cepa MC966A (tipo selvagem), KY73 (isogênica com MC966A mas deletada nos HXT1-7), BSY08 (isogênica com KY73 com o AGT1 deletado), BSY09 (isogênica com MC966A com o AGT1 deletado) e a EBY.VW4000 (hxt1-17 agt1 gal2-null), não cresceram em &#945;-MG como fonte de carbono. Além disso, estas cepas não transportaram &#945;-MG por difusão facilitada quando as células foram cultivadas em meio com maltose, levando-nos a concluir que os transportadores de hexoses não estavam envolvidos no transporte de &#945;-MG. Nós observamos que a cepa AP77-11B apresentou alta atividade de &#945;-metilglicosidase periplásmica quando as células foram cultivadas em &#945;-MG. Esta atividade enzimática foi ensaiada usando um método descrito primeiramente para invertase periplásmica, no qual as células eram incubadas com fluoreto de sódio, um inibidor da enolase, antes da incubação com &#945;-MG. Então, a glicose produzida durante a hidrólise do -MG poderia ser determinada. A atividade extracelular só está presente em células cultivadas em -&#945;MG. Células de-reprimidas não mostraram atividade de alpha-metilglicosidase. Os parâmetros cinéticos determinados para &#945;-metilglicosidase, indicaram que esta enzima tem baixa afinidade para o alpha-MG. Além do mais, a atividade específica da alpha-metilglicosidase periplásmica aumentou ao longo da curva de crescimento em &#945;-MG. Os resultados reportados mostraram que existem duas vias de utilização de &#945;-MG em Saccharomyces cerevisiae. Uma via é mediada pela Agt1p, responsável pelo transporte ativo de &#945;-MG. Na outra via, a &#945; -metilglicosidase é secretada para o espaço periplásmico das células. Então, a glicose produzida pela hidrólise do &#945;-MG é transportada pelos transportadores de hexoses por difusão facilitada. / Alpha-Methyl glucoside ( alpha-MG) transport in Saccharomyces cerevisiae was previously reported to be an active transport, a H+ -symport mediated by the Agt1p permease. Strain AP77-11B (a strain obtained in our laboratory) takes up 14C- alpha-MG by a mechanism which was ascribed to be facilitated diffusion since there is no H+-cotransport during the alpha-MG uptake. The HXT1-HXT17 there is no H genes belong to a family of hexose transporters in Saccharomyces cerevisiae. Therefore, we decided to investigate the possibility that -MG transport could be mediated by hexose transporters. We demonstrated that strains MC966A (w.t.), KY73 (isogenic to MC966A but hxt1-hxt7-null), BSY08 (isogenic to KY73 with AGT1 deleted), BSY09 (isogenic to MC966A with AGT1 deleted) and even strain EBY.VW4000 (hxt1-hxt17 agt1 gal2-null), were not able to grow on alpha-MG as the sole carbon source. Moreover, none of them presented alpha-MG transport by facilitated diffusion when the strains were grown on maltose leading us to conclude that the HXT glucose transporters were not involved in alpha-MG transport. We found that strain AP77-11B displayed a high periplasmic alpha-methylglucosidase activity when cells were grown on alpha-MG. This enzymatic activity was assayed using a method first described for periplasmic invertase in which cells were incubated with sodium fluoride, an inhibitor of enolase, prior to the incubation with alpha-MG. Then the glucose produced during alpha-MG hydrolysis could be accurately measured. The extracellular activity was present only in cells grown on alpha-MG. Glucose derepressed cells did not show periplasmic alpha-methylglucosidase activity.

O transporte de &#945;-metil glicosídio ( &#945;-MG) em Saccharomyces cerevisiae foi recentemente reportado como transporte ativo, do tipo simporte de &$945;-MG com H+ mediado pela permease Agt1p. A cepa AP77-11B (cepa selecionada em nosso laboratório) 14C-&#945;-MG pelo mecanismo descrito como difusão facilitada porque não existe co-transporte de H+ durante o transporte de &#945;-MG. Os genes HXT1-HXT17 pertencem à família dos transportadores de hexoses em Saccharomyces cerevisiae. Então, nós decidimos investigar a possibilidade que o transporte de &#945;-MG poderia ser mediado pelos transportadores de hexoses. Nós demonstramos que cepa MC966A (tipo selvagem), KY73 (isogênica com MC966A mas deletada nos HXT1-7), BSY08 (isogênica com KY73 com o AGT1 deletado), BSY09 (isogênica com MC966A com o AGT1 deletado) e a EBY.VW4000 (hxt1-17 agt1 gal2-null), não cresceram em &#945;-MG como fonte de carbono. Além disso, estas cepas não transportaram &#945;-MG por difusão facilitada quando as células foram cultivadas em meio com maltose, levando-nos a concluir que os transportadores de hexoses não estavam envolvidos no transporte de &#945;-MG. Nós observamos que a cepa AP77-11B apresentou alta atividade de &#945;-metilglicosidase periplásmica quando as células foram cultivadas em &#945;-MG. Esta atividade enzimática foi ensaiada usando um método descrito primeiramente para invertase periplásmica, no qual as células eram incubadas com fluoreto de sódio, um inibidor da enolase, antes da incubação com &#945;-MG. Então, a glicose produzida durante a hidrólise do -MG poderia ser determinada. A atividade extracelular só está presente em células cultivadas em -&#945;MG. Células de-reprimidas não mostraram atividade de alpha-metilglicosidase. Os parâmetros cinéticos determinados para &#945;-metilglicosidase, indicaram que esta enzima tem baixa afinidade para o alpha-MG. Além do mais, a atividade específica da alpha-metilglicosidase periplásmica aumentou ao longo da curva de crescimento em &#945;-MG. Os resultados reportados mostraram que existem duas vias de utilização de &#945;-MG em Saccharomyces cerevisiae. Uma via é mediada pela Agt1p, responsável pelo transporte ativo de &#945;-MG. Na outra via, a &#945; -metilglicosidase é secretada para o espaço periplásmico das células. Então, a glicose produzida pela hidrólise do &#945;-MG é transportada pelos transportadores de hexoses por difusão facilitada. / Alpha-Methyl glucoside ( alpha-MG) transport in Saccharomyces cerevisiae was previously reported to be an active transport, a H+ -symport mediated by the Agt1p permease. Strain AP77-11B (a strain obtained in our laboratory) takes up 14C- alpha-MG by a mechanism which was ascribed to be facilitated diffusion since there is no H+-cotransport during the alpha-MG uptake. The HXT1-HXT17 there is no H genes belong to a family of hexose transporters in Saccharomyces cerevisiae. Therefore, we decided to investigate the possibility that -MG transport could be mediated by hexose transporters. We demonstrated that strains MC966A (w.t.), KY73 (isogenic to MC966A but hxt1-hxt7-null), BSY08 (isogenic to KY73 with AGT1 deleted), BSY09 (isogenic to MC966A with AGT1 deleted) and even strain EBY.VW4000 (hxt1-hxt17 agt1 gal2-null), were not able to grow on alpha-MG as the sole carbon source. Moreover, none of them presented alpha-MG transport by facilitated diffusion when the strains were grown on maltose leading us to conclude that the HXT glucose transporters were not involved in alpha-MG transport. We found that strain AP77-11B displayed a high periplasmic alpha-methylglucosidase activity when cells were grown on alpha-MG. This enzymatic activity was assayed using a method first described for periplasmic invertase in which cells were incubated with sodium fluoride, an inhibitor of enolase, prior to the incubation with alpha-MG. Then the glucose produced during alpha-MG hydrolysis could be accurately measured. The extracellular activity was present only in cells grown on alpha-MG. Glucose derepressed cells did not show periplasmic alpha-methylglucosidase activity.

The glucose transport protein, GLUT1, is highly expressed in rapidly proliferating cells, including cancer cells, while decreased GLUT1 levels are found in diseases such as GLUT1 deficiency syndrome and Alzheimer’s. There is increased interest in developing GLUT1 inhibitors as novel anticancer therapeutics, and the discovery of compounds that directly stimulate GLUT1 function. This work investigates how small molecules stimulate and/or inhibit GLUT1-mediated glucose transport, either directly or through the AMPK pathway. Using sugar transport assays and docking analyses to explore Ligand–GLUT1 interactions and specificity of binding, we show that: 1) Ligands inhibit GLUT1 by competing with glucose for binding to the exofacial or endofacial sugar binding sites; 2) Subsaturating inhibitor concentrations stimulate sugar uptake; 3) Ligands inhibit GLUT1–, GLUT3– and GLUT4–mediated sugar uptake in HEK293 cells; and 4) Inclusion of a benzonitrile head group on endofacial GLUT1 inhibitors confers greater inhibitory potency. Furthermore, we investigated AMPK-regulated GLUT1 trafficking in cultured blood-brain barrier endothelial cells, and show that inhibition of GLUT1 internalization is not responsible for increased cell surface levels of GLUT1 observed with AMPK activation in these cells. This study provides a framework for screening candidate GLUT1 inhibitors for specificity, and for optimizing drug design and delivery. Our data on transport stimulation at low inhibitor concentrations support the idea that GLUT1 functions as a cooperative oligomer of allosteric alternating access subunits.