Cellulosic biomass is an incredibly abundant resource and a capable feedstock for production of energy, biofuels, and commodity chemicals. Current technologies for bioprocessing of cellulose utilize a three-step process in which enzymes capable of cellulose hydrolysis are expressed and purified, cellulose is hydrolyzed, and then product is formed in separate processes. This multi-step processing increase costs. As such, one approach to lowering these costs it to develop on consolidated system in which all three of these processes occur in a single step. Toward this aim, the three main goals of this dissertation are (1) characterization of a new hydrolytic enzyme and its application to fermentation of relevant sugars, (2) selection of proteins capable of intracellular cellobiose transport, and (3) development of a minimal set of cellulases capable of extensive hydrolysis under physiological conditions. A mixture of cellodextrins is produced by enzymatic hydrolysis of cellulose and Ced3A, a cellodextrinase, was shown to hydrolyze all of these completely to glucose and confer the ability to metabolize these sugars to E. coli when expressed. Activity on cellobiose, however, was lower than on other species. Co-expression of Cep94A, a cellobiose phosphorylase, and Ced3A was shown to improve the cellobiose metabolism of E. coli. In order to facilitate conversion of cellobiose to glucose by Cep94A, cellobiose must be transported into the cytoplasm. Three cellobiose permease enzymes, LacY, CP1, and CP2, were expressed in E. coli. It was shown that each protein has affinity for cellobiose transport and expression of each 126 allowed fermentation of cellobiose by E. coli strains expressing a cytoplasmic cellobiase. All three proteins are likely suitable for cellobiose transport during a consolidated bioprocess. Finally, a system of three cellulase enzymes Cel5H, Cel9R, and Cel48S were evaluated at E. coli physiological conditions and it was shown that extensive hydrolysis occurred at over half of the compositions tested. Additionally, when strains expressing cellulases were grown in binary culture with strains previously engineered for cellodextrin metabolism substantial product formation was observed, representing suitable performance of a consolidated cellulose bioprocess. This dissertation presents successful performance of all three components necessary for consolidated bioprocessing both individually and when working in tandem. Furthermore, the technologies developed in this dissertation demonstrate the capacity for consolidated bioprocessing of cellulose.

While the central role of spermatozoa in sexual reproduction and fertilization is well understood, many functional attributes of sperm have yet to be elucidated at the molecular level. One key to ultimately understand the molecular basis of sperm function is to comprehensively characterize its biochemical composition. This crucial information has been lacking, as molecular characterization of the sperm cell cannot be assessed by classic gene expression assays since mature spermatozoa are transcriptionaly inert. Whole-cell shotgun proteomic approaches have revolutionized the molecular analysis of sperm form and function. We have utilized improved methodologies to re-analyze the D. melanogaster sperm proteome and characterize five additional Drosophila species sperm proteomes. This methodology, which included a 1D SDS-PAGE prefractionation step, resulted in good reproducibility between biological replicates and high quality sperm proteomes. An interspecific analysis of the sperm proteomes revealed that despite variation in protein composition, Drosophila sperm proteomes have a consistent functional profile and 519 proteins were identified a being conserved across the melanogaster subgroup within a phylogenetic framework. Evolution of the sperm proteome was explored in Mus musculus through the utilization of targeted proteomic datasets completed, which provided subcellular localizations for sperm components. This study resulted in several novel findings, including evidence for accelerated evolution as well as an enrichment of positive selection on genes found in the cell membrane and acrosome. This may be a result of the selective pressures encountered by these membrane proteins during sperm development, maturation and transit through the female reproductive tract where the sperm cell membrane, and eventually the acrosome, are exposed to the extracellular milieu and are available for direct cell-cell interactions. These findings not only reveal the varying evolutionary pressures acting on a single cell type but also highlights the utility of the proteomics technique in clarifying protein interaction and evolutionary history.

Santrauka Visų ląstelės baltymų analizė – tai metodas teikiantis fenotipinės informacijos. Jis plačiai naudojamas grupuoti bakterijų kamienus, nustatyti jų pirminį identitetą. Rezultatų patikimumas daugeliu atvejų prilygsta DNR-DNR hibridizacijai – pagrindiniam genotipinės informacijos metodui. Tuo pačiu visų ląstelės baltymų analizės metodas yra greitai atliekamas ir pigus. Dėl šių priežasčių metodas plačiai naudojamas medicininės diagnostikos, o taip pat su maisto pramone susijusiose laboratorijose. Šiuolaikiniame pasaulyje pramonėje labai svarbūs yra termostabilūs fermentai, o Geobacillus genties bakterijų kamienai būdami termofilai, yra tokių fermentų producentai. Dėl šios priežasties Geobacillus gentis yra aktualus tyrimų objektas. Labai svarbu tiksliai identifikuoti kamienus produkuojančius tiriamus fermentus. Vienu iš pirminio identifikavimo būdų galėtų būti visų ląstelės baltymų analizės metodas. Šiame darbe buvo parodyta, kad Geobacillus genties kamienų tarpusavio panašumas gali būti nustatytas visų ląstelės baltymų analizės metodu. Tarp G. stearothermophilus rūšies kamienų visų ląstelės baltymų elektroforetinių profilių panašumas buvo apie 80% ir atitiko DNR-DNR hibridizacijos rezultatus. Taip pat sutapo ir 22 kamieno panašumas su tipiniu G. vulcani kamienu: WCPP parodė 84% panašumą, DNR-DNR hibridizacija – 93,9%. Pagal visų ląstelės baltymų analizės metodą gauti rezultatai paneigė G. stearothermophilus 10 DSM 13240 kamieno priklausomybę šiai rūšiai. Pagal panašumo... [toliau žr. visą tekstą] / Summary Electrophoretic whole-cell protein profile analysis (WCPP) – is a method providing phenotypic information. It is widely used to group bacterial strains and estimate their primal identity. In most cases reliability of this method is equal to DNA-DNA hybridization – that is the main method of genotypic information. WCPP is performed quickly and it is cheap. Therefore this method is widely used in laboratories of medical diagnostic and laboratories related to food industry. In modern world termostable enzymes are very important in industry, whereas thermophilic bacterial strains of the genus Geobacillus are producers of these enzymes. Consequently genus Geobacillus is an actual object of research. It is very important to strictly identify bacterial strains producing needed enzymes. One of the primal ways for identification of this genus could be WCPP. During this work it was shown that similarity of strains of the genus Geobacillus can be determined by WCPP. There was an 80% similarity among strains of G. stearothermophilus. These results matched with DNA-DNA hybridization of these strains. Also there were similar results of WCPP and DNA-DNA hybridization between strain 22 and typical strain of G. vulcani: 84% and 93,9% respectively. Results of WCCP denied dependence of strain G. stearothermophilus 10 DSM 13240 to this species. According to similarity dendrogram this strain should be assigned to G. vulcani because of its 97% similarity to this species. There were also... [to full text]

Antidepressants are generally believed to exert their clinical efficacy by enhancing 5-HT transmission. Interestingly, sustained administration of selective serotonin (5-HT) reuptake inhibitors (SSRIs) strongly suppresses in the first few days the firing activity of 5-HT neurons in the dorsal raphe nucleus (DRN), thereby severely hampering the increase of 5-HT in target regions. Remarkably, the firing activity of 5-HT neurons gradually recovers over the time course of treatment and this recovery is believed to be accounted for by the desensitization of 5-HT1A somatodendritic autoreceptors. Here, we sought to investigate whether additional mechanisms might contribute to the dynamic regulation of excitability of 5-HT neurons during the course of SSRI treatments. Borrowing from the well-described homeostatic strengthening of glutamatergic synapses onto cortical pyramidal neurons following prolonged periods of inactivity, we hypothesized that a similar homeostatic-like regulation of synaptic strength might be operant on 5-HT cells during an SSRI treatment. To test this possibility, we used whole-cell electrophysiological recordings on acute midbrain slices to monitor glutamatergic synapses onto 5-HT neurons. We found that a two-day treatment with the SSRI citalopram induced a robust reduction in both the amplitude and frequency of AMPAR-mediated mEPSCs. We also show that this depression in synaptic strength, induced by an SSRI, is transient since excitatory drive onto 5-HT neurons was enhanced by 7 days of treatments. Altogether, these results document a dynamic regulation of glutamatergic synaptic transmission during the time course of a prolonged treatment with an SSRI. Further elucidation of the cellular and molecular mechanisms driving this synaptic plasticity might identify novel pharmacological target to shorten the delay of antidepressant action.

Whole-cell biocatalysts are preferred in many biocatalysis applications. However, cell envelope often represents a formidable permeability barrier. As a result, reactions catalyzed by whole-cells are reportedly orders of magnitude slower than those of by their free enzyme counterparts. The present research addresses this critical issue by using membrane engineering approaches. Two E. coli strains with genetically altered outer membrane structures were used in the study, a lipopolysaccarides (LPS) mutant SM101 and a Braun's lipoprotein mutant E609L. The effects of outer membrane mutation on the permeability of substrates differing substantially in size and hydrophobicity were investigated by combining the mutant cells with model enzymes. The reduction of the outer membrane permeability barrier by these mutations led to significant accelerations (2 to 14 fold) in reaction rates of all whole-cell catalyzed reactions investigated. In the case of tetrapeptide, LPS mutation of the outer membrane can render the outer membrane completely permeable to substrate, a barrier-less condition that maximizes the reaction rate. For reaction rates of toluene dioxygenase (TDO)-catalyzed reactions, a dramatic increase of up to six fold was observed with the lipoprotein mutant for each of the three small, hydrophobic substrates tested. Mutations in either the LPS or in the Braun's lipoprotein are effective for accelerating reactions with UDP-glucose, resulting in a striking acceleration (up to 14 fold) of reaction rate. The magnitude of reaction rate acceleration was found to be dependent upon the substrate concentrations, the enzyme expression level, and on the nature of the mutations and substrates. In addition, the mutations have been demonstrated to be far more superior to common permeabilization procedures like freeze-thaw (FT) or treatment with the chelating agent EDTA (ethylene diamine tetraacetic acid). Importantly, lipoprotein mutant E609L exhibited a normal growth rate and expressed the recombinant multi-component enzyme as well as the isogenic parent. The exact nature of lpp lipoprotein mutation in E609L was further studied and deletion of lpp was successfully introduced into E. coli strain with different genetic background for whole-cell biocatalysis applications. An example was provided by introducing an lpp deletion into an E. coli O44K74 strain to achieve a higher yield for L-carnitine production. This research and the results outlined in this dissertation demonstrate a valid strategy for addressing permeability issues in whole-cell biocatalysis. The work also highlights a need for accessing substrate permeabilities in biocatalysis research and development.

Cells of the rodent medial entorhinal cortex (EC) possess cellular properties hypothesized to underlie the spatially periodic firing behaviors of 'grid cells' (GC) observed in vivo. Computational models have simulated experimental GC data, but a consensus as to what mechanism(s) generate GC properties has not been reached. Using whole cell patch-clamp and computational modeling techniques this thesis investigates resonance, rebound spiking and persistent spiking properties of medial EC cells to test potential mechanisms generating GC firing. The first experiment tested the voltage dependence of resonance frequency in layer II medial EC stellate cells. Some GC models use interference between velocity-controlled oscillators to generate GCs. These interference mechanisms work best with a linear relationship between voltage and resonance frequency. Experimental results showed resonance frequency decreased linearly with membrane potential depolarization, suggesting resonance properties could support the generation of GCs. Resonance appeared in medial EC but not lateral EC consistent with location of GCs. The second experiment tested predictions of a recent network model that generates GCs using medial EC stellate cell resonance and rebound spiking properties. Sinusoidal oscillations superimposed with hyperpolarizing currents were delivered to layer II stellate cells. Results showed that relative to the sinusoid, a limited phase range of hyperpolarizing inputs elicited rebound spikes, and the phase range of rebound spikes was even narrower. Tuning model parameters of the stellate cell population to match experimental rebound spiking properties resulted in GC spatial periodicity, suggesting resonance and rebound spiking are viable mechanisms for GC generation. The third experiment tested whether short duration current inputs can induce persistent firing and afterdepolarization in layer V pyramidal cells. During muscarinic acetylcholine receptor activation 1-2 second long current injections have been shown to induce persistent firing in EC principal cells. Persistent firing may underlie working memory performance and has been used to model GCs. However, input stimuli during working memory and navigation may be much shorter than 1-2 seconds. Data showed that input durations of 10, 50 and 100 ms could elicit persistent firing, and revealed time courses and amplitude of afterdepolarization that could contribute to GC firing or maintenance of working memory.

Antidepressants are generally believed to exert their clinical efficacy by enhancing 5-HT transmission. Interestingly, sustained administration of selective serotonin (5-HT) reuptake inhibitors (SSRIs) strongly suppresses in the first few days the firing activity of 5-HT neurons in the dorsal raphe nucleus (DRN), thereby severely hampering the increase of 5-HT in target regions. Remarkably, the firing activity of 5-HT neurons gradually recovers over the time course of treatment and this recovery is believed to be accounted for by the desensitization of 5-HT1A somatodendritic autoreceptors. Here, we sought to investigate whether additional mechanisms might contribute to the dynamic regulation of excitability of 5-HT neurons during the course of SSRI treatments. Borrowing from the well-described homeostatic strengthening of glutamatergic synapses onto cortical pyramidal neurons following prolonged periods of inactivity, we hypothesized that a similar homeostatic-like regulation of synaptic strength might be operant on 5-HT cells during an SSRI treatment. To test this possibility, we used whole-cell electrophysiological recordings on acute midbrain slices to monitor glutamatergic synapses onto 5-HT neurons. We found that a two-day treatment with the SSRI citalopram induced a robust reduction in both the amplitude and frequency of AMPAR-mediated mEPSCs. We also show that this depression in synaptic strength, induced by an SSRI, is transient since excitatory drive onto 5-HT neurons was enhanced by 7 days of treatments. Altogether, these results document a dynamic regulation of glutamatergic synaptic transmission during the time course of a prolonged treatment with an SSRI. Further elucidation of the cellular and molecular mechanisms driving this synaptic plasticity might identify novel pharmacological target to shorten the delay of antidepressant action.

This dissertation presents the design, fabrication, and testing of a microfluidic system to be used for whole-cell analysis. The study of cellular function and structure is essential for disease diagnosis and treatment. Microsystems developed to perform these bioanalyses add benefits such as requiring smaller samples and reagents, testing multiple samples in parallel, and supporting point-of-care testing, all of which increases throughput and reduces cost-per-analysis. Traditional methods for designing a microsystem use standard materials and techniques such as silicon, glass, photolithography, and wet and dry etching. This research is focused on utilizing materials and techniques that require less infrastructure, allow for a faster design-to-prototype cycle, and can integrate electrical and fluidic functionality to address a variety of possible applications. The microfluidic system presented in this thesis is comprised of multiple layers of Kapton, a polyimide available from DuPont. Kapton provides a biocompatible substrate that is flexible while maintaining structural stability and can be used in high temperature and other harsh environments. Microchannels with widths of 400 m and thru-hole fluidic vias less than 5 m in diameter are laser ablated through the flexible polyimide sheets using excimer and CO2 lasers. Electrical traces and contact pads are defined on the substrate by vapor deposition through reusable microstencils rather than with photolithography. The patterned layers are bonded using heat staking and then packaged with the addition of wires and a fluidic interface. Validation of the system for whole-cell analysis was first performed with impedance spectroscopy measurements collected on air, DI water, phosphate buffered saline, clusters of human cancer cells, and human cancer tissue samples. This was followed by testing the ability to use the device to control the movement and position of 10 m diameter microbeads and dissociated cells. As a whole, this research demonstrates the realization of a microfluidic system for whole-cell analysis based on non-standard fabrication materials and techniques.

No description available.

Antidepressants are generally believed to exert their clinical efficacy by enhancing 5-HT transmission. Interestingly, sustained administration of selective serotonin (5-HT) reuptake inhibitors (SSRIs) strongly suppresses in the first few days the firing activity of 5-HT neurons in the dorsal raphe nucleus (DRN), thereby severely hampering the increase of 5-HT in target regions. Remarkably, the firing activity of 5-HT neurons gradually recovers over the time course of treatment and this recovery is believed to be accounted for by the desensitization of 5-HT1A somatodendritic autoreceptors. Here, we sought to investigate whether additional mechanisms might contribute to the dynamic regulation of excitability of 5-HT neurons during the course of SSRI treatments. Borrowing from the well-described homeostatic strengthening of glutamatergic synapses onto cortical pyramidal neurons following prolonged periods of inactivity, we hypothesized that a similar homeostatic-like regulation of synaptic strength might be operant on 5-HT cells during an SSRI treatment. To test this possibility, we used whole-cell electrophysiological recordings on acute midbrain slices to monitor glutamatergic synapses onto 5-HT neurons. We found that a two-day treatment with the SSRI citalopram induced a robust reduction in both the amplitude and frequency of AMPAR-mediated mEPSCs. We also show that this depression in synaptic strength, induced by an SSRI, is transient since excitatory drive onto 5-HT neurons was enhanced by 7 days of treatments. Altogether, these results document a dynamic regulation of glutamatergic synaptic transmission during the time course of a prolonged treatment with an SSRI. Further elucidation of the cellular and molecular mechanisms driving this synaptic plasticity might identify novel pharmacological target to shorten the delay of antidepressant action.