The intracellular delivery of molecules to modulate signaling pathways and gene expression is a powerful approach to control stem cell fate decision. For applications in gene therapy and regenerative medicine, the use of genetic material and viral vectors raise concerns because stem cells persist throughout life, and long-term effects of uncontrolled genetic modifications could affect the cellular progeny. An alternative is to deliver directly peptides or proteins using cell-permeable peptides (CPPs) which have the ability of crossing the plasma membrane and carrying cargos into cells. CPPs can therefore be used to deliver factors to direct stem cells proliferation, survival and differentiation. This thesis describes an approach to control stem cell fate based on the delivery of a CPP-conjugated bioactive peptide. A first significant contribution from this work is the development of a flow cytometric assay to accurately quantify the uptake of a panel of CPPs. This study revealed that HIV-transactivator of transcription (TAT) and Antennapedia (Antp) offered the highest level of translocation in different cell types. The uptake was improved by treating the cells with a single, low-voltage electrical pulse that selectively enhances the amount of TAT-conjugated peptides and proteins delivered by at least an order of magnitude, without causing cellular toxicity or apoptosis. Subsequently, flow cytometry, confocal microscopy, capillary electrophoresis and mass spectrometry were used to examine the intracellular fate of TAT-conjugated peptides in order to define the parameters that limit their bioactivity and point to specific sequence modifications that can improve their efficacy. The advances described in this thesis were applied to the development of TAT-eIF2B, a peptide-inhibitor of glycogen synthase kinase-3 (GSK-3). TAT-eIF2B was found to be specific for GSK-3 and had a significant positive effect on the formation of neurospheres in embryonic stem cell cultures and on the survival of myeloid progenitors in cytokine-starved fetal liver cell cultures. On the other hand, GSK-3 inhibition reduced the number of neurospheres generated by human olfactory neuroepithelium cells due to lower proliferation and increased neuronal differentiation. In summary, this work describes the development of a peptide-based technology to deliver bioactive cargoes in cells, and it demonstrates its utility for modulating the activity of a master regulator of stem cell fate decision.

The intracellular delivery of molecules to modulate signaling pathways and gene expression is a powerful approach to control stem cell fate decision. For applications in gene therapy and regenerative medicine, the use of genetic material and viral vectors raise concerns because stem cells persist throughout life, and long-term effects of uncontrolled genetic modifications could affect the cellular progeny. An alternative is to deliver directly peptides or proteins using cell-permeable peptides (CPPs) which have the ability of crossing the plasma membrane and carrying cargos into cells. CPPs can therefore be used to deliver factors to direct stem cells proliferation, survival and differentiation. This thesis describes an approach to control stem cell fate based on the delivery of a CPP-conjugated bioactive peptide. A first significant contribution from this work is the development of a flow cytometric assay to accurately quantify the uptake of a panel of CPPs. This study revealed that HIV-transactivator of transcription (TAT) and Antennapedia (Antp) offered the highest level of translocation in different cell types. The uptake was improved by treating the cells with a single, low-voltage electrical pulse that selectively enhances the amount of TAT-conjugated peptides and proteins delivered by at least an order of magnitude, without causing cellular toxicity or apoptosis. Subsequently, flow cytometry, confocal microscopy, capillary electrophoresis and mass spectrometry were used to examine the intracellular fate of TAT-conjugated peptides in order to define the parameters that limit their bioactivity and point to specific sequence modifications that can improve their efficacy. The advances described in this thesis were applied to the development of TAT-eIF2B, a peptide-inhibitor of glycogen synthase kinase-3 (GSK-3). TAT-eIF2B was found to be specific for GSK-3 and had a significant positive effect on the formation of neurospheres in embryonic stem cell cultures and on the survival of myeloid progenitors in cytokine-starved fetal liver cell cultures. On the other hand, GSK-3 inhibition reduced the number of neurospheres generated by human olfactory neuroepithelium cells due to lower proliferation and increased neuronal differentiation. In summary, this work describes the development of a peptide-based technology to deliver bioactive cargoes in cells, and it demonstrates its utility for modulating the activity of a master regulator of stem cell fate decision.

Thesis (Ph. D.)--Brigham Young University. Dept. of Chemistry and Biochemistry, 2008. / Includes bibliographical references (p. 76-96).

No description available.

Dissertation (Ph.D.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.

Thesis (Ph.D.) - Simon Fraser University, 2004. / Theses (Dept. of Chemistry) / Simon Fraser University. Includes bibliographical references.

Thesis (M.S.)--University of Wisconsin--Madison, 1968. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Two failure modes related to water management in Proton Exchange Membrane fuel cells (dehydration and flooding) were investigated using electrochemical impedance spectroscopy as a diagnosis tool. It was hypothesised that each failure mode corresponds to changes in the overall stack impedance that are observable in different frequency ranges. This hypothesis was corroborated experimentally. The experimental implementation required new testing hardware and techniques. A four-cell stack capable of delivering individually conditioned reactants to each cell was designed, built, tested, and characterised under a variety of operating conditions. This stack is the first reported prototype of its type. The stack was used to perform galvanostatic, impedance measurements in situ. The measurements were made at three different temperatures (62, 70 and 80°C), covering the current density range 0.1 to 1.0 A cm−2 , and the frequency range 0.1 to 4 × 105 Hz. The recorded data represent the first reported set of measurements covering these ranges. The failure modes were simulated on individual cells within the stack. The effects on individual cell and stack impedance were studied by measuring the changes in stack and cell impedances under flooding or dehydration conditions. Dehydration effects were measurable over a wide frequency range (0.5 to 105 Hz). In contrast, flooding effects were measurable in a narrower frequency range (0.5 to 102 Hz). Using these results, separate or concurrent impedance measurements in these frequency ranges (or narrow bands thereof) can be used to discern and identify the two failure modes quasi-instantaneously. Such detection was not possible with pre-existing, do techniques. The measured spectra were modelled by a simple equivalent circuit whose time constants corresponded to ideal (RC) and distributed (Warburg) components. The model was robust enough to fit all the measured spectra (for single cells and the stack), under normal and simulated-failure conditions. Approximate membrane conductivities were calculated using this model. The calculations yielded a range from 0.04 to 0.065 S cm−1 (under normal humidification), and conductivities that deviated from these nominal range under flooding or dehydrating conditions. The highest conductivity value (was ∼0.10 S cm−1) was measured under flooding conditions at j = 0.4 A cm−2. The lowest conductivity (∼0.02 S cm−1) corresponded to a dehydrated cell at j = 0.1 A cm−2. These values fall within the ranges of published data for modern proton exchange membranes. The phenomenological and numerical results reported in this work represent the first demonstration of these techniques on a PEMFC stack under real operating conditions. They are also the basis of ongoing research, development, and intellectual property protection. / Graduate

The ubiquitously expressed pannexin 1 (Panx1) ion- and metabolite-permeable channel is capable of mediating ATP release in a multitude of cells and tissues. This leads to activation of nearby purinergic (P2X/P2Y) receptors in an autocrine/paracrine manner. Stimulation of P2 receptors also triggers Panx1 activation, leading to the formation of a positive feedback loop. Although the focus of Panx1 research has primarily been on its expression at the cell surface, there is robust and stable expression of Panx1 on intracellular membranes. Whether intracellular Panx1 was the consequence of direct diversion from the secretory pathway or internalization from the cell surface was unknown at the onset of my studies. I postulated that Panx1 internalization to these membranes would require a ubiquitous constitutively or episodically released stimulus to allow stable intracellular expression. ATP, a potent signalling molecule released via exocytosis (constitutive or regulated) or large pore channels, fit this criterion. My hypothesis was that ATP triggered Panx1 internalization to intracellular compartments. Upon elevation of extracellular ATP, I observed P2X7R-mediated non-canonical internalization of Panx1 via macropinocytosis. This involved upstream cholesterol-dependent P2X7R-Panx1 clustering via a physical interaction between P2X7R-Panx1 ectodomains and possible contribution of phospholipid (PA, PIP, PIP2) interactions localized to the Panx1 C-terminus. Physical P2X7R-Panx1 interaction may promote Panx1 association with actively endocytosing regions of the membrane. Internalized Panx1 was targeted to slow recycling Rab14/Rab11-positive endosomes in an Arf6-dependent mechanism. The data I presented here provides an additional negative feedback layer to P2X7R-Panx1 crosstalk in the many cell types where they are co-expressed. Further, this is the first evidence demonstrating that Panx1 surface expression is labile to changes in the cellular environment, which contributes to the understanding of the regulation of Panx1 and associated behaviours through trafficking mechanisms. / Graduate / 2018-06-20

Permeable reactive biobarrier (PRBB) is a flow-through zone where microorganisms degrade contaminants in groundwater. Discontinuous presence of contaminants in groundwater causes performance loss of a PRBB in removing the target contaminant. A novel enricher reactor (ER) - PRBB system was developed to treat groundwater with contaminants that reappear after an absence period. ER is an offline reactor for enriching contaminant degraders, which were used for augmenting PRBB to maintain its performance after a period of contaminant absence. The ER-PRBB concept was initially applied to remove benzene that reappeared after absence periods of 10 and 25 days. PRBBs without ER augmentation experienced performance losses of up to 15% higher than ER-PRBBs. The role of inducer compounds in the ER to enrich bacteria that can degrade a mixture of benzene, toluene, ethylbenzene, and xylene (BTEX) was investigated with an objective to minimize the use of toxic chemicals as inducers. Three inducer types were studied: individual BTEX compounds, BTEX mixture, and benzoate (a non toxic and a common intermediate for BTEX biodegradation). Complete BTEX removal was observed for degraders enriched on all three inducer types; however, the removal rates were dependent on the inducer type. Degraders enriched on toluene and BTEX had the highest degradation rates for BTEX of 0.006 to 0.014 day-1 and 0.006 to 0.012 day-1, respectively, while degraders enriched on benzoate showed the lowest degradation rates of 0.004 to 0.009 day-1. The ER-PRBB technique was finally applied to address the performance loss of a PRBB due to inhibition interactions among BTEX, when the mixture reappeared after a 10 day absence period. The ER-PRBBs experienced minimal to no performance loss, while PRBBs without ER augmentation experienced performance losses between 11% and 35%. Presence of ethanol during the BTEX absence period increased the performance loss of PRBB for benzene removal. PRBBs augmented with degraders enriched on toluene alone overcame the inhibition interaction between benzene and toluene indicating that toluene can be used as a single effective inducer in an ER. The ER-PRBB was demonstrated to be a promising remediation technique and has potential for applications to a wide range of organic contaminants.