Stem cells have the ability to reproduce themselves for a long period and differentiate into specific morphological and functional cells. The stem cells are an important material in the developmental biology, genomics, and transgenic methods, as well as in potential clinical applications, gene therapy and tissue engineering. The pluripotent stem cells will be a valuable source in numerous functional degenerated pathologies. Therefore, the objective of this research program was to establish transgenic porcine embryonic stem (pES) cell lines which can express green fluorescent protein (GFP) report gene stably for tracking after transplantation. We also developed a directed differentiation of pES into neural lineages and applied in rat Parkinson¡¦s disease model. Although the establishment of pluripotent ES cell lines from domestic species is much more difficult than that in murine species, our results had successfully isolated and established pES cell lines from pre-implantation blastocysts. Furthermore, we established the novel GFP-expressing pES cell lines (pES/GFP+), which were obtained by electroporation- mediated transfection with exogenous GFP gene. These pES/GFP+ cells exhibited pluripotent markers including Oct-4, AP, SSEA-4, TRA-1-60, and TRA-1-81 as that of human ES cells. The strategy of directed neural differentiation was to culture pES with neurogenic stimulators such as retinoic acid (RA), sonic hedgehog (SHH), and fibroblast growth factor (FGF). Upon directed differentiation toward neural differentiation, these pES-derived cells exhibited typical neuronal morphology and expressed neural lineage-specific markers such as nestin, NFL, MAP2, GFAP, A2B5, TH, ChAT, and GABA. These results showed the pES cells had the potential to differentiate into neural lineages. When pES/GFP+ cells were transplanted into the SD rat¡¦s brain, and their survival and development was determined by the non-invasive In Vivo Imaging System (IVIS 50), and the invasive fibered confocal Cellvizio® Imaging System (Cellvizio®). The results showed that fluorescent signals from pES/GFP+ cells on the injection site of SD rats¡¦ brain could be detected through the experimental period of 3 months. The level of fluorescent signals detected in treatment groups was two folds above that of the control group. Besides, the functional behavior recovery analysis by amphetamine-induced rotation test indicated the PD rat grafted with pES/GFP+ cells and their derived neural progenitors showed no significant recovery of rotation rate in these two treatments because a progressively increased relative rotation through 3 months duration. However, the relative rotation of PD rats grafted with the pES/GFP+-derived mature neurons, showed a stably decrease relative rotation and resulted in a functional recovery from Parkinsonian behavioral defects. Following 3 months completion of behavioral analyses, PD rats were sacrificed for immunohistochemical analysis. In the section of injected site without tumorgenesis and showed the survival and dopaminergic differentiation of grafted pES/GFP+ derived cells when stained with anti-TH and anti-DA. To our knowledge, there have been no reports of establishing GFP-expressing pES cell lines. These novel pES/GFP+ cell lines established in this study might serve as a non-rodent model and could benefit to the studies involving ES cell transplantation, cell replacement therapy, tissue regeneration, and actual approach for pre-clinical research due to their traceable capacity.

Fluorescence resonance energy transfer (FRET)-based biosensors have been designed to fluorometrically detect everything from proteolytic activity to receptor-ligand interactions and structural changes in proteins. While a wide variety of fluorophores have demonstrated effectiveness in FRET probes, several potential sensor components are particularly notable. Semiconductor quantum dots (QDs) are attractive FRET donors because they are rather bright, exhibit high quantum yields, and their nanoparticulate structure enables the attachment of multiple acceptor molecules. Fluorescent proteins (FPs) are also of particular interest for fluorescent biosensors because design elements necessary for signal transduction, probe assembly, and device delivery and localization for intracellular applications can all be genetically incorporated into the FP polypeptide. The studies described in this thesis elucidate the important parameters for concerted QD-FP FRET probe design. Experimental results clarify issues of FRET pair selection, probe assembly, and donor-acceptor distance for the multivalent systems. Various analysis approaches are compared and guidelines asserted based on the results. To demonstrate the effectiveness of the QD-FP FRET probe platform, a ratiometric pH sensor is presented. The sensor, which uses the intrinsic pH-sensitivity of the FP mOrange to modulate the FP/QD emission ratio, exhibits a 20-fold change in its ratiometric measurement over a physiologically interesting pH range, making it a prime candidate for intracellular imaging applications.

Oligo(ethylene glycol) groups have been used as substituents in poly(p-phenyleneethynylene)s (PPEs) to provide solubility, and to boost quantum yield. Properties such as water-solubility and increased quantum yield in aqueous solution make these conjugated systems promising for biosensory applications. In this thesis, a PPE containing a branched ethylene glycol side chain is synthesized as part of a polymer array for glycan biosensing. I also report that the same side chain can be put to use in a red-emissive polymer to lend water solubility. Another monomeric unit, containing ethylene glycol chains, is incorporated into a PPE to create an ampiphilic polymer. The versatility of these polymers allows them to be used for a variety of purposes, some of which will be described herein.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 19, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Thesis advisor: Dr. James E. Schoelz. Includes bibliographical references.

No description available.

Optical modulation has shown the selective and sensitive signal improvement in high background systems in cell imaging; however, cell applications are still limited due to biocompatibility and delivery issues. Fluorescent proteins have a variety of optically accessible states that make them ideal candidates for investigation of modulatability. Combining the optical modulation technique with the biocompatibility of fluorescent proteins is a major advance. This work focuses on evaluation fluorescent proteins and their optical states for modulation, as well demonstrations of cellular imaging. Herein, we evaluate a green fluorescent protein with interesting photophysical properties favorable for optical modulation. Positive for optical modulation, further investigation of the state dictating modulation reveals the presence of a slow component on the order of milliseconds. To better understand the mechanism responsible modulation, blue fluorescent proteins are created to modify the chromophore environment. Extraction of photophysics confirm the alteration timescales of the modulated state. Motivated by the ability to improve imaging and decode hidden dynamics, demodulation of these proteins demonstrates the selective recovery of signal in the presence of high cellular background. The continued investigation of several other fluorescent proteins identifies modulatable proteins across the visible wavelength region. Additionally, solvent environmental factors show varying timescales which, when combined with mutagenesis, suggest a cis/trans isomerization coupled with a proton transfer. This information of the properties dictating optical modulation allows for the engineering of improved modulatable proteins to study cellular dynamics.

As the move towards the miniaturization of many diagnostic and detection systems continues, the need for increasingly versatile yet sensitive labels for use in these systems also grows. Luminescent reporters provide us with a solution to many of the issues at hand through their unique and favorable characteristics. Bioluminescent proteins offer detection at extremely low concentrations and no interference from physiological fluids leading to excellent detection limits, while the vast number of fluorescent proteins and molecules available allows the opportunity to select a tailored reporter for a specific task. Both provide relatively simply instrumentation requirements and have exhibited great promise with many of the miniaturized systems such as lab-on-a-chip and lab-on-a-CD designs. Herein, we describe the novel employment of luminescent reporters for four distinct purposes. First off, by combining both time and wavelength resolution we have expanded the multiplexing capabilities of the photoprotein aequorin beyond duel-analytes, demonstrating the ability to simultaneously detect three separate analytes. Three semi-synthetic aequorin proteins were genetically conjugated to three pro-inflammatory cytokines (interleukins 1, 6, and 8) resulting in aequorin labeled cytokines with differing emission maxima and half lives to allow for the simultaneous detection of all three in a single solution through the elevated physiological concentration range. Secondly a semi-synthetic aequorin variant has been genetically enhanced to serve as an immunolabel and exhibited the ability to sensitively detect the acute myeloid leukemia marker, CD33, down to the attomole level in addition to improving aequorin imaging capabilities. In the third example, the aequorin complex was rationally, genetically split into two parts and attached to the termini of the cAMP selective cAMP receptor protein (CRP) creating a genetically fused molecular switch. The conformational change experienced by CRP upon the binding of cAMP translates into a loss of bioluminescent signal from aequorin and has shown the ability to respond linearly to cAMP over several orders of magnitude. Lastly, through custom design, a reagentless, portable, fluorescent fiber optic detection system has been developed, capable of being integrated into the body through a heart catheter. The system was able to respond to changes in potassium concentration selectively, reproducibly and reversibly with a fast response time of one minute.

Neurodegenerative disorders, including Alzheimer's and Parkinson's disease, and the development of neuroprotective agents have received significant research attention in recent years. Development of novel imaging techniques to study the biological mechanisms involved in the progression of these disorders have become an area of research interest. The design of novel small molecule imaging probes in combination with modem imaging techniques may provide information on neuroprotective binding site• interactions and would assist in the design of novel biological assay methods. Techniques to visualize physiological or pathophysiological changes in proteins and living cells have become increasingly important in biomedical sciences, especially fluorescent techniques. Fluorescent ligands in combination with sophisticated fluorescent imaging technologies are useful tools to analyze and clarify the roles of biomolecules in living cells, affording high spatial and temporal resolution. This study is based on the development of polycyclic fluorescent ligands, which may be used in the study of receptor-ligand and/or enzyme-ligand interactions, utilizing these fluorescently labeled ligands in combination with fluorescent imaging techniques. Fluorescent conjugates with high affinity for the• N-methyl-D-aspartate (NMDA) receptor, voltage gated calcium channels (VGCC) and/or the nitric oxide synthase (NOS) enzyme were designed and synthesised with the aim to directly measure binding of these novel molecules to receptors and/or enzymes. The first goal was to develop fluorescent ligands that exhibit similar inhibitory activity on NOS compared to the well-known selective neuronal NOS inhibitor 7-nitroindazole (7-NI). Polycyclic compounds, including amantadine and pentacycloundecane derivatives, were conjugated to fluorescent moieties that resemble the structure of 7-NI. It was thought that the lipophilic nature of the polycyclic compounds would increase the activity of the fluorescent moieties by facilitating increased blood brain barrier permeability and penetration through cell membranes. This would also potentially increase the selectivity of the novel conjugated compounds as selective neuronal NOS inhibitors, similar to 7-NI. The results from the NOS inhibition studies indicated that the novel fluorescent conjugates (5-14) inhibited the NOS enzyme at micromolar concentrations. Although none of the novel fluorescent polycyclic compounds were found to be more potent than 7-NI (IC50 = 0.11 11M), the indazole pentacyclorindecane (5), the coumarin-adamantane (7), the dansyl-adamantane (8), and the cyanoisoindole-adamantane (11) conjugates, exhibited IC5o values below 1 uM. These compounds could possibly be used as molecular probes in the development of high-throughput screening or competitive NOS displacement assays. Further studies on isoform selectivity will elaborate on the potential of these compounds as fluorescent molecular probes. The aforementioned fluorescent derivatives were further developed resulting in a series of novel fluorescent polycyclic conjugates with potent NOS inhibition indicating the potential of these compounds as neuroprotective agents. Due to the polycyclic structure's inherent inhibitory activity towards the NMDA receptor and VGCC we evaluated these derivatives as possible multifunctional neuroprotective agents acting on various neuroprotective targets. In the biological studies it was observed that four adamantane fluorescent compounds (7, 8, 10, 11) exhibited a high degree of inhibitory activity against the NOS enzyme and NMDA receptor and blocked VGCC. The fluorescent compounds were further able to scavange detrimental neurodegenerative free radicals. In silica studies also predicted a high degree of oral bioavailability and that these novel compounds should be effectively transported across the blood brain barrier. Taking the positive findings on the inhibition of the NMDA receptor and VGCC activity of the novel fluorescent polycyclic ligands into account we focused on the expansion of this series. This resulted in the synthesis of a series of fluorescent derivatives utilizing adamantane-3-aminopropanol as an intermediate to extend the chain length between the adamantyl and fluorescent moieties, to potentially reduce sterical hindrance and increase activity. These novel adamantane-3-aminopropanol fluorescent ligands were also evaluated for inhibition of the NMDA receptor and VGCC. The coumarin-, dansyl- and cyanoisoindole adamantane-3-aminopropanol fluorescent conjugates (15, 16, 19) displayed significant VGCC inhibition, with the dansyl (16) and di-nitrobenzene (20) fluorescent derivatives exhibiting NMDA receptor antagonistic activity. All these compounds showed improved activity when compared to known NMDA receptor and VGCC inhibitors in this class. Generally it was observed that the increased chain length analogues had improved VGCC inhibition and NMDA receptor activity when compared to their directly• conjugated counterparts. This led to the conclusion that an increase in chain length might indicate deeper immersion into the NMDA receptor and VGCC which may be necessary for stronger interaction with their putative binding sites. The dansyl analogue, N-[3-(1-adamantylamino)propyl]-5- dimethylaminonaphthalene-1-sulfonamide (16), was further used as a fluorescent NMDA receptor ligand in a fluorescent competition assay, utilizing known NMDA receptor inhibitors to demonstrate the possible applications of these novel fluorescent analogues and their benefit over the use of hazardous and expensive radioligand binding studies. Further investigation on the application of these derivatives, especially on the NOS enzyme and the NMDA receptor, will develop their potential as fluorescent ligands in the study of neurodegeneration and may also yield novel therapeutic agents against neurodegenerative disorders. / PhD (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2012

Neurodegenerative disorders, including Alzheimer's and Parkinson's disease, and the development of neuroprotective agents have received significant research attention in recent years. Development of novel imaging techniques to study the biological mechanisms involved in the progression of these disorders have become an area of research interest. The design of novel small molecule imaging probes in combination with modem imaging techniques may provide information on neuroprotective binding site• interactions and would assist in the design of novel biological assay methods. Techniques to visualize physiological or pathophysiological changes in proteins and living cells have become increasingly important in biomedical sciences, especially fluorescent techniques. Fluorescent ligands in combination with sophisticated fluorescent imaging technologies are useful tools to analyze and clarify the roles of biomolecules in living cells, affording high spatial and temporal resolution. This study is based on the development of polycyclic fluorescent ligands, which may be used in the study of receptor-ligand and/or enzyme-ligand interactions, utilizing these fluorescently labeled ligands in combination with fluorescent imaging techniques. Fluorescent conjugates with high affinity for the• N-methyl-D-aspartate (NMDA) receptor, voltage gated calcium channels (VGCC) and/or the nitric oxide synthase (NOS) enzyme were designed and synthesised with the aim to directly measure binding of these novel molecules to receptors and/or enzymes. The first goal was to develop fluorescent ligands that exhibit similar inhibitory activity on NOS compared to the well-known selective neuronal NOS inhibitor 7-nitroindazole (7-NI). Polycyclic compounds, including amantadine and pentacycloundecane derivatives, were conjugated to fluorescent moieties that resemble the structure of 7-NI. It was thought that the lipophilic nature of the polycyclic compounds would increase the activity of the fluorescent moieties by facilitating increased blood brain barrier permeability and penetration through cell membranes. This would also potentially increase the selectivity of the novel conjugated compounds as selective neuronal NOS inhibitors, similar to 7-NI. The results from the NOS inhibition studies indicated that the novel fluorescent conjugates (5-14) inhibited the NOS enzyme at micromolar concentrations. Although none of the novel fluorescent polycyclic compounds were found to be more potent than 7-NI (IC50 = 0.11 11M), the indazole pentacyclorindecane (5), the coumarin-adamantane (7), the dansyl-adamantane (8), and the cyanoisoindole-adamantane (11) conjugates, exhibited IC5o values below 1 uM. These compounds could possibly be used as molecular probes in the development of high-throughput screening or competitive NOS displacement assays. Further studies on isoform selectivity will elaborate on the potential of these compounds as fluorescent molecular probes. The aforementioned fluorescent derivatives were further developed resulting in a series of novel fluorescent polycyclic conjugates with potent NOS inhibition indicating the potential of these compounds as neuroprotective agents. Due to the polycyclic structure's inherent inhibitory activity towards the NMDA receptor and VGCC we evaluated these derivatives as possible multifunctional neuroprotective agents acting on various neuroprotective targets. In the biological studies it was observed that four adamantane fluorescent compounds (7, 8, 10, 11) exhibited a high degree of inhibitory activity against the NOS enzyme and NMDA receptor and blocked VGCC. The fluorescent compounds were further able to scavange detrimental neurodegenerative free radicals. In silica studies also predicted a high degree of oral bioavailability and that these novel compounds should be effectively transported across the blood brain barrier. Taking the positive findings on the inhibition of the NMDA receptor and VGCC activity of the novel fluorescent polycyclic ligands into account we focused on the expansion of this series. This resulted in the synthesis of a series of fluorescent derivatives utilizing adamantane-3-aminopropanol as an intermediate to extend the chain length between the adamantyl and fluorescent moieties, to potentially reduce sterical hindrance and increase activity. These novel adamantane-3-aminopropanol fluorescent ligands were also evaluated for inhibition of the NMDA receptor and VGCC. The coumarin-, dansyl- and cyanoisoindole adamantane-3-aminopropanol fluorescent conjugates (15, 16, 19) displayed significant VGCC inhibition, with the dansyl (16) and di-nitrobenzene (20) fluorescent derivatives exhibiting NMDA receptor antagonistic activity. All these compounds showed improved activity when compared to known NMDA receptor and VGCC inhibitors in this class. Generally it was observed that the increased chain length analogues had improved VGCC inhibition and NMDA receptor activity when compared to their directly• conjugated counterparts. This led to the conclusion that an increase in chain length might indicate deeper immersion into the NMDA receptor and VGCC which may be necessary for stronger interaction with their putative binding sites. The dansyl analogue, N-[3-(1-adamantylamino)propyl]-5- dimethylaminonaphthalene-1-sulfonamide (16), was further used as a fluorescent NMDA receptor ligand in a fluorescent competition assay, utilizing known NMDA receptor inhibitors to demonstrate the possible applications of these novel fluorescent analogues and their benefit over the use of hazardous and expensive radioligand binding studies. Further investigation on the application of these derivatives, especially on the NOS enzyme and the NMDA receptor, will develop their potential as fluorescent ligands in the study of neurodegeneration and may also yield novel therapeutic agents against neurodegenerative disorders. / PhD (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2012

This thesis describes the development of novel fluorescent and photocaged lipids, and their application as tools to probe the morphological effects of ceramide (Cer)-mediated membrane reorganization in supported lipid bilayers. Cer is a sphingolipid found in eukaryotic cells that plays a key role in regulating biological processes such as apoptosis, cell-to-cell communication, differentiation and some types of pathogenesis. Sphingolipid and cholesterol-rich lipid rafts in the plasma membrane are thought to be the point of origin for many of this lipid second messenger’s effects. Cer is formed in the exoplasmic leaflet of the plasma membrane via the enzymatic hydrolysis of sphingomyelin. The compositional complexity of biological membranes has prompted the adoption of simpler model systems to study the effects of Cer generation. When it is directly incorporated into model membranes, Cer segregates into highly ordered domains with physical properties that are distinct from those of the surrounding fluid environments. However, enzymatic generation of Cer induces complex and dynamic membrane heterogeneity that is difficult to interpret and reconcile with its direct incorporation. Here I describe the synthesis of 4-nitrobenzo-2-oxa-1,3-diazol-7-yl (NBD)-labelled cholesterol (Chol) and Cer analogs, and their use as probes in model membranes exhibiting liquid-disordered (Ld) and liquid-ordered (Lo) phase coexistence. The Chol probes reproduce the modest enrichment of Chol in Lo membrane domains as well as the Cer-induced displacement of cholesterol. One of the NBD Chol probes is used to provide direct visualization of Chol redistribution during enzymatic Cer generation, and assists in identifying new features as Cer-rich regions. The NBD-labelled Cer quantifies membrane order using orientational order parameter measurements derived from polarized total internal reflection fluorescence microscopy (pTIRFM) images. The probe reports on changes in membrane order upon enzymatic generation of Cer, and indicates a significant increase in the molecular order of Ld membrane regions that is consistent with the redistribution of Chol into these areas. The probe also identifies de novo Cer-rich domains as areas of particularly high molecular order. In the final project area, 6-Bromo-7-hydroxycoumarin-4-ylmethyl (Bhc)-caged Cers are shown to release Cer rapidly and efficiently upon irradiation with near-visible UV light. The caged lipids are then incorporated into supported membranes and photolyzed to release Cer with a high degree of spatial and temporal control. Controlled Cer generation is then used to drive protein-ganglioside clustering in lipid bilayers.