In this thesis, the development and implementation of fluorescence lifetime imaging microscopy that integrates time correlated single photon counting (TCSPC) and a confocal microscope will be described. The TCSPC method has high detection efficiency, with a time resolution limited only by the transit time spread of the detector, and directly delivers the decay functions in the time domain. TCSPC can also be used to obtain images that indicate the fluorescence resonance energy transfer (FRET) effect between critical fluorophores, an important method distinguish the difference between binding and co-localization. Estimation of distances between RET fluorophore pairs can also be established. Additionally, the effects of ion concentration, oxygen concentration, pH value, ..etc. can also be revealed.

<p>The aim of this diploma work is to improve selectivity and sensitivity in DNA-chips by utilizing fluorescence resonance energy transfer (FRET) between conjugated polyelectrolytes (CPEs) and fluorophores.</p><p>Leclerc and co-workers have presented successful results from studies of super FRET between fluorophore tagged DNA and a CPE during hybridisation of the double strand. Orwar and co-workers have constructed a DNA-chip using standard photo lithography creating a pattern of the hydrophobic photoresist SU-8 and cholesterol tagged DNA (chol-DNA). This diploma work will combine and modify these two ideas to fabricate a improved DNA-chip.</p><p>Immobilizing of DNA onto surface has been done by using soft lithography. Hydrophobic pattern arises from the poly(dimethylsiloxane) (PDMS) stamp. The hydrophobic pattern will attract chol-DNA that is adsorbed to the chip. Different sets of fluorophores are covalently bound to the DNA and adding CPEs to the complex will make FRET occur between CPE and bound fluorophore.</p><p>We will here show that the specificity in DNA hybridization by using PDMS patterning was high. FRET clearly occurred, especially with the CPEs as donor to the fluorophore Cy5. The intensity of FRET was higher when the fluorophore and the CPE were conjugated to the same DNA strand. The largest difference in FRET intensity between double stranded and single stranded complexes was observed with the CPE tPOMT. Super FRET has been observed but not yet fully proved. The FRET efficiency was lower with the fluorophore Alexa350 as donor compared to the Cy5/CPE complex. Most of the energy transferred from Alexa350 was extinguished by quenching.</p>

Mutations in BEST1, the gene encoding for Bestrophin-1 (Best1), cause five, clinically distinct inherited retinopathies: Best vitelliform macular dystrophy (BVMD), adult-onset vitelliform macular dystrophy (AVMD), autosomal recessive bestrophinopathy (ARB), autosomal dominant vitreoretinochoroidopathy (ADVIRC), and retinitis pigmentosa (RP). Little is known regarding how BEST1 mutations cause disease and why mutations cause multiple disease phenotypes. Within the eye, Best1 is a homo-oligomeric, integral membrane protein that is exclusively localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Here, it regulates intracellular Ca2+ signaling and putatively mediates anion transport. Since defects in localization and oligomerization are known to underlie other channelopathies, we investigated how mutations causal for BVMD, AVMD, ARB, ADVIRC, and RP impact the localization and oligomerization of Best1. We generated replication-defective adenoviral vectors encoding for WT and 31 mutant forms of Best1 associated with these five diseases and expressed them in confluent, polarized Madin-Darby canine kidney and/or RPE cells. Localization was assessed via immunofluorescence and confocal microscopy. Oligomerization was examined using live-cell fluorescence resonance energy transfer (FRET) as well as reciprocal co-immunoprecipitation experiments. We report that all 31 BVMD, AVMD, ARB, ADVIRC, and RP mutants tested can reciprocally co-immunoprecipitate with and exhibit comparable FRET efficiencies to WT Best1, indicative of unimpaired oligomerization. While all RP and ADVIRC mutants were properly localized to the basolateral plasma membrane, many but not all AVMD, ARB, and BVMD mutants were mislocalized to intracellular compartments. When co-expressed with WT Best1, mislocalized mutants predominantly co-localized with WT Best1 in intracellular compartments. Studies involving four ARB truncation mutants reveal that the first 174 amino acids are sufficient to mediate oligomerization with WT Best1 and that amino acids 472-585 are not necessary for proper trafficking. We conclude that, although mislocalization is a common result of BEST1 mutation, it is not an absolute feature of any individual bestrophinopathy. Moreover, we show that some recessive mutants mislocalize WT Best1 when co-expressed, indicating that mislocalization cannot, on its own, generate a disease phenotype, and that the absence of Best1 at the plasma membrane is well tolerated.

Somatostatin (SST) is a peptide hormone that was originally identified in the hypothalamus and subsequently found throughout the central nervous system and in various peripheral organs. Generally classified as an inhibitory factor, SST is secreted by endocrine, neuronal and immune cells and acts to regulate cell secretion, neurotransmission and cell proliferation. There are five receptor-subtypes known to engage SST, termed SSTR1-5, all belonging to the superfamily of G-protein coupled receptors (GPCRs). Within the past few years, there has been a prepondef8:llce of evidence to suggest the importance of GPCR dimerization in receptor-biogenesis, regulation and pharmacology. It has been previously reported in our laboratory, that human (h) SSTR5 homo- and heterodimerizes with hSSTR1 in an agonist-regulated manner. However, it was unclear as to the contribution of each subtype in the formation of the hSSTR1/hSSTR5 heterodimer, the possible molecular determinants involved and the effects of heterodimerization on the pharmacology of the receptors. Furthermore, the dimerization properties of other hSSTRs including their heterodimerization remain undetermined. Here, we demonstrate that agonist binding to hSSTR5 and not hSSTR1 modulates the formation of the heterodimer, with particular emphasis on its carboxyl-terminal tail in specifying the interaction. We also determined the mechanics of the hSSTR2 homodimer, unlike the previous hSSTRs investigated, forms constitutive dimers that dissociate into monomers following activation with agonist. This feature is important for receptor trafficking, as preventing their dissociation impairs agonist-mediated endocytosis. Lastly, we investigated the heterodimerization of hSSTR2 and hSSTR5, an interaction that, like the hSSTR1/hSSTR5 heterodimer, is subtype-specific, requiring selective-activation of hSSTR2 and not hSSTR5. The heterodimer exhibited enhanced signalling characteristics including, prolonged activation of MAP kinases and an increase in the induction of the cyclin-dependent kinase inhibitor p27Kip1. These enhanced properties of the heterodimer conferred an extended growth inhibitory response. Dimerization of GPCRs, with particular emphasis on heterodimers, generates novel receptors with unique properties distinct from those of the individual receptor monomers/homodimers. An understanding on the mechanisms involved in GPCR dimerization could provide a rationale in future drug design.

Imaging tools in cellular and molecular biology have long relied on organic fluorophores to observe microorganisms or various cell constituents. The advent of semiconductor nanoparticles known as quantum dots (QDs) has offered the possibility to use this new class of fluorescent probes with very advantageous optical properties in cell biology. The imaging of transmembrane potential and ionic currents is of significant importance for monitoring the activity of the cell. It remains possible with relatively complicated instruments and methods such as patch clamping. A complementary approach to view the dynamics of ion channels with modern and efficient fluorophores is therefore of great interest to the field of biology in general. / We developed a construct based on the FRET signal between QDs and organic fluorescent dyes to monitor the conformational changes of voltage gated sodium channels. The amino acid histidine was used as a "landing platform" for QDs and the bacterial sodium channel NaChBac was chosen for testing. This study focused on the preliminary steps of the project and aimed to characterize the electrophysiological behavior of the histidine-tagged channel. The whole-cell configuration of patch clamping was the tool we used to understand the differences between the wild-type and the histidine-tagged variants of the channels. We also explore the possibility to land QDs on the histidine tag.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Not embargoed. Vita. Bibliography: 198-212.

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The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. The E. coli Tat machinery minimally consists of three integral membrane proteins: TatA, TatB and TatC. A popular model of Tat translocation is that cargo first interacts with a substrate binding complex composed of TatB and TatC and then is transported across the inner membrane through a channel comprised primarily of TatA. The most common method for observing the kinetics of Tat transport, a protease protection assay, lacks the ability to distinguish between individual transport sub-steps and is limited by the inability to observe translocation in real-time. Therefore, a real-time FRET based assay was developed to observe interactions between the cargo protein pre-SufI, and its initial binding site, the TatBC complex. The cargo was found to first associate with the TatBC complex, and then, in the presence of a membrane potential (∆psi), migrate away from the initial binding site after a 20-45 second delay. Since cargo migration away from the TatBC complex was not directly promoted by the presence of a ∆psi, the delay likely represents some preparatory step that results in a transport competent translocon. In addition, the Tat system has long been identified as a potential biotechnological tool for protein production. However, much is still unknown about which cargos are suitable for transport by the Tat system. To probe the Tat system’s ability to transport substrates of different sizes and shapes, 18 different cargos were generated using the natural Tat substrate pre-SufI as a base. Transport efficiencies for these cargos indicate that not only is the Tat machinery’s ability to transport substrates determined by the protein’s molecular weight, as well as by its dimensions. In total, these results suggest a dynamic translocon that undergoes functionally significant, ∆psi-dependent changes during translocation. Moreover, not every protein cargo can be directed through the Tat translocon by a Tat signal peptide, and this selectivity is not only related to the overall size of the protein, but also dependent on shape.

The use of microfluidic tools to develop two novel approaches to surface-based oligonucleotide hybridization assays has been explored. In one of these approaches, immobilized oligonucleotide probes on a glass surface of a microfluidic channel were able to quantitatively hybridize with oligonucleotide targets that were electrokinetically injected into the channel. Quantitative oligonucleotide analysis was achieved in seconds, with nM detection limits and a dynamic range of 3 orders of magnitude. Hybridization was detected by the use of fluorescently labeled target. The fluorescence intensity profile evolved as a gradient that could be related to concentration, and was a function of many factors including hybridization reaction rate, convective delivery speed, target concentration and target diffusion coefficient. It was possible to acquire kinetic information from the static fluorescence intensity profile to distinguish target concentration, and the length and base-pair mismatches of target sequences. Numerical simulations were conducted for the system, and fit well with the experimental data. In a second approach, a solid-phase nucleic acid assay was developed using immobilized Quantum Dot (QD) bioprobes. Hybridization was used to immobilize QDs that had been coated with oligonucleotides having two different sequences. The hybridization of one oligonucleotide sequence conjugated to a QD (a linker sequence) with a complementary sequence that was covalently attached to a glass substrate of a microfluidic channel was shown to be an immobilization strategy that offered flexibility in assay design, with intrinsic potential for quantitative replacement of the sensing chemistry by control of stringency. A second oligonucleotide sequence conjugated to the immobilized QDs provided for the selective detection of target nucleic acids. The microfluidic environment offered the ability to manipulate flow conditions for control of stringency and increasing the speed of analytical signal by introduction of convective delivery of target sequences to the immobilized QDs. This work introduces a stable and adaptable immobilization strategy that facilitates solid-phase QD-bioprobe assays in microfluidic platforms.