More than one-third of all Magnetic Resonance Imaging (MRI) scans employ image-enhancing contrast agents to increase the differential signal intensity between diseased and normal tissue. Because current clinical contrast agents exhibit low relaxivity (mM-1 s-1), low dose efficiency, and rapid secretion, we have designed a group of protein-based MRI contrast agents with multiple gadolinium binding sites. In this study, the developed purification method for Class ProCA-3 agents allows for a quick and cost-effective way to abstract up to 109 mg of pure, soluble protein from a 1L E. Coli cell pellet devoid of DNA or RNA “contamination” for extensive animal studies. Circular dichroism far-UV spectra ensure the metal stability of the agents, revealing maintenance of their native α-helical structure in the presence and absence of metal ions. Furthermore, substantial evidence supports the high dose efficiency of these agents, exhibiting up to five folds higher relaxivity than their analogous commercial competitors.

Research toward a multiplexed nucleic acid biosensor that uses quantum dots (QDs) as donors in a fluorescence resonance energy transfer (FRET) assay is described. Optical fibers were modified with mixed films composed of different colours of QDs and different oligonucleotide probes that served as scaffolds for the hybridization of the corresponding target nucleic acid sequences. Fluorescent dyes that were suitable as acceptors for each QD donor were associated with hybridization and provided an analytical signal through FRET-sensitized emission. Different detection channels were achieved through the combination of different donors and acceptors: green emitting QDs with Cyanine 3 or Rhodamine Red-X; and red emitting QDs with Alexa Fluor 647. A detection channel that used the direct excitation of Pacific Blue complemented the FRET pairs. One-plex, two-plex, three-plex and four-plex hybridization assays were demonstrated. A sandwich assay format was adopted to avoid target labeling. Detection limits were 1-10 nM (1-12 pmol) and analysis times were 1-4 h. Single nucleotide polymorphisms were discriminated in multiplexed assays, and the potential for reusability was also demonstrated. Non-selective interactions between QDs and oligonucleotides were characterized, and routes toward the optimization of the QD-FRET hybridization assays were identified. A basic model for multiple FRET pathways in a mixed film was also developed. In addition to the advantages of solid-phase assays, the combination of QDs and FRET was advantageous because it permitted multiplexed detection using a single excitation source and a single substrate, in the ensemble, and via ratiometric signals. Spatial registration or sorting methods, imaging or spatial scanning, and single molecule spectroscopy were not required. The research in this thesis is expected to enable new chip-based biosensors in the future, and is an original contribution to both bioanalytical spectroscopy and the bioanalytical applications of nanomaterials.

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. 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. 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. 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.

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.

The quaternary status of G protein-coupled receptors (GPCRs) is important, unknown and controversial. Estimates of size from numerous pharmacological, biochemical and biophysical studies range from monomers to octamers. Accounts of stability vary from constitutive oligomers to a spontaneous, ligand-regulated interconversion between monomers and dimers. In the present investigation, the oligomeric size of GPCRs in live Chinese hamster ovary (CHO) cells has been examined by two methods. Both are based on the efficiency of Förster resonance energy transfer (FRET) between fluorophore-tagged receptors, as determined from emission spectra via spectral deconvolution. In the first, the apparent FRET efficiency (Eapp) was measured for cells expressing eGFP- and eYFP-tagged M2 muscarinic receptors at different ratios of acceptor to donor. Eapp then was related to the pair-wise efficiency (Ep) according to a model that enumerates all pathways for the transfer of energy between single donors and acceptors within an oligomer of given size (n). Each value n returned a distinct and well-defined value of Ep. Fluorescence lifetime imaging provided an independent estimate of Ep that was in close agreement with the model-based value when n = 4, identifying the M2 receptor as a tetramer. In the second approach, the M1 and M2 muscarinic receptors and the β1 and β2 adrenergic receptors were tagged with GFP2 and eYFP, and the value of Eapp was estimated for each pixel in the image of a cell. The distributions of Eapp from 34–40 cells expressing each receptor were compared with those predicted for populations of dimers, trimers and tetramers, the latter configured as a square and a rhombus. In each case, the combined data were well described in terms of a rhombus. Distributions obtained for the M2 and β2 receptors were not affected by agonists or inverse agonists, nor was there evidence for appreciable numbers of dimers or larger oligomers. Taken together, the results suggest that GPCRs of Family 1 exist largely or wholly as constitutive tetramers.

The quaternary status of G protein-coupled receptors (GPCRs) is important, unknown and controversial. Estimates of size from numerous pharmacological, biochemical and biophysical studies range from monomers to octamers. Accounts of stability vary from constitutive oligomers to a spontaneous, ligand-regulated interconversion between monomers and dimers. In the present investigation, the oligomeric size of GPCRs in live Chinese hamster ovary (CHO) cells has been examined by two methods. Both are based on the efficiency of Förster resonance energy transfer (FRET) between fluorophore-tagged receptors, as determined from emission spectra via spectral deconvolution. In the first, the apparent FRET efficiency (Eapp) was measured for cells expressing eGFP- and eYFP-tagged M2 muscarinic receptors at different ratios of acceptor to donor. Eapp then was related to the pair-wise efficiency (Ep) according to a model that enumerates all pathways for the transfer of energy between single donors and acceptors within an oligomer of given size (n). Each value n returned a distinct and well-defined value of Ep. Fluorescence lifetime imaging provided an independent estimate of Ep that was in close agreement with the model-based value when n = 4, identifying the M2 receptor as a tetramer. In the second approach, the M1 and M2 muscarinic receptors and the β1 and β2 adrenergic receptors were tagged with GFP2 and eYFP, and the value of Eapp was estimated for each pixel in the image of a cell. The distributions of Eapp from 34–40 cells expressing each receptor were compared with those predicted for populations of dimers, trimers and tetramers, the latter configured as a square and a rhombus. In each case, the combined data were well described in terms of a rhombus. Distributions obtained for the M2 and β2 receptors were not affected by agonists or inverse agonists, nor was there evidence for appreciable numbers of dimers or larger oligomers. Taken together, the results suggest that GPCRs of Family 1 exist largely or wholly as constitutive tetramers.