Glycerol kinase (GK) from Escherichia coli is allosterically controlled by fructose 1,6-bisphosphate (FBP) and the glucose-specific phosphocarrier protein IIAGlc of the phosphotransferase system. These controls allow glucose to regulate glycerol utilization. Fluorescence spectroscopic and enzyme kinetic methods are applied to investigate these allosteric controls in this study. The linkage between FBP binding and GK tetramer assembly is solved by observation of homo-fluorescence energy transfer of the fluorophore Oregon Green (OG) attached specifically to an engineered surface cysteine in GK. FBP binds to tetramer GK with an affinity 4000-fold higher than to dimeric GK. A region named the coupling locus that plays essential roles in the allosteric signal transmission from the IIAGlc binding site to the active site was identified in GK. The relationship between the coupling locus sequence in Escherichia coli or Haemophilus influenzae GK variants and the local flexibility of the IIAGlc binding site is established by fluorescence anisotropy determinations of the OG attached to the engineered surface cysteine in each variant. The local flexibility of the IIAGlc binding site is influenced by the coupling locus sequence, and in turn affects the binding affinity for IIAGlc. Furthermore, the local dynamics of each residue in the IIAGlc binding site of GK is studied systematically by the fluorescence anisotropy measurements of OG individually attached to each position of the IIAGlc binding site. The fluorescence steady-state anisotropy measurement provides a valid estimation of the local flexibility and correlates well with the crystallographic B-factors. Steady-state kinetics of FBP inhibition shows that the data are best described by a model in which the partial inhibition and FBP binding stoichiometry are taken into account. Kinetic viscosity effects show that the product-release step is not the purely rate-limiting step in the GK-catalyzed reaction. Viscosity effects on FBP inhibition are also discussed.

To understand protein stability and the mechanism of protein folding, it is essential that we gain a better understanding of the ensemble of conformations that make up the denatured state of a protein. The primary goal of the research described here was to see what we might learn about the denatured state using fluorescence. To this end, tryptophan was introduced at five sites in Ribonuclease Sa (RNase Sa): D1W, Y52W, Y55W, T76W, and Y81W. The fluorescent properties of the denatured states of these five proteins were studied and compared to the fluorescent properties of eight model compounds: N-acetyl-tryptophan-amide (NATA), N-acetyl-Ala-Trp-Ala-amide (AWA), N-acetyl-Ala-Ala-Trp-Ala-Ala-amide (AAWAA), and five pentapeptides based on the sequence around the original tryptophan substitutions in RNase Sa. Regardless of the denaturant, λmax for the proteins and model compounds differed very little, 349.3 ± 1.2 nm. However, significant differences were observed in the fluorescence intensity at λmax (IF), suggesting that IF is more sensitive to the immediate environment than λmax. The differences in IF are due in part to quenching by neighboring side chains. More importantly, IF was always significantly greater in the protein than in its corresponding pentapeptide, indicating that the protein exerts an effect on the tryptophan, which cannot be mimicked by the pentapeptide models. Acrylamide and iodide quenching experiments were also performed on the model compounds and proteins. Significant differences in the Stern-Volmer quenching constant (KSV) were also observed between the proteins and between the proteins and their corresponding pentapeptides. Importantly, the KSV for the protein was always less than in its corresponding pentapeptide. These data along with the IF data show that non-local structure in the unfolded state influences tryptophan fluorescence and accessibility. In summary, these and our other studies show that fluorescence can be used to gain a better understanding of the denatured states of proteins.

The thesis is divided into two major parts. The first part focuses on the localisation of probes in lipid/polymeric bilayers and in GM1 micelles. Included in this thesis is a new approach based on electronic energy transfer/migration (FRET/DDEM), which efficiently determines transversal positions of fluorescent molecules in lipid bilayers. This approach has been used to locate newly synthesized lipid probes in DOPC bilayers. The label was introduced at the end of sn-2 acyl chains of variable length. Analytical models accounting for FRET exist for a limited number of basic geometries. Here, a combination of FRET and Monte Carlo simulations enables the localisation of probes in bicelles and in bilayers containing pores, i.e. in lipid systems with variable curvature, or in non-homogenous lipid systems. This approach has been used to test whether conical-like fluorescence probes have an increased affinity to highly curved regions, which would enable preferential labelling of membrane pores. A simplified FRET model has been applied to localize 2-pyridones, a class of potential drugs, in GM1 micelles. Since the localisation of drugs within nanoparticles might influence the release kinetics and loading efficiency, knowledge about the drug location is highly relevant. It turned out that all derivatives were localised at the core-shell interface of GM1 micelles. The second part of the thesis focuses mainly on the estimation of lipid nanodomain size by means of FRET, which still remains the most powerful method in this field. Limitations of FRET in the determination of domain size have been explored. We showed that the limitations of FRET are mainly caused by a low probes affinity to either the liquid-ordered or liquid-disordered phase. In the continuing work we provided a detailed dynamic and structural study of crosslinker-triggered formation of nanodomains. Here, two different domains have been revealed, i.e. i) domains whose size grows with increasing amount of added cholera toxin (CTxB), and to which CTxB binds tightly; ii) domains formed in membranes containing a slightly increased amount of sphingomyelin (as compared to i) whose size does not change during titration by additional CTxB and to which CTxB binds less tightly. / Disertace je rozdělena do dvou hlavníchčástí. Prvníčást se zabývá lokalizací značek v lipidových/polymerních dvojvrstvách a v GM1micelách. V práci prezentujeme nový přístup založený na přenosu/migraci elektronické energie (FRET/DDEM), jež umožňuje efektivně určovat vertikální pozici fluorescenčních molekul uvnitř lipidové dvojvrstvy. Tato metoda byla použita k lokalizaci nově syntetizovaných lipidových značek značených na konci sn-2 acylového řetězce s různou délkou v DOPC dvojvrstvách. Analytické modely popisující FRET existují pouze pro limitovaný počet základních geometrií. Kombinace FRETu s Monte Carlo simulacemi nicméně umožňuje lokalizaci značek v bicelách a v dvojvrstvách obsahujících póry, tj. v lipidových systémech s proměnlivým zakřivením a v nehomogenních lipidových útvarech. Tento přístup umožnil např. zjistit, zda kuželovitětvarované značky mají zvýšenou afinitu k vysoce zakřiveným oblastem dvojvrstvy, což by umožnilo preferenční značení pórů. Lokalizovány byly rovněž tři deriváty 2-pyridonů(potencionálních léčiv) v GM1micelách za použití jednoduchého modelu zohledňujícího FRET mezi donory a akceptory nacházejícími se v micelách. Lokalizace léčiv v nanočásticích ovlivňuje kinetiku uvolňování (release kinetics) a množství látky solubilizované v micelách (loading efficiency). Druhá část se především zabývá určováním velikostí lipidových nanodomén pomocí FRETu, který stále zůstává nejvíce výkonnou metodou v této oblasti. Zkoumány byly limitace FRETu v určování lipidových nanodomén. Ukázalo se, že tato omezení jsou především způsobena nízkou afinitou značek buď k Lonebo k Ldfázi. V navazující studii jsme poskytnuli detailní dynamickou a strukturní studii formace nanodomén indukované crosslinkerem. Objevili jsme dva typy domén: a) domény, jejichž velikost se zvětšuje s rostoucím množstvím přidaného cholera toxinu (CTxB) a k nimž se CTxB váže pevně a b) domény vzniklé v membránách se zvýšeným množstvím sfingomyelinu (ve srovnání s a)), jejichž velikost se nemění během titrace dodatečným CTxB a k nimž se CTxB váže méně pevně. / This thesis has been elaborated within the framework of the Agreement on JointSupervision (co-tutelle) of an International Doctoral Degree Programmebetween Charles University in Prague, Czech Republic and the Department of Chemistry at Umeå University, Sweden.

Diabetes mellitus affects 25.8 million Americans (8.3%) and over 300 million people worldwide. Clinical trials indicate that proper management of blood glucose levels is critical in preventing or delaying complications associated with diabetes. Thus, there is a common need to monitor and manage blood glucose properly for people with diabetes. However, the patients’ compliance for recommended monitoring frequency is low due to the pain and inconvenience of current standard finger-pricking tests. To promote patient adherence to the recommended self-monitoring frequency, non-invasive/ minimally invasive glucose testing approaches are needed. Luminescent microparticle sensor is an attractive solution. For these sensors to be deployed in vivo, a matched optical system is needed to interrogate dermally-implanted sensors. This research project investigated the light propagation in skin and the interaction with implants using Monte Carlo modeling. The results of the modeling were used to design an optical system with high interrogation and collection efficiency (40~300 times improvement). The optical system was then constructed and evaluated experimentally. A stable skin phantom mimicking the optical properties of human skin was developed as a permanent evaluation medium to minimize the use of animals. The optical properties of the skin phantom matched the maximum published values of human skin in scattering and absorption over the spectral range of 540~700nm in order to avoid overestimation of the capability of the system. The significant photon loss observed at the connection between the designed system and a commercial spectrometer was overcome using two optimized designs: a two-detector system and a customized low-resolution spectrometer system. Both optimization approaches effectively address the photon loss problem and each showed good SNR (>100) while maintaining a sufficient system resolution for use with fluorescent materials. Both systems are suitable for luminescence measurement, because broad bands of the luminescent spectrum are of interest. In the future, either system can be easily modified into a more compact system (e.g. handheld), and it can be directly coupled to an analog-to-digital converter and integrated circuits offering potential for a single compact and portable device for field use with luminescent diagnostic systems as well as implanted sensors.

Lévy flights and Lévy walks are two mathematical models used to describe anomalous diffusion(i.e. those having mean square displacements nonlinearly related to time (as opposed to Brownian motion)). Lévy flights follow probability distributions p(|r|) yielding infinite mean square displacements since some rare steps are very long. Lévy walks, however, have coupled space-time probability distributions penalising very long steps. Both Lévy flights and Lévy walks are dominated by a few long steps, but most steps are much, much smaller. The semi-experimental part ofthis work dealt with how fluorescent probes moved in systems of cationic starch and latex/solutions of dodecyl trimethyl ammonium bromide, respectively. Visually, no Lévy walks couldbe detected. However, mathematical regression suggested enhanced diffusion and subdiffusion. Moreover, time-dependent diffusion coefficients were calculated. Also examined was how Microsoft Excel could be used to generate normal diffusion as well as anomalous diffusion. / Lévyflygningar och Lévypromenader är matematiska modeller som används för att beskriva anomal diffusion (i.e. dessa då medelvärdet av kvadratförflyttningarna är icke-linjärt relaterat tilltiden (till skillnad från Brownsk rörelse)). Lévyflygningar följer sannolikhetsfördelningar p(|r|)som ger oändliga medelkvadratförflyttningar eftersom vissa steg är väldigt långa. Lévypromenader,å andra sidan, har kopplade rum-tid-sannolikhetsfördelningar som kraftigt reducerar demycket långa stegen. Både Lévyflygningar och -promenader domineras av ett fåtal långa steg ävenom de flesta steg är mycket, mycket mindre. Den semiexperimentella delen av detta arbetestuderade hur fluorescerande prober rör sig i katjonisk stärkelse respektive latex/lösningar avdodecyltrimetylammoniumbromid. Inga Lévypromenader kunde ses. Emellertid taladematematisk regression för att superdiffusion och subdiffusion förelåg. Tidsberoende diffusionskoefficienter beräknades också. I detta arbete undersöktes även hur Microsoft Excel kan användas för att generera både normal och anomal diffusion.

<p>Current techniques for nucleic acid analysis often involve extensive sample preparation that requires skilled personnel and multiple purification steps. In this dissertation, we introduce an on-chip, isothermal, post-hybridization labeling and signal amplification technique that can directly interrogate unmodified DNA and RNA samples on a microarray format, eliminating the need for microarray sample pre-processing. </p><p>We name this technique Surface Initiated Enzymatic Polymerization (SIEP), where we exploit the ability of a template independent DNA polymerase called Terminal Deoxynucleotidyl Transferase (TdT) to catalyze the formation of long single-stranded DNA (ssDNA) chain from the 3'-end of a short DNA primer, which is tethered on the surface, and TdT's ability to incorporate unnatural reporter nucleotides, such as fluorescent nucleotides. We hypothesize that polymerization of a long ssDNA chain while incorporating multiple fluorescent nucleotides on target DNA or RNA hybridized to probe printed on a surface will provide a simple and powerful, isothermal method for on-chip labeling and signal amplification. </p><p>We developed the SIEP methodology by first characterizing TdT biochemical reaction to polymerize long homopolymer ssDNA (> 1000 bases) starting from the 3'-OH of ten bases oligonucleotides. We found that the preferred monomers (deoxynucleotide, dNTP) are dATP and dTTP, and that the length of the ssDNA extension is determined by the ratio of input monomer (dNTP) to initiator (short oligonucleotides). We also investigated TdT's ability to incorporate fluorescent dNTPs into a ssDNA chain by examining the effect of the molar ratios of fluorescent dNTP to natural dNTP on the initiation efficiency, the degree of fluorophore incorporation, the length and the polydispersity of the polymerized DNA strand. These experiments allowed us to incorporate up to ~50 fluorescent Cy3-labeled dNTPs per kilobase into a ssDNA chain. With the goal of using SIEP as an on-chip labeling method, we also quantified TdT mediated signal amplification on the surface by immobilizing ssDNA oligonucleotide initiators on a glass surface followed by SIEP of DNA. The incorporation of multiple fluorophores into the extended DNA chain by SIEP translated to a up to ~45 fold increase in signal amplification compared to the incorporation of a single fluorophore.</p><p>SIEP was then employed to detect hybridization of DNA (25 bases), short miRNA (21 bases) and long mRNA (1400 bases) by the post-hybridization, on-chip polymerization of fluorescently labeled ssDNA that was grown from the 3'-OH of hybridized target strands. A dose-response curve for detection of DNA hybridization by SIEP was generated, with a ~1 pM limit of detection (LOD) and a 2-log linear dynamic range while the detection of short miRNA and fragmented mRNA targets resulted in ~2 pM and ~10 pM LOD, respectively with a 3-log linear dynamic range.</p><p>We further developed SIEP for colorimetric detection by exploiting the presence of negatively charged phosphate backbone on the surface as target DNA or RNA hybridizes on the immobilized probe. The net negative charge on the surface is further increased by TdT catalyzed polymerization of long ssDNA. We then used positively charged gold nanoparticles as reporters, which can be further amplified through electroless metallization, creating DNA spots that are visible by eye. We observed an increase of 100 fold in LOD due to SIEP amplification.</p><p>Overall, we demonstrated the use of SIEP methodology to label unmodified target DNA and RNA on chip, which can be detected through fluorescence signal or colorimetric signal of metallized DNA spots. This methodology is straightforward and versatile, is compatible with current microarray technology, and can be implemented using commercially available reagents.</p> / Dissertation

While unprotected amino aldehydes are typically not isolable due to imine formation and consequent polymerization, stable unprotected aziridine aldehydes are useful and available reagents. Moreover, reversible hemiacetal and hemiaminal formation enable these compounds to reveal both their electrophilic and nucleophilic functional groups. This exceptional arrangement allows for aziridine aldehyde dimers to participate in and disrupt the mechanisms of an array of well-known organic reactions, including isocyanide-based multicomponent reactions. The scope and selectivity patterns of aziridine aldehyde induced amino acid or peptide macrocyclization have been investigated. A small library of constrained tri-, tetra-, and penta-peptide macrocycles – representing the most difficult cyclic peptides to synthesize – has been prepared. The scope of aziridine aldehyde participation in multicomponent reactions was also expanded to Ugi and Passerini reactions that do not employ tethered amine and acid functional groups. In order to facilitate cellular imaging of peptide macrocycles a fluorescent isocyanide reagent was prepared and applied to prepare mitochondrial targeting macrocycles. Thioester isocyanide reagents were synthesized to enable rapid assembly of cycle-tail peptides through ligation technology.

With the rapidly increasing interest in the field of glycomics, which is the comprehensive study of the roles carbohydrates play in a living system, urgent need for developing quick and highly selective carbohydrate sensors is growing. The boronic acid group, with its electron-deficient structure (6 valence electrons with an open shell), acts as a Lewis acid with high intrinsic affinity towards Lewis bases such as fluoride, cyanide and hydroxyl groups. Specifically, formation of a 5- or 6- membered ring between the boronic acid moiety and a1,2- or 1,3-diol in aqueous solution has been fully explored as a strategy of carbohydrate sensor design. Along this line, those binders were termed as ¡°boronolectins¡± because of their similar functions as lectins. One challenge in developing boronic acid-based carbohydrate sensors is to enhance the discriminating ability among various carbohydrate analytes with diverse building blocks and complex linkage patterns. One approach is using polypeptide or oligonucleotide as a backbone or scaffold with functionalized boronic acid moiety to create a molecular library, and then selecting binders with favorable properties. The work presented here includes three general research parts: synthesis of a naphthalimide-based boronic acid-conjugated thymidine triphosphate (NB-TTP), fluorescence property studies of NB-TTP incorporated DNA (NB-DNA), and cellular imaging studies using NB-TTP: 1) 4-Amino-1,4-naphthalimide (Nap) was chosen as the fluorophore because of its relatively long excitation and emission wavelengths, and stability. The synthesis of naphthalimide-based boronic acid (NB) followed similar route as previously published work. Tethering of boronic acid moiety and TTP was accomplished through Cu(I)-catalyzed azide-alkyne cyclization (CuAAC), known as click chemistry. The synthesized NB-TTP showed fluorescence enhancements at long wavelength (¦Ëem: 540 nm) upon sugar addition. 2) NB-TTP was incorporated into DNA through Klenow fragment-catalyzed primer extension reactions. Different DNA sequences were designed with varying number and spacing for NB-TTP incorporation. The preliminary study provided certain insight into several factors that affect the fluorescent properties of different NB -DNA. 3) NB-TTP was added into Hela cell culture medium to study its cell imaging properties. With the observation under fluorescent microscope, it was demonstrated that NB-TTP showed good cell membrane permeability and significant accumulation in cell nucleus.

La pollution des milieux aquatiques par les métaux lourds demande une évaluation du risque des effets toxiques chez des espèces aquatiques. En particulier, le cadmium est connu pour être un métal non essentiel pour le fonctionnement cellulaire et toxique à faible concentration. Environ 25 000 tonnes de cadmium par année sont rejetées dans l'environnement via les activités industrielles, d'extraction de métaux, par l'emploi de pesticides et autres. Dans ce contexte, tant la formation que l'activité de l'appareil photosynthétique représente une cible importante pour les contaminants métalliques parce que l'inhibition des processus biochimiques et biophysiques de la photosynthèse affectent en entier la physiologie des plantes. Dans cette étude, la biosynthèse de la chlorophylle et la fluorescence chlorophyllienne ont été utilisées afin d'évaluer les effets toxiques du cadmium sur la formation de l'appareil photosynthétique et son activité. Dans un premier temps, l'effet toxique du cadmium a été évalué sur l'activité photosynthétique de l'algue verte. Dans cette partie, la fluorescence chlorophyllienne des algues est utilisée comme une approche pour évaluer le risque de toxicité du cadmium. Une altération de la capacité photosynthétique induite par les métaux lourds se reflète dans le rendement et la cinétique de fluorescence. Deux souches de cette espèce, une souche «sauvage» (CC-125 WT mt+) avec paroi cellulaire et une souche «mutante» (CC-400 CW 15 mt+) sans paroi, ont été employées pour étudier les effets de la paroi cellulaire sur l'absorption du cadmium et son effet toxique après 24 et 48 heures de traitement. Les courbes dose-réponse obtenues pour ces deux souches ont permis d'identifier une grande sensibilité des effets toxiques du cadmium pour les algues sans paroi par rapport à celles avec paroi en présence d'une même concentration nominale de cadmium. Les paramètres photosynthétiques de fluorescence ont indiqués que le site d'inhibition au cadmium a été au complexe enzymatique du dégagement d'oxygène associé au photosystème II. Dans la seconde partie, afin d'évaluer la capacité à synthétiser la chlorophylle dans un environnement contaminé au cadmium, la phototransformation de la protochlorophyllide (PChlide) en chlorophyllide (Chlide) par l'enzyme protochlorophyllide oxydoréductase (POR) a été étudié chez l'algue unicellulaire Chlamydomonas reinhardtii. Une souche mutante ne synthétisant pas la chlorophylle à l'obscurité a du être utilisé afin d'obtenir des cellules étiolées, c'est-à-dire que les chloroplastes de ces cellules ne sont pas encore formés. La fluorescence à basse température (77 °K) est l'outil permettant de suivre dans le temps la phototransformation des pigments et la formation des photosystèmes. Les spectres de fluorescence ont permis d'évaluer un certain retard dans la phototransformation de la protochlorophyllide en chlorophyllide ce qui a une répercussion sur la formation des photosystèmes. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Cadmium, Chlamydomonas reinhardtii, Chlorophillide, Chlorophylle, Cytométrie de flux, Fluorescence, Métaux lourds, NADPH-protochlorophyllide oxydoréductase, Photosynthèse, Protochlorophillide, Toxicité.

Oil from flaxseed (Linum usitatisssimum L.) contains hydrophobic cyclic peptides or cyclolinopeptides (CLs) comprising eight or nine amino acids. These bioactive compounds have potential therapeutic applications and may be used as scaffolds for increased utility. Two steps were undertaken to increase the potential utility of these compounds. Initially multigram quantities of flax CLs were highly enriched from flax oil. Subsequently new synthetic procedures were developed for modification of the CLs through the methionine group (Met). Finally, the utility of the modified CLs was tested in a number of applications. CLs were recovered from a crude oil extract that contain five CLs (CLA, CLC, CLE, CLJ and CLK). Oxidation of this mixture reduced the complexity of the mix to just three CLA, CLJ and CLK. CLJ and CLK were enriched then characterized by NMR and MS-MS methods. CLs containing methionine sulfoxide groups (Mso), CLC and CLE were isolated from crude mixture then selectively reduced to afford Met containing analogs: CLB and CLE'. The Met of modified CLs was used as a point for attachment of tags and couplers for various applications. Cyclic peptide modification through Met groups has not been reported previously. Synthetic methods were devised to introduce activating functional groups such as -CN, -COOH, -OH and -NH2 to the sulfur atom of Met. The modified CL conjugates were characterized using spectrometric techniques including 1D and 2D NMR spectrometry, as well as mass spectrometry. After activation the CLs were covalently linked to molecules or materials of interest including fluorescence tags (coumarin), affinity chromatography media and bovine serum albumin (BSA) for production of polyclonal antibodies. Fluorescence studies were performed in methanol, ethanol, dimethylformamide and acetonitrile to study the solvent effect. CLs attached to solid affinity matrix showed specific binding to apolipoprotein A1 after incubation with chicken serum. These CLs also act as hapten and have been used to couple BSA to produce polyclonal antibodies. Met modification was a satisfactory approach to produce a range of useful peptide products where more conventional methods of molecule attachment are not available.