G-Protein Coupled Receptors (GPCRs) are a class of membrane-bound receptor proteins capable of relaying a biological signal across a cell membrane through a solely conformational change in their transmembrane domain. Previous work has shown that helical foldamers composed of achiral monomeric units can be used in an analogous manner to relay stereochemical information on the nano-scale through the conformational control of screw-sense preference. While this work has produced some highly successful examples of signal relay, mimicking the function of GPCRs, its reliance on screw-sense responsive NMR probes has restricted further development into membrane environments. This thesis describes the successful development of a pyrene based screw-sense responsive fluorescence probe and its subsequent use in the development of a series of membrane-based GPCR mimics. This thesis has also details the preliminary steps towards the development of light-responsive controllers of screw-sense preference for nano-scale signal relay devices.

Fluorescent protein labelling is a powerful tool for the sensitive visualization of proteins in living cells, allowing the elucidation of their localization, trafficking and ultimately their cellular function. We have developed a novel labelling technique based on the genetic fusion of a protein of interest to a small helical peptide sequence containing two Cys residues (dC10). This tag can undergo an efficient reaction with small fluorogenic labelling agents composed of a fluorophore and a dimaleimide core (dM10) that confers high reaction specificity, and quenches the latent fluorescence through photo-induced electron transfer, until both of its maleimide groups have formed robust covalent bonds with the tag Cys thiol groups. Our initial efforts at intracellular protein labelling demonstrated the importance of the selectivity of the labelling reaction, which is dependent on the reactivity of the dC10 tag. To that end, we re-engineered the dC10 tag through rational protein design. Mutant libraries were prepared through combinatorial mutation at specific positions of the helical tag sequence, and screened for their fluorogenic reactivity. In this way, we identified a novel sequence for a next-generation dC10 tag that confers 10-fold greater selectivity that we then applied to in cellulo labelling. Subsequent mechanistic studies revealed the basis for this dramatic increase in reactivity. Current applications of this powerful labelling technique, including the site-specific chelation of lanthanide ions for NMR spectroscopy and site-specific covalent heavy-atom labelling for X-ray crystallography, will also be discussed.

An assay based on Nile red fluorescence was developed for quantitative analysis of triglycerides, a common cellular component with important biological functions and is routinely analyzed for diagnosis of metabolic disorders and as an important feedstock of food industry and biodiesel production. Based on studies on the Nile red fluorescence of pure, binary, and ternary systems of triglycerides, ethanol, and water, 20% ethanol aqueous solution was determined to be the most suitable solvent for lipid fluorescence measurement. Excellent linearity was established for lipid samples in the range of 0.1- 0.5 mg/ml with several different lipid standards and vegetable oils. Results also suggest that the fluorescence of triglycerides was not sensitive to the fatty acid composition of lipids. This finding is important since it implies that the assay could potentially be used for the measurement of triglyceride content of different oil crops without causing significant variations. The results of this method were then verified by comparing with the results of the conventional gravimetric methods. The results of the fluorescence assay were consistently lower than that of the gravimetric method by approximately 10%. This phenomenon was tentatively attributed to the fact that the gravimetric method measures the total amount of lipophilic materials in samples while the fluorescence assay is selective to glycerides. Attempts were also made to apply this assay to estimate the lipid content of green alga Neochloris oleoabundans. However, the results were less than ideal due to the existence of interfering components in the extract of microalga samples that could significantly repress the fluorescence of lipids.

Les récepteurs nucléaires sont membres d'une famille de protéines activées par des ligands et qui régulent la transcription de nombreux gènes. Le récepteur nucléaire RevErbα est constitutivement inhibiteur de la transcription de gènes cibles via le recrutement du complexe corépresseur NCor-HDAC3 (Nuclear CoRepressor et Histone DeAcetylase 3). Ce complexe joue un rôle important dans le contrôle de l'horloge circadienne et de la glucogenèse via la régulation de la transcription des gènes codant pour les enzymes G6Pase (Glucose-6-Phosphatase) et PEPCK (PhosphoEnol Pyruvate Carboxy Kinase). Ces deux enzymes sont impliquées dans la glucogénèse qui est dérégulée en cas de diabète de type 2. Ce travail est basé sur l'investigation de l'interaction du récepteur nucléaire RevErbα avec deux corépresseurs: son partenaire établit NCor et SMRT (Silencing Mediator for Retinoid and Thyroid Receptors). Malgré ce qui est rapporté dans la littérature, c'est à dire que SMRT et RevErbα n'interagissent pas fonctionnellement in vivo, nous avons choisis d'étudier cette interaction à cause de la similarité de séquence entre les deux corépresseurs, mais également parce que les peptides des deux corépresseurs ont été rapportés comme interagissant avec d'autre récepteurs nucléaire. Afin d'étudier ces interactions, nous avons utilisé deux techniques complémentaires basées sur l'émission de fluorescence: Une technique in vitro qui est l'anisotropie de fluorescence et une technique de microscopie de fluorescence in cellulo appelée Number & Brightness. En plus de cette étude de l'interaction entre le récepteur nucléaire et les corépresseurs, nous nous sommes intéressé à déterminer l'effet de plusieurs ligands sur cette interaction. Trois ligands ont été testés: l'hème qui est rapporté comme étant le ligand naturel de RevErbα et deux ligands synthétiques et non naturels de RevErbα (SGN et SD7). Grâce à l'anisotropie de fluorescence (in vitro), nous avons confirmé et quantifié l'interaction entre le domaine de liaison au ligand (LBD) de RevErbα et un peptide NCor contenant le domaine d'interaction majeur (ID1 pour RevErbα et nous avons déterminé l'effet des trois ligands sur cette interaction. Nous avons également quantifié l'interaction entre RevErbα et d'autres peptides provenant de NCor et correspondant aux autres domaines d'interaction (ID2 et ID3) pour sa liaison à RevErbα. Nous avons déterminé que l'hème et le SD7 ont un effet déstabilisateur de la liaison de RevErbα à NCor in vitro, alors que le ligand SGN améliore la stabilité de complexe. Nous avons également confirmé une interaction entre RevErbα et un peptide corépresseur issu de SMRT. Afin d'étudier l'interactions des protéines pleine taille dans un contexte plus fonctionnel, nous avons utilisé le 2 photons-2 couleurs Number & Brightness. C'est une technique de microscopie à fluorescence basée sur la fluctuation de l'intensité de fluorescence pour étudier les interactions spécifiques de RevErbα avec NCor et SMRT pleine taille in cellulo ainsi que l'effet des ligands, précédemment mentionné, sur ces interactions. Dans les conditions choisies pour notre étude, nous avons déterminé que RevErbα interagit fortement avec NCor in cellulo et que cette interaction est améliorée par le ligand SGN. En revanche, l'hème et le SD7 n'ont pas d'effet observable sur ce complexe. Nous avons également montré pour la première fois que RevErbα forme des complexes avec le corépresseur SMRT pleine taille in cellulo. La continuité de ce travail pourrait être ciblée sur l'identification de ligands qui augmenterait le recrutement du complexe corépresseur NCor-HDAC3 sur RevErbα, menant ainsi à la diminution de l'expression des gènes cibles. En définitive, un ligand tel que celui-ci pourrait être d'un grand intérêt dans la recherche de la diminution de glucose dans le sang dans les cas de diabètes de type 2. / Nuclear receptors are members of ligand-inducible factors that regulate the transcription of many genes. Nuclear receptor RevErbα constitutively inhibits the transcription of target genes via the recruitment of the corepressor complex NCor-HDAC3 (Nuclear CoRepressor and Histone DeAcetylase 3). This complex plays an important role in controlling the circadian clock and glucogenesis via regulation of the transcription of the G6Pase (Glucose-6-Phosphatase) and PEPCK (PhosphoEnol Pyruvate Carboxy Kinase) genes, both coding for proteins involved in glucogenesis, which is central to type 2 diabetes. Here we have investigated the interaction of the nuclear receptor RevErbα with two corepressors: its establish partner, NCor and SMRT (Silencing Mediator for Retinoid and Thyroid Receptors). Despite literature reports that SMRT and RevErbα do not interact functionally in vivo, we choose to study this interaction because of sequence similarity between the two corepressors, because peptides of both corepressors were reported to interact with other nuclear receptors. To investigate these interactions, we used two complementary fluorescence techniques: In vitro assays based on fluorescence anisotropy and an in cellulo fluorescence microscopy technique called Number & Brightness. In addition to the interactions between the CoRs and the RN, we were interested in determining the effects of several ligands on these interaction. Three ligands were tested: heme, which is reported to be the natural ligand of RevErbα and two synthetic and non-naturals ligands of RevErbα (SGN and SD7). By fluorescence anisotropy (in vitro) we confirmed and quantitated the interaction between purified RevErbα Ligand Binding Domain (LBD) and an NCoR peptide containing the major interaction domains (ID1) for RevErbα and revealed the effect of the three ligands on this interaction. We quantitated as well, the interaction between RevErbα and other peptides from NCor corresponding to the other interaction domains (ID2 and ID3) for it binding to RevErbα. We found a destabilizing effect of heme and SD7 binding to RevErbα on it interaction with NCor in vitro, whereas the ligand SGN enhanced the complex stability. We also confirmed an interaction between RevErbα and a SMRT peptide corepressor in vitro. In order to examine these interactions in a more functionally relevant context using full length proteins, we used 2 photon 2 colors Cross Number and Brightness (N&B), an fluorescence microscopy technique based on fluorescence intensity fluctuations to study specific interactions of full length RevErbα with NCor and SMRT in cellulo as well as the effect of several ligands above mentioned. Under the conditions of our studies, we find that RevErbα and NCor interact strongly in cellulo, and we observed a slight enhancement of this interaction by the SGN ligand. No effect of heme or SD7 was observed on the complex. We show as well for the first time that RevErbα forms complexes with the full length SMRT corepressor in cellulo. Future extensions of these studies could be aimed at identifying ligands that enhance the recruitment of the corepressor complex NCor/HDAC3 by RevErbα, thus leading to a decrease expression of the target genes. Ultimately such a ligand could be of interest in the quest to decrease blood glucose levels in type 2 diabetes.

The diffraction limit restricts conventional light microscopes to approximately 250 nm laterally and 500 nm axially, these limits being first proposed by Abbe in 1873. Despite this, optical microscopes have found many applications in biological research and single cells that are 10 - 100 um in size. Furthermore by coupling the non-invasive nature of a light microscope with highly sensitive fluorescent probes, fluorescence microscopy has also become a standard imaging technique. Recent advances in fluorescence microscopy now provide a number of methods to circumvent the Abbe diffraction limit, with many techniques becoming prevalent over the last 10 years including direct Stochastic Optical Reconstruction Microscopy (dSTORM). A dSTORM system has been constructed and calibrated using a commercially available inverted florescence microscope and total internal reflection florescence (TIRF) imaging. dSTORM relies on the ability to switch sparse subsets of fluorophores and temporally separate them. Provided the spatial separation is sufficient between any member of a subset, the average error with which the emission can be localized is much less than size of the emission profile itself. The underlying mechanism for this switching is detailed based on the principle of photoinduced electron transfer (PET). The switching characteristics of the common florescent dye Alexa Fluor 568 are investigated and shown to be controlled by a number of factors including the excitation intensity and concentration of the primary thiol cysteamine beta-MEA. A number of parameters are defined, including the dye switching rate, for a given set of physical parameters. U2OS cells are labelled for the microtubule protein Tubulin using immunofluorescent labelling strategies. A direct comparison is made between diffraction limited TIRF images and dSTORM reconstructed images, with an average width for microtubules determined to (58.2 ± 8.1) nm. Further measurements are made by labelling the Rab5 effector Early Endosome Antigen 1 (EEA1). From this the aspect ratio for early endosomes is determined to be 1.68 ± 0.7 with an average radius of (45.8 ± 18.8) nm. The point spatial distribution of EEA1 is investigated by using the linearised form of Ripley's K-function H(r) and the null hypothesis of complete spatial randomness tested. EEA1 is shown to cluster at radius of 58.7 nm on individual endosomes, thought to be due to the well defined binding domains present on early endosomes for EEA1. Further evidence suggests that clustering is also exhibited at another maximum of approximately 500 nm when looking at an ensemble of EEA1 and early endosomes.

>Magister Scientiae - MSc / Sea anemones (Order Actinaria) are a diverse order from the Class Anthozoa. They are found in all marine habitats at all depths and their symbiotic relationships play an important role in energy transfers especially in the benthic-pelagic community. The evolutionary background and phylogenetics of the class is poorly understood due to a lack of correspondence between taxonomic and molecular data (Daly et al. 2008). Therefore, a deeper exploration into Cnidarian molecular biology is needed to establish these as an evolutionary model organism. Gene discovery from various marine invertebrates has facilitated the recovery of anti-cancer drugs, antibiotics and reporter genes (Faulkner, 2000; Allen and Jaspars, 2009). The most commercially lucrative products from sea anemones are fluorescent and chromoproteins (FP/CP), which are used as non-invasive real-time reporter genes. The applications for these proteins are extensive and range from monitoring cellular processes such as protein localisation and interactions to imaging (Alieva et al. 2008). Therefore, novel FP and CPs have potential for commercialization. The aims of the project were to analyze basic molecular diversity of the sea anemones Pseudactinia varia, Pseudactina flagellifera and Bunodosoma capensis and evaluate a new screening method to isolate novel FP and CPs. To assess the basic molecular diversity, of the sea anemones and their associated symbionts 16S rRNA and 18S rRNA clone libraries were generated. The sea anemones used in this study clustered together with those of the Family Actiniidae. The bacterial associations observed based on the closest relative BLAST analysis were dominated by Proteobacteria (gamma, alpha and epsilon) as well as Bacteroides. The associate bacterial symbionts possibly produce compounds that range from polyunsaturated fatty acids, polyhydroxyalkanoates to anti-microbial compounds that aid the host in various processes. In order to screen for FPs and CPs from sea anemones three types of cDNA libraries were generated to be screened either by sequence based or activity based approaches. Novel primers were designed which could be applied for the screening of a variety of Anthozoans. A positive control was also designed and synthesised in order to test the capability of the designed primers and optimise the amplification. Although amplicons were generated from gDNA and cDNA libraries from each of the sea anemones they were found to be non-specific products. The detection limit is likely to be the limiting factor. The construction of an activity based library was not achieved due to technical constraints, which highlights the need for new molecular tools in this field or improvements to the existing ones. / National Research Foundation (NRF)

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22489号 / 農博第2393号 / 新制||農||1076(附属図書館) / 学位論文||R2||N5269(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 近藤 直, 准教授 小川 雄一, 教授 飯田 訓久 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

The complexity of cell membranes is far from being only a simple assembly of lipids and proteins separating cells from the surrounding environment. Each of the thousands of different membrane components performs its specific role in cellular functions, since a multitude of biological processes is mediated by membranes. The understanding of the molecular basis of these processes is one of the important aims of current biological research. Our research employing single- molecule fluorescence methods (e.g. FCS, FCCS, FLIM-FRET) has made a contribution to the knowledge of membrane lateral organization or mechanism of membrane fusion. Furthermore, we revealed the mechanism of membrane activity of a small natural compound. As native cell membranes are very complex structures, we performed the experiments on simplified model lipid membranes that allow studying lipid-lipid or lipid-protein interactions at the molecular level in a controlled way. The first part of this thesis deals with the mode of action of a membrane active secondary metabolite didehydroroflamycoin (DDHR). We demonstrated that DDHR is a pore-forming agent and that this activity is influenced by the presence of cholesterol. Direct visualization of intrinsic fluorescence of DDHR revealed its preferential partitioning into membrane areas...

In the framework of the quasi-static approximation (QSA), some theoretical studies were conducted within the local response approximation (LRA). In these studies, certain plasmonic and plexcitonic systems were proposed, and their spectroscopic properties investigated. The QSA allows us to study metal nanoparticles (MNPs) and inter-particle distances that are small compared to the wavelength of light in the medium surrounding the MNPs, while the LRA enables us to utilize the bulk dielectric response of the metal in consideration. We have studied the following properties in detail: localized surface plasmon resonances (LSPRs), plasmon-induced transparency (PIT), and plasmon-enhanced fluorescence (PEF), while exciton-induced transparency (EIT) has only been partly studied. LSPR and PIT are properties of plasmonic systems while PEF and EIT are properties of plexcitonic systems. Both PIT and EIT are forms of electromagnetically-induced transparency. We started by constructing a geometry-based theoretical model that predicts the LSPR formula of any member of a certain group of single MNPs, using the LSPR for the most complex MNP geometry in the group. The model shows that from the LSPR of a nanorice, one could predict the LSPRs of concentric nanoshells, solid and cavity nanorods and nanodisks, respectively, and solid and cavity nanospheres. These formulae serve as quick references for predicting LSPRs since they can easily be compared to LSPRs obtained from spectral analysis. Likewise, we studied LSPR in addition to PIT in a nanoegg-nanorod dimer. We proposed this dimer in order to investigate how the interplay between plasmon coupling and MNP sizes affects PIT in complex geometries such as nanoeggs. Our result shows that the formation of PIT dips — regions in the dimer spectra where little or no incident radiation is absorbed by the dimer — are strongly-dependent on the nanorod size, due to the dependence of the plasmon coupling strength on the half-length of the nanorod. We investigated the phenomenon of PEF using a nanoegg-emitter system and a nanorod-emitter system, respectively. Emitters are organic or inorganic materials whose radiative decay rates increase dramatically when placed near a MNP subjected to plasmon excitation. Our theoretical results show that the choice of the MNP-emitter system to use depends on both the intrinsic quantum yield of the emitter and the antenna efficiency of the MNP. Theory shows that PEF is more substantial when the former is very low, and it will always occur if the latter is greater than the former. A nanorod-emitter system should serve as the preferred choice, due to the relatively easier synthesis of nanorods compared to nanoeggs, and the large longitudinal polarizability of nanorods as a result of the lightning rod effect. However, our theoretical model also shows that a nanoegg-emitter system can rival the PEF parameters obtained in a nanorod-emitter system, due to an increase in the Purcell factor of the emitter with increasing core-offset of the nanoegg, resulting from the presence of dipole-active modes in the nanoegg. / Thesis (PhD (Physics))--University of Pretoria, 2020. / University of Pretoria / National Research Foundation (NRF) / Physics / PhD (Physics) / Unrestricted

Orientador: Valdecir Faria Ximenes / Banca: Rodrigo Cardoso de Oliveira / Banca: Marinonio Lopes Cornelio / Banca: Paulo Noronha Lisboa Filho / Banca: Aguinaldo Robinson de Souza / Resumo: A proteína presente no plasma sanguíneo e conhecida como albumina humana possui inúmeras funções fisiológicas e propriedades como o transporte de fármacos e metabólitos na corrente sanguínea. Neste sentido, quando se estuda as características farmacocinéticas de um novo fármaco, entre as propriedades estudadas, busca-se elucidar a capacidade de ligação do mesmo na albumina e caracterizar o sítio de ligação da mesma. Neste projeto, estudamos as características espectroscópicas e biofísicas do corante vermelho Congo (VC) como potencial sonda para caracterização dos sítios de ligação da albumina e caracterização de agregados amiloidais. Os ensaios de supressão de fluorescência e dicroísmo circular induzido (ICD) revelaram uma forte associação entre VC e a albumina de soro bovina (BSA). Ensaios de deslocamento de sondas fluorescentes e alteração do espectro de ICD revelaram que o corante VC pode se ligar nos sitios I e II da BSA. Agregados proteicos com características de fibrilas amiloidais foram preparados por aquecimento da BSA a 70°C e caracterizados por fluorescência de tioflavina-T e espalhamento de luz Rayleigh. Observamos um deslocamento no sinal de ICD do VC ligado aos agregados. A monitoração do sinal de ICD em função do aumento de temperatura revelou uma típica curva de alteração de fase da proteína. Considerando que, quando presente, um sinal de ICD pode ser bastante específico e não sujeito às influências espectrais dos compostos presentes, propomos que esta técnica ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The protein present in blood plasma and known as human albumin has countless physiological functions, including transporting drugs and metabolites in the bloodstream. In this regard, when studying pharmacokinetic characteristics of a new drug, between the properties studied, it is common to elucidate its binding capacity in albumin and to characterize the binding site of the drug. In this project, we studied the spectroscopic and biophysical characteristics of Congo Red (CR) dye as a potential probe to characterize the albumin binding sites and amyloid aggregates. The fluorescence quenching tests and induced circular dichroism (ICD) showed a strong association between CR and bovine serum albumin (BSA). Displacement tests of fluorescent probes and change in the ICD spectrum revealed that the CR dye may bind to sites I and II of BSA. Protein aggregates with features of amyloid fibrils were prepared by heating the BSA at 70 °C and characterized by thioflavinT fluorescence and Rayleigh light scattering. We observed a shift in the ICD signal CR connected to the aggregates. When monitoring the ICD signal as a function of temperature, a typical phase change curve was obtained. Considering that, when present, an ICD can be a quite specific signal and not subject to spectral influences of the compounds, we proposed that this spectroscopic technique could be used to aid in the elucidation of the binding sites of the albumin and for the studies of the formation of amyloid aggregates in ... (Complete abstract click electronic access below) / Doutor