|
|
Study of Biomolecular Interactions with Surface Plasmon Resonance Biosensors
Šípová, Hana ; Homola, Jiří (advisor) ; Houska, Milan (referee) ; Skládal, Petr (referee)
Surface plasmon resonance (SPR) biosensors represent one of the most advanced sensing technologies for real-time studies of biomolecular interactions. In this thesis, methods for functionalization of SPR substrates were optimized and studied via spectroscopic methods. Effects related to the SPR sensor microfluidic interface on the measured biomolecular interactions were analyzed, and furthermore, means to decrease mass-transport limitations were proposed. Several SPR-based assays regarding the detection of nucleic acids were developed, which allow for the detection of physiologically relevant concentrations of nucleic acids as well as point mutations in a nucleic acid sequence. Assays for the determination of the enzymatic activity of HIV integrase and ribonuclease H were developed. These assays can be employed for the design and synthesis of molecules that function either as antiviral drugs or as gene-regulating agents.
|
|
|
Study of biomolecular interactions by the method of surface plasmon resonance
Bocková, Markéta ; Homola, Jiří (advisor)
Title: Study of biomolecular interactions by the method of surface plasmon resonance Author: Markéta Bocková Department: Department of Biochemistry, Faculty of Science Supervisor: Prof. Jiří Homola, Ph.D., DSc., Institute of Photonics and Electronics of the Czech Academy of Sciences Abstract: In this diploma work, a surface plasmon resonance (SPR) biosensor technology was exploited to study two different classes of molecular interactions: hybridization of partially complementary oligonucleotides (ONs) and interactions between enzymes and peptides involved in pathogenesis of Alzheimer disease (AD). In particular, the study of interactions between ONs was focused on determining kinetic parameters of the hybridization proces. It was found that the association equilibrium constant decreases with the increasing surface probe density. An SPR biosensor-based method for rapid and sensitive detection of the 17-HSD10 enzyme and its complex with amyloid β (Aβ) peptides was designed and developed. It was demonstrated that the developed method is capable of detecting 17β-HSD10/Aβs complex in real cerebrospinal fluid (CSF) samples and that CSF samples from AD patients exhibited elavated levels of 17β- HSD10/Aβs complex compared to the control groups. The research reported in this thesis resulted in two publications in...
|
|
|
Multifunctional biomolecular assemblies for parallelized analysis of biomolecular interactions
Bocková, Markéta ; Homola, Jiří (advisor) ; Brynda, Eduard (referee) ; Procházka, Marek (referee)
Title: Multifunctional biomolecular assemblies for parallelized analysis of biomolecular interactions Author: Markéta Bocková Department / Institute: Institute of Physics, Charles University Supervisor of the doctoral thesis: Prof. Jiří Homola, Ph.D., DSc., Institute of Photonics and Electronics, The Czech Academy of Sciences Abstract: Surface plasmon resonance (SPR) biosensors represent the most advanced optical method for the direct, real-time monitoring of biomolecular interactions without the need for labelling. This doctoral thesis aims to advance the SPR biosensor method and to expand its utility in the investigation of biomolecular interactions. This encompasses activities on two major fronts of SPR biosensor research - immobilization methods and biosensing methodologies. Methods for the immobilization of biomolecules were researched with the aim of enabling the immobilization of a broad range of biomolecules on the SPR biosensor surface in a spatially controlled manner. The development of novel biosensing methodologies was pursued in order to address the current limitations of SPR biosensors associated with non-specific adsorption and limited analyte transport, and thus to improve the accuracy and robustness of SPR biosensor measurements. Finally, advances in the development of immobilization...
|
|
|
Analysis of abiotic stress induced genes in rape
HOŠTIČKOVÁ, Irena
Breeding for abiotic stress tolerance is one of main topics in plant breeding. Oilseed rape breeding programs were for a long time focused on morphological and physiological parameters. In this thesis few experiments focused on identification of genes involved in abiotic stress reaction were performed using RT-qPCR (quantitative reverse transcription PCR). Simultaneously SPR (surface plasmon resonance) method were used as modern optical method facilitating very low native protein concentration even in presence of other substances. This method facilitates quantification of concrete proteins by binding them to specific antigen and in oilseed rape research it was not used by now. ERD10 protein was identified by SPR as protein involved in cold stress reaction (or acclimation). The results show ERD10 accumulation in standard conditions affects dynamics of its accumulation change during cold stress. In case we are searching for genotypes great in acclimation ability even during short and warm autumn SPR method should be suitable method for fast, easy and relatively cheap screening of large number of genotypes in breeding collections. Also genes LTI78, RCI2A, NRP1 and two genes for hypothetical proteins were analysed. Their relative expression during cold stress was markedly increased too. Very little is known about these genes and proteins nowadays therefor it will be interesting topic of our oncoming experiment. Relative expression of genes picked according to MALDI-TOF/TOF analysis results was also tested in microspore embryo regenerants stressed by simulated drought. Genes for lactoylglutathione lyase I, phospholipase D 1 and peroxiredoxin antioxidase were tested. In tolerant cultivar was markedly decreased gene expression of peroxiredoxin antioxidase in standard conditions and early stress. These gene will be subject for next research as potential marker for more tolerant genotypes selection.
|
|
|
Study of biomolecular interactions by the method of surface plasmon resonance
Bocková, Markéta ; Homola, Jiří (advisor)
Title: Study of biomolecular interactions by the method of surface plasmon resonance Author: Markéta Bocková Department: Department of Biochemistry, Faculty of Science Supervisor: Prof. Jiří Homola, Ph.D., DSc., Institute of Photonics and Electronics of the Czech Academy of Sciences Abstract: In this diploma work, a surface plasmon resonance (SPR) biosensor technology was exploited to study two different classes of molecular interactions: hybridization of partially complementary oligonucleotides (ONs) and interactions between enzymes and peptides involved in pathogenesis of Alzheimer disease (AD). In particular, the study of interactions between ONs was focused on determining kinetic parameters of the hybridization proces. It was found that the association equilibrium constant decreases with the increasing surface probe density. An SPR biosensor-based method for rapid and sensitive detection of the 17-HSD10 enzyme and its complex with amyloid β (Aβ) peptides was designed and developed. It was demonstrated that the developed method is capable of detecting 17β-HSD10/Aβs complex in real cerebrospinal fluid (CSF) samples and that CSF samples from AD patients exhibited elavated levels of 17β- HSD10/Aβs complex compared to the control groups. The research reported in this thesis resulted in two publications in...
|
|
|
Study of Biomolecular Interactions with Surface Plasmon Resonance Biosensors
Šípová, Hana ; Homola, Jiří (advisor) ; Houska, Milan (referee) ; Skládal, Petr (referee)
Surface plasmon resonance (SPR) biosensors represent one of the most advanced sensing technologies for real-time studies of biomolecular interactions. In this thesis, methods for functionalization of SPR substrates were optimized and studied via spectroscopic methods. Effects related to the SPR sensor microfluidic interface on the measured biomolecular interactions were analyzed, and furthermore, means to decrease mass-transport limitations were proposed. Several SPR-based assays regarding the detection of nucleic acids were developed, which allow for the detection of physiologically relevant concentrations of nucleic acids as well as point mutations in a nucleic acid sequence. Assays for the determination of the enzymatic activity of HIV integrase and ribonuclease H were developed. These assays can be employed for the design and synthesis of molecules that function either as antiviral drugs or as gene-regulating agents.
|
|
| |
|
|
Structural analysis of extrinsic proteins from the oxygen-evolving complex of photosystem II from higher plants
KOHOUTOVÁ, Jaroslava
All life on earth depends mainly on the presence of oxygen. Largest producers of oxygen are green plants, cyanobacteria and algae. Oxygen is released from the oxygenevolving complex of photosystem II during photosynthesis and it is used in cellular respiration of all life complexes. The oxygen-evolving complex of photosystem II has the same function in each photosynthetic organism, but it has a different composition and organization of extrinsic proteins; only PsbO protein is ubiquitous in all known oxyphototrophs. Until now only low resolution electron microscopy structural models of plant PSII and crystal structures of cyanobacterial PSII are available. Higher plant extrinsic proteins (PsbP, PsbQ and PsbR) are structurally unrelated, non-homologues to the cyanobacterial extrinsic proteins (PsbO, PsbU and PsbV) and this is the reason why it is not possible to predict arrangement of these proteins on the lumenal site of higher plant PSII. Recently, models differ mainly in the structure of the oxygen-evolving complex, which could be resolved by determination of the exact binding sites for extrinsic proteins. An other question evolves: if the difference in the oxygen-evolving complex composition is the result of evolution or adaptation of photosynthetic organisms to their environment. Structural knowledge of extrinsic proteins that could help to resolve the location and subsequently the function of extrinsic proteins is still incomplete. From this case,structural analysis, interactions and probably arrangement of proteins PsbP and PsbQ was studied and is described in detail in this thesis.
|
guest ::
