This thesis deals with the problematice of measuring membrane potential and monitoring the propagation of electrical activity of cells. For this purpose, fluorescence membrane voltage sensors have been developed to detect changes in the membrane potential by changing their fluorescence intensity. The practical part is focused on the study of the properties of the ASAP1 fluorescence probe, which was transfected into the HEK293 cell line, which are kidney cells from the human embryo. Cell membrane potential was changed using the patch-clamp technique.

Nuclear hormone receptors (NHR) are important transcription factors that regulate development and metabolism in the large group of animals. Caenorhabditis elegans contains 284 nuclear receptors, which is unusually large amount compared to receptors of Drosophila melanogaster (18) and humans (48). 15 receptors of the C. elegans have homologous receptor structure with receptors of D. melanogaster and mammals. The remaining 269 NHR are specific to nematodes and belong to the group of supplementary nuclear receptors (SupNRs), the evolutionary precursor of the HNF4 - an important transcription factor in humans. In this work we describe the nuclear hormone receptor nhr-97 C. elegans, whose expression and function have not yet been studied. The gene is encoded in the genome of C. elegans and is among SupNRs. Nhr-97 consists of two isoforms A and B, whose expression in C. elegans tissues is different. Localization of gene expression in vivo was determined using lines expressing nhr-97:: GFP. For the A isoform expression of nhr-97::GFP was localized in neurons in the pharynx and the tail, in the intestine and hypodermis, in isoform B in the pharynx, in neurons around the corpus of pharynx, the head mesodermal cell and in anal sphincter. Nhr-97 expression during development of C. elegans was determined by...

Formins are proteins facilitating formation of actin filaments. They affect structure of cytoskeleton and participate in cytokinesis and tip growth. There are 2 classes of formins in Arabidopsis thaliana, which include FH1 and FH2 (Formin Homology 1 and 2) domain. Formins of the class I have usually a transmembrane domain on N-terminus. Due to this fact they can interact with membranes. Some formins from the class II include PTEN domain (Phosphatase and Tensin Homolog) derived from sequences of PTEN proteins which has lost the function of phosphatase. It is assumed this domain can bind on a membrane via the phosphatase section or C2 domain. This thesis was focused on the formin AtFH13 from the class II in Arabidopsis thaliana and on its PTEN domain. There were analyzed differences between mutants and wild-types in length of roots in seedlings and in size of seeds and seed coats, and observed the effect of dexamethasone on the length of roots on AtFH13. PTEN domain of the formin was isolated from cDNA, cloned to a vector and fused with YFP. The tagged protein was visualized by the method of transient expression in epidermal cells in the leaves of Nicotiana benthamiana. No big differences were observed between plants mutant in the gene AtFH13 and wild-type in choice parameters. Dexamethasone did't influence...

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Mitochondria are dynamic organelles that function in a complex interconnected network within the cell. Neurons are sensitive and highly energy demanding cells in the brain which require a functioning mitochondrial network that is able to provide ATP and modulate calcium. Mitochondrial networks have yet to be explored which gives rise to the need for specific and efficient molecular tools. In this project, I designed and produced adeno-associated virus vectors carrying a fluorescent reporter gene for imaging mitochondrial networks under human synapsin 1 promoter to target neurons specifically. The design of each vector was conducted with careful consideration of the different components in the plasmid design that are important for optimal expression, which resulted in two constructs; one self-complementary adeno-associated virus vector that marks the mitochondria and one single-stranded that marks mitochondria and the membrane of neurons.  The modularity of viral vectors allows the usage of different serotypes which adapt the vector to the cell type and the model. For this project I chose the serotypes 1 for neurons in vitro and PHP.eB which suits in vivo models since it has better permeability to the blood brain barrier. The production was conducted in human embryonic kidney cells using the triple-plasmid transfection method, followed by extraction and purification. The existence of viral particles was verified through transmission electron microscopy and the DNA titer of the vector through quantitative polymerase chain reaction. The produced adeno-associated virus vectors were delivered into young brain organoids which were not able to express the reporter gene, probably due to not fully developed neurons. The fluorescent protein expression targeting specifically mitochondria and the membrane was however verified in the human embryonic kidney cells during the packaging stages.

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Recombinant DNA technology has been commonly used in a number of fields to synthesize new products or generate products with a new pathway. Conventional cloning methods are expensive and require significant time and labor; λ-PCR, a new cloning method developed in the Senger lab, has a number of advantages compared to other cloning processes due to its employment of relatively inexpensive and widely available materials and time-efficiency. While the amount of lab work required for the cloning process is minimal, the importance of accurate primer design cannot be overstated. The target of this study was to create an effective procedure for λ-PCR primer design that ensures accurate cloning reactions. Additionally, synthetic ribosome binding sites (RBS) were included in the primer designs to test heterologous protein expression of the cyan fluorescent reporter with different RBS strengths. These RBS sequences were designed with an online tool, the RBS Calculator. A chimeric primer design procedure for λ-PCR was developed and shown to effectively create primers used for accurate cloning with λ-PCR; this method was used to design primers for CFP cloning in addition to two enzymes cloned in the Senger lab. A total of five strains of BL21(DE3) with pET28a + CFP were constructed, each with the same cyan fluorescent protein (CFP) reporter but different RBS sequences located directly upstream of the start codon of the CFP gene. Expression of the protein was measured using both whole-cell and cell-free systems to determine which system yields higher protein concentrations. A number of other factors were tested to optimize conditions for high protein expression, including: induction time, IPTG concentration, temperature, and media (for the cell-free experiments only). Additionally, expression for each synthetic RBS sequence was investigated to determine an accurate method for predicting protein translation. NUPACK and the Salis Lab RBS Calculator were both used to evaluate the effects of these different synthetic RBS sequences. The results of the plate reader experiments with the 5 CFP strains revealed a number of factors to be statistically significant when predicting protein expression, including: IPTG concentration, induction time, and in the cell-free experiments, type of media. The whole-cell system consistently produced higher amounts of protein than the cell-free system. Lastly, contrasts between the CFP strains showed each strain's performance did not match the predictions from the RBS Calculator. Consequently, a new method for improving protein expression with synthetic RBS sequences was developed using relationships between Gibbs free energy of the RBS-rRNA complex and expression levels obtained through experimentation. Additionally, secondary structure present at the RBS in the mRNA transcript was modeled with strain expression since these structures cause deviations in the relationship between Gibbs free energy of the mRNA-rRNA complex and CFP expression. / Master of Science / Recombinant DNA technology has been used to genetically enhance organisms to produce greater amounts of a product already made by the organism or to make an organism synthesize a new product. Genes are commonly modified in organisms using cloning practices which typically involves inserting a target gene into a plasmid and transforming the plasmid into the organism of interest. A new cloning process developed in the Senger lab, λ-PCR, improves the cloning process compared to other methods due to its use of relatively inexpensive materials and high efficiency. A primary goal of this study was to develop a procedure for λ-PCR primer design that allows for accurate use of the cloning method. Additionally, this study investigated the use of synthetic ribosome binding sites to control and improve expression of proteins cloned into an organism. Ribosome binding sites are sequences located upstream of the gene that increase the molecule’s affinity for the rRNA sequence on the ribosome, bind to the ribosome just upstream of the beginning of the gene, and initiate expression of the gene. Tools have been developed that create synthetic ribosome binding sites designed to produce specific amounts of protein. For example, the tools can increase or decrease expression of a gene depending on the application. These tools, the Salis Lab RBS Calculator and NUPACK, were used to design and evaluate the effects of the synthetic ribosome binding sites. Additionally, a new method was created to design synthetic ribosome binding sites since the methods used during the design process yielded inaccuracies. Each strain of E. coli contained the same gene, a cyan fluorescent protein (CFP), but had different RBS sequences located upstream of the gene. Expression of CFP was controlled via induction, meaning the addition of a particular molecule, IPTG in this system, triggered expression of CFP. Each of the CFP strains were tested with a variety of v conditions in order to find the conditions most suitable for protein expression; the variables tested include: induction time, IPTG (inducer) concentration, and temperature. Media was also tested for the cell-free systems, meaning the strains were grown overnight for 18 hours and lysed, a process where the cell membrane is broken in order to utilize the cell’s components for protein expression; the cell lysate was resuspended in new media for the experiments. ANOVA and multiple linear regression revealed IPTG concentration, induction time, and media to be significant factors impacting protein expression. This analysis also showed each CFP strain did not perform as the RBS Calculator predicted. Modeling each strain’s CFP expression using the RBS-rRNA binding strengths and secondary structures present in the RBS allowed for the creation of a new model for predicting and designing RBS sequences.

There are two kinds of protein aggregates associated with Alzheimer’s disease: amyloid-beta and tau aggregates. Protein ligands are molecules with the ability to bind to these pathologicalprotein accumulations, and if the ligands are fluorescent, they can be used to detect the aggregates they’re bound to. The ligands can be selective and only bind to one kind of protein aggregate, or they can be general and bind to both kinds. PI-2620 is a ligand selective for tau aggregates, and in this thesis, three analogues of PI-2620 were synthesized and determined to be functional fluorescent protein ligands. Two of them, designated A1 and B1, were selective for amyloid-beta aggregates, while the third ligand, A2, obtained by ester-hydrolysis of A1, would bind to both tau and amyloid-beta aggregates. This finding suggests that introducing a charge to a ligand lowers its selectivity, since the staining experiments were carried out in a buffered solution at pH 7.4, where ligand A2 might be partially charged.

Techniques for conditional gene or protein expression are important tools in developmental biology and in the analysis of physiology and disease. On the protein level, the tunable and reversible expression of proteins can be achieved by the fusion of the protein of interest to a destabilizing domain (DD). In the absence of its specific ligand (Shield-1), the protein is degraded by the proteasome. The DD-Shield system has proven to be an excellent tool to regulate the expression of proteins of interests in mammalian systems but has not been applied in teleosts like the medaka. We present the application of the DD-Shield technique in transgenic medaka and show the ubiquitous conditional expression throughout life. Shield-1 administration to the water leads to concentration-dependent induction of a YFP reporter gene in various organs and in spermatogonia at the cellular level.

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