This diploma thesis deals with study of the rate-dependence of the magnitude of a current through the heart channel that conducts slowly activating component of delayed rectifier outward current (IKs). This property is very important for the IKs channel function. When other repolarizing currents are insufficient, but also when the heart rate accelerates, especially during elevated sympathetic tone, IKs provides so-called repolarization reserve, which prevents excessive lengthening of cardiac action potential repolarization. The IKs channel structure is encoded by the KCNQ1 (pore-forming -subunit) and KCNE1 (modulatory -subunit) genes. Mutations in these genes disrupt the physiological function of the IKs channel and cause inherited arrhythmogenic syndromes, especially long QT syndrome (LQTS). Such mutations include the c.926C>T (p.T309I) mutation in the KCNQ1 gene, which results in LQTS type 1 in heterozygous carriers. The theoretical part of the thesis provides basic information about the IKs channel and the patch clamp technique, this knowledge is necessary for the practical part. The experimental part is focused on cultivation of the CHO cell line and its transient transfection for subsequent electrophysiological measurements by whole-cell patch clamp technique to study the dependence of the IKs magnitude on stimulation frequency, both in the wild type channels (i.e. without mutation) and in those with cotransfected wild type and T309I subunits.

The master thesis deals with mechanical design of device used for stretching and fixing filtration fabric on plastics frames segments for disc filtration device. The thesis was solved in cooperation with IN-EKO Team company that deals with development and manufacturing of filter units. Solution is connected to already ended project FR-T13/699 (end of the year 2013). At the beginning is described the field of filtration devices in broader context with description of the overall functionality these devices and description of used elements in final design. Opening text is followed by concretization of task list and descriptions of concepts variants which are based on FEM analysis of behavior of stretched filtration fabric. The best concept is processed in detail as mechanical design along with pneumatic and PLC control design. Critical part of the design is stretching clamp which was experimentally verified by using experimental clamp in combination with testing machine. The whole design is developed for mass production of disc segments for disc filter which is projected in to the level of the automation.

In the beginning of this thesis is a short introduction into plasmid DNA which is in the form of a vector used in molecular biology. Plasmids can be used in the form of fluorescent probes to measure changes in membrane potential. Into their structure is added a dye called fluorophore. As an important representative of this thesis is a fluorescent probe ASAP1 which contains green fluorescent protein whose response to the membrane potential change is the decrease in the intensity of emitted light. The aim of this thesis was to make chemical transfection of this plasmid into the HEK293 cell line and carry out its characterization. In the work is also described the design of a method for the analysis of the time course of changes in fluorescence depending on the cell membrane depolarisation. In the end of this thesis is also desribed realized experiment including the discussion of aquired results.

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.

Astrocytes need to preserve constant volume in the face of osmolarity perturbations to function properly. To regain their original volume after hyposmotically induced swelling, they extrude intracellular electrolytes and organic osmolytes, such as inorganic ions, excitative amino acids or polyols, accompanied by osmotically driven water. This process is termed regulatory volume decrease and is ensured by various ion channels and transporters. Recently, much attention has been focused on the ubiquitous volume-regulated anion channels activated by cell swelling. VRACs are moderately outwardly rectifying with intermediary conductance, permeable to inorganic anions and organic osmolytes and sensitive to broad-spectrum anion channels blockers. Using patch-clamp technique we aimed to characterize VRACs in cultured cortical astrocytes isolated from neonatal Wistar rats and to elucidate the effect of intracellular Na+ on VRAC activity. In addition, we also intended to characterize these channels in situ in brain slices of 10 - 12 days old rats, focusing mainly on hippocampal astrocytes. To induce astrocytic swelling, we exposed astrocytes to hypotonic solution (250 mOsm). In agreement with previous findings, we showed that cultured cortical astrocytes activate VRAC currents upon exposure to hypotonic stress, which...

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder of the central nervous system characterized by loss of motor neurons and voluntary muscle degeneration. Astrocytes play a major role in regulation of the disease onset and progression due to their intimate association with neurons. Regulation of ionic homeostasis is one of their key functions and its failure has been linked to several neurological diseases. The aim of this thesis was to explore differences in membrane properties of astrocytes in ALS. To fulfill this aim, a double transgenic mouse strain with ALS-like phenotype and a specific expression of enhanced green fluorescent protein in astrocytes was generated. To phenotype this strain, two sensorimotor tests, wire grid hang test and rotarod test, were conducted. Immunohistochemistry was used to characterize the strain on a cellular level and to explore changes of specific ion channels. Functional properties of astrocytes were explored using the patch clamp technique. The double transgenic strain has the characteristic ALS-like phenotype and is comparable to the original strain with differences in symptom onset and progression between models and sexes. On the cellular level, there are characteristic ALS features, specifically loss of motor neurons and astrogliosis....

Increases in basolateral amygdala (BLA) activity drive avoidance-seeking behavior that may be associated with stress induced drug seeking. Activity of BLA pyramidal neurons is regulated by local and paracapsular gamma aminobutyric acid (GABA) interneurons. The lateral paracapsular interneurons (LPCs) border the external capsule, receive dense cortical/thalamic input and provide feed-forward inhibition onto BLA principle neurons. The GABAergic LPCs also express high concentrations of g-protein coupled µ-opioid receptors (MORs). Therefore, the effects of opiates on LPC activity and local GABA release were examined. Fluorescently double labeled LPCs were observed in glutamate decarboxylase (GAD) 65-mcherry/GAD67-green fluorescent protein (GFP) transgenic mice. Whole-cell electrophysiology experiments demonstrated that acute exposure to [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO; a synthetic selective MOR agonist), reduced LPC firing and spontaneous inhibitory postsynaptic current (sIPSC) frequency in LPCs, with no apparent effect on spontaneous excitatory currents (sEPSCs). Current injection induced firing in LPC neurons, but less effectively than in saline controls. Morphine-exposed mice (10mg/kg/day, across 5 days, 1-2 days off) had increased sIPSCs compared to saline-injected controls, as well as enhanced adenylyl cyclase (AC) activity. Together these data show that LPC neurons are a highly sensitive targets for opiate-induced inhibition, and that long-term opiate exposure results in impaired LPC excitability, possibly contributing to anxiety observed during opiate withdrawal.

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This thesis presents an active clamp forward converter for use in the Energy Harvesting From Exercise Machines project. Ideally, this converter will find use as the centerpiece in a process that links elliptical trainers to the California grid. This active clamp forward converter boasts a 14V-60V input voltage range and 150W power rating, which closely match the output voltage and power levels from the elliptical trainer. The isolated topology outputs 51V, higher than previous, non-isolated attempts, which allows the elliptical trainers to interact with a central grid-tied inverter instead of many small ones. The final converter operated at greater than 86% efficiency over most of the elliptical trainer’s input range, and produced very little noise, making it a solid choice for this implementation.

The obesity epidemic is an important global health concern. Obesity is associated with a number of diseases including type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), cardiovascular disease, and some cancers. Insulin resistance, a precursor to type 2 diabetes, is defined as an unresponsiveness of metabolic tissues to insulin, leading to long-term hyperglycemia and hyperinsulinemia. The fatty acid-induced model of insulin resistance indicates that an accumulation of lipid intermediates interferes with insulin signal transduction leading to insulin resistance. It is, therefore, important to examine means by which these lipid intermediates can be reduced to alleviate interferences in insulin signaling in the treatment of insulin resistance and type 2 diabetes. Exercise and metformin are two common interventions in patients with type 2 diabetes and obesity. They both commonly activate AMP-activated protein kinase (AMPK). AMPK contributes to a number of metabolic processes including increased glucose and fatty acid oxidation. However, the effects of AMPK activation on insulin sensitivity are currently not fully understood. This compilation of studies examined the insulin sensitizing effects of AMPK activation via metformin, exercise, and novel AMPK activator R419 in obese mice. In Chapter 2 we show that metformin increases AMPK phosphorylation of acetyl-CoA carboxylase (ACC) 1 Ser79 and ACC2 Ser212, resulting in increased fatty acid oxidation, decreased lipid content and improvements in hepatic insulin sensitivity. In Chapter 3 we show that exercise-induced improvements in insulin sensitivity occur independent of AMPK phosphorylation of ACC phosphorylation sites and independent of lipid content in the liver. Finally, in Chapter 4 we show that R419 improves skeletal muscle insulin sensitivity independent of AMPK and lipid content but improves exercise capacity via a skeletal muscle AMPK-dependent pathway in obese mice. These findings suggest that future studies examining the effects of AMPK activation in obesity will aid in our understanding of the mechanisms of insulin resistance and introduce methods of prevention and treatment of obesity and type 2 diabetes. / Thesis / Doctor of Philosophy (PhD)