The synthesis and characterization of simple mimics of pore forming antibiotics such as amphotericin B were explored. A sub-unit approach to the synthesis was employed which allowed for construction of a set of candidate structures. The targets are assembled by joining two "wall" units via a "linkage" unit with subsequent addition of polar head groups to either end of the structure. The wall units are macrocyclic diene tetraesters derived from maleic anhydride prepared by either acid catalyzed ester formation from diols or carboxylate substitution of dihalides (compounds 14, 15, 22, 23, 24, 30, 31, 34). Either set of reaction conditions limit the range of functionality possible in the starting diol or dihalide. Macrocycles 22, 23, and 24 were linked with m-xylylene dithiol via a 2:1 Michael addition reaction to give bis-macrocyclic alkene precursors. Alternatively, macrocycles 22 and 23 reacted with 3-thio-1-propanol and the mono-alcohol products were converted to iodides which were linked with 2R,3R-(+)-tartaric acid. Three types of polar head groups--neutral (1-thio-β-D-glucose and 3-thio-1-propanol), cationic (2-aminoethanethiol), and anionic (2-thioacetic acid)--were added to the bis-macrocyclic alkene precursors via Michael addition reactions. A total of fourteen candidate structures were prepared for transport evaluation. The activity of the fourteen mimics synthesized were determined by the pH-stat technique in which the transport of alkali metal cations across large unilamellar lipid bilayer vesicles were monitored by the collapse of a proton gradient. All the active compounds showed a zero order decay in proton gradient. Of the fourteen mimics surveyed, three had activities comparable to amphotericin B (compounds 51, 52, and 59). The other eleven compounds were not sufficiently active for further characterization. The "add back" experiments, the kinetic orders, and the alkali metal ion selectivity studies are consistent with the proposal that the mimics behave as pore formers. / Graduate

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The topic of this thesis is electrophysiological characterization of Kir2.1 membrane channel. Inward rectifier potassium channel Kir2.1 is located in muscular, heart and nerve cells and its dysfunction causes various diseases. Practical part of this stage is focused on cultivation of the HEK293T cell line that is used to transfection of the plasmid Kir2.1 and subsequent measurement of the ionic current through the electrophysiological method patch-clamp in whole-cell mode.

Neuroinflammation is prevalent in neurodegenerative diseases and plays a significant role in the central nervous system (CNS) innate immunity, which is the body’s first line of defense mechanisms against invading pathogens and injuries to maintain homeostasis. However, in neurodegenerative diseases, neuroinflammation becomes persistent alongside the subsequent damage to nearby neurons and affects CNS-resident immune glial cells, such as microglia and astrocytes. Accumulating evidence suggests that neuroinflammation is mainly characterized by the excessive activation of glial cells, thus causing abnormal changes in their microenvironment and release soluble factors that can promote or inhibit neuroinflammation. Currently, there is no effective treatment to cure these progressive neurological disorders. Therefore, it is critical to understand how neuroinflammation affects astroglia cell function and their biomechanical properties that change their behavior throughout disease progression. Astrocytes are the most predominant glial cell in the CNS and are critical in the development and maintenance of neuroinflammatory disorders. To date, very little is known regarding the role and specific function of Piezo1 mechanosensitive ion channel (MSC) in the CNS. Recently, Piezo1 expression was found to be upregulated in Lipopolysaccharide (LPS)-induced neuroinflammation in mouse astrocyte cultures. However, it is unknown whether the aberrant mechanical environment in astrocytes interplay with the mechanosensory function of Piezo1 and its current activity in neuroinflammatory conditions. In this study, we investigated Piezo1 mechanosensitive ionic currents by performing in vitro patch-clamp electrophysiology and calcium imaging. Our preliminary studies revealed that astrocytes derived from the mouse cerebellum stimulated with LPS or Piezo1 agonist, Yoda1, increased Ca2+ influx and further augmented when treated concurrently. We also found that electrophysiology recordings showed changes in mechanosensitive ionic currents and were comparable with our calcium imaging data indicating that MSCs are involved in neuroinflammation. Therefore, we postulated that Piezo1, a non-selective cation MSC that opens in response to mechanical force is a key mechanosensor involved in neuroinflammation by altered mechanical signals in C8-S astrocytes. Using an in vitro system of Mouse C8-S (Astrocyte type II clone), the goal of this study was to investigate if neuroinflammatory conditions upregulate Piezo1 calcium influx and current activity. We show that astrocytic Piezo1 regulates mechanotransducive release of ATP by controlling the mechanically induced calcium influx and current activation in LPS-induced astrocytes. Additionally, Piezo1 antagonist, GsMTx4 and Piezo1 siRNA significantly reduced the LPS-induced current, indicating that Piezo1 is involved in neuroinflammation. Our findings demonstrate that the activity of Piezo1 stimulated by neuroinflammatory conditions may be significant for the development of therapeutics to prevent or treat neuroinflammatory disorders and diseases.

Ca2+ is a ubiquitous and dynamic second messenger molecule that is induced by many factors including receptor activation, environmental factors, and voltage, leading to pleiotropic effects on cell function including changes in migration, metabolism and transcription. As such, it is not surprising that aberrant regulation of Ca2+ signals can lead to pathological phenotypes, including cancer progression. However, given the highly context-specific nature of Ca2+-dependent changes in cell function, delineation of its role in cancer has been a challenge. Hence, the role of store-operated Ca2+ entry (SOCE) in melanoma metastasis is still not fully elucidated. To address this, we examined UV-dependent metastasis, revealing a critical role for SOCE suppression. As previous literature demonstrated a role for cholesterol (CHL) in melanoma progression, our investigations corroborate this revealing UV-induced CHL biosynthesis as a critical mediator for UV-induced SOCE suppression and subsequent metastasis. However, SOCE suppression alone was both necessary and sufficient for metastasis to occur. Further, SOCE suppression facilitated UV-dependent differences in gene expression associated with increased invasion through altered glucose utilization. Functional analyses further establish that increased glucose uptake leads to a metabolic shift towards biosynthetic pathways critical for melanoma metastasis. Finally, examination of fresh surgically isolated human melanoma explants revealed CHL dependent low SOCE. Invasiveness could be reversed with either CHL biosynthesis inhibitors, pharmacological inhibition of terminal glycosylation enzyme, OGT, or pharmacological SOCE potentiation. In parallel, we demonstrate that Geranylgeranylpyrophosphate (GGPP) can function as a novel SOCE inhibitor either by saturation or prevention of transfer to membrane proteins; both of which lead to GGPP accumulation in the cytosol. Collectively, we provide evidence that Ca2+ signals can block invasive behavior, and suppression of these signals promotes invasion and metastasis. / Biomedical Sciences

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