Diseases such as epilepsy, pain, and neurodegenerative disorders are associated with changes in neuronal dysfunction due to an imbalance of excitation and inhibition. This work details a novel electroconvulsive seizure assay for C. elegans using the well characterized cholinergic and GABAergic excitation and inhibition of the body wall muscles and the resulting locomotion patterns to better understand neuronal excitability. The time to recover normal locomotion from an electroconvulsive seizure could be modulated by increasing and decreasing inhibition. GABAergic deficits and a chemical proconvulsant resulted in an increased recovery time while anti-epileptic drugs decreased seizure duration. Successful modulation of excitation and inhibition in the new assay led to the investigation of a cGMP-dependent protein kinase (PKG) which modulates potassium (K+) channels, affecting neuronal excitability, and determined that increasing PKG activity decreases the time to recovery from an electroconvulsive seizure. The new assay was used as a forward genetic screening tool using C. elegans and several potential genes that affect seizure susceptibility were found to take longer to recover from a seizure. A naturally occurring polymorphism for PKG in D. melanogaster confirmed that both genetic and pharmacological manipulation of PKG influences seizure duration. PKG has been implicated in stress tolerance, which can be affected by changes in neuronal excitability associated with aging, so stress tolerance and locomotor behavior in senescent flies was investigated. For the first time, PKG has been implicated in aging phenotypes with high levels of PKG resulting in reduced locomotion and lifespan in senescent flies. The results suggest a potential new role for PKG in seizure susceptibility and aging. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

The use of advanced functional polymer materials has gained an enormous impact during the past decades. Due to the fact that the physical properties of macromolecules are inherently dependent on their structure and connectivity on the nanoscale, precisely control over polymer architecture has been a longstanding goal for polymer chemists. The recent development of copper catalyzed azide-alkyne click chemistry provides a nearly quantitatively tool for macromolecular coupling. Through the combination of living polymerization and click chemistry, novel complex polymer architectures can be readily constructed, including star polymers, brush polymers, cyclic polymer and ladder polymers. While amphiphilic block copolymers have demonstrated their utility for a range of practical applications, the behavior of block copolymers that contain cyclic topologies remains largely unexplored due to limited synthetic access. In order to investigate their micelle formation, biocompatible cyclic amphiphilic poly(ethylene glycol)-block-polycaprolactone, c-(PEG-b-PCL), and tadpole shaped PEG-PCL, were synthesized by a combination of ring opening polymerization (ROP) and click chemistry. In addition, exactly analogous linear block copolymers have been prepared as control samples to elucidate the role of polymer architecture in their self-assembly and acid-catalyzed degradation. High purity homo-arm and mikto-arm poly(ethylene glycol) (PEG) stars were successfully prepared by the combination of epoxide ring openings and azide-alkyne click reactions. First, monohydroxy-PEG was modified via epoxide chemistry to bear one hydroxyl and one azide functionality at the same polymer chain end. An alkyne functionalized PEG chain was then coupled to the azide. Subsequently, the remaining hydroxyl could be reactivated by epoxide chemistry again to an azide and alcohol group. This enabled a step-wise coupling and reactivation of the end group to add a series of well-defined polymer arms onto a star polymer. The use of efficient reactions for this iterative route provided star polymers with an exact number of arms, and a tailorable degree of polymerization for each arm. Detailed characterization confirmed the high purity of multi-arm polyethylene glycol products. Novel cyclic brush-shaped polymers can be successfully prepared by using the CuAAC click coupling reaction. First, cyclic-shaped polymer bearing a single hydroxyl group can be synthesized by CuAAC click cyclization. After a one-step modification of the hydroxyl group by esterification with an azido-carboxylic acid, a “clickable†polymer ring was obtained. A linear polymer backbone with an alkyne functional group on every repeat unit was prepared by ATRP of acetoxystyrene followed by reduction to poly(4-hydroxystyrene) and esterification with pentynoic acid. Finally, by coupling multiple equivalents of the cyclic precursor onto the linear backbone, a cyclic brush-shaped polymer was prepared. This provides a highly efficient approach to prepare novel polymer architectures containing multiple cyclic components. / acase@tulane.edu

Philosophiae Doctor - PhD / Nitric oxide (NO) plays a key role as biological messenger in the biological system, however detection and quantification thereof has always posed significant problems. NOx is a principal constituent of air pollutants. There are seven oxides of nitrogen of which N2O, NO and NO2 are most important. NO is a free radical and reacts extremely fast with oxygen, peroxides and superoxides. It’s these reactions which are responsible for NO’s fleeting existence. The specific detection and quantification of NO still remains challenging. Most techniques rely on the measurements of secondary nitrite and nitrate species. Electrochemical techniques using ultra micro-electrode systems presented the possibility of direct detection of NO, offering a range of favourable characteristics; good selectivity towards NO, good sensitivity, fast response, long-term stability and ease of handling. Electrochemical detection of NO relies on the modification of electrode surfaces and exploiting the redox properties of NO. NO can either be oxidized or reduced electrochemically depending on the nature of the solution. Under cathodic current NO is reduced to nitrosyl, a highly unstable derivative of NO. These nitrosyls are subject to a serious of chemical reactions to eventually form nitrous oxide. Due to the interferences presented by the electrochemical reduction of NO, the electro oxidation of NO is therefore the methodology of choice for NO detection. The electrochemical oxidation of NO occurs at positive potentials around 800 mV vs. Ag/AgCl. However this potential range is not only favourable to NO oxidation but can lead to the oxidation of several other biological species. These interfering species are biologically present at concentrations higher than NO therefore selectivity is of the highest order when designing these electrode systems. Some nitric oxide sensors are limited in their sensitivity, stability and reproducibility. Direct electron transfer between redox proteins and conductive membrane layers has been scrutinized for years in an attempt to reproduce the mechanistic charge transfer processes for sensor application. However, literature reports have presented many arguments on the complexities associated with depositing these enzymes on electrode surfaces for the purpose of reproducing direct electron transfer at metalloprotein centres. The study sets out to design a material which could mimic the electrochemistry of denatured cytochrome-c. To achieve this it was imperative to design a polymer which could reproduce the electrochemistry of the ligands coordinated to the metal centre of the metalloprotein. A novel Schiff base was synthesized by cross-linking naphthalene to pyrrole to produce the monomer, N,N-bis((1H-pyrrol-2-yl)methylene)naphthalene-2,3-diamine). The monomer was electrodeposited on a screen print carbon electrode (SPCE) vs. Ag/AgCl and served as a supporting layer for denatured cytochrome-c. Cytochrome-c is classified as a metalloprotein. These metalloproteins possess metal centres which when denatured unfolds and allows access to the metal centre. Cytochrome-c was subjected to thermal denaturation which opened up the iron centre. The denatured metalloprotein was cross-linked to the ligand to reconstruct the heme centre environment. This was believed to facilitate the electrochemical activity of the system and allow for electrochemical analysis of these metalloproteins for sensor application. The redox behaviour of the sensors were modelled in phosphate buffer solution (PBS) with cyclic voltammetry. Electrochemical analysis reported the sensors to possess reversible electrochemistry with diffusion control characteristics. The sensor recorded a redox system in the negative potentials range. Following the establishment of the electrochemical profile of the sensor an attempt was made to produce a synthetic analogue of denatured cytochrome-c. Iron (II) was chelated to the monomer N,N-bis((1H-pyrrol-2-yl)methylene)naphthalene-2,3-diamine) to form an iron ligand complex. The complex was subjected to a series of characterization techniques which confirmed coordination to the metal centre. The iron ligand complex was electrodeposited on a SPCE over the potential window of -1 V and 1 V to model the electrochemical behaviour of the sensor. The material was found to be electroactive. Subsequent electrochemical analysis revealed the system to have electrochemical properties, analogous to that of the denatured cytochrome-c system. The sensor was applied in NO and NO2 studies and displayed an affinity towards NO. Based on extrapolated values it was postulated that the lower limit range for NO detection was in the range of 30 to 40 nM. The potentials recorded were lower than the reported oxidation potentials for nitric oxide. The sensor displayed stability and selectivity towards nitric oxide within a complex matrix. The complex matrix employed in this study was synthetic urine that was synthesised in the lab. The sensor displayed the capacity for linear range of NO detection with very low error margins. / 2021-09-01

In recent studies it was reported that the D-amino acid containing peptide HYD1 was used in the treatment of necrotic cell death in multiple myeloma cell lines and showed promising biological activity and in vivo activity. It was meaningful to explore strategies for increasing the therapeutic efficacy of HYD1, a linear peptide. These efforts led to the development of MT1-101 (cyclized peptidomimetics), a lead compound that showed increased in vitro activity and in vivo activity. MTI-101 was found to bind the cell adhesion molecule CD44 and induce programmed necrosis in myeloma cell lines. It was important to improve on the binding efficiency of the MTI-101 to this target and explore more cost effective ways to synthesize this peptide. This lead to developing Cyclic beta-hairpin-like peptoid scaffolds, which introduced diverse families of random peptoid-body libraries that will be screened to find small stable scaffolds that compete with and can replace antibodies as cell-surface targeting reagents. The synthesis of peptoids on solid-support can be more cost effective and a large library can be developed using a diverse library of primary amines. This initiated this thesis project to develop a generalized scheme for the synthesis of TRP-like primary amine peptoid side chains used in the cyclic beta-hairpin - Like scaffolds.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 121-137).

In this thesis, a system called Video Looping is developed to analyze human cyclic motions. Video Looping allows users to extract human cyclic motion from a given video sequence. This system analyzes similarities from a large amount of live footage to find the point of smooth transition. The final cyclic loop is created using only a few output images. Video Looping is useful not only to learn and understand human movements, but also to apply the cyclic loop to various artistic applications. To provide practical animation references, the output images are presented as photo plate sequences to visualize human cyclic motion similar to Eadweard Muybridge's image sequences. The final output images can be used to create experimental projects such as composited multiple video loops or small size of web animations. Furthermore, they can be imported into animation packages, and animators can create keyframe animations by tracing them in 3D software.

<p>Functional genomic analyses provide information that allows hypotheses to be formulated on protein function. These hypotheses, however, need to be validated using reverse genetic approaches, which are difficult to perform on a large scale and in diploid organisms. To address this problem, we developed a genetic screen to rapidly isolate lariat peptides that function as trans dominant inhibitors of protein function.</p> <p>We engineered intein proteins to genetically produce lariats. A lariat consists of a lactone peptide covalently attached to a linear peptide. Cyclizing peptides with a lactone bond imposes a constraint even within the reducing environment found inside of cells. The covalently attached linear peptide provides a site for fusing protein moieties. We fused a transcriptional activation domain to a combinatorial lactone peptide, which allowed combinatorial lariat libraries to be screened for protein interactions using the yeast two-hybrid assay.</p> <p>We confirmed that the intein processed in yeast using Western blot analysis. A chemoselective ring opening of the lactone bond with heavy water, followed by mass spectrometry analysis showed that ~ 44% of purified lariat contained an intact lactone bond. To improve the stability of the lactone bond, we introduced mutations into the engineered intein and analyzed their processing and stability by mass spectrometery. Several mutations were identified that increased the amount of intact lariat.</p> <p>Combinatorial libraries of lactone peptides were generated and screened using the yeast-two-hybrid interaction trap. Lactone cyclic peptides that bound to a number of different targets including LexA, Jak2, and Riz1 were isolated. A lactone cyclic peptide isolated against the bacterial repressor protein LexA was characterized. LexA regulates bacterial SOS response and LexA mutants that cannot undergo autoproteolyis make bacteria more sensitive to, and inhibit resistance against cytotoxic reagents. The anti-LexA lariat interacted with LexA with a dissociation constant of 37 µM by surface plasmon resonance. The lactone constraint was determined to be required for the interaction of the anti-LexA L2 lariat with LexA in the yeast-two-hybrid assay. Alanine scanning showed that only two amino acids (G8 and E9) in the anti-LexA L2 sequence (1-SRSWDLPGEY-10) were not required for the interaction with LexA. The interaction of the anti-LexA lariat with LexA in vivo was confirmed by chromatin precipitation of the lactone peptide-LexA-DNA complex. The anti-microbial properties of the anti-LexA lariat were also characterized. The anti-LexA lariat potentiated the activity of a DNA damaging agent mitomycin C and inhibited the cleavage of LexA, preventing the SOS response pathway from being activated.</p> <p>In summary, lariats possess desired traits for characterizing the function and therapeutic potential of proteins. The ability to genetically and chemically synthesize lariats allows the lariat transcription activation domain to be replaced by other peptide and chemical moieties such as affinity tags, fluorescent molecules, localization sequences, et cetera, which give them advantages over head to tail cyclized peptides, which have no free end to attach moieties.</p>

While unprotected amino aldehydes are typically not isolable due to imine formation and consequent polymerization, stable unprotected aziridine aldehydes are useful and available reagents. Moreover, reversible hemiacetal and hemiaminal formation enable these compounds to reveal both their electrophilic and nucleophilic functional groups. This exceptional arrangement allows for aziridine aldehyde dimers to participate in and disrupt the mechanisms of an array of well-known organic reactions, including isocyanide-based multicomponent reactions. The scope and selectivity patterns of aziridine aldehyde induced amino acid or peptide macrocyclization have been investigated. A small library of constrained tri-, tetra-, and penta-peptide macrocycles – representing the most difficult cyclic peptides to synthesize – has been prepared. The scope of aziridine aldehyde participation in multicomponent reactions was also expanded to Ugi and Passerini reactions that do not employ tethered amine and acid functional groups. In order to facilitate cellular imaging of peptide macrocycles a fluorescent isocyanide reagent was prepared and applied to prepare mitochondrial targeting macrocycles. Thioester isocyanide reagents were synthesized to enable rapid assembly of cycle-tail peptides through ligation technology.

The lymphatic system plays an important role in fluid and protein balance within the interstitial spaces. Its dysfunction could result in a number of debilitating diseases, namely lymphedema. Lymphatic vessels utilize both intrinsic and extrinsic mechanisms to pump lymph. Intrinsic pumping involves the active contraction of vessels, a phenomenon that is regulated in part by nitric oxide (NO) produced by lymphatic endothelial cells (LECs). NO production by arterial endothelial cells has been shown to be sensitive to both shear stress and stretch. Therefore, because of the unique mechanical environment of the LECs, we hypothesize that mechanical forces play an important role in regulation of the lymphatic pumping. Parallel-plate flow chambers and indenter-based cyclic stretch devices were constructed and used to apply mechanical loads to LECs. In addition, high-throughput micro-scale channels were developed and tested for shear experiments to address the need to increase the productivity and high- resolution imaging. Twenty-four hours treatment of LECs with different shear stress conditions showed a shear-dependent elevation in NO production. Moreover, 2.5 folds increase in cumulative NO was observed for stretched cells compared to the unstretched cells over six hours period. In conclusion, the upregulation observed in NO production under mechanical stimuli suggest new regulatory mechanisms that can be pharmaceutically targeted. These results provide an unprecedented insight into lymphatic pumping mechanism.

Autophagy is an evolutionarily conserved degradative eukaryotic cell pathway that plays a role in multiple cellular processes. One important function is as a key component of the cellular immune response to invading microbes. Autophagy has been found to directly target and degrade multiple intracellular bacterial species. In this thesis, I identify and characterize two distinct regulatory mechanisms for this pathway involving the second messengers: diacylglycerol and cyclic adenosine monophosphate (cAMP). Salmonella enteric serovar Typhimurium (S. Typhimurium) is a Gram-negative bacterial species that has been shown to be intracellularly targeted for degradation by autophagy. While targeting of this species has been previously shown to involve ubiquitination, this pathway accounts for only half of targeted bacteria. Here I show that ubiquitin-independent autophagy of S. Typhimurium requires the lipid second messenger diacylglycerol. Diacylglycerol localization to the bacteria precedes autophagy and functions as a signal to recruit the delta isoform of protein kinase C (PKC) in order to promote the specific autophagy of tagged bacteria. Furthermore, I have found that the role of diacylglycerol and PKC delta is not limited to antibacterial autophagy but also functions in rapamycin-induced autophagy indicating a general role for these components in this process. Multiple bacterial species have been found to be targeted by autophagy and while some have developed strategies that allow them to avoid targeting, no bacterial factor has yet been identified that is able to inhibit the initiation of this process. Here I show that two bacterial species, Bacillus anthracis and Vibrio cholera inhibit autophagy through the elevation of intracellular cAMP and activation of protein kinase A. Using two different bacterial cAMP-elevating toxins, I show that multiple types of autophagy are inhibited in the presence of these toxins. This is indicative of a general inhibitory function for these toxins and identifies a novel bacterial defence strategy. This work characterizes both a novel regulatory signal for the induction of autophagy and identifies a novel bacterial tactic to inhibit this process. Together the data presented in this thesis provide novel insight into the regulation of autophagy and offer potential targets for modulation of this process.