Obesity rates have risen steeply in recent decades. This is accompanied with increased prevalence of several obesity-related disorders including type 2 diabetes. Genetic, environmental, and lifestyle factors (increased caloric intake and/or decreased physical activity) predispose individuals to type 2 diabetes by decreasing the body's responsiveness to the pancreatic hormone insulin, a physiological phenomenon commonly referred to as insulin resistance. Insulin resistance occurs due to induction of inflammation characterized by increased secretion of pro-inflammatory cytokines from enlarged adipose tissue, endoplasmic reticulum stress, and oxidative stress associated with excess blood glucose. There is a strong correlation between insulin resistance and decreased circulating levels of adiponectin (APN), a hormone implicated in promoting insulin-like activities. Further, inflammation negatively affects both insulin and adiponectin levels. It is recognized that folding of APN 18mer-subunits (insulin-sensitizing oligomer) is hindered in obese and type 2 diabetic individuals. Likewise, formation of normal hexameric insulin complex is compromised in type 2 diabetes. Insulin biogenesis, packaging, and assembly are impaired and unable to compensate for high blood glucose levels. As insulin and APN are key metabolic hormones essential for proper glucose regulation, maintaining their correct folding and assembly is required for conserving overall metabolic homeostasis. This dissertation centers on investigating proper assembly pathways of APN and insulin isoforms to form the higher order complexes necessary for their function. The interaction between APN oligomers was studied in the presence and absence of zinc, previously shown to inhibit formation of disulfide bonds in APN. We observed that zinc protects APN from collapse under acidic conditions and likely stabilizes oligomers through high affinity histidine coordination. The interaction between oligomers was further assessed by analyzing conformational differences between oligomers through tryptophan fluorescence. Reduced oligomers were observed to have significant structural differences compared to oxidized oligomers indicated by changes in fluorescent intensities. The capacity of APN chaperone DSBA-L to promote assembly was also evaluated although no significant changes were observed. In addition, the interaction between zinc and insulin was assessed where we observed that in the presence of zinc, insulin is significantly protected from reduction and precipitation. Zinc formed large complexes with insulin under reducing environments to induce high structural stability of insulin oligomers. We then utilized the strong conformational stability of insulin to develop a novel insulin analog with properties to slowly release insulin in circulation and more quickly in the presence of high glucose concentrations. Insulin modification is at preliminary stages and requires further experimentation. Together, these results indicate that zinc plays a significant role in multimerizing properties to provide high stability towards APN and insulin structures. Zinc enhances multimerization of oligomers to both promote activity of APN and protect insulin from reduction and premature breakdown to monomers. Through this study we better identified the folding pathway of APN and elucidated the strong intermolecular forces involved in oligomer association. In addition, the multimerization pattern of insulin to large conformation complexes is observed to mediate protection under reducing conditions. This has implications in the development of new therapeutic options to promote insulin-sensitization and insulin activity to regulate plasma glucose levels. In addition, we propose the development of a novel insulin-analog to mimic physiological insulin secretion, currently unavailable in the market.

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This thesis presents the design, fabrication, and testing of a micromixing device intended for use in investigating protein dynamics on a microsecond timescale by Fourier transform infrared (FTIR) spectroscopy. Numerical modeling of flow was implemented to predict the influence of flow rates and geometric variations on mixing performance in three passive mixers. The simulation models were validated by experimental measurements using optical and infrared detection. The optimum level of mixing was observed in a multi-lamination mixer that combined thin filaments of differing fluids in an alternating manner. The multi-laminates were transferred onto polished calcium fluoride infrared-transparent optical windows by lithographic processing of an Epon-based polymer, SU-8. A rigid seal between two microchannels was accomplished through thermal bonding of an unexposed resist layer, which acted as a thermal epoxy under the influence of temperature. The multi-lamination mixer was used to study the changes in the secondary structure of beta-Lactoglobulin in deuterated phosphate buffer under varying physicochemical conditions by time-resolved FTIR spectroscopy using focal plane array detection. Upon a pH jump from pH 2 to neutral pH, a gradual loss of alpha-helical content, accompanied by an increase in random coils and turns was observed within 2 ms of mixing. In a second kinetic experiment, mixing of a neutral-pH solution of beta-Lactoglobulin with a 60% trifluoroethanol solution resulted in the formation of an alpha-helical intermediate with an accompanying increase in intramolecular beta-sheet structure within 500 mus of mixing. These results indicate that the multi-lamination mixer designed and fabricated in this study is well suited for investigations of protein dynamics on the micro- to millisecond timescale by time-resolved FTIR spectroscopy.

This research paper surveys the types of automatic gates, their advantages and drawbacks. New folding gates are analyzed; their moment of inertia during the movement is several times less than of common gates of the same width. Vibrations of common and folding automatic gates are also analyzed in the research. During the experimental survey a coefficient of tightness has been determined. Computer based program has been used for the analytical analyses. The most proper masses of construction knots and most optimum coefficients of tightness and suppression are chosen according to the optimum opening speed of the gates.

This study seeks to design algorithms that may be used to determine if a given lattice is a good approximation to a given rigid protein structure. Ideal lattice models discovered using our techniques may then be used in algorithms for protein folding and inverse protein folding. In this study we develop methods based on dynamic programming and branch and bound in an effort to identify “ideal” lattice models. To further our understanding of the concepts behind the methods we have utilized a simple cubic lattice for our analysis. The algorithms may be adapted to work on any lattice. We describe two algorithms. One for aligning the protein backbone to the lattice as a walk. This algorithm runs in polynomial time. The second algorithm for aligning a protein backbone as a path to the lattice. Both the algorithms seek to minimize the CRMS deviation of the alignment. The second problem was recently shown to be NP-Complete, hence it is highly unlikely that an efficient algorithm exists. The first algorithm gives a lower bound on the optimal solution to the second problem, and can be used in a branch and bound procedure. Further, we perform an empirical evaluation of our algorithm on proteins from the Protein Data Bank (PDB). / ix, 47 leaves ; 29 cm.

Self-folding polymer films were only recently proposed as an alternative method for the design of three-dimensional constructs. Due to the relative novelty of the approach, insufficient amount of data on the behavior of such systems is available in the literature. This study is bound to fill the gaps and give a deeper insight into the understanding of how and why different types of folding occur. In this study, four different types of folding of polymer bilayers are presented. Rectangles are one of the simplest geometrical forms and were therefore adopted as a convenient initial system for the investigation of the folding behavior of polymer bilayers. We chose PNIPAM for the active polymer, as it is a well-studied polymer with sharp Lower Critical Transition Temperature at around 33 C. For the passive layer, poly(methyl methacrylate) and poly(caprolactone) were chosen. The influence of different parameters of the system, such as polymer thickness and temperature was thoroughly investigated in order to be accounted for in later experiments. It was demonstrated that bilayers placed on a substrate start to roll from the corners due to quicker diffusion of water. Rolling from the long-side starts later but dominates at high aspect ratio. We showed that the main reasons causing a variety of rolling scenarios are (i) non-homogenous swelling due to slow diffusion of water in hydrogels and (ii) adhesion of polymer to a substrate until a certain threshold. Moreover, non-homogenous swelling determines folding in the first moments, while adhesion plays a decisive role at later stages of folding. After having understood the abovementioned basics, we decided to explore how those applied to more complex shapes. For the purpose, four- and six-arm stars were chosen, the main idea behind this being the creation of self-folding polymer capsules capable of encapsulation of microparticles and cells. Adjusting the polymer thickness and thus the radius of folding allowed creating structures, capable of reversible self-folding and unfolding. The possibility to reversibly encapsulate and release objects in the micro-range was demonstrated on the example of yeast cells. Noteworthy, the capsules were produced by means of the same process we used for the design of tubes – when compared to the folding of rectangles, it was the shape of the initial pattern and the folding radius that were changed; the mechanism was the same – simple one-step folding towards the center of the bilayer. Clearly the number of structures that can be generated by this method is fairly limited. The search for means to overcome this constraint led to the idea of hierarchical multi-step folding. Due to the edge-activation of the bilayers, the observed deformed shapes differ from the classical ones obtained by homogeneous activation. It was found that films could demonstrate several kinds of actuation behavior such as wrinkling, bending and folding that result in a variety of shapes. It was demonstrated that one can introduce hinges into the folded structure by proper design of the bilayer's external shape through diffusion without having to use site selective deposition of active polymers. Experimental observations led us to derive four empirical rules: 1) “Bilayer polymer films placed on a substrate start to fold from their periphery and the number of formed wrinkles/tubes decreases until the angle between adjacent wrinkles/tubes approaches 130°”; 2) “After the wrinkles along the perimeter of the film form tubes, further folding proceeds along the lines connecting the vertexes of the folded film”; 3) “The folding goes along the lines which are closer to the periphery of the films”; 4) „Folding of the rays may result in blocking of the neighboring rays if the angle between the base of the folded ray and the shoulders of the neighboring rays is close to 180°”. These rules were then applied to direct the folding of edge-activated polymer bilayers through a concrete example - the design of a 3D pyramid. One consequence of the second and third rules is that generally triangles are formed during the multi-step folding process. In order to create a cube, or any other 3D structure with non-triangular sides, an effective way to stop the folding along the lines, connecting neighboring vertexes had to be thought of. A possible solution would be the insertion of a rigid element inside the bilayer, perpendicular to the direction of folding. The solution of this problem was to design structures with pores. A pore would normally decrease the rigidity of a structure but in our case, a pore basically comprised an edge inside the structure and could thus form tubes which, as was already shown, exhibit much higher rigidity than a film. On the other hand, a pore, or many pores, would expose different parts of the active layer to the solvent and would strongly influence its swelling and, as a consequence, folding behavior. Hence, the influence of a pore on the swelling and the folding behavior of polymer bilayers had to be investigated. It was shown that pores of the right form and dimensions did indeed hinder the folding as intended. Instead, the polymer films took other ways to fold. As a result, despite the correctness of our reasoning, we failed to produce a cube by hierarchical folding of polymer bilayers. However, other sophisticated 3D objects were obtained, further increasing the arsenal of available structures, as well as giving an in-depth insight on the folding process.

Graphene is a promising material for many technological applications. To realize these applications, new fabrication techniques that allow precise control of the physical properties, as well as large scale integration between single devices are needed. In this work, a series of studies are performed in order to develop graphene based devices. First, using MD simulations we study the effects of irradiating graphene with a carbon ion atom at several positions and energies from 0.1 eV to 100 keV. The simulations show four types of processes adsorption, reflection, transmission, and vacancy formation. At energies below 10 eV the dominant process is reflection, between 10 and 100 eV is adsorption, and between 100 eV and 100 keV the dominant process is transmission. Vacancy formation is a low rate process that takes place at energies above 30 eV. Three types of defects were found: adatom, single vacancy, and 5-8-5 defect formed from a double vacancy defect. Also a bottom-up fabrication method is studied, in this method, the controlled folding of graphene structures, driven by molecular interactions with water nanodroplets, is analyzed considering the interactions with substrates such as SiO2, HMDS and IPA on SiO2. When the graphene is supported on SiO2, the attraction between graphene and the substrate prevents graphene from folding but if the substrate has HMDS or IPA, the interaction between graphene and the substrate is weak, and depending on the geometry of the graphene structure, folding is possible. Finally, to evaluate the characteristics of graphene based devices, we model the vibrational bending modes of graphene ribbons with different dimensions. The resonant frequencies of the ribbons and relations between the size of the ribbon and their resonant frequencies are calculated. The interaction of a graphene vibronic device with water and IPA molecules are simulated and demonstrate that this device can be used as a sensitive vibronic molecular sensor that is able to distinguish the chemical nature of the detected molecule. Also, the electrical properties of the graphene vibronic with armchair and zigzag border are calculated; the latter has the potential to generate THz electrical signals as demonstrated in this work.

Alpha-synuclein (AS) is a natively unfolded protein whose structure is extremely sensitive to its environment. The hallmark of Parkinson’s disease (PD) is aggregation and deposition of AS in inclusion bodies. Formation of misfolded AS monomers which are partially folded is the first and critical stage in fibrillation of AS and is a good target for designing therapeutic strategies. Characterization the biochemical properties of partially folded intermediates induced by fibrillization and anti- fibrillization agents will help to design drugs as new inhibitors of AS misfolding and aggregation. Nanopore analysis is an emerging technique for studying the molecular mechanism of protein misfolding. This technique was used to characterize the conformational change of AS in the presence of two groups of chemicals; anti-parkinsonian small molecules (dopamine and nicotine) and Parkinson’s developing toxin (Cu(II) and methamphetamine). Other biophysical techniques such as NMR spectroscopy and isothermal titration calorimentry (ITC) were able to confirm the nanopore analysis results and also to study other biophysical properties of the partially folded intermediates such as the binding constant of the interaction and the secondary structure content. The results from nanopore analysis showed that both groups of ligands shifted the blockade current peak of AS (centered at -86 pA) to lower blockade currents but in a different manner. Anti-parkinsonian drugs shifted the blockade current of AS to intermediate peaks between -40 to -80 pA but Parkinson developing toxins shifted the peak to a lower blockade current centered at -25 pA which suggests a more compact conformation. Thus nanopore analysis distinguished the different conformation induced by different ligands. Furthermore nanopore analysis with AS fragments showed that these ligands bind to different regions of AS. NMR spectroscopy of AS in the presence of dopamine and nicotine isomers was in agreement with the nanopore analysis and showed conformational changes of AS in a concentration dependent manner. CD spectroscopy results showed that the secondary structure of AS alone and in the presence of ligands was mostly random coil and suggests a loop formation model for the interaction of ligands with AS. The results of this thesis showed the application of nanopore analysis as a real-time and label-free technique to screen a library of ligands for designing misfolding inhibitors for PD treatment. The result of a synergic experiment with nicotine and caffeine showed that combination of these anti-parkinsonian small molecules would be a promising new drug for treatment of PD.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes numerous human pathologies. Biochemical evidence suggests that soluble misfolded proteins are retrotranslocated out of the ER, via the endoplasmic reticulum associated degradation (ERAD) pathway, for proteosome-mediated cytoplasmic degradation. Excess, misfolded- or insoluble proteins, are suggested to cause induction of “overflow” degradation pathways. For soluble proteins, overflow to vacuole-mediated destruction is suggested to occur via two Golgi-to-vacuole (Gvt) routes, the alkaline phosphatase (ALP), direct route, or, a carboxypeptidase Y- (CPY-), prevacuolar compartmentvacuole, indirect route, though only the CPY route is thought to degrade soluble proteins. Insoluble aggregate-containing structures are suggested to be degraded by engulfment by membranes of unknown origin and trafficking to the vacuole for destruction, via an autophagic pathway. To confirm biochemical evidence, wild-type (BY4742), autophagosome- (W303/ATG14), CPY- and autophagy pathway- (W303/VPS30), and proteosome (WCG/2) mutants of S. cerevisiae yeasts were transformed with a high expression pYES plasmid and mutant (Z) human alpha-1- proteinase inhibitor (A1PiZ), giving rise to the derivatives cells BY4742/Z, W303/ATG14/Z, W303/VPS30/Z and WCG/2/Z, respectively. Electron microscopy using gold labeling for A1PiZ, markers for the ER, the ERAD ER channel protein, Sec61, or the chaperone, binding protein (BiP), ALP for the ALP pathway, and CPY for the CPY pathway, was used. Overexpression of A1PiZ seems to result in targeting to the vacuole via a prevacuolar, CPY-like compartment (PVC, 200-500 nm), though CPY and A1PiZ appears not to colocalise, unconvincingly confirming collaborative biochemical data. Large amounts of A1PiZ localise in the cytosol, possibly indicating a largely proteasome-mediated degradation. ER-resident A1PiZ targeting to the vacuole seems also to occur by the budding of the ER and peripheral plasma membrane or ER membrane only. This occurs in all cells, but especially in ATG14 gene (ΔATG14) mutants, possibly indicating autophagosome-mediated degradation independence, in the latter mutants. The ATG14 mutation gave rise to crescent-shaped, initiating membranelike (IM-like) structures of approximately Cvt vesicle-diameter, possibly indicating that ΔATG14 blocks autophagosome- (500-1000 nm) and Cvt vesicle (100-200 nm) enclosure, after core IM formation. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.