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.

No description available.

No description available.

The automotive industry is adopting lightweight materials to improve emissions and fuel economy. Magnesium (Mg) alloys are the lightest of engineering metals, but work is required to assess their structural strength, especially for spot-welded applications. In the present research, fatigue behavior of magnesium spot-welds was characterized and compared with steel and aluminum spot-welds. A fatigue model was proposed to predict the failure location and crack initiation life in magnesium structures. The material under investigation, AZ31B-H24 Mg alloy, and its spot-welds were characterized from microstructural and mechanical perspectives. Microstructure and hardness of the base metal (BM) and different regions in the spot-welds were studied. Under cyclic loading, the BM had an asymmetric hysteresis loop. Cyclic behavior of magnesium spot-welds was measured using different specimen configurations, and the effect of geometrical factors on fatigue life was evaluated. A constitutive model was developed to model the asymmetric hardening behavior of wrought magnesium alloys under cyclic loading. An algorithm for numerical implementation of the proposed model was developed. The numerical formulation was programmed into a user material subroutine to run with the commercial finite element software Abaqus/Standard. The proposed model was verified by solving two problems with available solutions. A number of available fatigue models, as well as a new model proposed in this research were assessed by predicting fatigue life of magnesium spot-welds. The new model used a strain energy damage parameter. All models were evaluated by comparing the predicted and experimental fatigue lives for different Mg spot-welded specimens. The effect of considering the asymmetric hardening behavior of wrought magnesium alloys on the accuracy of the fatigue life prediction was not significant for the available experimental data. This was attributed to the limited experimental data on spot-welded specimens. The proposed material model and fatigue damage parameter were verified by simulating a real-life structure manufactured and fatigue tested by the US Automotive Materials Partnership. The results obtained from the proposed asymmetric model were compared with available symmetric simulation results and experimental data. The asymmetric material model along with the proposed damage parameter resulted in more accurate prediction of fatigue failure location and life.

In this thesis, cyclic biamperometry (CB) as a new method in electrochemistry, has been introduced and investigated. The hallmark of this method is the absence of a reference electrode which potentially allows simplification and miniaturization of the measurement apparatus. Similarities and differences of this method and cyclic voltammetry (CV) have been studied and it was shown that under conditions of using standard electrodes, CB has a better sensitivity and a lower detection limit than CV. A new equivalent circuit model for the cell has been proposed and parameters affecting the sensitivity of CB, such as keeping the concentration of one redox species in excess and having a larger W2 electrode, have been described. The redox cycling effect in biamperometric systems has been investigated and it is shown that improvements of at least two orders of magnitude in sensitivity can be achieved by using interdigitated electrodes (IDEs). In addition, an example for applications of this method, including biamperometric dead-stop titration of 1-naphthol with ferricyanide, has been presented and possible fields in which CB can be incorporated (e.g. monitoring the activity of alkaline phosphatase) have been illustrated. Finally, a few suggestions for future studies and further improvements have been outlined.

One of the limiting factors in gas turbine design is the allowable metal temperatures and loads in critical components. Specially designed superalloys are used when the conditions are most severe. One of these superalloys is Inconel 718. To be able to design components for higher temperature and higher loads, an accurate understanding and computational model of the material is needed. In this thesis the deformation mechanisms of Inconel 718 have been investigated and a theoretical basis for modelling in a large deformation context has been established. Finally a viscoplastic nonlinear kinematic hardening material model with an Armstrong-Frederick backstress evolution law has been implemented as a first step in describing the constitutive behaviour of the material Inconel 718.

One of the limiting factors in gas turbine design is the allowable metal temperatures and loads in critical components. Specially designed superalloys are used when the conditions are most severe. One of these superalloys is Inconel 718. To be able to design components for higher temperature and higher loads, an accurate understanding and computational model of the material is needed. In this thesis the deformation mechanisms of Inconel 718 have been investigated and a theoretical basis for modelling in a large deformation context has been established. Finally a viscoplastic nonlinear kinematic hardening material model with an Armstrong-Frederick backstress evolution law has been implemented as a first step in describing the constitutive behaviour of the material Inconel 718.

The study of coding theory aims to detect and correct the errors during the transmission of the data. It enhances the quality of data transmission and provides better control over the noisy channels.The perfect codes are collected and analyzed in the premises of the Hamming metric.This classification yields that there exists only a few perfect codes. The perfect codes do not guarantee the perfection by all means but just satisfy certain bound and properties. The detection and correction of errors is always very important for better data transmission.

In this thesis, cyclic biamperometry (CB) as a new method in electrochemistry, has been introduced and investigated. The hallmark of this method is the absence of a reference electrode which potentially allows simplification and miniaturization of the measurement apparatus. Similarities and differences of this method and cyclic voltammetry (CV) have been studied and it was shown that under conditions of using standard electrodes, CB has a better sensitivity and a lower detection limit than CV. A new equivalent circuit model for the cell has been proposed and parameters affecting the sensitivity of CB, such as keeping the concentration of one redox species in excess and having a larger W2 electrode, have been described. The redox cycling effect in biamperometric systems has been investigated and it is shown that improvements of at least two orders of magnitude in sensitivity can be achieved by using interdigitated electrodes (IDEs). In addition, an example for applications of this method, including biamperometric dead-stop titration of 1-naphthol with ferricyanide, has been presented and possible fields in which CB can be incorporated (e.g. monitoring the activity of alkaline phosphatase) have been illustrated. Finally, a few suggestions for future studies and further improvements have been outlined.

<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>