Thesis (M.A.)--Ohio University, June, 2005. / Title from PDF t.p. Includes bibliographical references (p. 56-62)

An infectious cDNA clone of Vero cell adapted Beaudette strain of IBV was constructed using in vitro assembly of cDNA fragments. The entire genome of IBV was RT-PCR amplified into seven fragments, with each piece overlapping about 10 nucleotides. The fragments were ligated and transcribed to synthesize RNA, which was transfected into BHK-21 cells. These cells were then overlaid onto IBV susceptible Vero cells. After five days transfection, the virus was successfully rescued from the transfected cells. The cDNA clone from our laboratory strain has a five nucleotide insertion not present in the originally sequenced virus, resulting in total genome size of 27,613 nucleotides. The infectious cDNA clone was further manipulated to demonstrate its potential as a gene transfer vector, by replacing the ORF5a open reading frame with enhanced green fluorescent protein. The recombinant infectious cDNA clone was also successfully rescued after three days transfection of BHK-21 cells followed by co-culturing with Vero cells. This study showed that the 5a protein, whose function is not known, is not necessary for in vitro IBV replication. This study also showed that the 5a ORF is a good candidate for an insertion site of recombinant genes for the development of IBV infectious cDNA clone as a gene transfer vector.

The physical properties (i.e., mass, particle diameter and composition) of apolipoprotein B (apoB)-containing lipoproteins (apoB-LP) are a major determinant of atherosclerotic cardiovascular disease (ASCVD) risk. The objective of this research was to investigate how nascent apoB-LP physical properties affect circulating lipoprotein profiles and risk of disease. Relationships between apoB-LP physical properties and arterial plaque formation in four genotypes of mice with apoB isoform specific clearance defects were investigated. Multivariate statistical analysis found that arterial lesions were most closely related to genetic background and apoB concentration related to delayed clearance rate. For defining the dietary effects on circulating lipoprotein profiles, the physical properties of lipoproteins in hamsters fed high-carbohydrate diets containing either 60% fructose or 60% cornstarch for 2 wk were studied. Fructose increased very low-density lipoprotein (VLDL) particle diameter and decreased low-density lipoprotein (LDL) particle diameter. Elevations in all high-density lipoprotein (HDL) fractions were observed in the fructose-fed group. Further investigation was made of whether changes to the physical properties in circulating lipoproteins resulted from changes to nascent particles in the assembly and secretion processes. Intermediate particles used for lipoprotein assembly were isolated from rough endoplasmic reticulum of hamster liver, and nascent VLDL were isolated from plasma after Triton WR-1339 injection of hamsters. A large, TG-rich apoB-deficient particle and a small, lipid-poor apoB-containing particle were isolated in each dietary setting. The diameter of first-step particles was larger in fructose feeding, which indicated that apoB degradation decreases and provides the basis for apoB oversecretion. Fructose feeding significantly increased the concentrations recovered from liver for these two particles and for nascent particles compared with chow or starch feeding. Collectively, these results demonstrate: 1) genetic factors can dictate metabolism, and metabolic conditions can critically affect the physical properties and further atherogenicity of apoB-LP; 2) changes in physical properties of circulating apoB-LP are derived from changes to the nascent particles; and 3) dietary factors can influence the assembly, secretion, and metabolism of apoB-LP. The findings of the research may provide a metabolic basis for the recognition of new targets that could regulate apoB-LP metabolism to prevent and treat ASCVD.

Iron-sulfur clusters are protein cofactors that are critical for all life forms. Elaborate multi-component systems have evolved for the biosynthesis of these cofactors to protect organisms from the toxic effects of free iron and sulfide ions. In eukaryotes, the Fe-S cluster assembly machinery operates in the matrix space of the mitochondria and contains a myriad of proteins that mediate sulfur, iron, and electron transfer to assemble Fe-S clusters on the scaffold protein ISCU2 and then distribute these clusters to target proteins. Our lab has recently described stable 3, and 4-protein complexes composed of the cysteine desulfurase NFS1, the co-chaperone ISD11, and ISCU2 (SDU), and NFS1, ISD11, ISCU2, and FXN (SDUF) subunits. In the latter, SDUF, FXN functions as an allosteric activator switching this assembly complex on for Fe-S cluster biosynthesis. Insufficient expression of the mitochondrial protein FXN leads to a progressive neurodegenerative disease, Friedreich's Ataxia (FRDA). In ~2% of patients, FRDA is caused by one of 15 known missense mutations on one allele accompanied by the GAA repeat on the other leading to a complicated phenotype that includes loss of Fe-S clusters. Here we present in vitro evidence that FRDA FXN variants are deficient in their ability to bind the SDU complex, their ability to stimulate the sulfur transfer reaction from NFS1 to ISCU2, and in their ability to stimulate the rate of cluster assembly on ISCU2. Here, in vitro evidence is presented that FXN accelerates the sulfur transfer reaction from NFS1 to ISCU2. Additionally, we present kinetic evidence that identifies the most buried cysteine residue, C104 on ISCU2 as the sulfur acceptor residue suggesting, FXN stabilizes a conformational change to facilitate sulfur delivery. Subsequent mutational studies suggest FXN binding to SDU results in a helix to coil transition in ISCU2 exposing C104 to accept the persulfide sulfur and thereby accelerating the rate of sulfur transfer. We further provide the first biochemical evidence that the persulfide transferred to ISCU2 from NFS1 is viable in Fe-S cluster formation. In contrast to human FXN, the Escherichia coli FXN homolog CyaY has been reported to inhibit Fe-S cluster biosynthesis. To resolve this discrepancy, a series of inter-species enzyme kinetic experiments were performed. Surprisingly, our results reveal that activation or inhibition by the frataxin homolog is determined by which cysteine desulfurase is present and not by the identity of the frataxin homolog. These data are consistent with a model in which the frataxin-less Fe-S assembly complex exists as a mixture of functional and nonfunctional states, which are stabilized by binding of frataxin homologs. Intriguingly, this appears to be an unusual example in which modifications to an enzyme during evolution inverts or reverses the mode of control imparted by a regulatory molecule.

Extracellular microenvironment, including its components and biophysical parameters such as matrix structure and stiffness, is a crucial determinant of cellular function. There exists interdependency between cellular behaviors and the extracellular matrix (ECM), whereby cells are constantly sensing and modifying their surroundings in response to physical stress or during processes like wound repair, cancer cell invasion, and morphogenesis, to create an environment which supports adaptation. To date, knowledge of the distinct regulatory mechanisms of this complex relationship is little, while the urge is evident as it plays a significant role in understanding tissue remodeling. Cells are observed to align with the parallel arrays of collagen fibrils found in tissues such as bone, tendon, and cornea, suggesting the importance of ordered matrices in defining cell functions. In this study, epitaxial growths of ordered two-dimensional collagen matrices were created, with parallelly aligned fibrils on muscovite mica, and novel triangular pattern matrix on phlogopite mica. Using Fluorescence and Atomic Force Microscopy, we were able to observe cell polarization along with stress fiber formation and matrix deformation at high resolution. Cells were observed to be able to penetrate between collagen fibrils and generate traction anisotropically to polarize. These ordered collagen matrices serve as an excellent model to study cellular remodeling of ECM in vitro, in which this fundamental apprehension of cell-matrix relationship is of crucial importance to manipulate the system and obtain desired cell functions.

The current project addresses the productivity improvement of a manual assembly line by making use of operations analysis in the framework of Lean production. A methodology is proposed that helps to improve the productivity of any production process. The methodology consists of selecting a product or product family to be studied followed by current process study. Once the existing process is documented, all the assembly tasks involved must be timed using time study techniques. Operations analysis enables the reduction of non-productive tasks and results in a set of standardized work elements along with the set of standard procedures for performing the operations. Assembly line balancing along with the associated operations analysis assists in constructing or re-configuring an assembly system, which is the key step in improving the overall performance of an assembly line. Following this approach, two manual assembly line configurations (single stage parallel line and five-stage serial line) are constructed for a case study. The results show that by changing over to the single stage assembly line configuration the operator productivity is doubled when compared to the existing assembly method.

The Mediator complex was originally discovered in the yeast Saccharomyces cerevisiae and has since then been shown to be required for transcriptional regulation both in vitro and in vivo. The Mediator complex also stimulates basal, unregulated transcription and serves as a bridge by conveying signals from promoter-bound transcriptional regulatory proteins such as activators and repressors to the RNA Polymerase II general transcriptional machinery. The Mediator consists of 21 subunits and can be divided into three distinct modules head, middle and tail. Despite the tremendous progress that has been achieved so far in characterizing the Mediator complex both functionally and structurally, many aspects of the complex are not yet well understood. The objective of this work is to achieve further understanding of the Mediator complex by studying the folding of different protein subunits, their interactions and how that affects assembly of the Mediator complex. In our first study we made a temperature-sensitive med21 mutant and used it to identify genes that can suppress the mutation when present in high copy number. Among the 10 genes that we identified, the strongest suppressors were Med7 and Med10, which encode Mediator subunits, and Ash1, which encodes a repressor of the HO gene. We also used 2-hybrid experiments and immunoprecipitation to study protein-protein interactions between Med21 and the Med4, Med7 and Med10 proteins which are all essential for viability and located within the middle domain of the Mediator complex. We found that the N-terminal 2-8 amino acids of Med21 are required for interactions with Med7 and Med10. These results led us to propose a model in which the N-terminal part of Med21 functions as a molecular switchboard where competing signals from various activators, repressors and mediator subunits are integrated prior to reaching the general transcription machinery. In our second study, we extended our studies of protein-protein interactions to another part of the mediator complex by studying the folding and the assembly processes of the mediator head domain subunits Med8, Med18 and Med20. Using purified proteins and a combination of several different methods such as immunoprecipitation, far-UV circular dichroism and fluorescence, we demonstrated that the Med8, Med18 and Med20 subunits are interdependent on each other for proper folding and complex formation.

Membrane proteins are key players in many biological processes. Since most membrane proteins are assembled into oligomeric complexes it is important to understand how they interact with each other. Unfortunately however, the assembly process (i.e. their social life) remains poorly understood. In the work presented in this thesis I have investigated when and how membrane proteins assemble with each other and their cofactors to form functional units. We have shown that that cofactor insertion in the hetero-tetrameric cytochrome bo3 occurs at an early state in the assembly process. We also found that the pentameric CorA magnesium ion channel is stabilised by different interactions depending on the magnesium ion concentration in the cell. These studies indicate that the assembly of a functional unit is a dynamic process, which is a result of many different forces. By studying the assembly of membrane proteins we have obtained a deeper insight into their function, which cannot be explained by static crystal structures. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

Due to the introduction of Restriction of Hazardous Substances (RoHS) directive Pb containing solders have been banned from the electronics industry and a reliable replacement for the Sn-Pb solder is being sought for by industry around the globe. Medical and Defense industries are currently exempt from the directive and use Sn-Pb solder in their manufacturing process. The switch to lead-free has led component manufacturers to use different lead-free solders, thus causing mixed solder joints of lead-free components with Sn-Pb paste. In this study, mixed assembly microstructures and mechanical properties were examined. Furthermore, six novel lead-free solders were prepared using SAC105 solder doped with elements: Ti, Ni, Mn, La, Ce and Y. The solidification microstructures, fracture behavior and wetting properties of these solders were evaluated to find a suitable replacement for SAC105 solder.

Due to the introduction of Restriction of Hazardous Substances (RoHS) directive Pb containing solders have been banned from the electronics industry and a reliable replacement for the Sn-Pb solder is being sought for by industry around the globe. Medical and Defense industries are currently exempt from the directive and use Sn-Pb solder in their manufacturing process. The switch to lead-free has led component manufacturers to use different lead-free solders, thus causing mixed solder joints of lead-free components with Sn-Pb paste. In this study, mixed assembly microstructures and mechanical properties were examined. Furthermore, six novel lead-free solders were prepared using SAC105 solder doped with elements: Ti, Ni, Mn, La, Ce and Y. The solidification microstructures, fracture behavior and wetting properties of these solders were evaluated to find a suitable replacement for SAC105 solder.