Master of Science / Department of Biochemistry / John M. Tomich / Biologically based adhesives offer many industrial advantages over their chemically synthesized counterparts, not the least of which are reduced environmental impact and limited toxicity. They also represent a renewable resource. In addition, nanoscale biomaterials also show an incredibly large potential for biomedical uses, including possible drug delivery and novel wound bandaging, as well as tissue engineering. Understanding the adhesion mechanisms at work in peptide-based nanomaterials is key for producing viable industrial and clinical biomimetic compounds. Our previous work has shown that small hydrophobic oligopeptide segments flanked by short tri-lysine sequences display adhesion strength that is dependent on the formation of β-structure and large-scale association of monomers. In this study, three oligopeptides were synthesized based on putative amyloid fibril nucleation sites. Two of the sequences originate from the Alzheimer’s beta amyloid peptide Aβ1-40, while the third sequence comes from a nucleation site for islet amyloid polypeptide (IAPP). These peptides show unusual structural properties associated with adhesive ability. Furthermore, they represent a third category of requirements for β-structure formation. In addition, I report the first morphological evidence for the previously predicted structural mechanism underlying our previous peptide based adhesives.

Doctor of Philosophy / Department of Biochemistry / Anna Zolkiewska / Muscle satellite cells are the primary stem cells of postnatal skeletal muscle. Quiescent satellite cells become activated and proliferate during muscle regeneration after injury. They have the ability to adopt two divergent fates: differentiation or self-renewal. The Notch pathway is a critical regulator of satellite cell activation and differentiation. Notch signaling is activated upon the interaction of a Notch ligand present in a signal-sending cell with a Notch receptor present in a signal-receiving cell. Delta-like 1 (Dll1) is a mammalian ligand for Notch receptors. In this study, we found that Notch activity is essential for maintaining the expression of Pax7, a transcription factor associated with self-renewing satellite cells. We also demonstrated that Dll1 represents a substrate for several ADAM metalloproteases. Dll1 shedding takes place in a pool of Pax7-positive self-renewing cells, but Dll1 remains intact in differentiated myotubes. Inhibition of Dll1 shedding with a dominant-negative form of ADAM12 leads to elevated Notch signaling, inhibition of differentiation and expansion of the pool of self-renewing cells. We propose that ADAM-mediated shedding of Dll1 helps achieve an asymmetry in Notch signaling in initially equivalent myogenic cells and helps sustain the balance between differentiation and self-renewal. Pax7 plays a key role in protecting satellite cells from apoptosis. The mechanism of Pax7 protecting muscle satellite cells from apoptosis is not well understood. In the second part of this study, we show that Pax7 up-regulates manganese superoxide dismutase (MnSOD) at the transcriptional level, suggesting the involvement of MnSOD in Pax7-mediated cell survival. A specific chromosomal translocation involving the Pax7 gene and generation of a fusion protein Pax7-FKHR is found a childhood cancer, rhabdomyosarcoma. Furthermore, the level of the wild-type Pax7 is down-regulated in rhabdomyosarcomas. In the third part of this dissertation, we investigated the dominant-negative effect of Pax7-FKHR fusion protein on the wild-type Pax7, and found that the Pax7 protein level is down-regulated by Pax7-FKHR expression while the Pax7 mRNA level is not affected. We propose a specific microRNA-mediated inhibition of Pax7 mRNA translation by the oncogenic Pax7-FKHR fusion protein.

Doctor of Philosophy / Department of Chemistry / Paul E. Smith / In the past several decades, molecular dynamics (MD) simulations have played an important role in providing atomic details for phenomena of interest. The force field used in MD simulations is a critical factor determining the quality of the simulations. Kirkwood-Buff (KB) theory has been applied to study preferential interactions and to develop a new force field. KB theory provides a path from quantities determined from simulation data to the corresponding thermodynamic data. Here we combine KB theory and molecular simulations to study a variety of intermolecular interactions in solution. First, a force field for the computer simulation of aqueous solutions of alcohols is presented. The force field is designed to reproduce the experimentally observed density and KB integrals for a series of alcohols, allowing for an accurate description of alcohols’ activity. Other properties such as the translational diffusion constant and heat of mixing are also well reproduced. Second, the newly developed force field is then extended to more complicated systems, such as peptide or mini-proteins, to determine backbone dihedral potentials energetics. The models developed here provide a basis for an accurate force field for peptides and proteins. Third, we have then studied the surface tension of a variety water models. Results showed that different simulation conditions can affect the final values of surface tension. Finally, by using the Kirkwood-Buff theory of solution and surface probability distributions, we attempted to characterize the properties of the Gas/Liquid interface region. The same approach is then used to understand the relationship between changes in surface tension, the degree of surface adsorption or depletion, and the bulk solution properties.

Doctor of Philosophy / Department of Biochemistry / Subbaratnam Muthukrishnan / In order to grow and develop, insects must undergo a process of molting, wherein the old cuticle is replaced with a new one. A thin envelope layer has been predicted to act as a physical barrier between molting fluid chitinases and the site of new chitin synthesis ensuring selective protection of newly synthesized chitin. The factors that help the new exoskeleton withstand the deleterious effects of chitinolytic enzymes remain poorly understood. In the current study a mechanistic role for two proteins, Knickkopf (Knk) and Retroactive (Rtv), was explored in organization and protection of the newly synthesized procuticular chitin. Our study demonstrated colocalization of molting fluid chitinases (chitinase-5) with chitin in T. castaneum pharate adult elytral cuticle. Presence of chitinases in the new cuticle, disproved the old theory of the envelope being a protective barrier against chitinases. Confocal and transmission electron microscopic imaging of T. castaneum pharate adult elytral cuticle suggested that Knk protein selectively colocalizes with chitin in the new procuticle, organizes chitin into laminae and protects it from the activity of molting fluid chitinases. Down-regulation of Knk expression resulted in reduction of procuticular chitin, disruption of the laminar architecture of the procuticle and severe molting defects that are ultimately lethal at all stages of insect growth. The presence and activity of Rtv protein ensures the trafficking of Knk into the procuticle. Down regulation of Rtv transcripts showed molting defects and a significant decrease in chitin content similar to those following Knk dsRNA treatment. Confocal microscopic analysis revealed an essential role for Rtv in proper trafficking of Knk from epithelial cells to within the newly synthesized procuticule. Once released into the procuticle, Knk organizes and protects chitin from chitinases. The conservation of Knk and Rtv in all insect species suggests a critical role for these proteins in maintenance and protection of chitin in the insect exoskeleton.

Doctor of Philosophy / Department of Biology / Ruth Welti / The molecular mechanisms by which plants respond to environmental stresses to sustain growth and yield have great importance to agriculture. Lipid metabolites are a major element of plant stress responses. The model plant Arabidopsis thaliana is well-suited to study stress-driven compositional dynamics, metabolism, and functions of lipid metabolites. When Arabidopsis plants were subjected to wounding, infection by Pseudomonas syringae pv tomato DC3000 expressing AvrRpt2 (PstAvr), infection by Pseudomonas syringae pv. maculicola (Psm), and low temperature, and 86 oxidized and acylated lipids were analyzed using mass spectrometry, different sets of lipids were found to change in level in response to the various stresses. Analysis of plant species (wheat versus Arabidopsis), ecotypes (Arabidopsis Columbia 0 versus Arabidopsis C24), and stresses (wounding, bacterial infection, and freezing) showed that acylated monogalactosyldiacylglycerol was a major and diverse lipid class that differed in acyl composition among plant species when plants were subjected to different stresses. Mass spectrometry analysis provided evidence that oxophytodienoic acid, an oxidized fatty acid, is significantly more concentrated on the galactosyl ring of monogalactosyldiacylglycerol than on the glycerol backbone. A mass spectrometry method, measuring 272 lipid analytes with high precision in a relatively short time, was developed. Application of the method to plants subjected to wounding and freezing stress in large-scale experiments showed the method produces data suitable for lipid co-occurrence analysis, which identifies groups of lipid analytes produced by identical or inter-twined enzymatic pathways. The mass spectrometry method and lipid co-occurrence analysis were utilized to study the nature of lipid modifications and the roles of lipoxygenases and patatin-like acyl hydrolases in Arabidopsis during cold acclimation, freezing, and thawing.

Doctor of Philosophy / Department of Biochemistry and Molecular Biophysics / Lawrence C. Davis / Synthetic dyes are capable of producing the whole color spectrum on account of their structural diversity but this diversity poses challenges in the degradation of dyeing wastes. Laccases and peroxidases from bacterial or fungal sources and parts of plants in the presence of hydrogen peroxide (H₂O₂) plus a mediator have been exploited in the bioremediation of synthetic dyes. However, intact plants have not found much favor despite their phytoremediation potential. The goal of this research was to further clarify ways by which whole plants bring about decolorization of different types of synthetic dyes. Hydroponically cultivated plants from two dicot families namely Arabidopsis thaliana and sunflowers (Helianthus annuus) were exposed to representative dyes from several classes: monoazo (Methyl Red and Methyl Orange), disazo (Trypan Blue, Evans Blue and Chicago Blue 6B), and arylmethane (Brilliant Blue G, Bromocresol Green, Malachite Green and Phenol Red). Tests were done in presence or absence of externally added H₂O₂, with or without a free radical mediator, 1-hydroxybenzotriazole, using UV-Visible spectrophotometry. The initial rate of decolorization and the overall percentage decolorization was calculated for each dye in the different treatments. Decolorization of the dyes from different classes varied between plant species and depending on the treatment. Except for Methyl Red, all dyes required added H₂O₂ as well as mediator to achieve rapid decolorization. Added H₂O₂ was found to be the limiting factor since it was degraded by plants within a few hours. Both species were able to slowly decolorize dyes upon daily addition of fresh dye even in the absence of added H₂O₂ and mediator, provided that nutrients were supplied to the plants with the dye. A. thaliana was found to be more effective in dye decolorization per gram tissue than sunflower when treated under similar conditions. Analysis of the residual dye solution by ESI/MS did not reveal any potential by-products following the decolorization treatment with plants, suggesting that the plant roots might be trapping the by-products of dye decolorization and preventing their release into the solution. All these findings support the potential application of whole plants for larger scale remediation.

Doctor of Philosophy / Department of Biochemistry / Subbarat Muthukrishnan / The extracellular matrices of cuticle and peritrophic matrix of insects are composed mainly of chitin complexed with proteins, some of which contain chitin-binding domains. This study is focused on the identification and functional characterization of genes encoding proteins that possess one or more copies of the six-cysteine-containing ChtBD2 domain (Peritrophin A motif =CBM_14 =Pfam 01607) in the red flour beetle, Tribolium castaneum. A bioinformatics search of T. castaneum genome yielded previously characterized chitin metabolic enzymes and several additional proteins. Using phylogenetic analyses, the exon-intron organization of the corresponding genes, domain organization of proteins, and temporal and tissue-specificity of expression patterns, these proteins were classified into three large families. The first family includes 11 proteins essentially made up of 1 to 14 repeats of the peritrophin A domain. Transcripts for these proteins are expressed only in the midgut and only during feeding stages of development. We therefore denote these proteins as “Peritrophic Matrix Proteins” or PMPs. The genes of the second and third families are expressed in cuticle-forming tissues throughout all stages of development but not in the midgut. These two families have been denoted as “Cuticular Proteins Analogous to Peritrophins 3” or CPAP3s and “Cuticular Proteins Analogous to Peritophins 1” or CPAP1s based on the number of ChtBD2 domains that they contain. Unlike other cuticular proteins studied so far, TcCPAP1-C protein is localized predominantly in the exocuticle and could contribute to the unique properties of this cuticular layer. RNA interference (RNAi), which down-regulates transcripts for any targeted gene, results in lethal and/or abnormal phenotypes for some, but not all, of these genes. Phenotypes are often unique and are manifested at different developmental stages, including embryonic, pupal and/or adult stages. The experiments presented in this dissertation reveal that while the vast majority of the CPAP3 genes serve distinct and essential functions affecting survival, molting or normal cuticle development. However, a minority of the CPAP1 and PMP family genes are indispensable for survival under laboratory conditions. Some of the non-essential genes may have functional redundancy or may be needed only under special circumstances such as exposure to stress or pathogens.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Timothy P. Durrett / The direct use of vegetable oils as a biofuel suffers from problems such as high viscosity, low volatility and poor cold temperature properties. 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) have lower viscosity and freezing temperature than regular vegetable oils. However, by modifying their fatty acid composition, further improvement in their fuel properties is possible. Our goal was to develop plants that synthesize seed oils with further improved fuel properties. Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes acetyl-TAGs by the acetyl-CoA dependent acylation of diacylglycerol (DAG). Knowledge of the substrate specificity of EaDAcT for its acetyl-CoA donor and DAG acceptor substrates is important to generate the required acetyl-TAG composition in seed oil. A rapid method to quantify acetyl-TAGs was developed based on electrospray ionization mass spectrometry to gain information about the substrate specificity of EaDAcT. This method is as accurate and more rapid than the traditional radiolabeled substrate based assay and additionally provides information on acetyl-TAG molecular species present. Using this assay, EaDAcT specificity for different chain length acyl-CoA and DAGs was tested. It was found that although EaDAcT can use other short chain length acyl-CoAs as acyl donors, it has high preference for acetyl-CoA. Further, EaDAcT can acetylate a variety of DAGs with short, medium and long chain length fatty acids with high preference for DAGs containing unsaturated fatty acids. To generate acetyl-TAGs with lower molecular mass, EaDAcT was transformed into transgenic Camelina sativa lines producing high amounts of medium chain fatty acids (MCFAs). EaDAcT expression was also combined with the knockdown of DGAT1 and PDAT enzymes, which compete with EaDAcT for their common DAG substrate. High acetyl-TAG yielding homozygous T3 transgenic lines were generated but the incorporation of MCFAs into acetyl-TAGs was inefficient. A small increase in the viscosity of acetyl-TAGs from these lines was observed compared to acetyl-TAGs produced in wild type Camelina plant. The combined effect of insufficient lowering of molecular mass and increased fatty acid saturation levels of acetyl-TAGs might be responsible for this increased viscosity. Overall, it was concluded that the molecular mass and the saturation levels of fatty acids of acetyl-TAGs need to be considered at the same time in future attempts to further decrease their viscosity.

Doctor of Philosophy / Department of Biochemistry / Lawrence C. Davis / The fate and bioavailability of organic contaminants in plants is a major ecological and human health concern. Current wet chemistry techniques that employ strong chemical treatments and extractions with volatile solvents, such as GC-MS, HPLC, and radiolabeling, although helpful, degrade plant tissue resulting in the loss of spatial distribution and the production of artifacts. Synchrotron radiation infrared microspectroscopy (SR-IMS) permits direct analysis of plant cell wall architecture at the cellular level in situ, combining spatially localized information and chemical information from the IR absorbances to produce a chemical map that can be linked to a particular morphology or functional group. This study demonstrated the use of SR-IMS to probe biopolymers such as cellulose, lignin, and proteins in the root tissue of hydroponically grown sunflower and maize plants as well as to determine the fate and effect of several organic contaminants in those root tissues. Principal components analysis (PCA), a data compression technique, was employed to reveal the major spectral variances between untreated and organic contaminant treated root tissues. Treatment with 1H-benzotriazole (BT) caused alterations to the lignin component in the root tissue of plants. The BT was found in xylem and epidermal tissue of sunflower plants but not associated with any particular tissue in maize roots. 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) altered the pectin and polysaccharide structure in both maize and sunflower. SR-IMS revealed the reduction of DNTs to their aromatic amine form in the vascular and epidermal tissues at low concentration. At high concentration, DNTs appeared to be associated with all the plant tissues in maize and sunflower. Exposure of sunflower and maize to 2,6-dichlorophenol (2,6-DCP) caused alterations to the polysaccharide and protein component of the root tissue. In some cases, phenolic compounds were observed in the epidermal tissue of maize and sunflower roots. The results of this research indicate that SR-IMS has the potential to become an important analytical tool for determining the fate and effect of organic contaminants in plants.

Doctor of Philosophy / Department of Biochemistry / Karl J. Kramer / Subbarat Muthukrishnan / Chitin, a linear homopolymer of beta-1,4-linked N-acetylglucosamine, is the second most abundant biopolymer next to cellulose. It is the major structural polysaccharide in the insect’s exoskeleton and gut lining. An extensive study of two of the major genes encoding enzymes involved in chitin metabolism, chitin synthases (CHSs) and beta-N-acetylglucosaminidases (NAGs), was undertaken. CHS genes from the tobacco hornworm, Manduca sexta, and NAG genes from the red flour beetle, Tribolium castaneum, were identified and characterized. In general, chitin deposition occurs in two major extracellular structures of insects, the cuticle that overlays the epidermis, and the peritrophic membrane (PM) that lines the midgut. Only two CHS genes were identified in M. sexta using Southern blot analysis. Extensive expression studies of both M. sexta CHS genes, MsCHS1 and MsCHS2, suggest a strict functional specialization of these two genes for the synthesis of epidermal and PM-associated chitin, respectively. Furthermore, two alternatively spliced transcripts of MsCHS1, MsCHS1a and MsCHS1b, were identified. Analysis of the levels of these transcripts in different tissues and stages of development indicated that the MsCHS1a transcript predominates in the integument during the feeding and pupal stages, whereas the MsCHS1b transcript is more abundantly present in the tracheae, foregut, and hindgut during all developmental stages tested. Four genes encoding putative NAGs (TcNAG1, TcNAG2, TcNAG3, and TcNAG4) were identified by searching the Tribolium genomic database. The full-length cDNAs for all four NAGs were cloned and sequenced, and the exon-intron organizations were determined. Studies on developmental expression patterns of each gene indicated that they are expressed during most developmental stages with TcNAG1 being the predominant one. The function of each NAG was assessed by down regulating the level of each transcript at various developmental stages using RNA interference. Selective knock down of each transcript, without significant reduction in the expression levels of the other NAG transcripts, was verified and the resulting phenotypes were documented. Knockdown of TcNAG1 interrupted larval-larval, larval-pupal, and pupal-adult molting, and the insects were unable to completely shed their old cuticles.