In this study, we design different inscribed/circumscribed circular torus-type microchannels to investigate the stretching behavior of DNA molecules. Strain rate and relaxation time play an important role in DNA stretching. In order to perform an analysis of the coil-stretch transition of DNA, we develop a method of stretching DNA molecules by using £gPIV and CLSM measurements. £gPIV is designed to measure the velocity distribution, after which the local strain rate can be estimated. The hydrodynamic stretching of DNA molecules in the elongation flow is observed using a confocal laser scanning microscope (CLSM). The relaxation time of the DNA molecules is then estimated according to the CLSM images analysis. At present, our experiments using the electro-osmotic flow (EOF) driven at various electric fields and viscosities to stretch DNA molecules show how one can investigate the influence of hydrodynamic interactions in the case of stretching of DNA molecules.

The objectives of this study were to develop an in vitro direct adventitious bud induction and an organogenic callus induction and shoot regeneration system via floral organ segments culture for bromeliads, moreover, explore the effect of auxin on plantlet elongation of Guzmania. And further, apply the above micropropagation system to physical and chemical methods to induce somaclonal variances of bromeliad plantlets in vitro for mutation breeding. The explant sources of bromeliads and the components of culture medium were studied to develop a micropropagation system for bromeliads. The results indicated that the 1/3MS basal medium supplemented with a combination of 1.0 mg l-1 BA + 0.5-1.0 mg l-1 NAA, or a combination of 3.0 mg l-1 BA + 0.5 mg l-1 NAA, showed the highest frequency of direct initiation of adventitious buds derived from shoot apex and lateral bud explants of Aechmea fulgens var. fulgens and Guzmania 'Focus'. The best results of adventitious buds induction of the both species were found in the lower lateral bud explants, at 47.5% and 35%, respectively. In addition, the adventitious buds began to form on day 16 after the G. 'Focus' decapitated plantlets had been cultured in medium supplemented with 3.0 mg l-1 BA + 0.5 mg l-1 NAA. However, this phenomenon did not occur in case of undecapitated explants, where only protruding nodules appeared. Petal- and ovary-derived calli of A. fasciata and G. 'Hilda' were induced on 1/2MS basal medium supplemented with 1.0-1.5 mg l-1 2,4-D in combination with 1.0 or 0.5 mg l-1 NAA. Organogenic calli were cultured on medium with 1.0 mg l-1 NAA and 0.5 mg l-1 TDZ could be induced to differentiate and regenerate the adventitious buds. Furthermore, the number of adventitious buds proliferating at the base of the plantlets derived from G. 'Hilda' floral organs, cultured in media with different concentrations of IAA, IBA, NAA, and 8-azaadenine, was only 1-2 adventitious buds individually. This result shows that auxin can indeed suppress cytokinin-effects. The influence on plantlet elongation was greatest in the treatments using 0.5 mg l-1 NAA and 1.0 mg l-1 NAA. After 4 months culture, plantlets grew to 5.73 and 5.62 cm in height, that was 2.22 and 1.95 cm higher than the control, respectively. Plantlets of A. fasciata hardened under the middle (50 £gmol m-2s-1) light intensity condition had a higher survival rate, 95%, than that hardened at a low light intensity (1 £gmol m-2s-1; 17.5%). The maximum number of newly developing roots, up to 4.15 per shoot, was also observed at the same light intensity treatment. During transplantation, plantlets growing in coir fiber showed the best results in terms of plant growth within 6 months ex vitro culture. The average length of the plantlets was 22.0 cm, and an average of 19.3 leaves per plantlet was achieved. When calli of G. 'Hilda' treated by sodium azide, the survival rate was 0%. The survival rate of decapitated plantlet explants treated with 0.5 mM sodium azide for 60 minutes was 51.3%, about half-lethal dose. In addition to the survival rates of decapitated plantlet explants of A. fasciata, G. 'Hilda', G. 'Cherry', G. 'Luna' and G. 'Focus' irradiated by £^-ray showed 74.2-100% with the exception of the G. 'Focus' irradiated by 15 Gy, which dropped to 45.0%. At present, mutant plantlets showed a great deal of chimeras in leaf and were transplanted to potting media.

The GTPase elongation factor G (EF-G) is an important player in the complex process of protein synthesis by bacterial ribosomes. Although extensively studied much remains to be learned about this fascinating protein. In the elongation phase, after incorporation of each amino acid into the growing peptide chain, EF-G translocates the ribosome along the mRNA template. In the recycling phase, when the synthesis of a protein has been completed, EF-G, together with ribosome recycling factor (RRF), splits the ribosome into its subunits. We developed the first in vitro assay for measuring the average time of a complete translocation step at any position along the mRNA. Inside the open reading frame, at saturating EF-G concentration and low magnesium ion concentration, translocation rates were fast and compatible with elongation rates observed in vivo. We also determined the complete kinetic mechanism for EF-G- and RRF-dependent splitting of the post-termination ribosome. We showed that splitting occurs only when RRF binds before EF-G and that the rate and GTP consumption of the reaction varies greatly with the factor concentrations. The antibiotic fusidic acid (FA) inhibits bacterial protein synthesis by binding to EF-G when the factor is ribosome bound, during translocation and ribosome recycling. We developed experimental methods and a theoretical framework for analyzing the effect of tight-binding inhibitors like FA on protein synthesis. We found that FA targets three different states during each elongation cycle and that it binds to EF-G on the post-termination ribosome both in the presence and absence of RRF. The stalling time of an FA-inhibited ribosome is about hundred-fold longer than the time of an uninhibited elongation cycle and therefore each binding event has a large impact on the protein synthesis rate and may induce queuing of ribosomes on the mRNA. Although ribosomes in the elongation and the recycling phases are targeted with similar efficiency, we showed that the main effect of FA in vivo is on elongation. Our results may serve as a basis for modelling of EF-G function and FA inhibition inside the living cell and for structure determination of mechanistically important intermediate states in translocation and ribosome recycling.

The length of the primary root and the density of lateral roots determine the architecture of the root. In this thesis the effect of NAA, ABA and the NO donor SNP alone as well as the combination of ABA or NAA with SNP on lateral root development was investigated. The interaction between CPTIO, a NO scavenger, and NAA or SNP is also reported. Following preliminary experiments in which it was observed that the aerial part of the seedling influenced LR growth and that there was a possible inhibitory effect of light on cultured root tips, experiments were conducted with excised roots tips in the dark. NAA was shown to have the potential to initiate LRs across a wide concentration gradient with the total number of LRs and initiated lateral root primordia (LRP) remaining constant across the range of concentrations tested. Over the last decade, nitric oxide (NO), a bioactive molecule, has been reported to be involved in the regulation of many biological pathways. The presence of NO in the system provided via sodium nitroprusside (SNP), promoted LRP initiation based on the NAA concentration gradient; but without changing the total LR initiation, that is LRs plus primordia density remained constant along the concentration gradient of NAA. The absence of LR and LRP in the treatments of CPTIO (a NO scavenger) with SNP or NAA suggests that NO regulates LRP initiation triggered by NAA, which is in agreement with the recent paper published after the commencement of this study (Correa-Aragunde et al., 2006). In agreement with previous studies, ABA inhibited lateral root development by reducing LR density and the number of LRs. The experiments with fluridone, an ABA biosynthesis inhibitor, may indicate that endogenous ABA was at sufficient concentrations in the excised root tips to inhibit primordia initiation. In this study, evidence is presented for the first time to show that SNP can relieve the inhibitory effect of ABA on LR density and number of LRs suggesting the NO, released from SNP, acts downstream of ABA. Overall these data confirm a critical role for NO in LR initiation.

abstract: This research explores microbial chain elongation as a pathway for production of complex organic compounds in soils with implication for the carbon cycle. In chain elongation, simple substrates such as ethanol and short chain carboxylates such as acetate can be converted to longer carbon chain carboxylates under anaerobic conditions through cyclic, reverse β oxidation. This pathway elongates the carboxylate by two carbons. The chain elongation process is overall thermodynamically feasible, and microorganisms gain energy through this process. There have been limited insights into the versatility of chain elongating substrates, understanding the chain elongating microbial community, and its importance in sequestering carbon in the soils. We used ethanol, methanol, butanol, and hydrogen as electron donors and acetate and propionate as electron acceptors to test the occurrence of microbial chain elongation in four soils with different physicochemical properties and microbial communities. Common chain elongation products were the even numbered chains butyrate, caproate, and butanol, the odd numbered carboxylates valerate and heptanoate, along with molecular hydrogen. At a near neutral pH and mesophilic temperature, we observed a stable and sustained production of longer fatty acids along with hydrogen. Microbial community analysis show phylotypes from families such as Clostridiaceae, Bacillaceae, and Ruminococcaceae in all tested conditions. Through chain elongation, the products formed are less biodegradable. They may undergo transformations and end up as organic carbon, decreasing the greenhouse gas emissions, thus, making this process important to study. / Dissertation/Thesis / Masters Thesis Chemical Engineering 2018

The increasing applications of different nanomaterials in the myriad of nano-enabled products and their potential for leaching have raised considerable environmental, health and safety (EHS) concerns. As systematic studies investigating potential anomalies in the morphology and anatomy of crop plants are scarce, herein we report on the developmental responses of two agriculturally significant crop plants, maize (Zea mays L.) and cabbage (Brassica oleracea var. capitata L.), upon in vitro exposure to nanoparticles of citrate-coated silver (Citrate-nAg) and zinc oxide (nZnO). Analyses involve histology of the primary root morphology and anatomy using light microscopy, metal biouptake, moisture content, rate of germination, and root elongation. Comparative toxicity profiles of the ionic salts (AgNO3 and ZnSO4) are developed. Notably, we uncover structural changes in maize primary root cells upon exposure to Citrate-nAg, nZnO, AgNO3, and ZnSO4, possibly due to metal biouptake, suggesting potential for functional impairments in the plant growth and development. Citrate-nAg exposure results in lower Ag biouptake compared to AgNO3 treatment in maize. Microscopic evidence reveals 'tunneling-like effect' with nZnO treatment, while exposure to AgNO3 leads to cell erosion in maize root apical meristem. In maize, a significant change in metaxylem count is evident with Citrate-nAg, AgNO3, and ZnSO4 treatment, but not with nZnO treatment (p>0.1). In both maize and cabbage, measures of germination and root elongation reveal lower nanoparticle toxicity compared to free ions. As moisture data do not support osmotically-induced water stress hypothesis for explaining toxicity, we discuss other proximate mechanisms including the potential role of growth hormones and transcription factors. These findings highlight previously overlooked, anatomically significant effects of metal nanoparticles, and recommend considering detailed anatomical investigations in tandem with the standard developmental phytotoxicity assays (germination and root elongation) as the latter ones appear less sensitive for screening plant responses to nanomaterial insults.

The increasing applications of different nanomaterials in the myriad of nano-enabled products and their potential for leaching have raised considerable environmental, health and safety (EHS) concerns. As systematic studies investigating potential anomalies in the morphology and anatomy of crop plants are scarce, herein we report on the developmental responses of two agriculturally significant crop plants, maize (Zea mays L.) and cabbage (Brassica oleracea var. capitata L.), upon in vitro exposure to nanoparticles of citrate-coated silver (Citrate-nAg) and zinc oxide (nZnO). Analyses involve histology of the primary root morphology and anatomy using light microscopy, metal biouptake, moisture content, rate of germination, and root elongation. Comparative toxicity profiles of the ionic salts (AgNO3 and ZnSO4) are developed. Notably, we uncover structural changes in maize primary root cells upon exposure to Citrate-nAg, nZnO, AgNO3, and ZnSO4, possibly due to metal biouptake, suggesting potential for functional impairments in the plant growth and development. Citrate-nAg exposure results in lower Ag biouptake compared to AgNO3 treatment in maize. Microscopic evidence reveals 'tunneling-like effect' with nZnO treatment, while exposure to AgNO3 leads to cell erosion in maize root apical meristem. In maize, a significant change in metaxylem count is evident with Citrate-nAg, AgNO3, and ZnSO4 treatment, but not with nZnO treatment (p>0.1). In both maize and cabbage, measures of germination and root elongation reveal lower nanoparticle toxicity compared to free ions. As moisture data do not support osmotically-induced water stress hypothesis for explaining toxicity, we discuss other proximate mechanisms including the potential role of growth hormones and transcription factors. These findings highlight previously overlooked, anatomically significant effects of metal nanoparticles, and recommend considering detailed anatomical investigations in tandem with the standard developmental phytotoxicity assays (germination and root elongation) as the latter ones appear less sensitive for screening plant responses to nanomaterial insults.

The knee is the most complex joint in the human body. It consists of a system of muscle, bone, and ligaments that endures repetitive loading during daily and athletic activities. When this loading is excessive, damage  to the knee occurs leading to a decreased quality of life.The medial collateral ligament (MCL) is one of the 4 major ligaments known to be commonly injured in the knee. The risk of injury to the knee joint increases with the elderly and individuals who experience chronic dehydration. For this reason, the focus of this study is to compare different mechanical quantities that can be used to analyze damage to the MCL. In this study, a novel mechanical testing protocol is used to progressively induce damage in dehydrated rat MCLs by performing tensile tests. This involves stretching the ligaments along their longitudinal axes to consecutive and increasing displacements starting at a 0.4 mm displacement and in increments of 0.2 mm until complete failure occurs. The load and change in length that the ligament experiences are measured at each displacement. Three different methods were evaluated to determine subfailure and damage propagation in rat MCLs: changes in tangent stiffness and chord stiffness, and changes in the load value at the 0.4 mm displacement for each load-displacement curve. The findings of this study indicate that the tangent stiffness and load at the 0.4 mm displacement provide information of the early onset of damage propagation. The decrease in chord stiffness of the ligament does not indicate damage progression in the ligament, but rather is the sign of the imminent failure of the MCL.This study provides insightful data into understanding the subfailure damage in the MCL. / Master of Science

Indiana University-Purdue University Indianapolis (IUPUI) / Cell shape changes and signaling pathways are essential for the development and function of the lens. During lens development proliferating epithelial cells will migrate down to the equator of the lens, differentiate into lens fiber cells, and begin to elongate along the lens capsule. The Fibroblast Growth Factor (FGF) signaling pathway has been extensively studied for its role in lens fiber cell differentiation and elongation. However, the main mediators of FGF stimulated lens fiber cell elongation have not been identified. Adaptor proteins Crk and CrkL are SH2- and SH3-containing proteins that transduce signals from upstream tyrosine phosphorylated proteins to downstream effectors, including Ras, Rac1 and Rap1, which are important for cell proliferation, adhesion and migration. Underlying their diverse function, these two adaptor proteins have been implicated in receptor tyrosine kinase signaling, focal adhesion assembly, and cell shape. To explore the role of Crk and CrkL in FGF signaling-dependent lens development and fiber elongation, we employed Cre/LoxP system to generate a lens specific knockout of Crk/CrkL. This led to extracellular matrix defects, disorganization of the lens fiber cells, and a defect in lens fiber cell elongation. Deletion of Crk and CrkL in the lens also mitigated the gain-of-function phenotype caused by overexpression of FGF3, indicating an epistatic relationship between Crk/CrkL and FGF signaling during lens fiber cell elongation. Further studies, revealed that the activity of Crk and CrkL in FGF signaling is controlled by the phosphatase Shp2 and the defect observed in lens fiber cell elongation can be rescued by constitutive activation of the GTPases Ras and Rac1 in the Crk and CrkL mutant lens. Interestingly, the deletion of the GTPases Rap1 in the lens showed no obvious phenotype pertaining to lens fiber cell elongation. These findings suggest that Crk and CrkL play an important role in integrating FGF signaling and mediating lens fiber cell elongation during lens development.

No description available.