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The overall focus of this project has two parts: First, was to develop a protocol utilizing serum-free media formulations and defined plating and culture techniques to create functional in vitro myotubes derived from adult skeletal muscle satellite cells. The second was to manipulate the inherent muscle parameters such as force output and fatigue of these myotubes by employing exercise regimes or by small molecule application. The importance of serum-free medium use for in vitro cultures is becoming increasingly important in creating functional systems that can be validated for drug testing by the Food and Drug Administration (FDA). Also, the study of age related diseases as well as the potential for “personalized medicine” relies on the proliferation and maturation of satellite cells from adult derived tissue. For that purpose, a serum-free medium and culture system was designed to create mature striated myotubes in culture on a defined non-biological substrate N-1[3-trimethoxysilyl propyl] diethylenetriamine (DETA). These myotubes were evaluated by morphology, muscle specific protein expression, and by muscle functionality. After the thorough characterization of the resultant myotubes the functional output of the muscle was altered utilizing chemical means (creatine supplementation and PGC-1? agonists), chronic long term stimulation, and the use of PGC-1? deficient tissue. In this thesis presentation the utility of the newly developed medium formulation to create myotubes from a variety of adult derived muscle sources will be shown. A protocol in which to exercise skeletal muscle in vitro to alter endurance was developed and employed to manipulate skeletal muscle. Finally, small molecules were tested to validate this system for drug study use. This engineered system has the potential for high-throughput screening of drugs for efficacy and drug toxicity studies as well as general biological studies on muscle fatigue.

By a tracer study, using ⁸²Br, it is demonstrated that bromine is bound to serum proteins in vivo. ⁸²Br⁻ of high specific activity was injected into rabbits and serum removed one day later. Approximately ½% of the total ⁸²Br in the serum was found to be protein-bound at this stage. The application of various separation methods (electrophoresis, bromide exchange, denaturation followed by desalting) showed that one-third of the protein-bound bromine is loosely attached whereas two-thirds are firmly bound. After partial and complete enzymatic hydrolysis the bromine was found in the amino acid fraction. On the basis of the elution pattern of the amino acids on calibrated cation exchange resin columns it is concluded that the main portion of the radioactivity appeared to be associated with 3-bromo-L-tyrosine. Little, if any, bromine was observed in the serum lipids and in the thyroxine fraction isolated from serum proteins. / Thesis / Doctor of Philosophy (PhD)

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

The purpose of these studies was to determine if an immunomodulator was present in caprine serum. Controlled studies demonstrated that CSF-I, material fractionated from caprine serum possessed an immunomodulatory compound. Caprine serum was further fractionated into it?s peptidic components and a small contaminant of immunoglobulin G and albumin (Caprine serum fraction - immunomodulator 2, or CSF-I2). This was refined to a three peptidic isolate collectively identified as tri-peptidic immunostimulant or TPI. CSF-I2 does not possess antibacterial capabilities (as typically characteristic of a cationic peptide or defensin), does not contain a level of endotoxin sufficient to promote a pyrogenic response, and its functional ability to improve animal survival after an infectious challenge does not reside with molecular weight components greater than 10 kilodaltons, effectively excluding the immunoglobins, albumin, cytokines, and collectins. CSF-I2 was able to significantly reduce the mortality observed in chickens (from 80% to 13%) infected with Pasteurella multocida (Willeford et al., 2000), in mice (from 83% to 13.3%) infected with Salmonella typhimurium, and in canines (from 50% to 9.8%) diagnosed with parvovirus. CSF-I2 may well prove to provide prophylactic and therapeutic health benefits to humans. CSF-I2 may effectively combat pathogenesis when used as either an adjunct with conventional therapy (e.g., antibiotics) or when provided as the primary medicant.

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No description available.

No description available.

Amyloidogenesis refers to a process of protein misfolding and aggregation that leads to the formation of highly stable amyloid fibers. Amyloidogenesis may lead to loss of physiological protein function and/or formation of toxic intermediates, which are linked to mutliple human diseases. Amyloidogenesis is inhibited by plasma proteins, which function as extracellular chaperones by binding to stressed and misfolded proteins, including amyloidogenic peptides, and preventing their aggregation. This thesis focuses on the ability of human serum albumin (HSA), the main protein in human plasma, to inhibit amyloidogenesis, with emphasis on the molecular nature of the interactions between HSA and the amyloid β peptide (Aβ) associated with Alzhemier’s disease. HSA is as a key amyloidogenic regulator, a novel function for this protein that goes beyond the traditional HSA roles as plasma osmotic pressure regulator and as binder and carrier of endogenous and exogenous low molecular weight ligands. As a first step towards understanding the detailed molecular nature of these interactions, this thesis will focus on defining the key binding determinants in the interaction between HSA and Aβ peptides. Primarily, we will try to answer two main questions. First, which HSA residues are critical for the recognition of Aβ peptides and the prevention of Aβ aggregation? Second, which Aβ residues are mostly affected by HSA binding? Starting form our knowledge about the stoichiometry and affinity of the Aβ interactions at the level of HSA domains, Chapter 2 addresses the first question through successful applications of a reductionist approach, based on a combination of mutational comparative analyses and fatty acid (FA) competition. This strategy allowed us to identify a short HSA derived peptide that specifically recognizes Aβ and prevents its aggregation. In Chapter 3, we examine the effect of HSA on the pseudo-equilibrium state between Aβ monomers and protofibrils. Using Dark state Exchange Saturation Transfer (DEST), Saturation Transfer Difference (STD) and 15N T2 relaxation experiments, we show that Aβ peptides interact with HSA via a dual mechanism. First, selected residues in Aβ (1-40) monomers bind specifically but weakly to HSA (Kd = 0.1 - 1 mM). Second, HSA competes with Aβ monomers for the binding to the protofibrils, as indicated by an HSA-dependent decrease in the direct vs. tethered probabilities for contacts between Aβ monomer residues and the protofibril surface. The effect of HSA mimics that of dilution for the majority of the Aβ (1-40) residues involved in the cross-beta strands of amyloid fibrils. Finally, Chapter 4 will outline future investigations to address currently open questions about HSA dynamics, HSA-Aβ and HSA-FA interactions, for which we acquired preliminary data. / Thesis / Master of Science (MSc)

Biofouling is an ongoing problem in the development and usage of biomaterials for biomedical implants, microfluidic devices, and water-based sensors. Antifouling coatings involving surface modification of biomaterials is widely utilized to reduce unwanted protein adsorption and cell adhesion. Surface modification strategies, however, are reliant on the working material’s chemical properties. Thus, published procedures are often not applicable to a wide range of material classes. This constitutes a serious limitation in using surface modification on assembled multi-material devices, i.e on whole device modification. The objective of this research is to develop an antifouling coating with non-aggressive reaction conditions that can universally modify polymers and other material classes. Two strategies using polydopamine (PDA) as an anchor for polyethylene glycol (PEG) surface attachment were investigated: (1) PDA-PEG backfilled with bovine serum albumin (BSA), and (2) PDA-PEG with light activated perfluorophenyl azide (PFPA) conjugated to the PEG. Three materials varying in surface wettability were studied to evaluate the coatings for multi-material applications: porous polycarbonate membrane (PC), polydimethyl siloxane (PDMS), and soda lime glass cover slips. Atomic force microscopy (AFM) and ellipsometry studies revealed substantial structural differences of PDA. Differences in PDA surface roughness affected PEG grafting in solution (the first method), with higher PEG coverage achieved on PC with intermediate surface roughness to PDMS and glass. Radiolabeled Fg adsorption and E. coli adhesion experiments showed reduced fouling on all PDA-PEG modified materials when backfilled with BSA. The ability for BSA to penetrate the PEG layer indicated that low PEG grafting densities were achieved using this grafting-to approach. The use of a photoactive labeling agent, PFPA, to tether PEG was proposed to improve PEG grafting on PDA. The PFPA-PEG modification protocol was optimized by quantifying Fg adsorption. Two treatments of PFPA-PEG were required to fully block PDA active sites. Fg adsorption was not significantly improved on PFPA-PEG modified PC and glass when backfilled with BSA, indicating sufficient PEG coverage of PDA. High Fg adsorption on PFPA-PEG surfaces indicate that high density PEG brushes were still not achieved with this method. PDMS surfaces were damaged with this procedure due to increased surface handling in the protocol. This is the first, to our knowledge, successful demonstration of PFPA modification on PDA surfaces. Photopatterning of polymer-based materials can be achieved, providing opportunities for utilising new materials in cell patterned platforms. Due to low PEG coverage on PDA surfaces from solution and using PFPA, ultra-low protein adsorption cannot be achieved using these aqueous-based methods. Antifouling modifications using PDA and PEG should be applied for short-term cell studies. / Thesis / Master of Applied Science (MASc)