Mitochondria: Bioenergetics and Predictive Therapeutics Using Vibrational Spectroscopy

Biomedical research comprises one of the most important and well-funded areas in all of scientific research. Technological innovation is a relatively small component of all research overall, but the applications are of immense benefit. Incorporation of already existing technologies from other areas of research directly into a biomedical setting is a simple way to leverage human innovation into the highest applicability. Vibrational spectroscopy is one such technology which, while steadily seeing more and more use in biomedical imaging, is still underutilized. Such advanced microspectroscopic techniques can leverage their ability to analyze global information in a cellular system to higher throughput and increased efficiencies. Metabolism is a key global system for every cell type and organism. Energy production, metabolic pathway augmentation, metabolite analysis, and appraisal of overall metabolic poise are several ways in which characterization of metabolism can be used analytically. Each of these can provide valuable insights into cellular systems and associated conditions, namely illness and disease. Using Raman spectroscopy to characterize metabolism has led to several interesting results. First, analysis of drug candidacy has shown a novel mode for drug testing. Effectively evaluating cellular health based on several different techniques shows level compatibility across the techniques employed. Localized spectroscopic analysis provides further evidence for mitochondrial protection conferral. In a similar vein of research, targeted analysis of desiccation tolerant proteins, localized to mitochondria, likewise showed a protective mechanism. Finally, an analysis of substrate change directly on metabolism was carefully evaluated, with an outcome of visualizing effects of varying metabolic pathways upon overall global cellular metabolism. These studies provide a strong platform for spectrometabolic evaluation to be considered for a variety of advanced characterization techniques. Coupling this method with other emerging technologies has the potential to shift the standards for metabolic evaluation.Ph.D.Mechanical Engineering, College of Engineering & Computer ScienceUniversity of Michigan-Dearbornhttp://deepblue.lib.umich.edu/bitstream/2027.42/168173/1/Jason Solocinski - final dissertation.pdfDescription of Jason Solocinski - final dissertation.pdf : Dissertatio

Development of Lyoprocessing Technique to Enhance Biological Storage Outcomes

Dry state preservation at ambient temperatures (lyopreservation) is a biomimetic alternative to low temperature stabilization (cryopreservation) of biological materials. Lyopreservation is hypothesized to rely upon the creation of a glassy environment, which is commonly observed in desiccation-tolerant organisms. Non-uniformities in dried samples have been indicated as one of the reasons for instability in storage outcomes. The current study presents a simple, fast, and uniform surface tension based technique that can be implemented for lyopreservation of mammalian cells. The technique involves withdrawing cells attached to rigid substrates to be submerged in a solution of lyoprotectant and then withdrawing the samples at a specific rate to an inert environment. This creates a uniform thin film of desiccated lyoprotectant. The residual moisture contents at different locations in the desiccated film is quantified using a spatially resolved Raman microspectroscopy technique. Post-desiccation cellular viability and growth is quantified using fluorescent microscopy and dye exclusion assays. Cellular injury following desiccation is evaluated by bioenergetic quantification of metabolic functions using extracellular flux analysis and by a Raman microspectroscopic analysis of change in membrane structure. The technique developed here addresses an important bottleneck of lyoprocessing which requires the fast and uniform desiccation of cellular samples.Master of Science in EngineeringMechanical Engineering, College of Engineering & Computer ScienceUniversity of Michigan-Dearbornhttps://deepblue.lib.umich.edu/bitstream/2027.42/143527/1/MasterThesis_Solocinski2.pdfDescription of MasterThesis_Solocinski2.pdf : Thesi

Effect of trehalose as an additive to dimethyl sulfoxide solutions on ice formation, cellular viability, and metabolism.

Cryopreservation is the only established method for long-term preservation of cells and cellular material. This technique involves preservation of cells and cellular components in the presence of cryoprotective agents (CPAs) at liquid nitrogen temperatures (−196 °C). The organic solvent dimethyl sulfoxide (Me2SO) is one of the most commonly utilized CPAs and has been used with various levels of success depending on the type of cells. In recent years, to improve cryogenic outcomes, the non-reducing disaccharide trehalose has been used as an additive to Me2SO-based freezing solutions. Trehalose is a naturally occurring non-toxic compound found in bacteria, fungi, plants, and invertebrates which has been shown to provide cellular protection during water-limited states. The mechanism by which trehalose improves cryopreservation outcomes remains not fully understood. Raman microspectroscopy is a powerful tool to provide valuable insight into the nature of interactions among water, trehalose, and Me2SO during cryopreservation. We found that the addition of trehalose to Me2SO based CPA solutions dramatically reduces the area per ice crystals while increasing the number of ice crystals formed when cooled to −40 or −80 °C. Differences in ice-formation patterns were found to have a direct impact on cellular viability. Despite the osmotic stress caused by addition of 100 mM trehalose, improvement in cellular viability was observed. However, the substantial increase in osmotic pressure caused by trehalose concentrations above 100 mM may offset the beneficial effects of changing the morphology of the ice crystals achieved by addition of this sugar

Dietary reference values for sodium

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) derived dietary reference values (DRVs) for sodium. Evidence from balance studies on sodium and on the relationship between sodium intake and health outcomes, in particular cardiovascular disease (CVD)-related endpoints and bone health, was reviewed. The data were not sufficient to enable an average requirement (AR) or population reference intake (PRI) to be derived. However, by integrating the available evidence and associated uncertainties, the Panel considers that a sodium intake of 2.0 g/day represents a level of sodium for which there is sufficient confidence in a reduced risk of CVD in the general adult population. In addition, a sodium intake of 2.0 g/day is likely to allow most of the general adult population to maintain sodium balance. Therefore, the Panel considers that 2.0 g sodium/day is a safe and adequate intake for the general EU population of adults. The same value applies to pregnant and lactating women. Sodium intakes that are considered safe and adequate for children are extrapolated from the value for adults, adjusting for their respective energy requirement and including a growth factor, and are as follows: 1.1 g/day for children aged 1\u20133 years, 1.3 g/day for children aged 4\u20136 years, 1.7 g/day for children aged 7\u201310 years and 2.0 g/day for children aged 11\u201317 years, respectively. For infants aged 7\u201311 months, an Adequate Intake (AI) of 0.2 g/day is proposed based on upwards extrapolation of the estimated sodium intake in exclusively breast-fed infants aged 0\u20136 months

Imaging of Scleral Collagen Deformation Using Combined Confocal Raman Microspectroscopy and Polarized Light Microscopy Techniques.

This work presents an optospectroscopic characterization technique for soft tissue microstructure using site-matched confocal Raman microspectroscopy and polarized light microscopy. Using the technique, the microstructure of soft tissue samples is directly observed by polarized light microscopy during loading while spatially correlated spectroscopic information is extracted from the same plane, verifying the orientation and arrangement of the collagen fibers. Results show the response and orientation of the collagen fiber arrangement in its native state as well as during tensile and compressive loadings in a porcine sclera model. An example is also given showing how the data can be used with a finite element program to estimate the strain in individual collagen fibers. The measurements demonstrate features that indicate microstructural reorganization and damage of the sclera's collagen fiber arrangement under loading. The site-matched confocal Raman microspectroscopic characterization of the tissue provides a qualitative measure to relate the change in fibrillar arrangement with possible chemical damage to the collagen microstructure. Tests and analyses presented here can potentially be used to determine the stress-strain behavior, and fiber reorganization of the collagen microstructure in soft tissue during viscoelastic response