Production & analysis of cyclopropanated metabolites from Streptomyces species
NÝ Chadhain, Biology; Christy West, Chemical Engineering; Jim Davis, Chemistry

Bacteria of the genus Streptomyces are among nature’s most accomplished chemists. Bacterial secondary metabolites and their synthetic derivatives have served as a source for many of the antifungals, antimicrobiocidals and statins on the market today. Bacterial genome sequences have revealed that a single strain can contain biosynthetic pathways for as many as 30 different natural products. Synthesis of these molecules is usually encoded by polyketide synthase (PKS) or nonribosomal peptide synthetase (NRPS) cassettes. The focus of most studies is on bioactive molecules such as antibiotics, antifungals, pesticides etc. However, many of the molecules produced by secondary metabolite pathways are also of interest from a bioenergetic point of view, particularly those containing cyclopropanated moieties. Examples include the antifungal compound FR-900848 that is produced by Streptomyces fervens and contains 5 cyclopropane groups, and U-106305, which is produced by another Streptomyces species, which contains 6 cyclopropane rings. Due to the highly strained nature of the cyclopropane ring structure cycloproanated molecules can serve as high energy intermediates in metabolism or potentially as model biofuels. We will identify PKS pathways in the sequenced genomes of Streptomyces species. These pathways will be cloned into cosmid vectors in order to express the cloned genes and produce the cyclopropanated metabolites. Students will work in collaboration with Dr. Sinead Ni Chadhain (Dept of Biology), Dr. Kevin West (Dept of Chemical Engineering) and Dr. James H. Davis, Jr. (Dept of Chemistry to isolate and characterize these molecules in terms of their physical and chemical properties related to their use as biofuels. Measuring the thermophysical properties of these molecules (such as enthalpy of combustion, melting point, vapor pressure, density and viscosity) will allow for a throughout understanding of whether or not these compounds would be compelling candidates for use as biofuels.

Identification of fluorescent proteins in the pulmonary vasculature
Silas Leavesley, Chemical Engineering; Tom Rich, Pharmacology; and Diego Alvarez, Medicine

An ongoing challenge in fluorescence microscopy is detection of fluorescence signals in highly autofluorescent samples. It is even more challenging to quantitatively measure multiple fluorescence signals in the presence of high autofluorescence. We have previously developed a hyperspectral analysis approach to separate spectral components of specific fluorophores from sample autofluorescence. The effectiveness of this approach has been demonstrated by detecting the presence of fluorescently-labeled cells in highly autofluorescent lung tissue, due to proteins including elastin and collagen that fluoresce with specific spectral patterns, making it ideal for testing the strengths and limitations of hyperspectral imaging and analysis. Our previous results indicate that this approach can be used to detect fluorescent labels within a highly autofluorescence environment. We are currently optimizing this approach for the quantitative analysis of multiple fluorescent proteins, using both epifluorescent and confocal microscope systems, to estimate intracellular protein kinetics using F÷rster Resonance Energy Transfer (FRET). The objective of this study is multifold, incorporating biological fluorescent protein assay development, hardware optimization, analysis algorithm development, and software modeling. We are developing biological assays using a FRET reporter to assess the levels and subcellular distribution of cyclic adenosine monophosphate (cAMP) in cultured cells. The goal of these assays is to understand the influence of subcellular cAMP distribution on the endothelial barrier integrity. To facilitate these measurements, we are applying our hyperspectral imaging approach to quantitatively assess multiple fluorescent protein concentrations. This involves characterizing and optimizing widefield and confocal hyperspectral microscopy systems. Analyzing the resultant image data involves a combination of spectral analysis algorithms and quantitative image processing tools, to calculate and track subcellular FRET levels over time. Finally, we are combining this approach with a software model to understand the effects that a changing subcellular microenvironment may have on fluorescent protein properties – such as quantum yield – and the resultant effects on a dual fluorescent protein FRET reporter. Students participating in this research will be exposed to a combination of biological cell culture and transfection techniques (supervised by Drs. Alvarez and Rich), cell signaling theory (Alvarez and Rich), microscopy techniques (Leavesley and Rich), hyperspectral imaging techniques (Leavesley), quantitative and spectral image processing (Leavesley), and software modeling (Leavesley and Rich). Students will also be responsible for weekly progress updates and the presentation of their work at a summer research forum.

Regulating protein actions by the development of protein phosphatase
David Forbes, Chemistry; Richard Honkanen, Biochemistry and Molecular Biology

Numerous essential biological processes, including the regulation of transcription and mitosis require the protein kinase-mediated phosphorylation of target proteins at serine and threonine residues, which leads to activation, inactivation, degradation, or subcellular localization of the specific target. The actions of these kinases are opposed by the serine/threonine phosphoprotein phosphatases (PPPs). Although much is known about serine/threonine kinases, less data are available on the PPPs. One PPP-family phosphatase is protein phosphate 5 (PP5), a ubiquitously expressed enzyme with recently identified roles in stress-induced signal-transduction networks. Because purified full-length PP5 (499 residues) has low basal activity, it is believed that the catalytic activity and substrate specificity of PP5 depends upon formation of complexes with protein partners. Studies showing that PP5 is a negative regulator of Raf-1, forms heterocomplexes with the glucocorticoid receptor and the chaperone Hsp90, suggest that PP5 may play a role in cellular proliferation and stress-induced signaling pathways. However, the complete substrate profile of PP5 remains unknown. As a result, the identification of selective inhibitors of PP5 activity should be useful for elucidating PP5 action in eukaryotic cells, identifying PP5 binding partners, and for discovery of potential therapeutic agents for human cancers. To date, several natural compounds, which inhibit the activity of PP1, PP2A, and PP5 have been identified, including cantharidin, fostriecin, calyculin A, tautomycin, and okadaic acid. Based upon structural and computer modeling studies, a key relationship within the structural motif has been identified. Systems exhibiting inhibitory activity have as part of their structural motif oxygen functionality relatively disposed at the C-1 and C-4 positions. The development of a method, which rapidly and efficiently assembles a host of novel drug candidates while preserving this key relationship, is ideal. Using cantharidin as a model of a core-inhibitor, modifications using well-established techniques will allow the productions of novel compounds that will be tested for inhibitory activity against PP5. Computer models of binding will also assist in the design and development of PP5 specific inhibitors. Students will work in conjunction with Dr. Forbes (Chemistry) and Dr. Honkanen (Biochemistry and Molecular Biology) on both the laboratory and modeling components of this project

Thermoregulatory responses and heat shock proteins
David Nelson, Mechanical Engineering; Boettcher*, Biology

Radiofrequency radiation, specifically in the millimeter wave range at 35 GHz, has a penetration depth that will reach into the vascular bed of skin tissue where skin blood flow is present. When the radiofrequency radiation happens upon the skin tissue, it is absorbed and presents as heat. In response to this heat, the thermoregulatory systems in the skin tissue increase the blood flow to that area. We would like to show that millimeter waves would provide us with a way of easily assessing skin blood flow rates. The long-term goal of this study is to develop a method of measuring skin blood flow rates based on a model created by examining the heating of the skin tissues with RF radiation at 35 GHz with "blood flow" present in the tissue and to assess the impact of this method in terms of stress response of the tissue. REU students will collaborate with Dr. Nelson (Mechanical Engineering) and Dr. Boettcher (Biology) on the development and assessment of methods for skin blood flow rates.

Tissue engineering of contractile small-caliber blood vessels
Saami Yazdani, Mechanical Engineering; Diego Alvarez, Medicine

Diseases of small and medium caliber arteries account for the majority of deaths in the western countries each year. Over 500,000 coronary bypass grafts and 50,000 peripheral bypass grafts are performed annually in the US alone. However, up to 30% of the patients who require arterial bypass surgery lack suitable autologous conduits such as small caliber arteries or saphenous veins, which remain the gold standard tissue for coronary bypass surgery. Synthetic grafts, such as polytetrafluoroethylene (PTFE) or Dacron have been used successfully to bypass large caliber high-flow blood vessels.  However, these grafts invariably fail when used to bypass small-caliber (low flow blood) vessels due to increased thrombogenicity and accelerated intimal thickening resulting in early graft stenosis and occlusion.  Therefore, the goal of this project is to develop a small-caliber tissue engineered graft by seeding endothelial cells onto the lumen of a decellularized porcine artery and developing methods to re-populate the media with smooth muscle cells .  Endothelial cell and smooth muscle cell functions will be assessed by endothelial nitric oxide synthase (eNOS) and smooth muscle actin proteins, which are involved in cell motility, structure and function.  Students will work in conjunction with Dr. Yazdani (Mechanical Engineering) and Dr. Alvarez (Medicine) to culture cells, develop bioreactors and perform cell seeding and analysis.

Toxicity: Understanding Nanoparticle Fate and Protein Interactions
Andrew Whelton, Civil Engineering; Sean Powers, Marine Sciences; and Anne Boettcher, Marine Biology

 Poly(amidoamine) (PAMAM) dendrimers are nanoscale materials being explored for a variety of biomedical applications and their toxicity has investigated in vivo. Dendrimers are being pursued for vehicles that enable targeted drug delivery, controlling the therapeutic release rate. The binding mechanisms between the drug and delivery vehicle (and the vehicle’s toxicity) are critically important in technology development. In addition to their medical applications, PAMAM materials are also being investigated as nontoxic oil spill dispersant chemicals to apply following large scale incidents such as the Deepwater Horizon and Exxon Valdeez. As dispersants however, environmental fate and aquatic toxicity of PAMAM dendrimers has gone relatively unstudied. This project will examine the role of functional group and size (G3-G6) on dendrimer environmental fate. Lethal and sub-lethal effects will be assessed using LC-50 toxicity assays and biomarker analyses.  Lessons learned from this project will have direct relevance to their fate in the environment.

Proteomic Detection Methods
Lewis Pannell, Mitchell Cancer Institute

The proteomics group of the Mitchell Cancer Institute works on methods to identify diseases at an early stage when they are more treatable.  While they have multiple early detection projects in cancer, two potential projects for the summer program involve the detection of diseases in neonatal and early childhood development.  This research is in association with the USA Children and Women’s Hospital adjacent to the MCI.  Samples from patients will be prepared for analysis and processed using the equipment within the proteomics facility, including some statistical analysis of the results.  The group is highly interactive and the student will be exposed to, and help with, multiple projects within the group.