National Aeronautics and Space Administration (NASA)/American Society for Engineering Education (ASEE) Summer Faculty Fellowship Program, 1993, volume 1

The JSC NASA/ASEE Summer Faculty Fellowship Program was conducted by Texas A&M University and JSC. The objectives of the program, which began nationally in 1964 and at JSC in 1965, are as follows: (1) to further the professional knowledge of qualified engineering and science faculty members; (2) to stimulate an exchange of ideas between participants and NASA; (3) to enrich and refresh the research and teaching activities of participants' institutions; and (4) to contribute to the research objectives of the NASA centers. Each faculty fellow spent at least 10 weeks at JSC engaged in a research project in collaboration with a NASA/JSC colleague. This document is a compilation of the final reports on the research projects completed by the faculty fellows during the summer of 1993

Functional analysis of microRNA-181a: identification of target proteins and application in HCC therapy

Hepatocellular carcinoma (HCC), or the cancer of the liver, is of great concern due to its poor patient outcome despite the various treatments available. It is imperative, therefore, that a novel, viable treatment method is developed such that patient survival rates may be improved from current statistics of less than 50%. The role of miRNAs in the regulation of gene expression and cellular development makes it an important player in cancer development process, as it is found that the aberrant expression of miRNAs is a typical feature of cancer cells or even pre-disposed cancer cells. MiR-181a has been shown to be an important miRNA involved in HCC. In this study, we investigated the potential effects of miR-181a in HepG2 cells and the mechanisms in which it works in controlling cell fate. As chemotherapy is widely used in liver cancer treatment, we also study the use of miR-181a along with chemotherapy (i.e. Cisplatin). Using iTRAQ-coupled 2D LC-MS/MS analysis, we report here the study of protein profile of HepG2 cells transfected with miR-181a and its inhibitor respectively. Three main types of cellular proteins including metabolic enzymes, protein binding and stress proteins displayed changes. The changes in the level of proteins (14-3-3σ, Hsp-90β and NPM1) involved in important cancer processes like cell growth were further supported by a Western blot analysis. MiR-181a was subsequently found to significantly increase HepG2 cell viability while inhibiting it displayed the opposite effect. Inhibiting miR-181a also sensitized HepG2 cells to cisplatin treatment and retards cell cycle progression by decreasing the proportion of cells in S and G2/M phases. We next investigated the reasons behind these observations at a molecular level. As miRNAs are known to regulate genes by binding to and targeting mRNAs, we first used bioinformatics to screen out potential cellular targets. Two important genes identified, cyclin-dependent kinase inhibitor 1B (CDKN1β) and transcriptional factor E2F7 (E2F7), which are involved in cell cycle and cell proliferation, were chosen to be further experimentally studied. In vitro validation via surface plasmon resonance (SPR) technique showed a positive binding between miR-181a and the seed regions of the 3’UTRs of the two putative mRNA targets, with dissociation constants being 272.5 ± 0.008 nM and 1.186 ± 0.009 uM for CDKN1β and E2F7 respectively. In vivo luciferase assay studies further validated the miR-181a:mRNA bindings, in both cases displaying significant decrease in luciferase activity when HepG2 cells were co-transfected with the 3’UTR-containing reporter plasmids and miR-181a. A positive binding, however, may not necessarily lead to a lowered expression of protein levels. A Western blot study on the expression levels of the two proteins, however, showed a decrease in the levels of CDKN1β and E2F7. Lastly, to gain an insight into the overall effects miR-181a has in HepG2 cells, a microarray analysis was performed. Cellular pathways important in cancer were studied and results show that miR-181a significantly activated the MAPK/JNK pathway by increasing the expression levels or activity of transcription factor activator protein 1 (AP-1). Inhibiting miR-181a, on the other hand, abolished this observation and significantly decreased expression levels or activity of hypoxia-inducible factors (HIF) and also significantly upregulated the expression levels or activity of SMAD2/3/4 proteins, possibly inducing a cancer-suppressing effect. Overall, miR-181a appears to activate mainly cancer-promoting pathways, and may act as an oncogene in HepG2 cells. Inhibiting it, on the other hand, activates mainly the tumour-suppressing pathways, making it a possible option for therapy. A separate microarray analysis on gene expression showed that one way in which miR-181a could have activated the SMAD, NFκB and MAPK pathways is via the significant increase in gene expression of bone morphogenetic protein receptor type II (BMPR2), a cellular receptor that mediates the signal transduction of these pathways. Our findings provide a new platform of identifying miRNA targets, in the process offering molecular evidence on the mechanism of action of miR-181a, including the beneficial effects of inhibiting miR-181a in HCC therapy.Open Acces

[¹⁸F]fluorination of biorelevant arylboronic acid pinacol ester scaffolds synthesized by convergence techniques

Aim: The development of small molecules through convergent multicomponent reactions (MCR) has been boosted during the last decade due to the ability to synthesize, virtually without any side-products, numerous small drug-like molecules with several degrees of structural diversity.(1) The association of positron emission tomography (PET) labeling techniques in line with the “one-pot” development of biologically active compounds has the potential to become relevant not only for the evaluation and characterization of those MCR products through molecular imaging, but also to increase the library of radiotracers available. Therefore, since the [18F]fluorination of arylboronic acid pinacol ester derivatives tolerates electron-poor and electro-rich arenes and various functional groups,(2) the main goal of this research work was to achieve the 18F-radiolabeling of several different molecules synthesized through MCR. Materials and Methods: [18F]Fluorination of boronic acid pinacol esters was first extensively optimized using a benzaldehyde derivative in relation to the ideal amount of Cu(II) catalyst and precursor to be used, as well as the reaction solvent. Radiochemical conversion (RCC) yields were assessed by TLC-SG. The optimized radiolabeling conditions were subsequently applied to several structurally different MCR scaffolds comprising biologically relevant pharmacophores (e.g. β-lactam, morpholine, tetrazole, oxazole) that were synthesized to specifically contain a boronic acid pinacol ester group. Results: Radiolabeling with fluorine-18 was achieved with volumes (800 μl) and activities (≤ 2 GBq) compatible with most radiochemistry techniques and modules. In summary, an increase in the quantities of precursor or Cu(II) catalyst lead to higher conversion yields. An optimal amount of precursor (0.06 mmol) and Cu(OTf)2(py)4 (0.04 mmol) was defined for further reactions, with DMA being a preferential solvent over DMF. RCC yields from 15% to 76%, depending on the scaffold, were reproducibly achieved. Interestingly, it was noticed that the structure of the scaffolds, beyond the arylboronic acid, exerts some influence in the final RCC, with electron-withdrawing groups in the para position apparently enhancing the radiolabeling yield. Conclusion: The developed method with high RCC and reproducibility has the potential to be applied in line with MCR and also has a possibility to be incorporated in a later stage of this convergent “one-pot” synthesis strategy. Further studies are currently ongoing to apply this radiolabeling concept to fluorine-containing approved drugs whose boronic acid pinacol ester precursors can be synthesized through MCR (e.g. atorvastatin)

A systems approach to understanding Dupuytren's disease

Introduction: Dupuytren's disease (DD) is an ill-defined fibroproliferative disorder affecting the palms of the hands of certain patient groups. Whether changes in DD fibroblasts are due to genetic alterations alone or related to metabolic dysregulation has not yet been investigated. Hypotheses: 1. DD is a disease of several networks rather than of a single gene. 2. DD may be investigated more effectively by employing systems biology. 3. Strict definition of cell passage number is important for the revelation of any DD phenotype. 4. Some of the differences between DD and healthy tissues reside in a difference in their respiratory metabolism. 5. Any such differences are akin the Warburg effect noted for tumour cells in the literature. Methods: We induced hypoxia in healthy and disease cells to test whether the difference in disease cell types and healthy is the same as the difference in control fibroblasts cultured in normoxia and hypoxia. We investigated both at the metabolic level (intracellular and extracellular) and at the transcript level. This study also employed Fourier transform infrared spectroscopy to permit profiling of cells: (1) DD cords and nodules against the unaffected transverse palmar fascia (internal control), (2) those (1) with carpal ligamentous fascia (external controls) (3) those in (1) against DD fat surrounding the nodule, and skin overlying the nodule. We then compared metabolic profiles of the above to determine the effect of serial passaging by assessment of reproducibility. Subsequently, a novel protocol was employed in carefully controlled culture conditions for the parallel extraction of the metabolome and transcriptome of DD-derived fibroblasts and control at normoxic and hypoxic conditions to investigate this hypothesis. Gas chromatography-mass spectrometry combined with microarrays was employed to identify metabolites and transcript characteristic for DD tissue phenotypes. The extracellular metabolome was also studied for a selected subset. The metabolic and transcriptional changes were then integrated employing a network approach. Results: Carefully controlled culture conditions combined with multivariate statistical analyses demonstrated metabolic differences in DD and unaffected transverse palmar fascia, in addition to the external control. Differences between profiles of the four DD tissue phenotypes were also demonstrated. In addition early passage (0-3) metabolic differences were observed where a clear separation pattern in clusters was observed. Subsequent passages (4-6) displayed asynchrony, losing distinction between diseased and non-diseased sample phenotypes. A substantial number of dysregulated metabolites involved in amino acid metabolism, carbohydrate metabolism and also metabolism of cofactors and vitamins including downregulated cysteine and aspartic acid have been identified from the integrative analyses. Metabolic and transcriptional differences were revealed between fibroblast cell samples (passage number 3) cultured in 1% and 21% oxygen. The hypothesis that the difference in disease and healthy cells maybe akin to the differences in healthy cells in normoxia and hypoxia was rejected as only a very small number of significant molecules from these studies coincided in perturbed fascia and disease samples. No lactic acid was observed and little difference in the pyruvate concentrations. Yet, upon perturbation several of these transcripts and metabolites involved in the afore-mentioned pathways were significantly dysregulated. Conclusion: Early, but not late, passage numbers of primary cells provide representative metabolic and transcript fingerprinting for investigating DD. A unique parallel analysis of transcript and metabolic profiles of DD fibroblasts and control, enabled a robust characterization of DD and correlation of parameters across the various levels of systemic description. The tools that should facilitate our understanding of these complex systems are immature, but the pleiotropy of the difference between healthy and DD tissue suggest the aetiology of a network-based disease.EThOS - Electronic Theses Online ServiceGBUnited Kingdo