17th Annual CBE Graduate Student Research Symposium

Friday, October 3, 2014

Keynote Lecture

Dr. Daniel K. Schwartz | Chair of the Department of Chemical and Biological Engineering and the Alfred and Betty Look Professor of Engineering at the University of Colorado Boulder

Single Molecule Tracking at Wet Interfaces

Interactions between molecules and surfaces lead to complex and highly-varied interfacial behavior, where heterogeneity may arise from spatial variation of the surface/interface itself or from molecular configurations (i.e. conformation, orientation, aggregation state, etc.). These phenomena greatly impact technologies and applications including biomaterials, separations (chromatography and membrane filtration), heterogeneous catalysis, and biosensing, among others. The direct observation of adsorption, interfacial diffusion, and desorption of individual fluorescent molecules permits the characterization of heterogeneous interfacial behavior in ways that are inaccessible to traditional ensemble-averaged methods. Moreover, spectral  information can be used to simultaneously track molecular configuration (aggregation or folding state). Single-molecule tracking experiments have traditionally been limited by small sample sizes (e.g. a few hundred molecules), leading to poor statistical significance and a lack of sensitivity to rare populations. However, new advances in high-throughput tracking methods now enable hundreds of thousands of molecules to be followed in a given experiment. This approach has recently been used to characterize heterogeneous molecule-surface interactions including: multiple modes of diffusion and desorption associated with both internal and external molecular configuration, intermittent interfacial transport, spatially dependent interactions, and others.

Dan Schwartz is Chair of the Department of Chemical and Biological Engineering and the Alfred and Betty Look Professor of Engineering at the University of Colorado Boulder. He has been a professor at CU-Boulder since 2001. He was previously an Assistant and Associate Professor of Chemistry at Tulane University from 1994-2000. Dan received his Bachelor’s Degree (in Chemistry and Physics) and his PhD (in Physics) from Harvard University in 1984 and 1991 respectively, and subsequently performed postdoctoral fellowships in Chemical Engineering at UC Santa Barbara and Physical Chemistry at UCLA. Dan’s research interests focus on interfacial phenomena with specialties in surface modification, singlemolecule microscopy, biomaterials, surfactant phenomena, biotechnology, liquid crystals, biosensing, and biomimetic catalysis. He has published more than 160 peer-reviewed manuscripts that have been cited over 5000 times. His recognitions include the NSF CAREER award, the Dreyfus Foundation Teacher-Scholar award, the CU-Boulder Faculty Assembly Award for Excellence in Research, and election as a Fellow of the American Physical Society and a Fellow of the American Chemical Society. Dan has been a Senior Editor of the journal Langmuir, the American Chemical Society’s journal of surfaces and colloids since 2004. He was the founding Director of the Tulane Science Scholars program (an enrichment program for High School students) and of the Summer Research Experience for Undergraduates Program in Functional Materials at CU-Boulder. He also developed a general education course at CU-Boulder, Creative Technolog, that has communicated state-of-the-art technology concepts to more than 6,000 non-science undergraduates to date.

Graduate Student Lectures

Charles Jones | Doctoral Student in Professor Pfeifer's Group

Genetic Vaccines

Genetic vaccines offer a treatment opportunity based upon successful gene delivery to specific immune cell modulators.  Driving the process is the vector chosen for gene cargo packaging and subsequent delivery to antigen presenting cells (APCs) capable of triggering an immune cascade.  As such, the delivery process must successfully navigate a series of requirements and obstacles associated with the chosen vector and target cell.  In this work, we present the development and assessment of a hybrid gene delivery vector containing biological and biomaterial components.  Each component was chosen to separately design and engineer gene delivery in a complimentary and fundamentally distinct fashion.  A bacterial (Escherichia coli) inner core and a biomaterial (poly(beta-amino ester))-coated outer surface allowed the simultaneous application of molecular biology and polymer chemistry to address barriers associated with APC gene delivery which include cellular uptake and internalization, phagosomal escape, and intracellular cargo concentration.  The approach combined and synergized normally disparate vector properties and tools, resulting in increased in vitro gene delivery beyond individual vector components or commercially-available transfection agents.    Furthermore, the hybrid device demonstrated a strong, efficient, and safe in vivo humoral immune response when compared to traditional forms of antigen delivery.  In summary, the flexibility, diversity, and potential of the hybrid design were developed and featured in this work as a platform for multivariate engineering at the vector and cellular scales for new applications in gene delivery immunotherapy. 

Panagiotis Mistriotis | Doctoral Student in Professor Andreadis'Group

Rejuvenating Senescent Mesenchymal Stem Cells: Implication for Vascular Tissue Engineering

The regenerative capacity of adult stem cells is known to decline with aging while aged stem cells enter a senescent state, which further impairs their function. Notably we recently discovered that ectopic expression of Nanog delayed senescence and restored the contractile function of adult ovine Mesenchymal Stem Cells (MSC). Here we report that Nanog does not only delay but can also reverse senescence of human MSC. In addition, we identified a novel mechanism through which Nanog restores the myogenic differentiation potential and contractile function of senescent MSC. 

To study the effects of Nanog on senescent stem cells, tissue specific MSC such as hair follicle and bone marrow were isolated and transduced with a tetracycline regulatable vector that carries the Nanog gene. This system allows Nanog expression only when cells are treated with the tetracycline analogue, Doxycycline (Dox). To mimic stem cell senescence, cells were serially passaged until their proliferation capacity was seized (late passage, LP). Subsequently Dox was added to the culture medium and the stem cell myogenic capacity was evaluated and compared to early passage (EP) cells.

Our results show that senescent stem cells fail to respond to myogenic stimuli as evidenced by the loss of contractile proteins such as smooth muscle actin (ACTA2). In addition LP-MSC fail to polymerize ACTA2 into filaments and show decreased force generation capacity. Interestingly, Nanog expression in senescent MSCs restores the response to myogenic stimuli and induces ACTA2 expression, polymerization and force generation. Furthermore, we report that the loss of myogenic capacity in LP-MSCs is mediated through the decreased expression of critical myogenic transcription factors such as SRF. On the other hand, we show that Nanog restores myogenesis by reversing the loss of SRF and enhancing the transcriptional activity of the SRF DNA binding site (CArG box).

Taken together our data provide novel insight on the mechanism of stem cell senescence and propose a strategy to regain the lost properties, thereby increasing the potential of stem cells from aged donors for cellular therapy and tissue regeneration.