2010 Summer Researchers Participants-HHMI Undergraduate Program - Carnegie Mellon University

2010 HHMI Summer Researchers Participants

Karen Akasaka

Karen Akasaka
Mentor: Dr. Justin Crowley

Katherine Bonnington

Katherine Bonnington
Mentor: Dr. Gordon Rule

Kelsey Briggs

Kelsey Briggs
Mentor: Dr. Stefan Zappe

Development of Polymeric Microcapsules for Neural Stem Cell Culture and Tissue Engineering

Tissue engineering (TE) was prominently defined as "an interdisciplinary field that applies the principles of engineering and life sciences towards the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ". While the potential of TE is enormous, current technologies for designing TE constructs have difficulty reproducing the complexity of native tissues and organs. In general, our lab aims to address complexity in TE through development of material systems for creation of polymeric microcapsules that contain cells and serve as individual elements of complex TE constructs. We hypothesize that we can develop microcapsular environments which are capable of influencing cell fate in support of a therapeutic approach. Specific aim of this project was to develop microcapsules, based on hyaluronic acid (HA) and methylated collagen (MC), that support expansion of neural stem cells (NSCs) and their specific neuronal differentiation, respectively. Microcapsules were generated based on agarose gel templating and complex coacervation. We identified optimum agarose, HA, and MC concentrations allowing for complexation of the natural polymers and melting of the agarose. Fate of cultured NSCs was characterized through immunostaining. We have also explored the possibility of joining individual capsules via covalent crosslinking of the components of the capsular walls and worked on functionalization of surfaces upon which capsules might be arranged towards complex TE constructs. Our work shows that HA/MC-based microcapsules are capable of enhancing phenotype maintenance and neuronal differentiation of cultured NSCs, respectively. Our preliminary results regarding attachment of capsules to each other and to functionalized surfaces represent important first steps towards generation of complex, modular TE constructs.

Ian Campbell

Ian Campbell
Mentor: Dr. John Woolford

Sruthi Reddy Chintakunta

Shruti Reddy Chintakunta
Mentor: Dr. Justin Crowley

Suhl A Choi

Suhl A Choi
Mentor: Dr. Mark Bier

Si Won Choi

Si Won Choi
Mentor: Dr. Kris Dahl

 

Katherine Chong

Katherine Chong
Mentor: Dr. Bruce Armitage

Adrian Chow

Adrian Chow
Mentor: Dr. Marlene Behrmann

Lianne Cohen

Lianne Cohen
Mentor: Dr. Jonathan Jarvik

Targeted Evolution of scFv dimers for cytosolic expression using somatic hypermutation in Ramos cells

 Currently most single chain variable fragments (scFv’s) produce a brighter fluorescent signal outside of the cell than in the cytoplasm. To improve the activity and brightness of an scFv, we will be using Ramos cells, a human B-cell line, to evolve in a directed method, dNC138. dNC138 is a dimer of light chains, connected with a flexible linker and with flexible extensions on both the N-terminus and C-terminus. Ramos cells undergo somatic hypermutation in genes that are transcribed, using activation-induced cytidine deaminase, AID, to mutate the Ig regions at a rate of approximately 10-3 creating mostly random mutations. A “dumbbell” construct, which consists of one protein domain, green fluorescent protein (GFP) on the exterior of the cell and another, dNC138, in the cytoplasm, separated by a transmembrane domain, will be used to express the scFv in the Ramos cell line. This enables dNC138 to be optimized for expression in the cytosol of a mammalian/human cell. For this project, the dumbbell construct with dNC138 will be placed under the control of the Tet-on inducible system, allowing the transcription and therefore mutation of the target gene to be regulated. Phoenix cells will be used to package the dumbbell construct into viral particles which will then stably infect the Ramos cells. After each round of expression and mutation, the Ramos cells will be sorted for the brightest mutants by fluorescence-activated cell sorting (FACS). We hope that these mutants of dNC138 will provide a better reporter for labeling cytosolic proteins.
Nicole Dangelo

Nicole Dangelo
Mentor: Dr. Newell Washburn

Investigation of Cell Differentiation as a Function of Dynamic Substrate Rigidity Through the use of Stimulus-Responsive Hydrogels

It has been discovered that cells will probe a surface and will differentiate based on the mechanical properties of the substrate. Prior research has been conducted using static hydrogels and the cell’s response to these surfaces has been well documented. However, how the cells respond to a dynamic environment has yet to be investigated. It has been hypothesized that materials with switchable mechanical properties can be used to guide cell differentiation in tissue engineering applications, providing an environment that contains cues which evolve as new tissue is established. Another potential medical application is to reduce the amount of stem cells needed by gaining a better understanding of adult cells and how to change their function while the cells are undergoing normal cell growth and division. The goal of this project is to develop a dynamic, biocompatible hydrogel that will change mechanical properties, such as stiffness and viscosity, upon exposure to UV light, while also being able to revert back to its original state via a different wavelength of UV light. The second goal is to determine how the cells change as a function of their environment and if there is a critical point beyond which the cells will no longer change. Each of the gels components must be synthesized using materials that have been shown to be biocompatible. Once the cells have been shown to change, the idea is to place them back inside the body. Any materials that have been in contact with the cell have a potential to be transferred into the body. Therefore, increasing the biocompatibility of the material will reduce the adverse side effects of the cell differentiation. This project will explore the synthesis, purification, and characterization of a hydrogel with UV-switchable mechanical properties such that the cell’s response to a dynamic environment can be discovered and applied to future medical applications.

Lynley Doonan

Lynley Doonan
Mentor: Dr. John Woolford

Molly Evans

Molly Evans
Mentor: Dr. Subha Das

Adam Foote

Adam Foote
Mentor: Dr. Adam Linstedt

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Katherine Fu
Mentor: Dr. Christopher Bakkenist

Siping He

Siping He
Mentor: Dr. A Javier Lopez

Andre Hersan

Andre Hersan
Mentor: Dr. Mark Macbeth

Mark Holfelder

Mark Holfelder
Mentor: Dr. David Yaron

 

 Mike Khan

Mekail Khan
Mentor: Dr. Aaron Mitchell

Jean Kim

Jean Kim
Mentor: Dr. Umamaheswar Duvvuri

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Margaret Kim
Mentor: Dr. David Hackney

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Maria Kobidze
Mentor: Dr. Mark Macbeth

Determination of Adenosine Deaminase That Acts on RNA Activity on DNA

Adenosine deaminase that acts on RNA (ADAR) catalyzes a point mutation of adenosine to inosine, which is essential for correct neuronal function, by editing post-transcriptional double stranded RNA. Its catalytic domain is structurally similar to the catalytic domains of cytidine deaminases, including that of Activation-Induced Deaminase (AID), which acts to deaminate double stranded DNA. In yeast, AID edits the CAN1 gene which encodes for arginine permease, a plasma membrane protein responsible for arginine uptake. Editing of the CAN1 gene leads to an inability of the yeast to take up arginine. This confers resistance to the antibiotic canavanine, which is structurally similar to arginine and is also taken up by arginine permease. The similarity in the catalytic domains between AID and ADARs suggests that ADARs can catalyze the deamination of DNA as well as dsRNA. Preliminary results show that human ADAR2 (hADAR2) deamination activity on DNA also may confer resistance to canavanine, implying that hADAR2 can indeed deaminate DNA. The goal of this project is to describe the extent to which hADAR2 acts on DNA by determining the CAN1 mutation frequency of yeast with overexpressed hADAR2 in comparison to AID and empty vector controls, in addition to a negative control in which the hADAR2 catalytic domain has a point mutation that prevents its function. The nature of the mutations will also be determined by sequencing.

Sefa Kploanyi

Sefa Kploanyi
Mentor: Dr. Mark Macbeth

Eun Hwa Lee

Eun Hwa Lee
Mentor: Dr. David Whitcomb

Janet Lee

Janet Lee
Mentor: Dr. Marie Defrances

WEndy Li

Wendy Li
Mentor: Dr. Chien Ho

Ex Vivo Labeling of Immune Cells with Iron Oxide Particles

Currently, the clinical “gold standard” for detecting graft rejection following heart transplantation is biopsy, which is not only invasive, but also prone to sampling errors. We propose that cardiac rejection can be measured and detected through Magnetic Resonance Imaging (MRI), circumventing the need for biopsies, by tracking the accumulation of immune cells at the site of organ rejection.  Dr. Ho’s group focuses on improving immune cell labeling efficiency ex-vivo and in-vivo in order to further understand the role that each type of immune cell plays in cardiac rejection and to detect cardiac rejection at an earlier stage. This project will focus specifically on improving efficiency of labeling and detecting non-phagocytic T-cells ex-vivo so that they can be more easily tracked in-vivo through MRI. We will use iron-oxide particles containing a terminal carboxyl group (IOPC) provided by the Industrial Technology Research Institute of Taiwan (ITRI).  We believe that modifying ITRI-IOPC particles by conjugating a cationic amine group (to form ITRI-IOPC- NH2) can increase their efficiency in labeling T-cells. Completion of the project will involve the isolation, culturing, and labeling of T-cells and macrophages from rats; the synthesis of ITRI-IOPC-NH2 particles from ITRI-IOPC particles; and the analysis of labeled immune cells through MRI and Transmission Electron Microscopy (TEM). We found that both T-cells and macrophages are efficiently labeled by ITRI-IOPC-NH2 particles, and that the modifications made to the ITRI-IOPC particle increased our ability to detect T-cells through MRI.

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Heather Lynn
Mentor: Dr. Qiuhong He

Breast Cancer Migration via BDNF Endothelial Expression Outside of Blood Vasculature

While cancerous cells are known to migration via the body’s circulatory and lymphatic system, we have proposed that these cells could possibly migrate long distances outside of the vasculature. This proposed method of metastasized breast cancer migration is similar to that already observed by neural progenitor cells in the adult brain.  The neural signal of BDNF, which is released from blood vessels, may guide the neurons along the brain vascular to their intended destination. The hypothesis proposed is that metastasized tumor cells may migrate in the same manner in the body using signals released by the endothelial cells to move outside of the blood vessel and lymphatic system. The main methods of testing this hypothesis are twofold. The primary investigation is to construct a 3D blood vascular network of endothelial cells (fluorescent protein labeled) and image fluorescently tagged cancerous cells migration in vitro. This 3D blood vessel network expressed BDNF and GFP, so it may be imaged using time lapse microscopy. The breast cancer cell line used (MDA-MB-123) expresses RFP or GFP, so the cells are visible in contrast to the 3D vasculature network. The second method of investigation is to use luciferase imaging of live mice to view the migration of metastasized tumor cells in vivo, as well as study GFP-labeled MDA-MB-231 cells or MMTV-tva/RCAS-PyMT tumor cell migration in fresh tumor tissues.

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Janice Lyu
Mentor: Dr. Jonathan Minden

Elizabeth McCarty

Elizabeth McCarty
Mentor: Dr. Justin Crowley

Steven Nguyen

Steven Nguyen
Mentor: Dr. Jeffrey Hollinger

Gel Scaffolds in Bone Tissue Engineering

The regeneration of bone is promoted by proliferation and differentiation of osteocytes and is further influenced by the presence of supportive scaffolds as well as signaling molecules such as platelet-derived growth factors (PDGF) and bone morphogenetic protein (BMP). In other words, cells alone cannot produce new bone; they require signaling molecules to differentiate into bone cells and biodegradable scaffolds to have a biocompatible medium to grow on. Before administration of these biomaterials into patients, the in vitro results and techniques must be verified; this is the emphasis of our research. The specific focus area of the research was the biological properties and pharmacokinetics of hydrogel based scaffolds: Corgel™, Matrigel™, and four varieties of sodium alginate based gels (FMC). Our experiments were conducted with human mesenchymal stem cells (hMSC); the significance of using hMSCs was their tendency to differentiate into bone cells. We hypothesized that while the hMSCs along with growth factors PDGF and/or BMP were grown within the matrix of the different gel scaffolds for particular time frames, the gels would exhibit properties of biocompatibility and facilitate growth factor release as well as the proliferation and differentiation of the hMSCs. The hMSCs were grown in osteogenic growth media until confluent enough for experimentation. Five key assays were performed on the cells with several repeats and under various biological conditions (i.e. growth factor concentration); these assays included Live/Dead®, Picogreen®, alkaline phosphatase (ALP), calcium, and enzyme-linked immunosorbent assays (ELISA). The assays determined cell viability within the gels, proliferation, differentiation of the hMSC’s into bone cells, calcium concentration, and growth factor release. The results indicated that the hydrogels are very biocompatible, and we hope that they can serve as functional delivery mechanisms in clinical bone grafting.

Frank Olechnowicz

Frank Olechnowicz
Mentor: Dr. Catalina Achim

Anna Park

Anna Park
Mentor: Dr. John Woolford

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Bernard Parker
Mentor: Dr. Tomasz Kowalewski

Mass Spectroscopic Studies of Porous Nanocarbon Material from Block Copolymer Precursors

The Kowalewski lab has developed new nanographene structures with unique properties over the past few years, with unique electronic properties and the ability to be functionalized in a regular manner. The nanographenes are prepared by creating a block copolymer of polyacrylonitrile and poly(n-butyl acrylate), which self-assembles into a regular structure. The material is then converted to porous nanographene using pyrolysis. The small size of the resulting nanographitic domains means that the chemical and electronic properties of the material are significantly affected by the configuration of nanographene edges, with nitrogen atoms on two of them. This gives them improved electronic properties (due to the extra lone electron pairs) as well as the ability for other functional groups to be added to the structure. Such nanographenes have potential uses in energy storage, transistors, and biosensors, due to their electronic properties, which can change in the presence of other molecules and functional groups. Laser desorption ionization-time of flight mass spectrometry (LDI-TOF MS) was used in a novel way to characterize these nanographenes. In order to determine its utility, the effect of laser power on the LDI-TOF spectra was investigated in studying several different nanographene samples. Several advanced curve fitting techniques were applied to determine the best fit for the data. There was found to be a weak correlation between increasing laser power and maximum nanographene mass detected. The use of C60 (fullerene) was also investigated as a standard for LDI-TOF MS. C60 is relatively inert, is compatible with the nanographenes, and was expected to have a clear peak in the mass spectra. Several methods of spiking samples with C60 were attempted. From this, the LDI-TOF spectra were determined to be accurate. However, several of the methods attempted were shown to be inadequate as the C60 most likely oxidized/fragmented during the pyrolysis procedure.

Joshua Patent

Joshua Patent
Mentor: Dr. David Yaron

Subha Patibanda

Subha Patibanda
Mentor: Dr. Kausik Chakrabarti

Rachel Pferdehirt

Rachel Pferdehirt
Mentor: Dr. Newell Washburn

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Joshua Plotnik
Mentor: Dr. John Woolford

Jewel Pothen

Jewel Pothen
Mentor: Dr. Newell Washburn

Devin Prior

Devin Prior
Mentor: Dr. Chien Ho

Effect of Intralipid on In Vivo Iron-Oxide Particle Labeling of Immune Cells in a Rat Model

Cellular MRI is a powerful technique for studying a range of diseases and treatments. The Ho laboratory is developing a new approach to use cellular MRI to monitor organ rejection by imaging infiltration of macrophages into a rejecting heart (Ye, et al. 2008). This method is based on labeling immune cells with iron-oxide particles, particularly macrophages which will internalize the particles, thus allowing the cells to be detected in vivo by MRI. Iron oxide particles can have very short blood half-life because they are quickly taken up by Kupffer cells of the liver.  It is hypothesized that iron-oxide blood clearance can be delayed by using other agents cleared by the Kupffer cells prior to injection of the iron-oxide particles. If more macrophages take up the iron-oxide particles, the sensitivity for detecting macrophage infiltration into the rejecting grafts will be improved. In this project, we investigate the effect of intralipid, a clinically approved fat supplement, on the blood half-life of micron-sized iron-oxide particles (MPIO) and compare the effect on macrophage cellular labeling by using flow cytometry.

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Mohit Raghunathan
Mentor: Dr. Stephanie Tristram-Nagle

Elizabeth Record

Elizabeth Record
Mentor: Dr. Bita Moghaddam

Sang Ah Roh

Sang Ah Roh
Mentor: Dr. A. Javier Lopez

Kelvin Rojas

Kelvin Rojas
Mentor: Dr. Philip LeDuc

Anna Romanova

Anna Romanova
Mentor: Dr. Nathan Urban

Jonathan Snider

Jonathan Snider
Mentors: Karen Stump and Dr. Bruce Armitage

Balaji Srinivas

Balaji Srinivas
Mentor: Dr. David Hackney

Shriya Venkatesh

Shriya Venkatesh
Mentor: Dr. Brooke McCartney

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Young Yeo
Mentor: Dr. David Hackney

Cindy Zhu

Cindy Zhu
Mentor: Dr. Brooke McCartney

Alessandra Zimmermann

Alessandra Zimmermann
Mentor: Dr. Subha Das