Independent Student Research Projects at BCA Involving Microscopy
Anthony Arena, AAST 2011
The Effect of Polyclonal Antibodies on Streptococcus mutans Biofilm Formation
The purpose of this study was to test the effect of polyclonal antibodies on oral bacterial biofilm formation. Antibody serum therapy for microbial diseases has been a more recent topic in microbiology, and its application on oral bacteria has been scantly written about. Streptococcus mutans, one of the leading cariogenic strains, secretes a protein, autoinducer-2, and colonizes to form biofilms. The bacteria in these biofilms secrete an enzyme, glycosyltransferase, that allow bacteria to here to each other and the substrate, in this case, teeth. This study used an immunoglobulin G antibody, ab31181, that inhibits the function of glycosyltransferase, and studied its effects on S. mutans growing on calcium hydroxyapatite and porcelain fused to metal (PFM) discs. Various concentrations of antibody were added to bacterial cultures growing on both porcelain fused to metal (PFM) and calcium hydroxyapatite discs. A dose response relationship was found: higher concentrations of antibody reduced biofilm formation for both hydroxyapatite and PFM samples. Data was found to be statistically significant for the hydroxyapatite (p<0.05). This shows that antibody therapy may be a viable option for the prevention of dental caries. Future research would need to be conducted clinically to test the effects on humans and its practical implications.
Regina Cai, AAST 2011
Reducing Infectivity by Blocking Virus-Cell Interaction
In this study, the interaction between antibodies and viruses is investigated with regard to how entry to the cell might be prevented by blocking key molecules on the viral surface. Vesicular stomatitis virus (VSV) uses a protein on its surface, called the g protein, to gain access to the cells it is designed to infect. This protein has been co-opted for the construction of viral vectors, defective viral particles engineered for the purpose of delivering genes to target cells and organs. Vectors were constructed based on the lentiviral framework, containing the green fluorescent protein (GFP) as the genetic payload and an integrated VSVg on the viral surface. This was accomplished by constructing a transient packaging cell line using 293T cells via delivery of three plasmids. The interaction of these viruses with human epidermal keratinocytes in tissue culture was studied. It was hypothesized that the anti-VSVg antibodies will bind to the surface proteins of the VSVg virus, thus inhibiting the infectivity of the virus, as determined by a reduction in the number of green fluorescent cells identified by flow cytometry. A transmission electron microscope (TEM) will be used to image the viral vector in aqueous solution.
Junyoung Choi, AAST 2011
Hydrophobic and Amphiphilic Nanoencapsulation of Paclitaxel for in vitro Drug Delivery
Paclitaxel (PTX) is a potent, chemotherapeutic agent that induces apoptosis by binding to the Bcl-2 protein and to the beta-subunit of tubulin. Due to its limited solubility, PTX is commercially delivered with Cremophor-EL solvent, collectively called Taxol. However, Taxol has been shown to cause adverse side effects in patients, including severe hypersensitivity reactions and death. Therefore, the goal of this experiment was to develop new excipients for PTX delivery, using self-assembled nanoparticles to increase the antitumor effect while minimizing cytotoxicity. Two types of novel, nanoparticulate excipients were engineered: amphiphilic and hydrophobic. This study is the first comprehensive investigation analytically comparing amphiphilic and hydrophobic PTX encapsulation. Previously, the amphiphilic encapsulation technique of PTX was explored, using fluorescent glycol-chitosan nanoparticles (GCF-PTX) shown to reduce in vitro cytotoxicity while maintaining efficacy. Afterward, hydrophobic conjugates, formulated with glycol chitosan and 5beta-cholanic acid, were also engineered for the encapsulation of PTX (CGC-PTX). Conjugates were tested for their effects on human breast cancer cells and human dermal fibroblast cells (control group). Additionally, Taxol inclusion complex and Cremophor-EL solvent were also tested on cells to compare the cytotoxicities of the excipients. The viability was conducted in various concentrations of the nanoaggregates and inclusion complexes using the MTS assay. It was found that Cremophor-based treatments were extremely toxic to both cell lines (p < 0.001). CGC was found to be nontoxic to both cell lines while CGC-PTX was toxic only to breast cancer cells (p < 0.01). It was also concluded that CGC-PTX was more effective in downregulating breast cancer cells than the previous GCF-PTX (p < 0.05). As a result, it was concluded that hydrophobic nanoparticulate treatments of breast cancer are highly practical and less cytotoxic than the commercially-available treatment, a promising result in the targeted drug delivery of chemotherapeutic agents to localized cancer cells.
Julie Chong, AEDT 2013
Electrochemical Synthesis and Characterization of Titania Nanotubes for Photovoltaic Applications
In a world of impending energy crisis, dye synthesized solar cells (DSSC) are an increasingly popular option of viable alternative energy. The purpose of this experiment is to electrochemically synthesize titania nanotubes (TNTs) of varying diameters and to determine the effect of adding the TNTs to dye synthesized solar cells. TNTs were synthesized at three voltages (50V, 55V, and 60V) in .5% HF in ethylene glycol solution. The nanotubes were then imaged and measured with a scanning electron microscope. The diameter of the 50V TNTs averaged 99.6 nm and the diameter of the 60V TNTs averaged 127.1 nm. The nanotubes were peeled from the TiO2 foil they were anodized on and placed onto the TiO2 paste on the conductive glass of the DSSC. The glass, with the TNTs on it, was then sintered for 20 minutes at 400 degrees Celsius. Fruit juice will be used to dye the solar cell. Results are pending and will be presented.
Samantha Dargis, AAST 2013
Change in Morphology and Antioxidative Properties of Irradiated Bean Plants
The focus of this experiment was to study the effect that gamma-irradiation has on the plant species Vigna unguiculata. Plant height, leaf production, stomata presence in leaves, and mitochondria levels numbers were all measured in plants that had been exposed to radiation as seedlings. The exposure levels varied from .15 KR to 40 KR. Results show that mitochondrial area was significantly larger in plants that had been exposed to 10 KR than the control. This indicates a greater ability within the plant cell to deal with the large amounts of energy of radiation . While the control plants grew at a significantly faster average rate than the treated plants, the 10 KR plants produced significantly many more leaves than the control plants (p<0.05). These results are significant in the farming community, as radiation is used on crops to prevent disease and pest infestations.
Elizabeth Dente, AMST 2012
The Benzoin-Based Complex: A New Frontier for Skin Repair
The wound repair properties of the organic compound benzoin and the antioxidant olive fruit extract, in combination, have never before been observed. In addition, the ability of benzoin to have wound healing properties on the skin has not been scientifically proven. Additionally, olive fruit extract contains the powerful antioxidant hydroxytyrosol, which can prevent the damage caused by oxidative stress and free radicals, which are induced in skin by a severe wound. Matrix metalloproteinase-9 (MMP-9), an enzyme in the MMP family, is involved in the degradation that occurs when the skin is wounded and as it ages over time. When the activity of MMP-9 is inhibited, the skin can maintain a healthier structure and recover from wounds more rapidly. In addition, fibronectin, collagen and elastin are proteins that promote wound repair and skin elasticity. The model cell lines used were HaCaT, a human keratinocyte cell line, which originates in the epidermis layer of skin, and dermal fibroblasts, which originate in the dermis of skin. It was hypothesized that the combination of benzoin and olive fruit extract would decrease the levels of MMP-9 in human keratinocytes and increase the levels of fibronectin, collagen, and elastin in dermal fibroblasts in a dose-dependent manner. An MTS assay showed that all concentrations of benzoin and olive fruit extract used were nontoxic. Furthermore, the combination of benzoin and olive fruit extract induced statistically significant proliferation in the HaCaT cell line (p<.05). Olive fruit extract reduced 2,2-diphenyl-1-picrylhydrazyl (DPPH) in a dose-dependant manner, exhibiting its exceptional antioxidant ability at all concentrations. Statistically significant enzyme-linked immunosorbent assay (ELISA) results showed that MMP-9 levels in HaCaT decreased as the concentrations of benzoin and olive fruit extract increased, both alone and combined (p<.05). Benzoin and olive fruit extract, when combined, demonstrated a trend towards a modest increase of fibronectin in human keratinocytes as well as significant upregulation of collagen and elastin in dermal fibroblasts. A scratch assay was also performed to qualitatively observe the wound healing abilities of the treatment. The assay demonstrated that benzoin and olive fruit extract caused faster recovery (coverage of cells across the scratched region) compared to the control. These results suggest that benzoin and olive fruit extract, when applied to skin, could lessen the degradation done to the skin in a severe trauma. In the future, the combination of benzoin and olive fruit extract deserves further recognition for its wound healing effects on the skin as a result of its ability to downregulate MMP-9 and upregulate fibronectin, collagen and elastin in skin cells.
Ysa Esquilin, AMST 2012
mtDNA Migration: The Role of Exosomes in Horizontal Gene Transfer
Exosomes are membrane-bound vesicles, ranging in size from 40 to100 nm in diameter, that are released by a variety of cell types. Recent studies suggest that exosomes are a novel method of intercellular communication that contain a variety of biologically active molecules, including mitochondrial DNA (mtDNA). The focus of this study was to determine if exosomes carrying mtDNA of one cell type would fuse with the plasma membrane of another cell type, releasing non-native mtDNA into the cytoplasm. Such transfers of mitochondrial genes could explain the mechanism of degenerative diseases affecting mitochondria-rich tissues such as Parkinson s disease and Alzheimer s disease. Exosomes were isolated from human fibrosarcoma (HT1080) cells, lysed and sequenced to confirm the presence of mtDNA. Transmission electron microscopy was used to confirm the production of exosomes. Intact exosomes derived from HT1080 cells expressing a fluorescently-labeled membrane protein CD63 were added to cultures of human alveolar basal epithelial cells (A549) and fluorescent microscopy was used to determine if the vesicles would be absorbed. The HT1080 exosomes were found to contain mtDNA and were taken up by A549 cells, supporting the hypothesis that exosomes may have the potential to participate in horizontal gene transfer. Future studies will focus on the functionality of exosomal mtDNA following transfer.
Alina Fiato, AAST 2013
A Closer Look at Biofilms
This research builds upon studies of hydrocarbonoclastic bacteria and their role in the cleanup of oil spills. In looking into the mechanisms used by bacteria that consume hydrocarbons, one of specific interest was the formation of a biofilm. When hydrocarbonoclastic bacterium Cornyebacterium was studied, the shape and dimensions of the colonies formed suggested the formation of a biofilm. To further investigate the influence of an oil spill and the oil content in local waters, samples sand and water from Spring Lake beach in New Jersey were studied and analyzed to identify possible hydrocarbonoclastic bacteria. The results yielded two hydrocarbonoclastic bacteria samples that were found to form a biofilm-like mass in media. A scanning electron microscope was then used to analyze these two formations.
Michelle Guo, AAST 2013
Investigating the Ultrastructure of Stem Cells to Evaluate Pluripotency States
Clearly distinct states of pluripotency have been described both in vivo and ex vivo. While mouse embryonic stem cells (ESC) are considered naive and exist in a more immature state of pluripotency, human ESC are considered primed or prone to differentiate. The metabolic states of these stem cells may provide an explanation to these differences. Formerly believed to have mitochondria with minimal function, human and mouse stem cell mitochondria are now more closely being analyzed with respect to their roles in stem cell fate and nuclear reprogramming. It was hypothesized that because primed human ESC are more prone to differentiate, their mitochondria would resemble those of somatic cells and be more elongated and larger than naive mouse ESC mitochondria. Human and mouse ESC were analyzed using TEM microscopy. Morphometry with Image J software revealed that mouse ESC are 2.36 times greater in area, 1.65 times greater in perimeter, and 1.78 times greater in aspect ratio than human ESC. Human ESC are rounder and smaller, suggesting that primed stem cell mitochondria may be less functional than naive stem cell mitochondria. This is consistent with previous discoveries that human ESC mitochondria are functioning although the majority of cellular energy is produced through glycolysis. Mouse ESC may rely upon oxidative phosphorylation in addition to glycolysis to meet energy needs. Future research will include biochemical assays to quantify oxygen consumption and levels of oxidative phosphorylation and glycolysis in mouse and human ESC. Analysis of mouse primed epiblast-derived stem cells (EpiSC) and human naive stem cells will be performed to demonstrate whether all stem cells of the same state (naive or primed) exhibit the same mitochondrial function and morphology regardless of species. In order for stem cells to be utilized in future clinical treatments, their pluripotency and mitochondrial functionality must be understood.
Andrew Hahm, ATCS 2013
Chemical Synthesis of Bismuth Telluride/Sulfide Core/Shell Nanorods For Thermoelectric Devices
Bismuth telluride is ubiquitous in its application to thermoelectric devices because of its high Seebeck coefficient, an important value in determining the thermoelectric effectiveness of a material. Bismuth telluride nanoparticles have since shown promise in further increasing this Seebeck coefficient. Because thermoelectric devices by definition have the capability of converting heat energy into an electric current, research into bismuth telluride nanoparticles, and the maximization of the Seebeck coefficient in general, shows promise in energy conservation applications. This experiment investigates the synthesis method and thermoelectric characterization of bismuth telluride/sulfide core/shell nanorods, particularly as pertaining to branching, an attribute that contributes to differences in the Seebeck coefficient. Bismuth chloride was placed in a solution of biomolecular surfactant L-glutathionic acid (LGTA) before reacting with telluric acid, allowing for the creation of a sulfide shell. Increasing the molar ratio between LGTA and bismuth chloride was expected to stimulate further branching of the nanorods, which may be confirmed using electron microscopy.
Evan Hess, AAST 2011
Soft lithography is a promising technique in the field of green chemistry, due to its applications in surface science. This experiment involves the production of self assembled monolayers through soft lithography and how they can assist in the effort to be environmentally conscious. Self assembled monolayers of thiols were fabricated on gold surfaces and observed using an electron microscope. Pictures taken reveal how a single layer of thiols can be as effective, if not more, than a thick layer in protecting a surface from deterioration. In addition, using a single layer can immensely reduce wasted materials, since less material is used in coating a surface when forming a self-assembled monolayer.
George Iwaoka, AMST 2012
Investigating UBM Degradation Products as a Possible Therapeutic Treatment for Regenerative Medicine Using a P19 Cell Model
Urinary Bladder Matrix (UBM) is the decellularized product of the extracellular matrix of a pig bladder and it has been shown in prior studies to promote soft tissue regeneration. However, UBM s mechanism of action necessary for the migration, differentiation, and proliferation of stem cells at the site of the reconstruction is poorly understood. Pluripotent P19 embryonic carcinoma cells were introduced as a model cell line to elucidate these events in vitro. Three cell cultures were established from the P19 cells: undifferentiated P19 cells, UBM-differentiated P19 cells, and spontaneously-differentiated P19 cells. Novel results from this experiment demonstrated that UBM induces differentiation in these cells along several lineages. Immunocytochemistry demonstrated cells exposed to UBM for 5 days were positive for MAP2 (Neuron), O1(Oligodendrocyte), and GFAP (astrocytic) proteins. Spontaneously differentiated cells were only positive for GFAP protein (astrocytic). Untreated P19 cells remained positive for the pluripotentcy marker SSEA-1. Proliferation and migration assays were conducted investigating two aspects of tissue reconstruction against UBM. All three cell cultures had increased viability across the concentrations of exogenous UBM (p<0.05) except UBM differentiated P19 cells at lower concentrations (25, 50 g/mL)which demonstrated reduced viability (p<0.05). Migration results against UBM showed an increase by up to 2053% in undifferentiated P19 cells (p<0.05) and a 414% (p<0.05) increase in spontaneously differentiated cells while UBM differentiated P19 cells demonstrated reduced chemotaxis (p<0.05). The study also found evidence that each cell type utilizes different signaling pathways for chemotaxis and proliferation though the details are yet to be understood.
Dongyoun Jang, AAST 2011
Understanding the Nucleation of Calcium Carbonate Crystals through Self-Assembled Monolayers
Calcium carbonate (CC) crystalline structures, also commonly known as calcite, occur naturally within organisms such as diatoms and sea sponges. Nanoscale studies of calcite formations within these organisms have shown that these organisms have stable, ordered structures. Such structural properties of the CC are favorable for engineering projects associated with constructing nanoscale materials that are both cheap and efficient. This research focused on emulating the nucleation of amorphous calcium carbonate, or ACC, to crystal CC under the use of SAM templates terminated by corresponding thiols. Attaching the ACC to a hydroxyl-terminated template, an ACC SAM can be created in which the amorphous stage can be maintained. Under the view of the Scanning Electron Microscope, results show that with the introduction of water and direct contact with a carboxyl-terminated, nucleating template, the ACC can be morphed into crystalline CC. Adding ions and doping the hydroxyl-terminated template will change the morphology of the crystalline and thus lead to varying structural properties and a better emulation of natural crystalline formation. Through the study of how natural CC forms, there will be great improvement in future engineering endeavors in the nanotechnology field.
Woncheol Jeong, AAST 2013
Iron Pyrite Solar Cell Fabrication
Solar energy has been increasingly gaining attention in the world as one of the few viable methods of widespread energy generation, but is hindered by a high initial cost of materials, namely high purity silicon. Economies of scale cannot be depended upon to lower cost, as high purity silicon is also in high demand within the computing industry. Therefore, the solution can be found by moving away from the usage of high purity silicon as a base material; we choose to Iron pyrite, FeS2, has a cost that is at least eight orders of magnitude lower than that of high purity silicon, due to its ubiquity within mining extract, and a suitable band gap for use within a photovoltaic cell (Eg = 0.95 eV), (Wadia, 2009). Iron Pyrite was synthesized on fluorine-doped tin oxide glass substrate through electrochemical deposition, a scalable method of production.
Briette Karanfilian, AVPA 2010
Astrocytes: What Signals Are Yours Sending?
Astrocyte function in the brain is gaining increasing attention. Once thought to play the role of only a structural component of the brain, it is now known that these star-shaped cells with many processes can regulate chemical signals between neurons, they can control the flow of blood in the micro-circulation, and they, themselves, secrete chemical signals called cytokines. Having such significance it is possible that manipulating their activity or using them in transplant has enormous potential in brain disorders. Understanding how they respond on micro and nano-topographies, more like their natural environment than a plastic cell culture dish, is thus of immediate importance. This experiment explores the use of nano-surfaces and their effect on these special cells. Nanosurfaces are surfaces which can have various topographical features such as ridges, needles, or cones in which the varying patterns found on the surfaces are smaller than cells themselves. The surfaces used in this experiment were 1cm2, made of silicon, and had nano-topographies of ridges of varying heights (Varying from 50-600 nm in height). Smooth surfaces acted as the control. IL-6 cytokine production was monitored as this is an inflammatory cytokine produced by astrocytes with significant repercussion in the diseased brain. Mouse astrocytes were grown on the surfaces in 24-well plates and they were imaged using SEM for alterations in orientation caused by the topographies. Viability was determined using the conversion of tetrazolium to formazan through the release of NADH and IL-6 production was measured using ELISA. Cell alignment was significantly affected by topography (p<0.05); cell orientation along the ridges was significantly altered as the height of the ridges increased. Cytokine production results are pending. There was no significant effect on cell viability.
Ji-Sung Kim, AAST 2014
Effect of poly(NIPAm/MAA) nanoparticle membranes on expression of beta-amyloid protein
Diabetics have an increased risk of developing Alzheimer s disease due to their frequent insulin injections. Furthermore, these conventional insulin injections assume that one standard insulin dosage is appropriate for all tissues of the body. This false assumption causes both hypoglycemia and hyperglycemia; both of which promote the expression of beta-amyloid in the brain, a protein critical to the pathology of Alzheimer s disease. As a result, an insulin delivery system which can respond to local levels of glucose in the body is necessary. The proposed solution is a glucose-sensitive, poly(NIPAm/mAA) nanoparticle membrane system. The system relies on a glucose/glucose oxidase reaction which activates the poly(NIPAm) to release insulin. This allows a localized treatment of insulin deficiencies. The nanoparticles were created using a polymerization reaction and were purified and cast into an ethyl cellulose ethanol solution with glucose oxidase and catalase, creating the membrane. Results of the efficacy of the system are pending.
Paul Kim, AEDT 2013
Fabrication and Characterization of Functionalized Multi-wall Carbon Nanotube Filters for Cd(II) Ion Removal
Industries like electroplating and metal refinery in developing countries dump wastewater containing cadmium ions into bodies of water that communities depend on for food and water. Cadmium ions accumulate in organisms, like humans, causing kidney and bone tissue problems. This research uses carbon nanotubes to filter harmful ions from contaminated water. Functionalized (–COOH and –CONH2) and non-functionalized multi-wall carbon nanotube (MWCNT) films were prepared onto mixed cellulose (MCE) filters (0.20 micron pore) by vacuum filtration of corresponding aqueous MWCNT suspensions dispersed by sodium dodecylbenzene sulfonate. This study revealed that –CONH2 filter rejected 0.549 of the total cadmium ions at 10 mg/L compared to 0.379 rejection ratio of the –COOH filter and 0.198 rejection rate of the normal filter. The thicknesses of the films were measured by scanning electron microscopy and used to normalize the rejection ratios for the –COOH and the -CONH2 functionalized filters, for the same thickness as the non-functionalized film. The normalized rejection ratios were 0.517 for –COOH and 0.924 for –CONH2 both at 10 mg/L, increasing the rejection ratio by 36% and 68% respectively. The positive charge of the –CONH2 functionalized MWCNT may have caused the filter to remove the greatest amount of cadmium ions.
Era Kovanxhi, AMST 2011
The Effect of Pronathocyanidins on E. coli Adherence to Small Intestine Cell Line
There are many reports on the health benefits of consuming cranberries. Cranberries contain compounds called proanthocyanidins, which have A-type and B-type linkages. These proanthocyanidins have also been suggested to prevent adhesion of P-fimbriated uropathogenic Escherichia coli to uroepithelial cells. Fimbriae are filamentous proteins on the surface of bacterial cells that may behave as adhesins for specific adherence, involving many lock-and-key bonds between complementary molecules on each cell surface. How do B-type and A-type fimbriae differ from each other? How does concentration of proanthocyanidins play a role? It has been found that grape seed extract yields a higher concentration of B-type proanthocyanidins than those isolated from cranberry extract. Cranberry extract has a greater concentration of A-type proanthocyanidins. The goal of this study was to compare the effects of A-linked and B-linked proanthocyanidins on the structure and function of the fimbirae of Escherichia coli (ATCC 25922, 29194, 49161) and to also observe the effect of the proanthocyanidins on the adhesion properties of the fimbriae on small intestine cells (CCL-241).
Michael Knyszek, ATCS 2013
Growth of Single Crystalline Potassium Tantalate for Spintronics Applications
As research into spin transport electronics continues, the search for suitable materials to act as solid-state devices continues as well. The objective of this experiment was investigation of the creation of potassium tantalate, a solid-state single crystalline semiconductor to be used in conjunction with spin transport electronics research. Potassium carbonate and tantalum oxide were the primary components melted in a 20mL platinum crucible in a high temperature muffle furnace. The components were heated to liquid state with excess potassium carbonate used as a flux, and soaked for several hours before being super-cooled. This non-dopant experiment produced roughly 3mm bulk potassium tantalate crystals, as proven through EDX sample analysis. Further proof of the materials identity was found in SEM imaging where it exhibited crystal-like properties such as uniformity. However, in order for the material to be useful in development of giant magnetoresistance devices, the bulk crystal needs to sport ferromagnetism, which theoretically could be solved by using cobalt cations as a dopant. A second experiment was run where cobalt(II) carbonate was added to the initial mixture before entering the furnace but has yet to show any substantial results. More experimentation is necessary in order to determine the conditions required successfully grow doped single crystalline potassium tantalate.
Won Ik Lee, AAST 2011 and Hong Joon Park, AAST 2012
Second Life: Novel and Interspecies Reprogramming of Induced Pluripotent Stem Cells
Induced pluripotent stem cells (iPSCs) have immense potential for regenerative medicine and tissue repair. One approach to generate such cells from mammalian somatic cells involves the expression of four defined transcription factors, Klf4, c-Myc, Oct3/4, and Sox2. However, current reprogramming strategies through retroviral transduction lead to tumorigenesis, and the difficulty of maintaining human iPSC pluripotency further discourages clinical use. Thus, the purpose of this project was to develop a novel method of reprogramming that precludes tumorigenesis and facilitates iPSC colony maintenance. We hypothesized that polyethyleneimine (PEI) transfection using four murine Yamanaka factors in human fibroblasts should allow for the overcoming of both major challenges. After transfecting with DNA-PEI complexes, a number of fluorescence and enzyme immunoassays indicated optimal ratios for PEI-based reprogramming. Moreover, our results demonstrate that generation of iPSCs can be accomplished interspecially by reprogramming human fibroblasts to pluripotency with murine genes. This was confirmed by morphological and alkaline phosphatase analyses and immunohistological staining of the unique human interspecies iPSC colonies, which showed that they do not differ in form or function from traditional human iPSCs. Most importantly, it was shown that the pluripotency of human interspecies iPSCs can be preserved on leukemia inhibitory factor (LIF) instead of the conventional feeder layer, greatly promoting ease of maintenance. Thus, we demonstrate that PEI transfection is an efficient, viable method of iPSC generation without the risk of insertional mutagenesis and present the novel development of human interspecies iPSCs, providing a promising future for the clinical use of iPSCs.
Dennis London, AAST 2010
Control of Astrocyte Cell Growth Using Multiwalled Carbon Nanotubes
The goal of this research was to determine whether it is possible to control the direction of astrocyte cell growth using functionalized multiwalled carbon nanotubes (MWNTs). These nanotubes are available with a variety of surface charges and functional groups attached, such as amine and carboxyl groups. Astrocytes are characteristic star-shaped glial cells in the brain and spinal cord, which can be easily grown in cell culture, unlike neurons. The growth of astrocytes was studied morphologically using light and scanning electron microscopy, as a function of MWNT concentration, charge and functional group. Assays were done with MWNTs attached to a surface as well as with MWNTs dispersed in a media. The mechanisms through which MWNTs enter astrocytes were also studied using transmission electron microscopy, and it was found that MWNTs both passively penetrate the cell membrane and are actively endocytosed in vesicles. For the MWNTs in solution assay, effective concentrations of MWNTs were found to be between 1.95 g mL and 31.3 g mL. It was found in both assays that astrocytes grew in three-dimensional filaments when in the presence of MWNTs, as opposed to controls which grew uniformly and flatly over the substrate. Astrocytes in the experimental group grew over each other and formed connections with other astrocytes in their own plane and also with astrocytes above and below them. In addition, MWNTs clumps caused astrocyte clumps to form around them. Astrocytes outside clumps and filaments underwent cell death and fragmentation. This result suggests that astrocytes grow preferentially on carbon nanotubes, but astrocytes that are not in clumps or filaments are unable to survive in the presence of carbon nanotubes. This provides the possibility of controlling astrocyte cell growth to heal spinal cord and other injuries of the central nervous system, in which astrocytes form scar tissue, thus preventing nerve regeneration. In addition it opens the possibility for targeted drug delivery to astrocytes using carbon nanotubes. Targeted drug delivery would allow for the destruction of gliomas (a cancer arising in astrocytes) using existing chemotherapy drugs without destroying healthy cells.
Joshua Meier, AAST 2014
The Missing Genome: Mitochondrial DNA Deletions in Stem Cells
Induced pluripotent stem cells (iPSCs) have great potential for medical use based on their similarities to embryonic stem cells (ESCs), but are easier to obtain based on their ability to be generated from somatic cells. While iPSCs seem promising due to their qualities of pluripotency and self-renewal, recent research has shown that iPSCs differ from ESCs with a much higher rate of apoptosis, decreased rate of proliferation, and premature aging. It was hypothesized that this phenomenon of rapid senescence is triggered by a large deletion in the mitochondrial DNA (mtDNA) of iPSCs. The presence of this deletion was then investigated using polymerase chain reaction on the mtDNA of iPSCs generated using different methods, their source cells, and the mtDNA from a patient known to have the deletion as a control. In addition, a standard cancer line was also tested and found not to have the deletion. Finally, transmission electron microscopy was utilized in order to determine whether any morphological differences exist between the mitochondria of iPSCs and their source cells. The results demonstrate that iPSCs have mtDNA deletions and morphological changes that are correlated with the methods used to generate them. Future research will include analysis of other cells lines in order to determine the different mechanisms in which iPSC generation causes damage to the mitochondria. The results suggest that presence of mtDNA deletions may be a useful diagnostic test for determining whether novel methods of developing stem cells have medical potential.
Jae Seong No, AAST 2011
Synthesis and Characterization of Nanoparticles for Hydrogen Gas Generation
Catalysis is an important process that allows scientists to induce and speed up chemical reactions that would otherwise have occurred less efficiently. According to recent studies, noble metal nanoparticles have been discovered to have a unique ability to catalyze different chemical reactions. One such reaction is the generation of hydrogen from ammonia-borane. Hydrogen generation is a relatively new topic that scientists have been studying due to their interest in the application to fuel cells. Hydrogen may be used in the future to supply an alternative source of energy that would be much greener than present-day fuel sources. This research project will explore how the noble metal nanoparticles Ruthenium and Platinum affect the catalysis of hydrogen generation from an ammonia-borane complex. The supported metal catalysts will be synthesized by mixing personally prepared solutions of the noble metal nanoparticles in aluminum oxide (Al2O3) that will then be calcined at high temperatures. The sample will be then placed in a closed container with the ammonia-borane complex and water where the amount of hydrogen gas will be measured at set volume intervals by collecting the gas over water into an inverted graduated cylinder.
Andres Paez & Namrata Ramani, Engineering Majors (BCTS), 2010
Rapid Cycling Brassica rapa: A Novel Phytomining Plant
Many plants are capable of drawing specific heavy metal ions out of contaminated soil and converting them to solid metal by a process known as phytomining. These plants are referred to as hyperaccumulators. Brassica rapa has not been labeled a hyperaccumulator, however, known hyperaccumulators include Brassica napus, Brassica juncea, and Brassica oleracea. The goals of this experiment are to determine the ability of rapidly cycling Brassica rapa to hyperaccumulate gold; if so, to then compare hyperaccumulation when the plant is exposed to a gold ion solution as opposed to exposure to fully formed gold nanoparticles; and to determine localization of gold nanoparticles in root, stem and/or leaf tissue by transmission electron microscopy.
Yeonjin Park, AEDT 2013
Investigating the bacterial inhibition rate of related sugars and berries
Dental caries, or cavities, is a disease that affects the greater part of the world s population. These cavities are caused by the formation of dental plaque by oral bacteria. It is known that different strains of these bacteria are able to attach to one another and coaggregate. It has been discovered that varioussugars and berries can be used to inhibit the coaggregation of bacterial pairs, thus preventing further progressionto dental caries. The object of the research reported here was to identify which type of sugar and which berrieshave the most potential with regard to bacterial inhibition.Streptococcus mutans and Lactobacillus rhamnosus were cultured separately in the presence and absence of various sugars and berry extract and then mixed together and allowed to coaggregate. Absorbance values acquired from spectrophotometer were used to calculate thepercentage of inhibitionof coaggregationbased on the formula used by University of Eastern Finlandin a study on theantiaggregation potential of berry fractions.It was found that lactose produced 97% inhibition; D-galactose 90% inhibition; D-fructose 46% inhibition; and D-fucose 43% inhibition of aggregation as compared to control, untreated bacteria. Results from berry fractions are pending.
Supriya Rastogi, AMST 2011
A Novel Intracellular Target for Anti-Allergic Medications
Curcumin, a potent spice found in Asian cuisines, offers scant explanation of its anti-allergic properties. The primary cause of allergies is the release of histamine through a process called degranulation of mast cells. This study demonstrates that treating cells with curcumin (1 microgram/ml) significantly decreases the release of histamine (p<0.05), suggesting curcumin inhibits degranulation. Degranulation involves two pathways: calcium-independent microtubule translocation and calcium-dependent F-actin ring disassembly, both of which are mediated by protein RhoA. Cells treated with curcumin and stimulated to degranulate exhibited significantly lower levels of activated RhoA when compared to degranulating cells (p<0.05). To determine the pathway being affected by curcumin, its effect on microtubules, F-actin rings, and calcium levels was measured using Paclitaxel, phalloidin, and indo-1AM dye respectively, in rat basophilic leukemia (RBL-2H3) cells. Fluorescent microscopy and transmission electron microscopy (T.E.M.) were used to image histamine and histamine-secreting vesicles, respectively. Intriguingly, curcumin appeared to cause microtubule aggregation (p<0.05), increase disassembly of F-actin rings (p<0.05), have no effect on calcium levels, and keep histamine within the outskirts of the cell. Curcumin appears to inhibit degranulation by impeding functional microtubule polymerization. T.E.M. pictures provide evidence that curcumin promises to add some "spice" as a battling therapeutic drug, exemplifying how microtubules can be a novel intracellular target for anti-allergic medications.
Theresa Regetz, AAST 2013
Investigation of Fibroblast Cell Interaction with Different Surface Morphology of Titanium for Implant Applications
Titanium is the most compatible metal with biological systems due to its inertness and strong corrosion resistance; hence, it is a frequently used material for implants. One of the issues facing current implants is the extensive recovery time due to the cells slowly proliferating around the new implants. In this study, the in-vitro growth of fibroblasts on titanium substrates of varying surface textures will be observed to examine which topography simulates the healing process most effectively. Fibroblasts, found primarily around joints, bones, cartilage and adipose (or fat tissue) where titanium implants are most often placed, are used in this study because they make up the majority of connective tissue. The implant success, altered by surface topography, will be verified by the quality of healing via cell viability and morphology. By observing the cell viability, adhesion, density, morphology, and the levels of Interleukin-1B throughout the extracellular membrane, this experiment should determine which titanium surface texture will allow fibroblast cells to survive the best.
David Sadegh, AEDT 2010
Athletic Tape: How Structure and Elasticity Accelerate the Healing Process
The goal of this project was to determine which qualities make kinesiology tapes so much more effective than other athletic tapes in the accelerated healing of muscles and joints. Each tape was analyzed based on elasticity, chemical composition, and fabric structure. I used a Structural Stress Analyzer to test elasticity, obtaining a stress over strain curve for each sample. Using a Scanning Electron Microscope, I took pictures of the tapes to get a better understanding of the fabric and adhesive structure designs. I used the X-ray function of the microscope to find the percentage and distribution of chemical elements within the samples. Finally, I developed an anonymous clinical survey, administered by a collaborating doctor, to compare my data with patient and physician responses. I focused on the tapes which were agreed to be most effective and tried to find correlations with my results. Identifying the most influential healing factors would help verify which tape brands are most useful to athletes, workers, and even soldiers, who need quick and simple treatment of injuries to stay active and keep moving.
Jasmin Sadegh, AEDT 2009
Tissue Engineered Blood Vessels
The goal of my experiment is to seed various matrixes with cells to ultimately mimic the structure of a blood vessel. I seeded a mouse embryonic carcinoma cell line on various matrixes like macaroni and gel-like materials; the gel-like materials include an alginate solution and an agarose solution. I chose macaroni as a possible substrate due to the pre-formed vascular shape of some types. Microscopic pictures recorded the cells ability to adhere to the matrices. Using one particular cell growth medium, the cells were able to grow on macaroni as a substrate. On the native agarose gel, cells did not adhere well until supplements like collagen and polyethyleneimine (PEI) were added. The matrixes were also tested for their ability to form shapes. The agarose was wrapped around a tube; the alginate was shaped as it was solidified by release into a calcium chloride solution. Agarose proved to be easiest to manipulate into a tubular shape.
Varsha Subramaniam, AMST 2013
Genetic Identification Using Skin Fragments left on Latent Fingerprints
Short Tandem Repeats (STR) markers are genetic markers that are used in human identity testing by the method of multiplex Polymerase Chain Reaction (PCR). The assays measure the length of the PCR product and compare it to the sequenced allelic ladder. The STRs are highly polymorphic and can be used to type from very little template material. As in all forensic situations, the sample DNA to be tested undergoes varying level of degradation due to bacterial, biochemical or oxidative process and there are additional contaminants from the environment that has mixed in with the sample. Loss of signal is common outcome when using STR markers for such samples. Recently, using smaller sized PCR products from STR markers (called mini STRs), generated by moving the primers in as close to the STR repeat region as possible, has been shown to be more successful with degraded samples. It has been shown that human cells can be recovered from latent fingerprints. The DNA in these cells is expected to be in various stages of degradation. The current investigation is geared towards using miniSTRs to generate DNA fingerprinting profile from cells recovered from such latent fingerprints. As a first step towards this goal, the sensitivity of miniSTR s on the amount of DNA from invitro cultured human breast-cancer cells (MCF-7) was tested. Results show that PCR with a mini STR primer, D7S820, can amplify from samples that contain approximately 1 picogram of template DNA. Similar analyses are performed at other miniSTR loci, and samples are being tested from various simulated forensic environments.
Daniel Zhou, AAST 2010
Enhancing Transfection Using Functionalized Single-Wall Carbon Nanotubes and DNA Complexes
Two distinct fields of science, biology and nanotechnology has come together to lead the way in a new and exciting field called nanobiotechnology. Although numerous advancements have been made, there still exists a great potential for future breakthroughs. This project aims to improve our knowledge of how these two fields interact. With that said, this project incorporates the use amine functionalized single-wall carbon nanotubes (f-SWNT) as possible novel mode of transfecting cells. The idea behind this project stems from the basis that DNA is negatively charged. The positively charged f-SWNTs would, in theory, neutralize the DNA s charge, forming an overall neutral complex capable of entering a cell. This method of transfection was compared to standard transfection protocols using polyethyleneimine and DNA. The transfecting targets were HepG2 cells, a type of epithelial liver cancer cell line. The results had disproved the original hypothesis. However, though the f-SWNT:DNA complexes took a longer time to transfect the HepG2 cells than the PEI:DNA solutions, it appeared they two methods of transfection yielded similar results given enough time.