Organizational Unit:

Undergraduate Research Opportunities Program

Permanent Link

https://hdl.handle.net/1853/70802

Parent Organization

Organizational Unit

Office of Undergraduate Education

ArchiveSpace Name Record

https://finding-aids.library.gatech.edu/agents/corporate_entities/1180

Full item page

Publication Search Results

Now showing 1 - 10 of 149

An investigation of the mechanism behind Adjuplex and its effect in adjuvant combinations

(Georgia Institute of Technology, 2020-12) Vogel, Gabriela Maria

This research thesis contributes to the field of biomedical engineering, specifically to the following topics: immunotherapy, drug delivery, and adjuvant-vaccine work. By utilizing RAW-Blue cells to observe IFN-β production, NF-κB production, and overall activation of TLRs, we were able to show the effects dosage and combinations of adjuvants have on immune activation. While investigations summarized in this thesis show that Adjuplex in combination with other common adjuvants such as MPLA and CpG decreases the production of IFN-β, this did not indicate that Adjuplex suppresses the immune response that known adjuvants normally induce. Additionally, Luminex assays, ELISA assays, and flow cytometry experiments were performed to learn more about the mechanism behind Adjuplex’s ability to produce an immune response and more in depth on how Adjuplex interacts with other adjuvants in combination. Results showed that there was synergy between Adjuplex and GLA when looking at cytokines produced through the inflammasome pathway. Therefore, future work should expand on this finding and experiments should focus on further validating this finding by looking at other markers in the inflammasome pathway. Characterization of the type of adaptive immune response Adjuplex produces as an adjuvant is of critical relevance to the development of synergistic or suppressive adjuvant combinations and the optimization of vaccines.
Biofilm Matrix Properties in Pseudomonas aeruginosa under Bacteriophage Treatment

(Georgia Institute of Technology, 2020-12) Butler, James H.

Biofilms are a specialized structural formation that some bacterial species form when growing in certain environments. One such environment is the human lung, where the bacteria Pseudomonas aeruginosa forms biofilms that infect the lungs of cystic fibrosis patients. Techniques used to destroy biofilms have been studied previously, and one promising technique involves the use of bacteriophage. Phage are small, bacteria-targeting viruses that when introduced to biofilms cause the lysis, or destruction of bacterial cells and in some cases, the subsequent destruction of the biofilm. While the impact of phages on biofilms is well known, the changes in physical properties such as extracellular polymeric substance (EPS) distribution and pH distribution in biofilms treated with phage has not been examined in detail. This study seeks to establish pH distribution in Pseudomonas aeruginosa biofilms using confocal microscopy and the pH sensitive molecular probe CSNARF4, as well as SYPRO ruby biofilm for the staining of all EPS proteins. Both phage-treated and untreated biofilm samples with the separately added stains of CSNARF4 and SYPRO ruby biofilm matrix were imaged using confocal microscopy and evaluated with a microtiter plate reader, respectively. It was discovered that biofilms treated with phage exhibited little reduction in average pH across all depths into the biofilm, while there was a pronounced increase in protein release into the EPS upon cell death. These results provide greater insight into the effect phages have when being used to treat biofilms and elucidates points of improvement in biofilm treatment.
Alginate Core-Shell Scaffolds for CAR T cell Manufacturing

(Georgia Institute of Technology, 2020-12) Mousavi Karimi, Zahra

Cancer patients with advanced-stage disease develop resistance to traditional therapies such as chemotherapy and radiation therapy, leading to the necessity of a novel technology that is both specific and efficient to the patient’s cancer. Genetically engineered T cell receptors can recognize and bind to antigens expressed on tumor cells and kill them. As a result, T cell immunotherapies have been established as a new strategic cancer therapy. The promising success of T cell immunotherapies for treatment of cancer and other diseases demonstrate the need for scalable manufacturing processes for product commercialization. Unlike traditional chemotherapies, cell-based immunotherapies are composed of living entities, and hence they are different in their development, properties and regulatory pathways in comparison to traditional drugs, which are simple chemicals. Current T cell manufacturing techniques are complicated processes that do not account for the complexity of the lymph nodes, where T cells expand rapidly in response to disease. Consequently, it is difficult to produce enough cells for quality control assays and meet the Good Manufacturing Practices (GMP) guidelines. Thus, we hypothesize that creating a novel microenvironment that can mimic the lymph nodes will enhance expansion of T cells and allow this promising treatment to reach more patients in the clinic. To do so, we proposed using alginate which has been widely used for cell encapsulation. We prepared and optimized alginate core-shell scaffolds to provide an environment for close cell-cell contact and communication as well as protect T cells against stress for CAR T cell signaling and expansion. After expansion, the scaffolds were dissolved through both enzymatic and physical dissociation. This method will eventually allow the modified T cells to be placed in a bioreactor, allowing this technique to be commercialized by companies in the cell manufacturing industry.
Developing a Luminal Perfusion System for Human Intestinal Organoids

(Georgia Institute of Technology, 2020-12) Slyman, Raleigh James Hudson

Human intestinal organoids (HIOs) are 3-dimensional aggregates of cells that can replicate the structure and function of the human gastrointestinal system. HIOs generally resemble fetal tissue, so efforts have been made to mature them such that they replicate adult gastrointestinal physiology. Previous studies have shown HIO maturation following an injection of E. coli into the HIO lumen but did not attempt to regulate E. coli populations thereafter. This thesis presents a perfusion system which has the potential to modulate a luminal bacteria population through fluidic transfer. The system consists of a double-barrel glass capillary connected to a pressure-based pump, which allows for high temporal control of fluid flow. The system has been shown to regulate the concentration of diffusible dye molecules in the lumen and partially regulate E. coli distributed heterogeneously in the lumen. If volumetric flow control and incubator compatibility are implemented, the new proposed system may enable long-term study of HIO-E. coli interaction.
Dissociable Effect of Stimulus Fluency on Accuracy and Confidence in Perceptual Decision Making

(Georgia Institute of Technology, 2020-12) Webber, Alexis Kaitlyn

Perceptual decision making relies on collecting evidence from stimuli to make an accurate judgement. The decision is accompanied by a sense of confidence based on the same evidence. Although the accuracy and confidence of a decision are often correlated, it is important to understand cases when they are dissociable. The goal of this study was to investigate qualities of the stimuli that lead to these cases. A dot motion experiment was performed where participants were asked to judge the direction of the motion and report their subsequent confidence in the decision. The motion of the dots was varied in two ways: the strength of signal was controlled by adjusting the tilt difference and the noise of the signal was controlled by adjusting the coherence of the motion. It was discovered that in cases with high coherence and low or medium tilt, confidence was higher, but accuracy was lower than cases with low or medium coherence and high tilt. From this it was concluded that coherence has more of an effect on confidence while tilt has more of an effect on accuracy, causing a dissociation between accuracy and confidence. These findings are important because they provide insight into cases of over and under confidence that can allow for us to better gauge the validity of the confidence judgement as it relates to accuracy depending on the qualities of signal strength and noise.
Characterizing Excitatory and Inhibitory Neuron Responses to Dark and Bright Stimuli in the Visual Cortex of Awake Mice

(Georgia Institute of Technology, 2020-08) Meyer-Baese, Lisa

An increased understanding in mouse primary visual cortex (V1) will allow us to better characterize the internal electrical and neural circuit mechanisms of the visual process. So far, most studies directly recording single neurons have been mainly performed in anesthetized animals. This study is novel in that it aims to extend these findings to the awake visual cortex, and to multiple retinotopic locations in V1 of mice. To characterize how excitatory and inhibitory neurons in V1 respond to bright and dark visual stimuli the mice were presented with input stimulus of different colored bars, ranging in luminance defined by Michelson Contrast. Their response to these stimuli was recorded using multi-shank probes that were placed in V1. Once neurons were identified as being inhibitory and excitatory, their response properties were then be tied back to the different input stimuli. Results do not indicate any statistically significant differences between the response of the two classes.
Characterizing the Dynamics of Macrophage Polarization and Signaling

(Georgia Institute of Technology, 2020-05) Forsmo, James Edward

Apart from their primary functions in innate immunity and phagocytosis, macrophages are critical regulators of inflammation through their ability to adopt polarization states, a spectrum of phenotypes in which macrophages change their morphology and produce large amounts of pro/anti-inflammatory cytokines and biomolecules. Dysregulated macrophage polarization has been implicated in numerous chronic inflammatory diseases. However, as a result of the tightly-regulated nature of macrophage polarization, it is difficult to design immunomodulatory strategies for modulating macrophage polarization without a robust understanding of the temporal dynamics involved. As a result, there is a need for a deeper understanding of polarization dynamics that can be used in conjunction with computational modeling to develop better immunomodulatory strategies. In this thesis, RAW 264.7 murine macrophages are subject to a number of different stimulation strategies in order to temporally characterize how these cells translate inputs (such as pro-inflammatory cytokines and biomolecules) into a key marker of pro-inflammatory polarization output (iNOS expression over time). Specifically, I measured iNOS expression primarily through immunocytochemistry performed in either 96-well microwell plates or in PDMS microfluidic devices for three-dimensional (3D) culture experiments. Performing polarization experiments in microfluidic devices revealed that 3D culture environments exhibit differences in polarization dynamics. In particular, I found that the M1 response to lipopolysaccharide (LPS) stimulation is always transient regardless of whether macrophages are re-stimulated with fresh LPS-containing medium. Subsequently, I investigated re-stimulating with an M1 cytokine that activates different pathways than LPS to upregulate pro-inflammatory genes, referred to in this thesis as an orthogonal stimulus. Interestingly, further re-stimulation with an orthogonal stimulus, which upregulates iNOS through a separate pathway, such as interferon-γ, can extend M1 polarization and reach greater levels of iNOS expression than any single stimulus alone, in an order and dose-dependent fashion. I also showed that culturing macrophages in a three-dimensional environment within microfluidic devices delays their response to LPS but exhibits the same critical maximum of iNOS expression, suggesting a potential role for the extracellular matrix in regulating polarization dynamics. This thesis contributes novel findings to our understanding of macrophage polarization and proposes new avenues of investigation into the dynamics of other important cytokines in both 2D and 3D culture environments. These results may inform the development of novel models for understanding the temporal dynamics of macrophage polarization and direct further work into modulating polarization states for the treatment of inflammatory diseases.
AxoNet: A Deep Learning-based Tool to Count Retinal Ganglion Cell Axons

(Georgia Institute of Technology, 2020-05) Ritch, Matthew D.

In this work, we develop a robust, extensible tool to automatically and accurately count retinal ganglion cell axons in optic nerve (ON) tissue images from various animal models of glaucoma. We adapted deep learning to regress pixelwise axon count density estimates, which were then integrated over the image area to determine axon counts. The tool, termed AxoNet, was trained and evaluated using a dataset containing images of ON regions randomly selected from whole cross sections of both control and damaged rat ONs and manually annotated for axon count and location. This rat-trained network was then applied to a separate dataset of non-human primate (NHP) ON images. AxoNet was compared to two existing automated axon counting tools, AxonMaster and AxonJ, using both datasets. AxoNet outperformed the existing tools on both the rat and NHP ON datasets as judged by mean absolute error, R2 values when regressing automated vs. manual counts, and Bland-Altman analysis. AxoNet does not rely on hand-crafted image features for axon recognition and is robust to variations in the extent of ON tissue damage, image quality, and species of mammal. Therefore, AxoNet is not species-specific and can be extended to quantify additional ON characteristics in glaucoma and potentially other neurodegenerative diseases.
Anti-CRISPRs Oligonucleotides Facilitate Cell Type-Specific Control of Nanoparticle Cas9 Gene Editing

(Georgia Institute of Technology, 2020-05) Fitzgerald, Jordan Paul

While CRISPR-Cas systems represent a powerful research tool, in order to become clinically viable significant challenges woud have to be overcome. One of the largest issues facing the breadth of CRISPR technologies is the lack of cell type specific gene editing outside of hepatocytes. Controlling the cell type-specific activity of CRISPR-based drugs would enable new Cas9 therapies. Natural anti-CRISPRs can inhibit gene editing, suggesting that synthetic anti-CRISPRs delivered to ‘off target’ cells could limit undesired gene editing in vivo. This report shows that anti-CRISPRs termed inhibitory oligonucleotides (iOligos), which target single guide RNA (sgRNA) via a Rnase H15 independent mechanism, can modulate gene editing in adult mice. By delivering iOligos to hepatocytes, Cas9 hepatocyte editing is blocked, altering the tropism of a nanoparticle so it preferentially edits genes in the spleen. This represents a technology by which it is possible to markedly suppress gene editing in hepatocytes without reducing splenic editing. Synthetic anti-CRISPRs can improve the reach and specificity of Cas9 therapies in adult mammals without improvements in drug delivery vehicles like new nanoparticles.
Development of a microscale aqueous two-phase system bacterial culture platform

(Georgia Institute of Technology, 2020-05) Pavlidis, Despina

Development of a bacterial culture platform which accounts for the characteristic human gut oxygen gradient could allow for improved understanding of the gut microbiome and its implications in human health as well as targeted therapies for related diseases. This work presents a microscale bacterial culture platform in an aqueous two-phase system (ATPS) with integrated oxygen-sensing microbeads. Generation of the oxygen gradient is explored through the culture of the facultative bacterium, Escherichia coli (E. coli), within the platform. Growth characterization of GFP-expressing E.coli O157:H7 within the platform is achieved through time lapse measurements of oxygen consumption and GFP fluorescence, as well as plate counting data. Phase fluorimetry measurements are collected through the oxygen-sensing microbeads placed at the bottom of the culture wells. Results indicate that E.coli was able to grow in the ATPS culture environment and that near anaerobic conditions were achieved at the bottom of the culture wells. Oxygen concentration information is restricted to the bottom of the culture wells due to microbead placement; however, in future iterations, this platform could accommodate other types of bacteria such as aerobic and anaerobic bacteria.