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PROFILING EXTRACELLULAR VESICLES IN CIGARETTE SMOKE-INDUCED BLOOD-BRAIN BARRIER DAMAGE AND ESTABLISHING AN INDUCED PLURIPOTENT STEM CELL VASCULAR MODEL
No full textCigarette smoke exposure adversely affects neurovascular health and is linked to disorders such as stroke, vascular dementia, and Alzheimer's disease. In this thesis, we use cigarette smoke extract (CSE) and cell culture models to assess the contributions of cigarette smoke exposure and extracellular vesicles (EVs) to blood-brain barrier (BBB) damage. Specifically, we investigate the viability and morphology of brain microvascular endothelial cells (BMECs) under exposure to different concentrations of CSE, as well as BBB marker proteins including tight junction marker ZO-1, cytoskeletal marker VE-cad, and glucose transporter GLUT-1. Additionally, EVs were collected from CSE-treated BMECs, characterized, and used to treat BMECs to gain insight into how EVs affect the BBB. Lastly, we establish a model of endothelial cell-pericyte interactions. Induced pluripotent stem cells (iPSCs) were differentiated into endothelial cells (CD31+) and pericytes (NG2+) and combined into a three-dimensional model. EVs from iPSC-derived endothelial cells (iECs) were then introduced into the model to investigate endothelial cell communication with pericytes
Learning to Synthesize Images with Multimodal and Hierarchical inputs
No full textIn recent years, the field of image synthesis and manipulation has experienced remarkable advancements driven by the success of deep learning methods and the availability of Web-scale datasets. Despite this progress, most current approaches predominantly rely on generating images based on simplistic inputs such as text and label maps. While these methods have demonstrated an impressive capability in generating realistic images, there persists a notable disconnect between the intricate nature of human ideas and the simplistic input structures employed by the existing models. In this thesis, we present our research towards a more natural way for controllable image synthesis inspired by the coarse-to-fine workflow of human artists and the inherently multimodal aspect of human thought processes. We consider the inputs of semantic and visual modality, as well as the varying levels of hierarchy under each modality. For the semantic modality, we introduce a general framework for modeling semantic inputs of different levels, which includes image-level text prompts and pixel-level label maps as two extremes and brings a series of mid-level regional descriptions with different precision. For the visual modality, we explore the use of low-level and high-level visual inputs aligning with the natural hierarchy of visual processing. Specifically, we model the low-level process as an image manipulation problem, characterized by the pixel-wise alignment between the output and input; while the high-level process is cast into a reference-based generation where the goal is to transfer information from reference images to the generated images at the granularity of objects or concepts. Additionally, as the misuse of generated images becomes a societal threat, we introduce our findings on the trustworthiness of deep generative models in the second part of this thesis. After that, I will discuss some potential future research directions
DNA DAMAGE DRIVES ANTIGEN DIVERSIFICATION THROUGH MOSAIC VSG FORMATION IN TRYPANOSOMA BRUCEI
Get PDFTrypanosoma brucei evades the host humoral immune system through antigenic variation, using a dense Variant Surface Glycoprotein (VSG) coat to cover the parasite surface in a single antigen then swapping it to avoid immune clearance. Parasites encode thousands of VSG-encoding genes, however most of these are not intact. Parasites must extend their repertoire of viable VSGs to maintain long term, chronic infections.
The main mechanism of VSG diversification appears to be mosaic formation, where multiple VSGs are recombined into new antigens. I created a new tool, VSG-AMP-seq, which allows me to characterize VSG diversification in unprecedented detail. Using VSG-AMP-seq, I characterized mosaic formation after a DNA double strand break introduced by Cas9. AnTat1.1, a VSG with multiple homologous family members, recombines with homologous VSG donors for DNA repair at the break site within its coding sequence, keeping the resulting VSG in frame. Most VSGs have homologous families and can diversify through mosaic formation.
I further demonstrated that mosaic VSGs are formed by templated insertions that copy donor VSG sequence from within the genome. The recombination sites themselves are regions of short perfect homology, suggesting microhomology plays a role in DNA repair. The location of the donor VSG within the genome appears to have little influence on its accessibility as a donor.
Mosaic VSGs matching those I detected in vitro from AnTat1.1 are formed in vivo in WT mice within tissues and in blood in immunocompromised μMT mice. The immune response which clears AnTat1.1 from WT mouse blood appears to select recombination events with larger insertions replacing most of the VSG N- terminus. However, the recombination sites detected still match microhomology observed in vitro.
Finally, I demonstrated that the position within AnTat1.1 which produces the most mosaic VSG recombination events occurs at the unstructured apex of the VSG directly facing host antibody and provides substantial immune evasion. Overall, this thesis provides unprecedented insight into VSG diversification – an important, often neglected feature of antigenic variation. Here I present the experimental groundwork for multiple new avenues of investigation into antigen diversification to further dissect the mechanisms driving mosaic-generating DNA repair
IN VITRO BIOMATERIALS CELL CULTURE SYSTEMS: INVESTIGATING THE INFLUENCE OF STIFFNESS AND STRESS RELAXATION ON CELL MIGRATION, MICROGLIA ACTIVITIES, AND ALZHEIMER'S DISEASE MODELING
No full textThe extracellular matrix (ECM) surrounding cells provides both biochemical cues and biomechanical cues that regulate cell behaviors and fate. However, there is not a full understanding of the impacts of stiffness and viscoelasticity on cell migration, microglia activities and functions, and neurodegenerative diseases like Alzheimer’s disease (AD). Also, the mechanical properties of the matrix have often been overlooked in the design of in vitro cell culture models. This dissertation investigates the relationship between the mechanical properties of ECM and cellular behavior, with a focus on its implications for AD modeling.
After a brief introduction to the applications of alginate hydrogel in tissue engineering, cell migration, Mechanotransduction, AD, and brain mechanical changes, the dissertation discusses three main projects. Chapter 2 explores the impact of stress relaxation on different modes of cell migration in both 2D and 3D environments. This work demonstrates that faster stress relaxation significantly regulates cell migration behavior and identifies key cellular processes involved in this response. This project also explores the usage of alginate hydrogel for different types of in vitro cell culture models and establishes a platform for later projects. Chapter 3 studies the role of altered brain mechanics in AD pathogenesis. Through comprehensive mechanical characterization of brain tissues of AD mouse models, the study reveals increased brain stiffness with AD progression, particularly in regions associated with cognitive decline. Furthermore, using a tunable hydrogel system mimicking the mechanical properties of healthy and AD brains, the research demonstrates that increased stiffness influences microglial behavior, leading to increased reactive oxygen species production, impaired amyloid-beta(Aβ) phagocytosis, and upregulation of neuroinflammatory markers. Chapter 4 continues the study on AD, and engineers a viscoelastic 3D AD-blood-brain barrier (BBB)-on-a-Chip microfluidic device. This advanced platform integrates iPSC-derived AD brain organoids with a functional BBB model within a tunable viscoelastic alginate hydrogel system. By recapitulating key aspects of the AD brain microenvironment and enabling independent manipulation of both neural and vascular components, this model provides a powerful tool for investigating AD pathogenesis and evaluating potential therapeutic interventions
Pig-to-Human Kidney Xenotransplantation: Designing the First Patient Education Visuals
Get PDFKidney transplantation is considered the most effective treatment for end-stage renal disease, however, there is a severe shortage of available kidneys. Nearly 90,000 people in the US are waiting for a kidney transplant, but only 25,000 kidney transplants occur annually. Each day, 17 people die waiting for an organ transplant. In response to the organ shortage, Johns Hopkins is at the forefront of research into pig-to-human kidney xenotransplantation, with human clinical trials expected to begin as early as 2025.
As clinical trials approach, it is critical to provide kidney transplant waitlist candidates with adequate education so they can consider xenotransplant, which is a novel, complex treatment option. To educate patients prior to clinical trials, Johns Hopkins plans to conduct a series of three one-hour, physician-led patient information sessions to educate kidney transplant waitlist candidates about xenotransplantation. A prototype of visual materials to be used during the first of three educational sessions was designed and created to accompany the following topics: 1) background on xenotransplantation, 2) immune system and xenotransplant rejection, 3) genetic modification, and 4) where pig kidneys come from and wrap up. Three types of visuals were designed: standalone images, sequential image series, and 2D animations. Ninety individual visual assets were created and arranged into slide layouts and storyboards. A draft slideshow with 130 color illustration and storyboard slides was developed and iterated upon based on formative feedback from transplant surgeons and patient educators.
The result of this thesis is a 34-slide PowerPoint presentation consisting of 14 standalone illustrated slides, four sequential image series, and three 60-second 2D animations. Four additional slides and one animation storyboard were created as supplemental material. These are the first visuals depicting kidney xenotransplantation designed specifically for kidney transplant waitlist candidates.
Future directions for this project include conducting an IRB-approved study to evaluate the effectiveness of these visuals for the target audience, expanding the prototype to include additional topic areas, and adapting these visuals for web and print. This project successfully created a framework for patient education that can be built upon as xenotransplantation moves into accepted clinical practice
Investigating the Role of Complement Factor B in Lung Fibroblasts
Get PDFComplement proteins have versatile functions: the serum protein provide immunity through the complement cascade while the intracellular proteins have emerged as a critical regulator of cell physiology through non-canonical cell signaling. Intracellular complement proteins in non-immune cells are under investigation. In lung fibroblasts, Complement Factor B (CFB) is found in abundance, even compared to immune cells. We aimed to understand the role of CFB in lung fibroblasts. We knocked down CFB in lung fibroblast cells using siRNA, validated the knockdown, and determined the effect of CFB on fibroblast proliferation, migration in response to a scratch wound, IL-6 and IL-8 expression, chemotaxis assay, and expression of fibrosis markers. CFBKD showed no difference in proliferation compared to Scrambled. CFBKD did not alter overall migration in the scratch wound assay, although the wound width of CFBKD cells trended higher than the wound width of Scrambled during the first 24 hours. CFBKD showed no effect on fibrosis markers in the setting of TGFB-1 mediated fibrosis. CFBKD cells produced less IL-6 and IL-8 at both the mRNA and protein levels and induced less migration in dHL60 cells in a scratch wound assay. In the presence of exogeneous CFB and C5-antibody, CFB still produced lower IL-6 levels, indicating result is independent of MAC complex. While CFB does not affect proliferation, overall wound healing, or expression of fibrosis markers, the mechanism of the difference in cytokine levels under CFBKD conditions, as well as other effects of CFBKD on lung fibroblasts should be considered in the future
Evaluation of Augmented Reality for Collaborative Environments
Get PDFThe intersection of intelligent analytics and climbing is relatively nascent. Various forms of climbing have begun to grow exceedingly fast in recent years, with a combination of three sub-categories even having become adopted into the Olympic games (i.e. bouldering, speed climbing and lead climbing). With a growing community, injuries due to the inherent health risks of climbing will undoubtedly grow as well. We aim to address these health concerns by helping lay the groundwork for future preventive technologies that help climbers better understand and plan their routes.
Multiple works have applied mobile or projector-based augmented reality or tools such as computer vision and deep learning to help with problems such as climbing route difficulty assessment, games for user retention in the commercial setting and fall protection via wall monitoring. However, few if any, examine collaborative problems in climbing with real-time, augmented reality systems using modern, head-mounted displays.
To this end, we frame our examination of the climbing space as a collaborative, layout and design task, thereby targeting collaborative, route setting as the specific application space. We present ClimbAR, an open-source, collaborative, real-time, augmented reality application running natively on the Hololens 2 that allows climbers to virtually and collaboratively, set climbing holds in their physical environments. Due to the necessary infrastructure required to develop ClimbAR, we also present its core, Hololens 2 synchronization functionality as a separate, open-source platform called SynchronizAR. Finally, we present the qualitative results of demonstrating ClimbAR at two climbing gyms as well as the quantitative results of analyzing SynchronizAR's spatial alignment accuracy through two proto-user studies. We find an average rotational alignment error of 12.8338 degrees and an average translational alignment error of 0.0385 meters when using SynchronizAR for collaborative layout tasks involving two users
CONFLICTS OF INTEREST, ETHICAL THEORY, AND RESEARCH ADMINISTRATION
Get PDFThis thesis used a systematic review of conflict of interest (COI) and financial conflicts of interest (FCOI) standards across a sampling of 18 high education institutions. COIs are defined variously within this thesis in addition to between the peer institutions from which information for this thesis was gathered. Furthermore, these are an important area of study due to the fact that COIs and FCOIs play an important role in ensuring that research is conducted in an ethical manner. Without these definitions and mitigation policies, one would be in the dark in regard to conducting research in an ethical fashion.
This thesis also analyzed ethical theory as it pertains to research administration, particularly on consequentialism, deontology, and virtue ethics. This thesis showed how COIs are identified, categorized, and mitigated. Furthermore, objective COI and FCOI standards were looked at insofar that objective COI and FCOI standards should be universalizable. Across research administration, ethical theory should be seen as a way to ensure research is conducted ethically and in accordance with federal regulations which upholds both The Belmont Report and The Common Rule. Therefore, this thesis aimed to uncover what each institutions’ COI and FCOI policies contain, how are they impacted by ethical theory, how they are universalizable, how they are affected by public and institutional policies, and why this analysis is important.
In short, this thesis is singular insofar that other papers do not deeply delve into both ethical theory and conflicts of interest in such a way that one can analyze how objective COI and FCOI standards are impacted by ethical theory and are therefore universalizable
YOUNG (18-29) BLACK AMERICAN VOTERS: MID-TERM ELECTIONS, WHY THEY MATTER AND THE IMPACT THEY HAVE
Get PDFThe Black American vote has been very important to elections to in Congress and both major political parties have sought to acquire the vote over the years. However, since 2008 the young (18-29) Black American vote has sprung to prominence as it has been used to decide almost every federal election since then. The key to the young (18-29) Black American vote is determining the best way to get out the vote among the group and motivate them to participate in the electoral process. Not surprisingly, there is an enormous amount of scholarly literature on the Black American vote and its impact on elections; surprisingly there is not nearly as much scholarly research on the young (18-29) Black American vote.
Through several case studies, this paper aims to examine the young (18-29) Black American vote, specifically its impact on mid-term elections and the legislative agenda. Beginning with a historical overview of Black American politicians in Congress and the rise of Black American politicians in Congress. Next, the paper looks at the importance of mid-term elections on the legislative agenda by studying three important mid-term elections, 2002, 2010, and 2018. Finally, the paper moves on to examining why young (18-29) Black Americans vote and why it’s important that they do.
The conclusion of this paper confirms that the empowerment of more Black Americans to run for political office and address the concerns of black and brown communities comes from the increase of the young (18-29) Black American voters, that mid-term elections determine the legislative agenda which sets the tone for the country, and proves that mid-term elections are, at times, more consequential than presidential elections
THE RELENTLESS PURSUIT OF DIFFERENCE: THE SUBVERSIVE ROLE OF ALTERITY IN THE RELIEFS AND ARCHITECTURE OF SARGON II'S (721 – 705 BCE) ROYAL PALACE AT KHORSABAD
No full textWhat is Assyria without the other? This question encapsulates the dichotomous relationship between the Neo-Assyrian Empire’s desire to ideologically define itself against the other while simultaneously relying on the presence of the other to define and sustain itself. Through three case studies focused on the Palace Terrace at Sargon II’s (721 – 705 BCE) royal palace at Khorsabad, I examine the human and non-human other to argue for a deep-seated anxiety regarding the potential threat the world beyond Assyria posed to the empire. In chapter one, I discuss how the proliferation of non-Assyrian tribute procession scenes was used to apotropaically reinforce the obedience of non-Assyrian peoples in the palace and, ideologically, within the empire itself. Furthermore, I argue that the repetition of these scenes in the palace indicates a ritualization of tribute procession scenes during the reign of Sargon II. In chapter two, I explore the role of the non-human other (plants, animals, and architecture) through a programmatic analysis of a hunt scene carved in relief in Room 7. I argue that Room 7’s relief program is a celebration of Sargon’s kingship. Furthermore, I argue that the room’s position, and relief program suggest that the room is the ideological “seat” of power at Khorsabad. Chapter three investigates architecture's role in constructing “otherness” through a spatial, architectural, and iconographic analysis of Monument X, an architectural monument heavily influenced by Syro-Anatolian architectural practices. I argue that Monument X, in conversation with the Assyrian procession scene on Façade L, is a representation of “treasure” brought back to Khorsabad following Sargon’s campaigns in the Syro-Anatolian region. These three case studies reveal the integral role of the human and non-human other in visually defining Neo-Assyrian imperial identity. Moreover, this dissertation demonstrates a more nuanced approach to the study of alterity in the field of Neo-Assyrian art and architecture. The inclusion of the non-human other in studies of alterity is rare in the Assyrian context. My research treats images of the human and non-human other as active, subversive agents in relation to the ostensible message of Assyrian hegemony that is communicated in the palace’s relief program