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Establishing and Validating Novel Highly Multiplexed Antibody-Profiling Platforms Integrating Linear Epitope Reactivities and Virus Neutralization Activities to Better Understand Humoral Immunity Against Hemagglutinin Protein of Influenza Virus
No full textInfluenza A virus (IAV) is a highly contagious respiratory pathogen that affects millions of people globally each year, causing significant morbidity and mortality. Despite the widespread use of vaccines, protection against IAV remains limited by factors such as relatively low vaccine efficacy, rapid viral mutation, and the lack of broad and durable immunity. Immune responses to influenza are influenced by a range of by a range of vaccine, virus, and host factors, including virus strains in the vaccine formula and circulation, age, health status, prior exposure, and immunization history, making it challenging to design universally effective vaccines. A critical step toward improving our understanding of protective immunity is identifying the specific viral targets that elicit functional antibody responses. While neutralizing antibodies directed against the viral hemagglutinin (HA) protein are central to protection, the contribution of linear epitopes within HA to functional neutralization is not fully understood. We used data from two complementary high-throughput platforms: the InFlux neutralization assay, which uses a whole-virus barcoded HA library to assess neutralizing activity across diverse IAV strains, and the HAscan PhIP-Seq library, which profiles peptide-level antibody binding across the HA protein. By comparing these datasets, we aim to map the relationship between antibody binding to linear HA epitopes and their ability to neutralize viral infection. This integrative approach provides new insights into the functional relevance of linear epitopes in the context of vaccine-induced humoral immunity and supports efforts to develop more effective, broadly protective influenza vaccines
Identification of Proteins that Interact with Cellular Retinol Binding Protein 1 (Crbp1) In Cardiac Myocytes and Mouse Hearts
No full textRetinoic acid (RA), the active metabolite of vitamin A, is a critical multi-gene master regulator of cardiac development and function. Despite the established importance of RA signaling in heart physiology, the precise mechanisms governing retinol metabolism in cardiomyocytes remain poorly understood. In this study, we attempt to investigate the interactome of Cellular Retinol Binding Protein 1 (Crbp1), a key intracellular chaperone of retinol, to identify enzymatic partners involved in RA biosynthesis in the heart. Using a combination of Crbp1 overexpression, immunoprecipitation, proximity labeling with TurboID, and mass spectrometry-based proteomic profiling in neonatal rat ventricular myocytes (NRVMs), several candidate interactors of Crbp1 were identified. Notably, Dhrs4, Aldh9a1, and Hsd17b4 were significantly enriched in the Crbp1 affinity pulldown and form a functional network suggestive of roles in retinol and retinaldehyde metabolism. In addition to these candidates, the proteomic analysis revealed sets of previously unrecognized Crbp1-associated proteins including Mlip and Prdm2, broadening the understanding of the cardiac retinoid metabolic landscape. These findings provide new insight into the molecular architecture of retinol metabolism in cardiomyocytes and suggest potential targets for precise therapeutic intervention and development of novel therapies to combat heart failure conditions marked by RA deficiency
Longitudinal Relationship between Clofazimine Exposure, Skin Pigment Changes, and Plasma Clofazimine Levels Among Adults Treated for MDR-TB in India
Full text linkThis study investigated the longitudinal relationship between clofazimine therapy, skin pigmentation changes, and plasma clofazimine levels among adults treated for multidrug-resistant tuberculosis (MDR-TB) in India. 697 skin images from a cohort of 91 patients were analyzed. The images were rated using a Munsell Soil-Color chart 10YR hue scale. For each image, a combination of value and chroma was recorded, which was assigned ITA, a, b, and L values as measures of skin pigmentation. Baseline skin tones were defined as skin without the effect of clofazimine, determined by pre- or post-treatment images. The change of each patient’s skin tone was arithmetically and geometrically normalized based on their individual baseline ITA, a, b, and L values to capture within-individual variability over time. It was observed that ITA and the arithmetic normalization method best captured skin tone changes over time. The average time required for peak change in skin pigmentation was between 16 and 18 months after treatment initiation. In contrast, plasma clofazimine trough levels peaked earlier, around 8 months. Stratified analysis revealed no significant sex- or underweight-related differences in time to peak skin pigmentation. However, diabetic patients exhibited significantly earlier skin tone changes. Correlations between skin tone measures and plasma clofazimine levels were consistently weak (R2 < 0.03), suggesting that skin pigmentation is a poor surrogate for plasma clofazimine exposure. Slightly stronger, yet still modest, correlations were observed in diabetic individuals. These findings emphasize that the standard practice of direct blood sampling and plasma drug level measurement remains essential for accurately assessing clofazimine exposure. Overall, this study provides an understanding of how clofazimine affects skin pigmentation and shows that there are complexities to predict plasma clofazimine drug levels by measuring skin tone changes
DISCOVERY AND CHARACTERIZATION OF NOVEL PLASMODIUM FALCIPARUM PLASMA BIOMARKERS
No full textThe increasing prevalence of the gene deletions of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) has led to widespread failures in rapid diagnostic tests (RDTs), posing a serious challenge to malaria diagnosis and control. To address this issue, this study focuses on identifying alternative plasma protein biomarkers that can serve as new RDT targets. DIA mass spectrometry was adopted to identify P. falciparum proteins in the plasma of infected patients, providing a pool of potential biomarker candidates. MutateX and AlphaFold are applied to assess the functional impact of genetic variations in these proteins, ensuring the selection of stable targets. Additionally, structural and splicing variations in stage-specific P. falciparum proteins are explored by integrating single-cell transcriptomic data with alternatively spliced protein isoforms, providing insights into their expression dynamics and suitability as biomarkers. By combining proteomics, structural modeling, and transcriptomics, this study aims to discover robust plasma biomarkers that can improve malaria diagnostics and mitigate the impact of Pfhrp2 deletions on RDT accuracy
Label-Free Optical Biosensing for Malaria and Osteoarthritis
No full textThis thesis presents two complementary, label-free optical biosensing platforms—spatially offset Raman spectroscopy (SORS) and quantitative phase imaging (QPI)—and demonstrates their utility across disparate biomedical challenges. In Chapter 2, we establish a material-agnostic theoretical foundation for SORS in turbid media by deriving closed-form expressions for photon sampling depths and optimal source–detector geometries via Monte Carlo simulations. Building on this, Chapter 3 introduces pulse-correlated SORS, in which Raman acquisition is dynamically gated to the cardiac cycle. In silico results reveal that simple Fourier-based pulse correlation selectively amplifies blood-specific Raman features and markedly improves classification accuracy among uninfected, ring-stage, and gametocyte-stage malaria infections, reducing critical false negatives in advanced stages.
Chapter 4 translates the SORS framework into osteoarthritis research by mapping depth-dependent enzymatic degradation of bovine cartilage. By coupling Fick’s law diffusion models with the Monte Carlo-derived photon distribution, we obtain high-fidelity, zone-specific measurements of glycosaminoglycan depletion that correlate strongly with depth-resolved effective moduli from compression and shear testing. Finally, Chapter 5 introduces QPI as a label-free optical modality for malaria vector surveillance. We show that Plasmodium oocysts in mosquito midguts exhibit distinct refractive-index signatures that can be imaged without staining, and validate these findings through one-to-one registration with mercurochrome-stained brightfield microscopy.
Together, these studies demonstrate the power of label-free biophotonics to probe molecular and structural signatures noninvasively, whether through subsurface vibrational contrast or refractive-index mapping, and lay the groundwork for portable, high-throughput diagnostics in global health and musculoskeletal care. Future work will focus on experimental validation, melanin-compensation strategies, machine-learning–driven automation, and hybrid optical modalities to translate these approaches into point-of-care devices
BUBBLES IN THE BRAIN AND LICKING ROCKS A COLLECTION OF WRITINGS
No full textThis collection of personal essays handles two overarching themes: family medical history, with a focus on brain aneurysms and aphasia, and geology, with explorations into its quirky characters and interesting elements. Pieces range from emotional to humorous
IMPROVED COOKSTOVES: GLOBAL DEVELOPMENT, INDIGENOUS TECHNOLOGY, AND HEALTH
Full text linkThis thesis analyzes six decades of the history to explore the transformation of cookstove technology within the context of global development and international aid, as well as indigenous efforts aimed at improving efficient cooking and health. Billions of people utilize cookstoves daily for food preparation and heating. This thesis aims to answer the questions of how and why cookstove technology evolved in the 20th century.
Using published articles, local case studies, oral histories and archival sources covering six decades of local and international efforts to improve cookstove technology, this thesis describes social, economic and political factors leading to changes in cookstove design in global settings. It places modifications to cookstoves both within the context of external efforts on the part of NGOs and intergovernmental bodies to provide foreign aid as well as local efforts to improve health and wellbeing. Additionally, it seeks to utilize cookstove technology to explore or highlight wider changes to developmental assistance from the 1950s to the present. The history of cookstove modification is a part of a larger development narrative in low- and middle-income settings. With each modification the stove was remolded to meet a purpose (i.e. decreased fuel consumption, improved air quality). This thesis also aims to identify discrepancies in intended purpose between foreign developers and local populations. Despite sixty years of development work, uptake of “improved” cookstoves remains inconsistent. Cookstove modifications occurred because of competing priorities by international donors, local government and the populations who utilized these devices.
This project leverages several resources that allow for longitudinal assessments of the adaptation of cookstoves and cooking fuel to understand how recurrent agendas including appropriate technology, environmentalism, developmental economics, women’s rights and global health influence modification of stove technology and at times compete. This thesis serves to build on previous work concerning developmental economics in the early 20th century as well as the appropriate technology movement in the mid-20th century. It defines the introduction of cookstoves not as a singular event but rather a technology that morphs, reconstructs, and helps contextualize our understanding of technology and development efforts in the 20th century
MOLECULAR DETERMINANTS OF PLASMODIUM INFECTION: INDEPENDENT STUDIES ON THE ROLE OF MOSQUITO ENOLASE AND P. BERGHEI SERA5 IN PARASITE TRANSMISSION AND DEVELOPMENT
No full textMalaria, caused by the Plasmodium parasite, is a major parasitic disease that keeps devastating public health, with a significant impact on the sub-Saharan African region. The Plasmodium parasite relies on both vector-derived and parasite-derived factors to initiate and maintain the transmission of malaria. Here, we investigate two molecular determinants, a parasite-derived cysteine protease (SERA5) and mosquito-derived enzyme (enolase), necessary for malaria transmission.
RNAi-mediated gene knockdown was employed to silence the mosquito enolase gene and investigate its role as a mosquito host factor aiding the Plasmodium parasite invasion after infecting the enolase-deficient mosquitoes with the standard membrane feeding approach. Gene silencing efficiency was determined by qPCR, and the role of the microbiota in modulating immune response in the leaky gut environment was determined by using the septic and aseptic mosquito treatment. Across all biological replicates, the silencing efficiency was determined, ranging from 90.8% to 98.3%. The results demonstrated a reduction in oocyst burden in enolase-deficient mosquitoes, as well as validated the role of microbiota in immune activation against the Plasmodium parasite, resulting in the upregulation of immune genes such as Tep1, LRIM1, FBN9, and LRRD7 under septic conditions only. This suggests that enolase is essential in maintaining the midgut integrity of the mosquito, and in turn shields the Plasmodium parasite from immune invasion. The disruption of enolase led to a leaky gut syndrome in the mosquito, which influenced microbial-mediated immune response against the parasite.
PbSERA5’s role after sporozoite exit from oocysts was examined by performing a genetic cross between an mCherry-wildtype and a GFP-PbSERA5 KO parasites. The results from the fluorescence assays and qPCR-based quantification demonstrated a ratio of 2:1 wildtype and mutant in salivary glands, supporting Mendelian segregation. The downstream infection assay showed a reduced blood-stage infection level of the PbSERA5-KO mutant compared to the wildtype parasite in a ratio of 1:2. The outcome of this study suggests that PbSERA5 may play a role in facilitating mouse host hepatocyte invasion by sporozoites, hence providing insight into the cascade of molecular events needed for liver-stage infection.
The findings of these studies provide an understanding of the transmission biology of the malaria parasite and suggest potential malaria intervention strategies for global malaria control
Cellular Fractionation and Reconstitution of Immune Subcellular Vesicles for Synthetic Biology
No full textOrganelles are essential for the survival, proliferation, and differentiation of eukaryotic cells and carry out cell-specific functions that influence phenotype. Cell phenotype is reflected in the abundance and activity of organelles; for instance, energy-intensive cells such as skeletal muscle fibers have greater mitochondrial density and activity due to their metabolic requirements. Despite the importance of organelles for determining cell function and phenotype, relatively little attention has been devoted to isolating and repurposing organelles as a tool for synthetic biology and cellular engineering. The top-down approach of isolating organelles from biological cells and reconstituting them to form synthetic cells dates back to the early 1970s, but this field has seen little development and attention since the late 1990s. To enable the generation of reconstituted synthetic cells, whole cells can be split into cytoplasts (enucleated cells) and karyoplasts (membrane-wrapped vesicles containing the nucleus and a small volume of cytoplasm) through density gradient centrifugation in the presence of cytochalasin B, an agent that destabilizes the cellular cytoskeleton and prevents actin polymerization. Cytoplasts from one cell type can be fused to whole cells or karyoplasts from another cell type to generate cybrids (cytoplast-whole cell) or reconstituted cell (cytoplast-karyoplast) hybrids that exhibit different characteristics depending on the donor cells. Cybrids and reconstituted cell hybrids have unexplored potential as research tools and therapeutics for diseases including metabolic disorders and cancer. For example, cybrids could be used to treat mitochondrial disorders by fusing cytoplasts containing healthy mitochondria to patient hematopoietic stem cells to generate cybrids with enhanced mitochondrial function. Prior studies have successfully generated cybrids and reconstituted cells with hybrid phenotypes via more rudimentary methods and performed initial characterization, including proliferation and gene expression assays. However, rigorous functional characterization of these products is lacking and the protocols used to generate cytoplasts, karyoplasts, and reconstituted cells vary significantly in their methods and results, warranting further investigation. In this thesis, a streamlined method to generate cytoplasts, karyoplasts, and reconstituted cybrids or cell hybrids is presented. Initial optimization experiments were performed to facilitate improved fractionation product yield, identification and staining, storage conditions, and characterization of reconstituted cell products
EXPLORATION OF GENE REGULATION IN B-1 CELL DEVELOPMENT, MAINTENANCE, AND FUNCTION
No full textB-1 cells are innate-like B cells that play a key role in natural antibody production, a first line of defense in immunity. However, the genes that selectively regulate their development, maintenance, and function remain to be more fully explored. This study is based on the search for genes inducing changes in B-1 cell frequencies in the spleens of 641 mouse strains, each harboring one known gene knockout, compared to their wild-type controls (about 200 mice) generated as part of the NIH-supported Knock Out Mouse Program and analyzing a total of 56 distinct cell populations using two multicolor flow cytometric panels. Seven mouse strains in which B-1 cell frequencies in the spleen were significantly and selectively altered were identified. Among these, mice lacking Ppp2r5a stood out as having shown significant reductions in spleen B-1 cells in both male and female mice. Ppp2r5a encodes a regulatory subunit of protein phosphatase 2A (PP2A), a regulator of cell growth and division. To confirm and expand these findings, for this thesis I retrieved mice lacking Ppp2r5a and conducted further flow cytometric analysis, which confirmed the selective B-1 cell deficiency in spleen and bone marrow in these mice. In addition, we observed significant decreases in serum IgM and IgG levels in PP2A-/- female but not in male mice. Thus, our work identifies Ppp2r5a as a gene necessary for normal B-1 cell self-renewal and their differentiation into antibody-secreting cells