Morningside GSBS Dissertations and Theses

Permanent URI for this collection

https://hdl.handle.net/20.500.14038/6

ABOUT THIS COLLECTION

Since the school's inception in 1979, students in the Morningside Graduate School of Biomedical Sciences (GSBS) at UMass Chan Medical School have contributed more than a thousand doctoral dissertations and masters theses to the field of biomedical sciences. This collection makes this body of work accessible to our students, faculty, potential recruits, the citizens of Massachusetts, and the world.

HOW TO SUBMIT

Review the submission guidelines and complete all requirements and the Morningside GSBS online permission form
Navigate to eScholarship@MassChan and select the Log In link in the upper right. Choose "UMass Chan Login".
Select the + (plus sign) on the left to open up the New menu and select Item
Choose the Morningside GSBS Dissertations and Theses collection
Fill in submission form and deposit
You will receive an email with a persistent link to your submission when it is posted.

QUESTIONS?

Contact escholarship@umassmed.edu with your questions.

Browse

The Role of MDC1-53BP1-H2AX in SSDNA Gap Management: Unveiling RAD51 Essentiality in BRCA1-Deficient Cancer
(UMass Chan Medical School, 2024-10-18) Lee, Silviana; Sharon Cantor; Morningside Graduate School of Biomedical Sciences
BRCA1/2-deficient cancers are hypersensitive to genotoxic agents and toxicity has traditionally been attributed to the inability to repair or prevent DNA double stranded breaks (DSBs). Here, we attribute the toxicity to an inability to protect single stranded DNA (ssDNA) replication gaps. We re-asses the roles of canonical DSB damage responders 53BP1-MDC1-H2AX and investigate their role in unperturbed conditions during replication. MDC1, like 53BP1, is enriched in the chromatin of BRCA1-deficient cells. Its loss, like 53BP1, confers resistance to poly (ADP)-ribose polymerase (PARP) inhibitors and translesion synthesis (TLS) inhibitors by gap suppression and reducing PARP1 activity. In BRCA1-deficient cells, RAD51 fails to form nuclear “foci” in response to DNA damage, supporting a model that PARPi sensitivity is due to a loss of RAD51 function in homologous recombination (HR). 53BP1 loss, which counteracts HR by blocking end resection, enhances PARPi resistance and recovers RAD51 foci, further linking these outcomes. Unexpectantly, we find that RAD51 is essential and enriched in the chromatin of BRCA1-deficient cells, while deletion of 53BP1 alleviates this enrichment and dependency. The same pattern evolves along with PARPi resistance following loss of MDC1 or H2AX. We link these factors to 53BP1 nuclear bodies associated with ssDNA gaps in BRCA1-deficient cells. Unlike 53BP1 loss however, loss of MDC1 and H2AX in BRCA1 deficient cells does not restore RAD51 foci, further uncoupling HR from PARPi resistance. Collectively, we propose a model that gaps necessitate RAD51 chromatin enrichment for cell survival which limits its availability for other functions and reveals a targetable dependency.
OTTR: New Tricks for an Old Dog – An Updated View of tRNAs and their Fragments
(UMass Chan Medical School, 2024-12-11) Gustafsson, Hans Tobias; Oliver J. Rando; Morningside Graduate School of Biomedical Sciences
Playing an essential role in the central dogma of molecular biology – bringing amino acids to the elongating polypeptide chain during translation – transfer RNAs (tRNAs) were long considered to be solely part of the translation machinery. However, discoveries made in the last two decades have indicated that enzymatically cleaved tRNA-derived fragments (tRFs) are ubiquitous in cells. These findings group them with other small RNAs (such as piRNA, miRNA, snoRNA, etc.) as biologically active molecules that regulate diverse cellular processes. Accurate deep sequencing of tRNAs and their fragments has historically been constrained by their structural complexity and extensive posttranscriptional modifications. In this work, I first benchmark a novel cloning protocol (OTTR) for sequencing tRNAs to characterize intact tRNAs and tRFs in yeast and mouse tissues. I show that OTTR captures a wide variety of tRF species in the sample. And, unlike standard cloning methods, I also show that OTTR captures both 5’ and 3’ fragments at similar levels, giving us the most accurate picture of tRF levels to date. Given our lab’s interest in transgenerational epigenetic inheritance, I also used OTTR on mature mouse spermatozoa. My data demonstrate that mammalian sperm carries a far more complex payload of tRFs than previously appreciated, including both 5’-tRFs and 3’-tRFs derived from the majority of tRNAs. These results force us to alter our understanding of the mature sperm RNA payload and the potential epigenetic functions it may carry.
Developing an siRNA-Based Approach for the Treatment of SOD1-Related Amyotrophic Lateral Sclerosis
(UMass Chan Medical School, 2024-07-10) Rivera Flores, Iris Valeria; Anastasia Khvorova; Morningside Graduate School of Biomedical Sciences
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, and current treatments are ineffective. Reduction of SOD1 is the primary clinical approach to treatment, with several strategies to silence SOD1 expression under preclinical and clinical investigation. Small interfering RNAs (siRNAs) are a new class of drugs for which potent and sustained modulation of gene expression has been demonstrated. The effectiveness of siRNA knockdown is modulated by its chemical modification properties. We evaluated the impact of chemical variance and siRNA length on target knockdown and identified a potent siRNA that modulates SOD1 expression in SOD1-G93A mice. When siRNA compounds are not homologous to a model species, performing preclinical studies is challenging. An alternative strategy to expand targetability against genes across different species is to adapt a potent human-targeting lead siRNA for the model species by changing the nucleotide sequence of the siRNA guide strand to match the target site in the model species. I systematically evaluated this strategy of “animalizing” the guide strand and found that the ability to generate species-active compounds based on human leads was highly affected by the target sequence and number of mismatches. The potency of a mouse-active compound generated from this strategy was significantly lower than a mouse-active compound identified from a focus screen. Although our strategy failed to generate a dog-active compound to target canine SOD1, a canine SOD1 focus screen identified potent siRNAs. Thus ,parallel screening for species-active compounds may be a more uniform strategy for the fast clinical progression of RNAi-based drugs.
Molecular Analysis of Neural Mechanisms Controlling Organismal Responses to Oxidative Stress
(UMass Chan Medical School, 2024-11-13) BISWAS, KASTURI; Michael M. Francis; Morningside Graduate School of Biomedical Sciences
Organisms have evolved protective strategies for limiting cellular damage and enhancing organismal survival during stress, in particular oxidative stress. My thesis work investigated previously underappreciated links between neuronal activity, neurotransmission, and oxidative stress responses in the nematode Caenorhabditis elegans. I demonstrated a requirement for neural activity in regulating organismal survival during oxidative stress using a genetic strategy for neuronal silencing. From a survey of evolutionarily conserved neurotransmitter systems, I showed that cholinergic transmission from motor neurons was important for protection from prolonged oxidative stress. I showed that prolonged oxidative stress mobilizes a widespread transcriptional response. A deficiency in cholinergic transmission strikingly reduces the scope of this response. I found that cholinergic involvement is primarily mediated through activation of the Gq-coupled muscarinic acetylcholine receptor GAR-3. Interestingly, gene enrichment analysis revealed a lack of upregulation of proteasomal proteolysis machinery in both cholinergic-deficient and gar-3 mutants, suggesting that cholinergic activation of GAR-3 may be critical for stress-responsive upregulation of protein degradation pathways. In parallel, I investigated the impact of oxidative stress on neuronal integrity and observed progressive neurodegeneration during prolonged oxidative stress. Curiously, I found that a deficiency in cholinergic transmission does not enhance the severity of neurodegeneration during the early stages of prolonged oxidative stress, perhaps suggesting that neurodegeneration and organismal survival are not precisely linked. My research provides key insights into neuronal modulation of antioxidant defenses through cholinergic activation of G protein-coupled receptor signaling systems and transcriptional activation of protective pathways, suggesting potential targets to combat oxidative damage and related inflammatory, systemic and neurodegenerative diseases.
Exploring Influenza Virus Structure and Dynamics through Cryo-Electron Tomography and Convolutional Neural Networks
(UMass Chan Medical School, 2024-09-10) Huang, Qiu Yu; Celia Schiffer; Mohan Somasundaran; Morningside Graduate School of Biomedical Sciences
Influenza viruses pose severe threats to global public health. As influenza virions are highly pleomorphic in shape, size, and organization, cryo-electron tomography (cryoET) is the optimal tool for depicting the 3D organization of pleomorphic influenza and visualize host-virus interactions. However, the low signal-to-noise ratio inherent to this technique as well as the massive manual effort required to analyze heterogenous influenza virions has resulted in limited resolution of previous cryoET studies. Addressing this issue, I developed a cryoET analysis pipeline that leveraged convolutional neural networks to quantitatively characterize the morphological architecture of the A/Puerto Rico/8/34 (PR8) influenza strain. This pipeline was used to successfully characterize influenza morphology, calculate glycoprotein density, and perform subtomogram averaging of influenza hemagglutinin (HA) and neuraminidase (NA). Applying this pipeline to the viral life cycle, I investigated influenza receptor binding through cryoET of PR8 influenza virions incubated with a sialic acid-containing receptor mimic. I observed a distinct rearrangement of HA organization and orientation. Compared to even inter-glycoprotein spacing of unliganded HA, receptor binding promotes clustering of HA to form a previously unseen trimer of HAs configuration. Subtomogram averaging resulted in several subnanometer resolution reconstructions of unbound and receptor-bound HA. Structural analyses revealed HA1-HA1 contacts between two proximal HA protomers that formed a symmetric interface orchestrated through hydrogen bonding. These data support previous studies which suggest the cooperation of 3-4 HAs for successful membrane fusion and further illustrate the dynamic nature of the influenza virion during cellular entry. Lastly, subtomogram averaging was used to determine full-length NA structures. These reconstructions unveiled the domain architecture of full-length NA and illustrated how substrate binding can affect NA structure. Better understanding influenza dynamics using structural virology may inform future structure-based drug design, antibody discovery, and innovative vaccine strategies.
Investigating the Function of Regulatory Factor Interactions in Human Gene Regulation
(UMass Chan Medical School, 2024-10-30) Gao, Mingshi; Zhiping Weng; Morningside Graduate School of Biomedical Sciences
Regulatory factors are essential for the precise spatial and temporal control of gene expression in humans. These factors include sequence-specific transcription factors (TFs) and non-sequence-specific proteins such as histone modifiers, chromatin remodelers, and cofactors. TFs recognize specific DNA motifs within cis-regulatory elements (CREs) and recruit other proteins to modulate CRE functions. Understanding how regulatory factors interact is critical for deciphering the mechanisms that control gene expression. Despite advances in technology that allow for the mapping of protein binding sites and interactions, the complete landscape of regulatory protein interactions remains largely unexplored. Genetic variants in CREs can disrupt the binding affinity of TFs and their partners, which in turn impacts gene expression and phenotypic outcomes in cells and organisms. This work leverages multiple omics data to prioritize and characterize the interaction between regulatory proteins. As a case study, we first identified YAP and TAZ as coactivators of AP-1 proteins and STAT3 during breast cell transformation. We then developed a predictive framework for protein-TF interactions by integrating genotype data with allele-specific binding (ASB) information. Our framework identified both known and novel interactions in the GM12878 cell line, demonstrating its robustness and utility. We further showed that genetic variants displaying ASB are highly informative for TF binding preferences and are enriched in regions associated with complex traits and disease heritability. Overall, this work provides valuable insights into the interplay of regulatory proteins and their influence on gene expression, contributing to the broader understanding of gene regulatory mechanisms.
Elucidating Migratory and Signaling Requirements For Intestinal B Cell Responses
(UMass Chan Medical School, 2024-10-11) Raso, Fiona; Andrea Reboldi; Morningside Graduate School of Biomedical Sciences
Peyer’s patches(PPs) are secondary lymphoid organs on the antimesenteric side of the small intestine that continuously sample commensals from the intestinal lumen. This exposure to bacterial stimuli promotes chronic germinal center(GC) reactions giving rise to memory B(MemB) cells and plasma cells(PCs). In PPs, most B cells undergo IgA class switch before entering the GC reaction. In the GC, B cells improve their B cell receptor(BCR) affinity and those successfully selected exit as MemB cells or PCs which next migrate into their survival niche, providing long-term protection. Despite the importance of IgA antibodies in gut homeostasis and for oral vaccination, little is known about how B cells undergo selection in the PP GC to become IgA+ PCs or how mucosal MemB cells are maintained for long-lived humoral immunity. Here we defined a role for the IgA BCR in GC B cell fitness and differentiation into MemB cells and PCs. We showed Iga-/- cells are outcompeted in PP GC and are reduced as MemB cells and gut-homing PCs in a mixed chimera setting. We determined IgA+ GC B cells have stronger BCR signaling, receive more T cell help, and are resistant to FasL-induced apoptosis. Additionally, we’ve identified the PP subepithelial dome(SED) as a survival niche for MemB cells. MemB cells express high levels of the migratory receptors GPR183 and CCR6 and genetically removing either migratory receptor altered the SED positioning of MemB cells. Advancing our understanding of how B cells populations undergo selection and maintenance will allow for better oral vaccine design.
Loss Of Ms4a6c Attenuates Tau-Mediated Neurodegeneration and Neuroinflammation
(UMass Chan Medical School, 2024-10-31) Luu, Thuyvan; Paul Greer; Program in Molecular Medicine
Alzheimer’s Disease (AD) is the most common form of dementia worldwide, robbing patients of their cognitive abilities, their memories, and ultimately their lives. Despite significant effort, current therapies for treating AD are highly limited in both number and efficacy, necessitating a greater understanding of the etiology underlying AD. Genome-wide association studies have revealed single-nucleotide polymorphisms in the Membrane Spanning 4a (MS4A) locus are among the strongest and most reproducible genetic modifiers of AD susceptibility. However, little is known about how MS4A family members contribute to AD since many variants localize to non-coding regions of the locus, and their effect on MS4A function is largely unknown. Addressing whether MS4A genes have protective or harmful effects during AD is critical for the advancement of any potential MS4A-based therapeutic approach. In this work, we have used targeted knockout mice, in which we have specifically deleted Ms4a6c, the mouse orthologue of the human MS4A gene most tightly linked to altered AD susceptibility, to determine the role of Ms4a genes in a mouse model of AD. We found that deletion of Ms4a6c protected against cognitive defects, ameliorated tau-mediated neurodegeneration and mitigated synapse loss. Additionally, we demonstrated that Ms4a6c was required for activation of a microglial transcriptional program associated with aberrant cytokine production and phagocytosis. This work reveals that loss of function of MS4A genes is protective against AD and contributes to our understanding of how MS4A proteins modulate AD pathogenesis.
INVESTIGATING THE ROLES OF ZNFX-1 IN PROPAGATING EPIGENETIC STATES ACROSS GENERATIONS IN C. ELEGANS
(UMass Chan Medical School, 2024-12-04) Durning, Daniel; Craig Mello; Morningside Graduate School of Biomedical Sciences
Epigenetic mechanisms of gene regulation allow organisms to transmit information to their progeny while retaining the same DNA sequence. One means by which epigenetic information is established and maintained is through germline Argonaute pathways. The Caenorhabditis elegans PIWI Argonaute PRG-1 engages thousands of endogenously encoded piRNAs within perinuclear nuage. Sufficient base-pairing between piRNAs and their mRNA targets can elicit a silencing response that is maintained transgenerationally by worm-specific Argonautes (WAGOs), even in the absence of continued PRG-1 activity. We previously described ZNFX-1 as a factor involved in the maintenance of piRNA-triggered inherited silencing. However, the functions of ZNFX-1 in balancing and maintaining inherited silencing and how it functions alongside other core components of germline small RNA pathways remain poorly understood. In this dissertation I explore how ZNFX-1 and PRG-1 work in parallel to promote properly directed silencing on endogenous genetic elements. I show that they function together to prevent thousands of endogenous mRNAs from becoming de-novo targets of small RNA templating. Surprisingly, I find that RNAi inheritance, while defective in znfx-1, is partially rescued in prg-1; znfx-1 double mutants. These data suggest that ZNFX-1 promotes but is not essential for the inheritance of silencing. I also report that znfx-1 mutants cause GLH-4—but not other nuage components analyzed to date—to become de-localized from the nuage. This includes not only null alleles but also catalytic point mutants, suggesting ZNFX-1 function contributes to GLH-4 localization. I hypothesize this mislocalization of GLH-4 contributes to some, but not all, of the small RNA phenotypes observed in znfx-1 mutants. Taken together, my findings shed light on how ZNFX-1 contributes to robust transmission of epigenetic information in C. elegans.
RIPK1-Caspase-8-Mediated Inflammation and Cell Death During Bacterial Infection and Neurodegenerative Disease
(UMass Chan Medical School, 2024-09-19) Zhang, Boyao; Egil Lien; Medicine; Morningside Graduate School of Biomedical Sciences
Inflammation and cell death play crucial roles in acute infection and neurodegenerative disease. Receptor interacting serine/threonine kinases 1 (RIPK1) and caspase-8 orchestrate the crosstalk among inflammation, apoptosis, pyroptosis, and necroptosis. Thus, careful regulation of these multimodal proteins is critical for proper immune function and tissue homeostasis. We identified splicing factor Raver1 as a key regulator of RIPK1 expression and function. Loss of Raver1 promotes alternative splicing on Ripk1, resulting in a truncated, dysfunctional variant (Splice I) and reduced functional RIPK1 levels. Raver1-deficient primary macrophages display impaired caspase-8-mediated pyroptosis and IL-1ß/IL-18 release in response to Yersinia or TAK1-blockade. Consequently, Raver1-deficient mice exhibit compromised antimicrobial responses and increased susceptibility to Yersinia infection. Additionally, Raver1 promotes the expression and function of the C-type lectin receptor Mincle via RIPK1 control. Our study highlights the role of Raver1 in modulating innate immune responses and a new layer of regulation during pyroptosis. RIPK1 activation is implicated in Alzheimer’s disease (AD), but its mechanisms in regulating neuroinflammation are not fully understood. Here we demonstrate that in primary microglia, neuroinflammatory stimuli enable RIPK1 to control caspase-8-mediated pyroptosis and IL-1ß release, as well as RIPK3-dependent necroptosis. Moreover, genetic deficiencies in RIPK1, RIPK3, MLKL, or caspase-8 differentially ameliorate neuroinflammation and spatial memory deficits in aged APP/PS1 mice, an AD model. These findings suggest that RIPK1 coordinates caspase-8 signaling with necroptosis axis in AD. Collectively, my work elucidates how RIPK1 orchestrates an intricate inflammatory cell death network during pathogenic infection and neurodegeneration.
Loss of Ms4a6c Ameliorates Male tgSOD1G93A Defects and Reveals Sexual Dimorphic Pathology
(UMass Chan Medical School, 2024-07-22) Mocarski, Kit (Katherine); Paul Greer, PhD; Robert Brown, MD, PhD; Morningside Graduate School of Biomedical Sciences
Amyotrophic lateral sclerosis (ALS) is a fatal, non-cell autonomous neurodegenerative disease targeting motor neurons (MN) that has a higher prevalence in males than in females. The MS4A gene cluster, including MS4A6A, has been associated with Alzheimer’s Disease in genome-wide association studies and is expressed in CNS myeloid cells. Here, we find MS4A6A is upregulated in human ALS spinal cord tissue along with its mouse ortholog, Ms4a6c, which is upregulated in spinal cords of the tgSOD1G93A mouse model. We find that knocking out Ms4a6c in tgSOD1G93A mice delays disease progression and extends survival in a male-specific manner. Through histological and transcriptional analyses, Ma4s6c-/- also improved MN loss and astrogliosis in males. Disease associated microglial expression and inflammation were reduced in both sexes by Ma4s6c knock-out. RNA sequencing of spinal cord tissue revealed a sexually dimorphic expression profile in tgSOD1G93A mice both with and without the Ma4s6c gene. In male but not female tgSOD1G93A Ma4s6c-/- mice, genes involved in angiogenesis and blood spinal cord barrier maintenance were over-represented as compared to tgSOD1G93A mice. Altogether, these findings implicate Ms4a6c in ALS and demonstrate fundamental sex differences that may play a role in the sex bias occurring in ALS.
Investigating the role of p16Ink4a in Alzheimer's Disease pathogenesis
(UMass Chan Medical School, 2024-10-07) Holloway, Kristopher; Hong Zhang, PhD; Pediatrics
Advancing age is the most significant risk factor for Alzheimer’s Disease (AD), but how aging contributes to AD development remains unclear. Human induced pluripotent stem cell (iPSC) technology has revolutionized AD modeling, enabling the study of AD cellular pathogenesis in patient-derived cells. However, the epigenetic landscape and aging clock are reset to the embryonic state in iPSCs during reprogramming. Thus, current iPSC-based models lack proper cellular aging and do not fully recapitulate AD hallmark pathologies, including amyloid- beta plaques and neurofibrillary tau tangles, observed in human AD brains. My thesis aims to introduce factors of cellular aging to improve iPSC-based modeling of AD pathogenesis. I focus on CDKN2A (p16Ink4a or p16), an important senescence regulator and aging biomarker. I demonstrated that robust induction of p16 in iPSCs reduces cell proliferation and triggers downstream pathways, including focal adhesion and extracellular matrix organization. The inducible approach enables temporal control of p16 expression during the differentiation of iPSCs to neurons. I found that inducible p16 increases tau phosphorylation in iPSC-derived neurons in a cell-autonomous manner, providing a direct link between p16 and an AD-related cell pathology. This robust system provides a powerful resource to improve AD disease modeling to advance our understanding of the impact of aging in AD and develop innovative AD therapeutic drug discovery.
Characterizing Colibactin Toxicity and the Resulting Cellular Response to DNA Damage in Mammalian and Bacterial Systems
(UMass Chan Medical School, 2024-09-03) Lowry, Emily; Amir Mitchell; Morningside Graduate School of Biomedical Sciences
The bacterial toxin colibactin, produced primarily by the B2 phylogroup of Escherichia coli, crosslinks DNA and can promote colon cancer in human hosts, where it has been extensively studied. A systematic approach to identify the DNA damage response to colibactin-induced toxicity has yet to be applied and colibactin toxicity in bacteria remains underexplored. Using a genome-wide CRISPR screen in colon cancer cells, I found that colibactin activates most DNA repair pathways with key roles for Fanconi anemia/interstrand crosslink repair and fork quality control pathways. I also conducted a genome-wide loss-of-function screen in E. coli that identified a key role for homologous recombination in repairing colibactin-induced damage. I determined that colibactin induces a mutational pattern in E. coli in A/T rich regions, as it does in colon cells, but that the resulting mutational signature differs in E. coli. I then predicted that long- term colibactin exposure will culminate in a genomic bias based on this mutational signature, which may be detected in colibactin-producing bacteria. I tested this prediction by analyzing thousands of E. coli genomes andfound that colibactin-producing strains show skewness in trinucleotide composition. Finally, I used a sensitive DNA damage reporter assay to find that cell-cell contact is not required in bacteria as was previously suggested for both bacteria and mammalian cells, and that this needs to be reevaluated in mammalian cells. Taken together, this work revealed the DNA damage response to colibactin-induced damage in both colon and bacteria cells, a bacteria-specific mutation pattern, and that cell-cell contact is not required in bacteria.
Investigating the role of the Non-Classical MHCII Molecule HLA-DO in Regulating Exogenous Antigen Presentation in B Cells
(UMass Chan Medical School, 2024-08-21) Ramesh, Karthik M.; Lawrence J. Stern; Morningside Graduate School of Biomedical Sciences; Pathology
In B cells, exogenous antigens internalized through the BCR are processed in the endocytic pathway and loaded onto class II MHC protein (MHCII) for presentation to CD4+ T cells. The non-classical MHCII molecule HLA-DM (DM) catalyzes peptide exchange, selecting peptides with high affinity to MHCII, while HLA-DO (DO) binds to and inhibits DM in a pH-dependent manner. DO expression increases the diversity of the MHCII self-peptide repertoire and, through its inhibition of DM, allows for the presentation of low-affinity self-peptides, but the effect on exogenous antigens is unclear. We wanted to understand if DO expression in B cells affects exogenous antigen presentation and determine whether DO promotes peptide loading in particular intracellular compartments. We used an immunopeptidome workflow to identify naturally processed endogenous, exogenous, and virus-derived peptides in DO-sufficient and DO-deficient B cells infected with influenza virus, and we used a T cell assay to track the presentation of a single DR1-bound epitope derived from influenza HA with altered pH of fusion. We found that DO increases the diversity of peptides presented in infected cells favoring presentation of DM-susceptible viral epitopes. Tracking a single epitope, we show that the absence of DO favors exogenous peptide presentation, driving loading to later endocytic compartments. Our study expands on the role of DO in modulating the MHCII peptidome via a DM-dependent peptide-selection mechanism that regulates presentation of exogenous antigens and helps to illuminate its role in humoral responses to infections.
STING Gain-of-Function in Endothelial Cells Impairs Wound Healing Responses
(UMass Chan Medical School, 2024-09-19) Chuprin, Jane Evelyn; Mehdi Rashighi, MD; Michael Brehm, PhD; Dermatology
STimulator of Interferon Genes (STING) gain-of-function (GOF) mutations, resulting in constitutive STING activation, have been linked to a rare autoinflammatory disease called STING-Associated Vasculopathy with onset in Infancy (SAVI). SAVI patients present with hallmark skin findings, including chilblains (cold-sensitive lesions on acral surfaces) and progressive ulcerative lesions. We used a murine model of SAVI, STING(V154M/WT)-(VM), to explore the impact of the VM mutant on wound repair using ultraviolet B (UVB) irradiation as a tool for skin injury. Following UVB-induced injury, we found that VM mice developed exacerbated skin inflammation that persisted for 21 days or more. Conversely, WT mice developed mild erythema and erosion, which resolved within 7 days. Despite a strikingly different phenotype, total immune cell infiltration in VM skin was the same as WT within the first 5 days post-UVB irradiation. However, there were differences in the immune composition, including a significant lack of macrophage expansion during healing in VM skin. Further, we discovered that the VM phenotype is independent of T-cell responses and type 1 interferon signaling, challenging prior expectations in the literature. To identify the cellular driver(s) of skin disease, we used busulfan chimera and conditional knock-in mouse models. We determined that STING GOF in endothelial cells was sufficient to induce ulcerative lesions in VM mice. The critical finding in this thesis work is that following UVB-induced skin injury, STING GOF mutation in endothelial cells prevented macrophage expansion and impaired wound healing responses.
Mapping the ALS Citrullinome: A Proteomic Perspective on Neurodegeneration, Aggregation, and Protein Dysfunction
(UMass Chan Medical School, 2024-09-08) Camille, Webb; Zuoshang Xu; Paul Thompson; Biochemistry and Molecular Biotechnology
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that is characterized by progressive motor neuron loss, muscle wasting, paralysis, and death. Ninety percent of the cases are sporadic (sALS), while the remaining ten percent are familial (fALS). A hallmark of ALS neuropathology is aberrant protein aggregation and inclusion body formation in neurons leading to eventual degeneration of motor neurons in the brain and spinal cord. Previously, we showed that protein citrullination (PC), a post-translational modification (PTM), and peptidyl arginine deiminase 2 (PAD2) expression are altered dynamically in the spinal cord during ALS disease progression, increasing in astrocytes while decreasing in neurons (PMID: 36076282, 38253209). Interestingly, the citrullinated proteins accumulate in myelin protein aggregates, suggesting a role of PC in protein aggregation. Here, we applied proteomic methods to identify citrullinated proteins in an ALS mouse model expressing mutant SOD1G93A. The ALS citrullinome profiles disease progression distinctly from normal aging in mice and highlighted an increase in citrullinated glial proteins and a decrease in citrullinated neuronal proteins, validating PC as a marker of reactive astrogliosis and neurodegeneration. Additional analyses found that soluble citrullinated proteins were enriched in inflammation, membrane traffic and metabolic pathways, whereas for insoluble citrullinated proteins, myelin proteins were enriched. The findings in the soluble fraction were validated in analysis of two human ALS proteomic datasets. Among the highly citrullinated proteins in ALS are heat shock proteins and MBP. PC severely compromises these proteins’ structure and function. These results demonstrate the impact of PC in protein function, and furthermore, suggest that PC could provide candidate biomarkers of early-stage ALS and be targeted with novel ALS therapeutics.
Identification of WNK1 as a Therapeutic Target to Suppress IgH/MYC Expression in Multiple Myeloma
(UMass Chan Medical School, 2024-08-08) Ye, Tianyi; Michael Green; Michelle Kelliher; Morningside Graduate School of Biomedical Sciences
Multiple myeloma (MM) remains an incurable hematological malignancy demanding innovative therapeutic strategies. Targeting MYC, the notorious yet traditionally undruggable oncogene, presents an appealing avenue. This thesis aims to identify and characterize novel regulators of MYC expression as therapeutic targets in MM. Using a genome-scale CRISPR/Cas9 screen, we identify the WNK lysine deficient protein kinase 1 (WNK1) as a regulator of MYC expression in MM cells. Genetic and pharmacological inhibition of WNK1 reduces MYC expression and, further, disrupts the MYC-dependent transcriptional program. Mechanistically, WNK1 inhibition attenuates the activity of the immunoglobulin heavy chain (IgH) enhancer, thus reducing MYC transcription when this locus is translocated near the MYC locus. Furthermore, we show in MM cells possessing distinct translocation profiles that WNK1 inhibition also downregulates other oncogenes frequently translocated near the IgH locus, including CCND1, FGFR3, and NSD2, broadening its potential therapeutic implications. WNK1 inhibition profoundly impacts MM cell behaviors, leading to growth inhibition, cell cycle arrest, senescence, and apoptosis. Importantly, the WNK inhibitor WNK463 inhibits MM growth in primary patient samples as well as xenograft mouse models, and exhibits synergistic effects with various anti-MM compounds. Collectively, this study uncovers WNK1 as a promising therapeutic target in MM, and suggests the utility of IgH translocations as useful biomarkers.
Computationally Detecting Viral Infection and Characterizing Host-Virus Dynamics in scRNA-seq Datasets
(UMass Chan Medical School, 2024-07-26) Cao, Yuming; Manuel Garber; Genomics and Computational Biology
Viruses pose significant threats to human health, with their impacts varying by type. Advances in single-cell RNA sequencing (scRNA-seq) have enhanced our understanding of viruses and host responses by mapping human and viral transcripts within individual cells. However, ambient RNA contamination complicates the accurate identification of viral infections in scRNA-seq datasets. To address this, we introduced scVirusFinder, a method that uses a zero-inflated negative binomial model followed by a support vector machine classifier to identify virus-infected cells. This approach improves the detection of true viral infections in scRNA-seq datasets of virus infected cells. We applied this method to scRNA-seq data from nasal washes of healthy donors and those with acute influenza during the 2017-18 season. We identified seventeen cell populations, including a novel epithelial cell population with high MHC class II gene expression in infected individuals. Influenza virus infections were found in most cell populations, primarily in epithelial cells and major immune cells such as macrophages and neutrophils. Using viral reads from the scRNA-seq data, we discovered that each donor harbored a unique influenza variant with distinct non-synonymous mutations. Additionally, we observed interferon production and response in infected samples, with type III interferon particularly produced in infected ciliated epithelial cells. This study highlights the challenge of identifying infected cells from scRNA-seq datasets and provides a robust solution applicable to clinical samples, enhancing our understanding of viral infections and paving the way for therapeutic discoveries.
Racial Disparities and Trends in Anticoagulant Use among Ambulatory Care Patients with Atrial Fibrillation and Atrial Flutter in the United States from 2007-2019
(UMass Chan Medical School, 2024-08-08) Kan, Vincent; Matthew Alcusky; Emergency Medicine; Population and Quantitative Health Sciences
Introduction Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, significantly increasing the risk of stroke. The introduction of direct oral anticoagulants (DOACs) since 2010 has transformed anticoagulation therapy, offering an alternative to warfarin with improved safety profiles. Despite the increased adoption of DOACs, disparities in their use among different racial and ethnic groups in the United States remain understudied. Methods This study utilized a repeated cross-sectional design, analyzing data from the National Ambulatory Medical Care Survey (NAMCS) from 2007 to 2019. The study population included adults diagnosed with AF or atrial flutter (AFL). We analyzed the temporal trends of DOAC and warfarin use from 2007 to 2019. We examined the prevalence of DOAC versus warfarin use and assessed associations between race/ethnicity, patient characteristics, and DOAC utilization from 2011 to 2019. Multivariable modified Poisson regression models were used to calculate adjusted prevalence ratios (aPR) for the associations. Results From 2011 to 2019, NAMCS recorded 3,224 visits involving AF or AFL, representing a weighted estimate of 103.6 million visits. DOAC use increased significantly, with apixaban becoming the predominant anticoagulant by 2016. Non-Hispanic Black patients were less likely to use DOACs compared to non-Hispanic White patients over time (aPR 0.75; 95% CI, 0.63-0.90). Patients with Medicaid insurance were also less likely to use DOACs (aPR 0.14; 95% CI: 0.04-0.46). Conclusion Despite the shift from warfarin to DOACs for AF and AFL treatment, significant racial and socioeconomic disparities persist. Non-Hispanic Black patients and those with Medicaid insurance are less likely to use DOACs. These findings highlight the need for targeted strategies to ensure equitable access to advanced anticoagulant therapies.
Synthesis of Chemically Modified Nucleic Acids to Characterize and Inhibit APOBEC3 Enzymes
(UMass Chan Medical School, 2024-05-28) Hedger, Adam K; Jonathan K. Watts; Celia A. Schiffer; Biochemistry and Molecular Biotechnology; RNA Therapeutics Institute
APOBEC3 proteins (A3s) are enzymes that catalyze the deamination of cytidine (C) to uridine (U) in single-stranded DNA (ssDNA) substrates, playing a key role in innate antiviral immunity. However, incomplete A3 restriction of viruses is shown to cause drug resistance, and erroneous deamination of host DNA is strongly implicated in many cancers and therapy resistance. Small molecule drug discovery efforts have so far largely failed in finding potent and selective inhibitors of A3 enzymes. Recent structural advances have informed the design of “substrate mimicking” competitive oligonucleotide inhibitors, with potencies in the low-µM to -nM range. Nevertheless, further medicinal chemistry efforts are needed to improve the cellular stability and potency of these compounds. With that goal, in this thesis I report the first co-crystal structure of an active A3 enzyme bound to an oligonucleotide inhibitor at high-resolution, capturing the transition-state, and further informing inhibitor design. Next, I develop new and efficient synthesis routes to novel cytidine analogs for later incorporation into oligonucleotide inhibitors, including 2′-fluoroarabino- and 2′-difluoro-1,3-diazepinone nucleosides. Finally, I carry out a detailed structure-activity relationship of chemically modified oligonucleotide inhibitors against A3 enzymes. The resulting chemically optimized inhibitors from this work are the most potent reported to date, show greatly enhanced nuclease stability, and display inhibition in a cellular inhibition assay. In summary, my thesis work combines structural biology with nucleic acid chemistry to further A3 inhibitor design on three fronts, ultimately achieving the goal of generating potent and chemically stabilized inhibitors for use in cellular assays and beyond.

Browse

Recent Publications