Now showing items 1-20 of 1029

    • Tumor Microenvironmental and Cell-Intrinsic Mechanisms of Microtubule Stabilization in Breast Cancer

      Stemberger, Megan; Martin, Stuart S. (2024)
      Metastasis accounts for over 90% of breast cancer-associated mortality. Elucidating the pro-metastatic changes that occur within breast tumor cells and the surrounding tumor microenvironment (TME) is imperative for the development of antimetastatic therapies. This dissertation characterizes the effects of elevated H2O2 in the breast TME and cell-intrinsic alterations in the microtubule cytoskeleton for their impact on metastatic potential. H2O2 exposure induces -tubulin detyrosination and acetylation, two markers of poor patient prognosis, via a conserved Ca2+-dependent mechanotransduction pathway. H2O2 induces the formation of microtubule-based microtentacles (McTNs) which are enriched in detyrosinated-tubulin and acetylated-tubulin, but inhibits McTN-mediated functions of cell clustering and reattachment. This dissertation also elucidates the function of specific subsets of microtubules that have been post-translationally detyrosinated in breast cancer. Detyrosinated microtubules may provide an improved therapeutic target compared to current microtubule-stabilizing agents, which indiscriminately target all microtubules and are associated with broad side effects and inadvertently increase metastasis. Leveraging the recent discovery of the tubulin carboxypeptidase (TCP) enzyme, this study demonstrates the feasibility and functionality of lentiviral-based, constitutive TCP overexpression in mammary epithelial cells. TCP overexpression increases detyrosinated-tubulin, which is accompanied by morphological changes and enhanced cellular reattachment and migration. Collectively, this dissertation establishes that H2O2 signals in the TME induce microtubule stabilization and affect metastatic phenotypes, and provides the tools and preliminary data to continue investigating the therapeutic potential of targeting -tubulin detyrosination to reduce breast cancer metastasis.
    • Characterizing HIV-1 Genome Dimerization

      Yasin, Saif; Summer, Michael F. (2024)
      Like nearly all retroviruses, HIV-1 selectively packages two copies of its full-length genome after the formation of a dimer – a process essential not only to packaging but also reverse transcription and recombination. The dimerization process has been found to be mediated by the untranslated region of the HIV-1 transcript, the 5′-Leader, a nearly 400 nucleotide region of the RNA genome that is found to be responsible for regulating RNA fate and function. Structural methods on large RNAs like the dimeric leader (~800 nts) are notoriously limited due to the their inherent flexibility and size; however, more recent methodologies within the Summers lab have allowed us to characterize secondary structure domains within the intact leader. Using these defined structures, we can analyze how different domains of the leader influence RNA function and affect the viral life cycle. The goal of this thesis is to characterize the HIV-1 genomic dimer. We specifically looked at different domains and how they regulate dimerization and subsequent functional processes. We were specifically able to study the function of the 5′-polyadenylation signal, protein coding sequence downstream of the leader, the dimerization initiation site, and the major splice donor. We aimed to characterize processes such as dimerization, packaging, and even translation. Our work also sought to answer a long-standing question about the HIV-1 retroviral lifecycle: how could a single RNA transcript produce protein and also serve as the viral genome? We believe the process relies on the production of two different transcripts with different start sites, which modulate dimerization, but more importantly the structure at the 5′-cap which seems to subsequently dictate RNA fate. Overall, this work highlights the dynamic nature of RNA processes and how small changes in sequence can lead to dramatic changes in the HIV-1 lifecycle. We show that the role of dimerization within the HIV lifecycle is misunderstood, and still requires further characterization to understand how this structure dictates all its necessary functions. Our hope is that understanding the RNA processes involved in HIV-1 replication will allow the development of new therapeutic targets for treatment of the still ongoing HIV epidemic.
    • Characterization of Pseudomonas aeruginosa lipid A structural variants in cystic fibrosis

      Hofstaedter, Casey; Ernst, Robert K. (2024)
      Pseudomonas aeruginosa is the most common Gram-negative bacteria to cause chronic lung infection in people with cystic fibrosis (pwCF), leading to structural lung damage and progressive pulmonary decline. P. aeruginosa in the CF lung undergoes numerous genetic and phenotypic changes, adapting to the airway environment while establishing chronic infection. The work presented in this thesis characterizes one specific P. aeruginosa adaptation that occurs during CF lung infection: lipid A structural modification. Lipid A is the membrane anchor of lipopolysaccharide (LPS) (i.e., endotoxin), which comprises approximately 75% of the outer membrane of Gram-negative bacteria and is a potent agonist of toll-like receptor (TLR)-4, an innate immune receptor. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4 and the subsequent immune response. We hypothesize that lipid A structural alteration is beneficial for P. aeruginosa survival and pathogenesis by manipulating the host immune response during lung infection. Using a cohort of CF-derived P. aeruginosa isolates, we identify lipid A structural variation in isolates from 20% of pwCF. These lipid A structural alterations are driven by non-synonymous mutations in three lipid A genes: lpxO1, lpxO2, and pagL. We then characterize two P. aeruginosa lipid A enzymes encoded by lpxO1 and lpxO2 that can be mutated during chronic lung infection in CF. These two lipid A enzymes have distinct functions, mediating lipid A 2-hydroxylation in a site-specific manner. We also characterize P. aeruginosa isolates obtained from another inflammatory lung disease, diffuse panbronchiolitis, which result in the synthesis of structurally-distinct lipid A structures, suggesting that lipid A structural variation is not CF-specific. Lastly, we evaluate the impact of P. aeruginosa lipid A structure on host immune recognition and response. In vivo P. aeruginosa lacks PagL-mediated lipid A deacylation, which subsequently induces a stronger cytokine response. P. aeruginosa lipid A that lacks LpxO2-mediated 2-hydroxylation has reduced inflammatory potential, whereas LpxO1-mediated 2-hydroxylation has no measurable impact. This demonstrates distinct roles for each of the lipid A 2-hydroyxlation enzymes during in vivo P. aeruginosa infection. Taken together, P. aeruginosa lipid A structure plays an important role in pathogenesis during lung infection.
    • Advancing Genetic Studies in Latin American Populations: Enhancing Imputation and Investigating X Chromosome Associations with Alzheimer’s Disease

      French, Jennifer; O'Connor, Timothy D. (2024)
      Estimating genetic risk factors of diseases is challenging in diverse populations underrepresented in genomic studies. Genome-wide association studies (GWAS) are commonly used to discover genetic risk loci associated with health and disease. However, the majority of participants in GWAS are of European descent. These studies rely on imputation and many existing imputation reference panels are largely composed of individuals of European ancestry, resulting in lower imputation quality in underrepresented populations. The studies comprising this dissertation investigate how the composition of imputation reference panels affects imputation quality in four target Latin American cohorts. We achieve this through comparing imputation quality for chromosomes 7 and X when altering the imputation reference panel by: 1) increasing the number of Latin American individuals; 2) excluding either Latin American, African, or European individuals, or 3) increasing the Indigenous American (IA) admixture proportions of included Latin Americans. We found that increasing the number of Latin Americans in the reference panel improved imputation quality in the four cohorts; however, for some there were differences between chromosomes 7 and X. Finally, increasing Indigenous American (IA)-like admixture proportions in the reference panel increased imputation quality at different levels in the populations. The difference in imputation results between populations and chromosomes suggests that existing and future reference panels containing Latin American individuals are likely to perform differently in different Latin American populations, consistent with what we know of the varying population structure and ancestry proportions of the Latin Americans. As a further investigation, we imputed 87,393 variants on the X chromosome for 49,405 Latin American individuals and conducted a GWAS and X chromosome-wide association study (XWAS) in a Caribbean cohort. We identified 3 autosomal and 17 X-chromosome variants significantly associated with Alzheimer’s disease. Future studies are required to replicate X chromosome findings and include more robust X chromosome data for more diverse Latin American populations. This work highlights the importance of not treating Latin Americans as a homogenous population and taking population structure and the X chromosome into account when studying these populations.
    • Defining the Role of SLC35A2 in Cortical Development and Epilepsy

      Elziny, Soad; Crino, Peter B. (2024)
      Epilepsy is a common neurological disorder (3.4 million adults and 470,000 children) defined by recurrent seizures. Medically intractable (drug resistant) epilepsy affects approximately one-third of adults and 20-25% of children. Intractable epilepsy is often the result of germline gene mutations e.g., ion channels, kinases, neurotransmitter receptor subunits, identified in patient blood. Interestingly, recent studies have revealed somatic mosaicism associated with epilepsy in which variants are focally present in a subset of brain cells and cause epilepsy-associated focal malformations of cortical development (MCD). SLC35A2, was recently identified in a substantial fraction focal cortical dysplasia type 1a specimens. SLC35A2 encodes UGT-1, a transmembrane UDP-galactose transporter that facilitates movement of UDP-galactose from the cytosol to the lumen of the Golgi apparatus. Further, the variant allele frequency (VAF) in somatic SLC35A2 patients appears to correlate with severity in phenotype i.e., higher allelic burden is associated with greater morbidity. Patients exhibit a range of phenotypes including MRI confirmed FCD, intractable seizures, and intellectual disability. Germline variants in SLC35A2 are categorized under congenital disorders of glycosylation (CDG) and are implicated in an X-linked developmental and epileptic encephalopathy. All pathogenic variants, both somatic and germline, prevent UDP-galactose from being transported across the Golgi membrane and thus lead to aberrant glycosylated proteoglycans. Solute carrier families (SLCs) are the largest family of transmembrane transporters of sugars and a portion of these genes are implicated in epilepsy, neurodegenerative diseases, and autism spectrum disorder. To date, no study has addressed the effects of SLC35A2 knockout (KO) on neuronal morphology, protein glycosylation, or cortical lamination in a mouse model despite SLC35A2 mutations being recognized as a common cause of drug resistant epilepsy. I hypothesize that Slc35a2 KO results in disrupted neuronal Golgi structure, aberrant dendritic arborization, altered glycosylation profiles, aberrant cortical lamination, and disrupted network integrity. I will test this hypothesis under 4 specific aims: 1) To define the consequences of Slc35a2 KO in vitro on Golgi structure and dendritic arborization, 2) To demonstrate that Slc35a2 KO results in aberrant glycosylation profiles in mouse neurons, 3) To demonstrate that Slc35a2 KO in vivo alters cortical lamination and network integrity in mice using in utero electroporation, and 4) To establish 2 conditional KO (cKO) mouse lines of Slc35a2 and demonstrate that they alter cortical architecture and network integrity.
    • Sex-Dependent Differences in Alcohol-Induced Alpha-Tubulin Acetylation in Different Regions of the Mouse Brain

      Elesinnla, Abosede; Kristian, Tibor (2024)
      Downstream products of alcohol metabolism can impact the level of acetylated alpha-tubulin through the production of acetyl-CoA and the effects of acetylase alpha-tubulin N acetyltransferase1 (ATAT1) and deacetylase histone deacetylase6 (HDAC6) expressions. To determine the impact of ethanol intake on the modulation of tubulin acetylation in the brain, we administered ethanol to mice for different durations and examined their brain tissues. We used wild-type and HDAC6 null adult male and female mice. The control groups received PBS, and the treatment groups received ethanol (20% in PBS, 2g/kg) intraperitoneally. We analyzed the acetyl-CoA and CoA metabolites using high-performance liquid-chromatography (HPLC) methods. Furthermore, we determined changes in the acetylated alpha-tubulin levels and the enzymes regulating alpha-tubulin acetylation by western blots. We observed sex-related changes in the ethanol-induced hyperacetylation of alpha-tubulin in mice cerebellum and hippocampus. These differences can be attributed to the changes in the ethanol-induced expression levels of the acetylase/deacetylase enzymes.
    • Insights into the Role of Bifidobacterium in Neonatal Intestinal Maturation

      Collins, Rebecca A.; Ma, Bing (2024)
      Infant mortality and morbidity rates are impacted by premature birth, a critical issue in neonatal health. The prevalence of preterm births and accompanying disorders, such as necrotizing enterocolitis (NEC), underscore the urgent need for improved remedies. The "leaky gut" phenomenon, characterized by decreased intestinal barrier integrity, lies at the heart of these difficulties. Understanding the role of u in gut health, particularly in metabolizing maternal breast milk, holds potential for intervention. Human Milk Oligosaccharides (HMOs), a key component in breast milk, promote the growth of beneficial Bifidobacterium species in the newborn gut, but further research into their genetic features is necessary for a better understanding. This study aims to isolate Bifidobacterium strains from preterm infant stool samples, characterize their carbon utilization attributes, and define their genetic content. The findings reveal significant phenotypic and genotypic diversity among strains, illustrating varied genetic content within Bifidobacterium species. Differences in carbon source consumption patterns suggest functional versatility across strains. These results emphasize the crucial importance of understanding Bifidobacterium's genetic characteristics and metabolic capabilities in supporting gut health, particularly in preterm infants, and pave the way for targeted interventions aimed at reducing gastrointestinal complications and improving outcomes in this vulnerable population.
    • Exploring the Role of the Intestinal Microbiome in IBD-Associated Arthritis

      Alizadeh, Madeline; von Rosenvinge, Erik C.; Ravel, Jacques, Ph.D. (2024)
      Inflammatory Bowel Diseases (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), are increasingly common and characterized by inflammation of the gastrointestinal (GI) tract. A subset of patients with IBD also develop non-intestinal symptoms, known as extra intestinal manifestations (EIMs). The most common EIM, IBD-associated spondyloarthritis (IAA), occurs in up to 40-50% of patients within 30 years of diagnosis, and can be incredibly debilitating. However, it is poorly understood why some patients develop EIMs, but others do not. GI mucosal and stool microbial richness are decreased in IBD and non-IBD spondyloarthritis, and some overlapping changes indicate key microbial factors may drive both conditions. IBD disease location can be predicted by microbial profiles, and IAA tends to be associated with location of disease involvement, suggesting the region-specific intestinal microbiome plays a role in IAA. However, the relationship between the microbiome and IAA remains unexplored. I hypothesized that within the colon, the regional microbiome promotes IAA. To test this hypothesis, I first assessed clinical characteristics of participants in the SPARC-IBD cohort, and found EIMs were associated with increased biologic cycling, and that IAA was associated with a number of clinical factors, including right-sided disease involvement. I then enrolled 182 patients with IBD (53 with IAA) scheduled for clinical colonoscopy and obtained tissue biopsies from across the intestinal tract. 16S V3V4 rRNA gene amplicon sequencing was generated from tissue samples, and a variety of statistical tools were used to assess differences between those with and without IAA. Sample clustering was predominantly participant driven, with similar taxa present across the colon. Alpha-diversity significantly differed based on IBD type (CD & IBD-type undetermined (IC) < UC). Models assessing taxa associated with IAA, accounting for IBD type, sex, and the interaction between these factors and taxa, further revealed sex- specific interactions, where females and males with IAA displayed decreased Roseburia intestinalis and enrichment of Corynebacterium, respectively. This is the first study to compare the intestinal mucosal microbiome in subjects with and without IAA, offering an increased understanding of the potential role of the microbiome in IAA, and is a pivotal first step in driving biomarker identification necessary for new precision treatment for IBD.
    • Family History, Genetic Risk Factors, and Risk of Multiple Primary Cancers

      He, Shisi; Berndt, Sonja I.; Mitchell, Braxton D. (2023)
      Multiple primary cancers (MPC), often called second cancers, occur when more than one tumor arises in a patient from different cellular origins at different sites or presents with different histologies or morphologies [1, 2]. With improvements in the early detection and treatment of cancer over the years, both the population of cancer survivors and the chance of developing a new primary cancer has grown [3-5]. However, the etiology of MPC is not well understood. Established and suspected risk factors for MPC include host-related factors (e.g., primary immune deficiency), medical and lifestyle factors (e.g., immunosuppression, chemotherapy), environmental exposures (e.g., arsenic), and genetic factors (e.g., Lynch syndrome)[1]. However, many of the established factors, such as radiation and chemotherapy, only account for a small fraction of the risk [6]. Most research investigating genetic factors for MPC has focused on known cancer syndromes (e.g., Li-Fraumeni syndrome) and rare genetic variants with little research on the contribution of common variants. To understand the heritable risk of MPC, the first project aimed to understand if a family history of cancer is a risk factor for MPC. I also tested if there was a linear relationship between the number of cancers in the family history and MPC. The second project aimed to find common genetic variants associated with MPC. Molecular factors, such as inflammatory factors, insulin-like growth factor, and telomere length (TL), are also hypothesized to play a role in the development of cancer. Telomeres are DNA-nucleoprotein complex at the termini of eukaryotic chromosomes that protect the chromosome from degradation [7]. Telomeres are important in cell division and senescence and critical for chromosomal stability. Progressive telomere shortening occurs naturally with aging, and telomere attrition has been associated with some age-related diseases [7]; however, the relationship with cancer is complex. Despite the expectation of higher cancer risk with shortened telomere length, studies of measured TL have not always found that to be true. A meta-analysis including 121 studies conducted on blood cells did not find associations between TL and overall cancer risk; however, they found both positive and negative associations when stratified by cancer type [8]. Previous studies focused on certain primary cancers, while the relationship between TL and the risk of MPC is not well understood. Association studies with measured TL may be more susceptible to confounding by environmental and lifestyle factors. TL is highly heritable [9, 10], and 197 common genetic variants have been found associated with the leukocyte TL [11]. The advantage of using genetically predicted TL is that it uses germline genotypes that are present from birth and are uncorrelated with environmental exposures. The third study aimed to understand whether genetically predicted TL is associated with the risk of MPC. In summary, the overall objective of this dissertation is to provide insight into the etiology of MPC. The specific aims of the study are to: 1. To examine the association between a family history of cancers and the risk of MPC 2. To identify common genetic variants associated with the risk of MPC 3. To examine the association between genetically-predicted telomere length and MPC To our knowledge, this dissertation project is one of the first studies to examine the role of family history, genetically predicted telomere length and common genetic variants in relationship to the risk of MPC. The results will contribute to our understanding of the etiology of MPC and may suggest biological mechanisms and potential biomarkers for future studies.
    • Altered Gene Expression Profiles and Immune Responses in a Murine Model of a Non-lethal Flame Burn with Pseudomonas aeruginosa Infection

      Kambouris, Adrienne Renee; Cross, Alan S. (2023)
      Humanity has lived with fires for millennia, but combat, domestic use, and recent wildfires have increased the risk of burn injuries. Worldwide, over 100,000 deaths occur each year due to burns. If these patients survive the burn wound itself, the most common causes of death are multiorgan failure and sepsis, often caused by infection by Pseudomonas aeruginosa (PA). Utilizing a 10% total body surface area (TBSA) non-lethal flame burn model in mice, a superimposed infection of PA caused 100% mortality within 36 hours post-burn. This effect was transient, as infection 72 hours post-burn resulted in survival. The hypothesis was that this mortality could be linked to changes in gene expression that altered host-pathogen interaction. NanoString™, a system that allowed us to develop a custom panel of probes, was utilized to measure Mus musculus and PA gene transcripts simultaneously in each sample. Sampling from the blood, spleen, liver, and skin, gene expression in the burn and infection condition (B/I) was significantly different in each tissue when compared to mice that were burned alone, infected alone, and neither burned nor infected (Sham). The expression of the anti-inflammatory gene, Il10 is significantly increased over time in the spleen; administering anti-IL-10 antibodies delayed mortality by one day. While Arg1 and Nos2 gene expression were not significantly altered, administering arginine concurrently with PA restored survival in our mouse model, likely due to an inhibition of both PA motility and growth. We also hypothesized that burn-induced neutrophil dysfunction allowed for PA proliferation. Neutrophils isolated from the seroma of burned mice had a decreased ability to produce antibacterial reactive oxygen species (ROS) compared to neutrophils in the circulation of the same mice. Surprisingly, naïve neutrophils in the circulation of burned mice had a decreased ability to kill PA, possibly due to their premature ROS production induced by a burn-generated DAMP, HMGB1, present in the serum of burned but not Sham mice. In conclusion, a non-lethal burn injury is sufficient to induce multi-faceted changes in the murine immune system that results in an increased susceptibility to lethal PA superinfection.
    • Pim kinase inhibitor enhances FLT3 inhibitor gilteritinib efficacy through GSK-3β activation and GSK-3β-mediated c-Myc and Mcl-1 proteasomal degradation

      Lee, Jonelle; Baer, Maria R. (2023)
      Acute myeloid leukemia (AML) with fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) has poor outcomes. FLT3-ITD drives constitutive and aberrant FLT3 signaling, activating STAT5 and upregulating the downstream oncogenic serine/threonine kinase Pim-1. FLT3 inhibitors have limited clinical efficacy. We previously showed that concurrent Pim and FLT3 inhibition increases apoptosis induction in FLT3-ITD-expressing cells through post-translational downregulation of Mcl-1. Here we further elucidate the mechanisms of action of this dual targeting strategy. Protein expression and turnover, cytotoxicity and apoptosis were measured in FLT3-ITD-expressing cell lines and AML blasts treated with FLT3 inhibitor gilteritinib and/or Pim inhibitors AZD1208 or TP-3654. Pim and FLT3 inhibitor co-treatment decreased c-Myc protein, prior to Mcl-1, increased turnover of both proteins, rescued by proteasome inhibition, dephosphorylated (activated) GSK-3β, and increased apoptosis and in vivo efficacy. GSK-3β inhibition prevented c-Myc and Mcl-1 downregulation and apoptosis. Pim and FLT3 inhibitor co-treatment of Ba/F3-ITD cells infected with T58A c-Myc or S159A Mcl-1 plasmids, preventing phosphorylation at these sites, did not downregulate these proteins, increase their turnover or induce apoptosis, consistent with GSK-3β activation and c-Myc T58 and Mcl-1 S159 phosphorylation as the mechanism of combination treatment. These data further support GSK-3β activation as a therapeutic strategy in FLT3-ITD AML.
    • Role of Social Determinants of Health on the HIV Testing and Treatment Cascade in Nigeria

      Mohanty, Kareshma; Stafford, Kristen (2023)
      Introduction: The Joint United Nations Programme on HIV and AIDS proposed that to achieve epidemic control of HIV by 2025; 95% of all people living with HIV are aware of their status, 95% of people diagnosed with HIV receive sustained antiretroviral therapy (ART), and 95% of all people on ART are virally suppressed (VLS). The 2018 Nigeria HIV/AIDS Indicator and Impact Survey (NAIIS) found that in Nigeria, while only 47% knew their status, 96% had received ART, and 81% had achieved VLS. Social determinants of health (SDH), like wealth index (WI), have been shown to play a significant role in HIV in western countries, but the evidence has been limited and mixed in Nigeria. Identifying political, social, cultural, demographic, economic, and behavioral indicators of SDH, can better explain and address the disparities in the HIV epidemic, especially in the testing and treatment cascade, that are preventing the UNAIDS targets from being met in Nigeria. Objective: Examine the role of singular and composite indicators of SDH on the 95-95-95 targets: HIV testing, receipts of ART, and VLS in people living with HIV in Nigeria. Additionally, examine if wealth modifies the relationship between SDH indicators and the three 95 targets. Methods: Using the World Health Organization-SDH framework and Factor Analysis, I constructed composite indicators of SDH for Nigeria from various population-level survey data sources. Scores from the sub-indices and Global Terrorism Index were categorized as low, medium, and high, and individual or states were assigned one of these categories. Subsequently, I examined the association of the composite and singular SDH factors with HIV testing, receipt of ART, and achievement of VLS through survey-weighted multivariable logistic regression. Additionally, I examined the significance of the SDH indicators with the testing and treatment outcomes, by each of the wealth quintiles. Results: Out of the seven sub-indices constructed, only Access to Public Services, Crime & Conflict, Government Corruption, and Government Performance met the internal reliability criterion (Cronbach alpha > 0.7). Global Terrorism Index was constructed based on the prescribed methodology. When examining HIV testing, the first target in the 95-95-95 UNAIDS strategy, medium levels of Government Corruption, lower/medium Government Performance, and high Terrorism was associated with lower testing. Unemployment, living in rural areas, and married before 18 years of age were significantly associated with lower odds of HIV testing. For receipt of ART, second 95-95-95 target, low/medium treatment coverage was associated with lower odds of being on treatment. Younger age, male sex, being single, and living in rural areas were the singular factors associated with lower receipt of ART. Finally, for the third 95-95-95 target, only singular SDH, like lack of condom usage during sex, CD4 count (<500), and ethnic languages were associated with lower VLS. Wealth modified the relationship between the social determinants and HIV testing and treatment, but the role was weak. Wealth may increase the gap between the lowest and highest wealth index strata; HIV-related disparities experienced might be more pronounced between the two ends. Conclusion: Understanding and addressing structural determinants like political stability, terrorism, gender equality, accessibility to public services, and treatment facility coverage, rather than individual-level behavioral factors, could help Nigeria achieve the 95-95-95 targets.
    • Impact of Host and Parasite Factors on Gametocyte Production in Plasmodium falciparum

      Vareta, Jimmy Amakwa; Laufer, Miriam K.; Takala-Harrison, Shannon (2023)
      Stalled progress in reducing the malaria burden over the past few years suggests the need to develop new interventions to augment existing ones, including interventions aimed at interrupting gametocyte transmission from humans to the mosquito vector. To develop and effectively apply interventions that target gametocytes, there is a need to understand patterns of gametocytemia observed between individuals. Gametocytemia varies by host age, season, symptom status, antimalarial drugs use, and complexity of infection; however, the underlying mechanisms of this variation are not fully understood. Complexity of infection may modulate gametocytemia in P. falciparum; however, mechanisms of how clone composition influences gametocyte production are not clear. Addressing this gap requires a genotyping assay that can detect and estimate relative clone frequency of gametocytes and asexual parasites in infections. The dissertation aims were two-fold: 1) To develop an amplicon sequencing assay to genotype P. falciparum mature gametocytes; and 2) To evaluate the impact of host and parasite factors and gametocyte production in P. falciparum infections. We identified a polymorphic region of the pfs230 gene as a marker to distinguish P. falciparum mature gametocyte clones. When evaluating the impact of host and parasite factors on gametocyte production and gametocytemia, we found that more complex and high parasite density infections were more likely to produce and harbor gametocytes. The proportion of infections that produced gametocytes were similar between age-groups and between symptomatic and asymptomatic individuals, but children and asymptomatic individuals were more likely to harbor gametocytes than adults and symptomatic individuals, respectively. These findings suggest that complexity of infection and parasite density may increase gametocyte production, but additional factors such as host immunity and duration of infection may contribute to the presence or absence of gametocytes after initiation of gametocyte production. Coupled with the development of the gametocyte genotyping assay which will be an important tool for studies aimed at understanding dynamics of gametocyte production in polyclonal infections, understanding the impact of host and parasite factors on gametocyte production and gametocytemia will help explain variation in gametocytemia observed between individuals. This knowledge could inform development and effective deployment of transmission interrupting interventions.
    • An Insight to Further Malaria Vaccine Development: PfSPZ Vaccine Correlates of Protection Appear to be Cross-Reactive Antibodies to Immunodominant Low-Complexity Epitopes

      Berry, Andrea; Takala-Harrison, Shannon (2023)
      Plasmodium falciparum circumsporozoite protein (PfCSP) coats the sporozoite surface and is the target of multiple malaria vaccines in development. Discovery of additional vaccine candidate antigens beyond PfCSP may lead to improved vaccines. To identify new antigens, we used peptide microarrays to map antibody responses to P. falciparum proteins in adults who received a whole organism sporozoite vaccine, PfSPZ Vaccine, and were protected or unprotected after controlled human malaria infection. We discovered antibody responses that correlate with protection, but further examination, including of two monoclonal antibodies derived from protected PfSPZ Vaccine recipients, suggests that some antibodies elicited by PfCSP cross-react with peptides representing non-CSP proteins, a demonstration of inter-protein cross-reactivity. This work provides evidence that PfSPZ Vaccine elicits inter-protein cross-reactivity, provides amino acid-specific detail of putative epitopes, and introduces opportunities for further exploration that may help to elucidate underexplored immunological mechanisms and inform development of next-generation malaria vaccines.
    • Nitric Oxide in Mucormycosis Pathogenesis

      Soare, Alexandra; Bruno, Vincent, Ph.D. (2023)
      Mucormycosis is classified by NIAID as an emerging disease and is caused by Mucorales fungi. The recent surge of mucormycosis cases among COVID-19 patients has thrust the disease and lack of available treatments into the spotlight. Clinical data suggests a lack of inflammatory responses during mucormycosis despite severe fungal angioinvasion and tissue necrosis. In this dissertation, I sought to characterize immune evasion mechanisms by Mucorales, focusing on the interaction between fungi and macrophages. Macrophages infected with Mucorales fungi block the production of nitric oxide, a free radical molecule with strong antimicrobial properties and an important signaling role in immunity. Despite the increased expression of Nos2 mRNA and inducible nitric oxide synthase (iNOS) protein in Mucorales-infected macrophages, these macrophages are unable to produce nitric oxide, even when stimulated with nitric oxide-producing stimuli (LPS and IFN-γ). My results suggest that Mucorales fungi prevent the accumulation of nitric oxide through at least 2 mechanisms: (1) removal of nitric oxide from the surrounding environment, and (2) depletion of nutrients required to make nitric oxide. Additionally, a potent nitric oxide-donor (DETA-NONOate) inhibits in vitro growth of Mucorales fungi indicating that nitric oxide may be have antifungal activity against Mucorales. At lower concentrations of DETA-NONOate that are unable to inhibit growth of Mucorales, I observed downregulation of mRNAs encoding Mucorales virulence proteins including Mucoricin, a ricin-like toxin that is critical for Mucorales pathogenesis. By downregulating these genes, nitric oxide could be attenuating the virulence potential of the fungus, rendering it less pathogenic. My research describes a new immune evasion mechanism by Mucorales fungi and presents nitric oxide as a potential therapeutic for mucormycosis.
    • Role of Galectin-3 in Airway Epithelial Barrier Integrity During Influenza A Viral Infection

      Iqbal, Muddassar; Vasta, Gerardo R. (2023)
      Influenza A virus (IAV) infects the airway and alveolar epithelia in humans, and causes seasonal influenza annually which is a major global health concern, along with COVID-19, besides exhibiting pandemic potential. In severe cases IAV infection causes acute respiratory distress syndrome (ARDS) resulting from increased alveolar permeability due to the disruption of cell-cell tight junctions. The detailed mechanisms involved, however, remain to be fully elucidated. Galectins are β-galactose-binding lectins implicated in diverse cellular functions as well as in pathogen infections, including those from respiratory viruses. In previous studies from our lab on a murine model, IAV infection enhanced galectin-3 (Gal-3) secretion in the bronchoalveolar fluid. In the present study, I investigated in vitro the potential role of the secreted Gal-3 in airway epithelial barrier function during IAV infection. The results thus acquired using the human airway epithelial A549 cells indicate that IAV infection leads to a significant desialylation of the cell surface, exposing the sub-terminal β-galactose ligands, with a concomitant increase in the binding of recombinant galectin-3 (rGal3). I detected potential Gal-3 receptors CD147, integrin-β1 and MUC1 on the surface of A549 cells, and observed an increase in the secretion of matrix metalloproteinases MMP2 and MMP9 after exposure of these cells to rGal3. Furthermore, I observed disruption of the A549 cell surface distribution of tight junction proteins occludin and ZO-1 upon both IAV infection and Gal-3 exposure, as well as significant increase in the monolayer permeability. Using rGal-3, I demonstrated its direct interaction with the A549 cell CD147 as well as integrin-β1 which possibly mediated the above-mentioned cellular effects and validated some of the key findings in A549 cells from this study to the primary small airway epithelial cells (SAECs). In the end by searching for the sequence variants in the Gal-3 encoding genes LGALS3 in two public genetic databases, I found three rare variants that might alter protein function and one that was associated with the phenotypes indicative of a role for Gal-3 in influenza outcome: including influenza vaccine, flu treatment, acute sinusitis, and emphysema.
    • Microtubules as a therapeutic target in Duchenne muscular dystrophy

      Vanegas, Camilo; Ward, Christopher, Ph.D. (2023)
      Microtubules (MT) are dynamic polymers of tubulin protein whose post-translational modifications regulate MT interactions with other proteins, including intermediate filaments and actin. This integrated cytoskeletal network performs a vast array of cellular functions, including cell movement, transport of cellular cargo, defining cell shape and stiffness, and transmitting mechanical information to proteins that generate biological signals (mechanotransduction). My work is in Duchenne muscular dystrophy, where the absence of dystrophin leads to the proliferation of microtubules marked by an increase in posttranslational (PTM) modifications to their tubulin including detyrosination (deTyr-tubulin) and acetylation (acetyl-tubulin). The consequences of this change are increased cytoskeletal mechanics (i.e., stiffness) of the muscle fiber and increased mechanotransduction through NADPH Oxidase 2 (Nox2) dependent reactive oxygen species (ROS) and calcium signals during contraction that together have been implicated in the contraction injury that drives the pathology. My work addressed two open questions related to the pathobiology of these microtubule changes independent of dystrophic disease (Aim 1; Chapter 2) and the potential for targeting their reduction as a therapeutic option for halting or slowing disease progression (Aim 2; Chapter 3).
    • Biological, psychological, and sociocultural contributions to pain processing in the central nervous system: A whole-person approach to understanding chronic pain

      Cundiff-O'Sullivan, Rachel; Colloca, Luana (2023)
      Introduction: Chronic pain affects approximately 20% of the global population, yet effective treatments remain elusive. This study employs a biopsychosocial model, recognizing the importance of all facets of life, to investigate the complex interactions of biological, psychological, and sociocultural factors influencing pain outcomes and pain processing in the central nervous system. Methods: This project utilized functional magnetic resonance imaging to assess pain severity, interference, and endogenous pain modulation via placebo analgesia in a sample of participants with temporomandibular disorder. Brain-age difference was estimated via a pre-trained Gaussian Process Model using cortical thickness as a predictor of pain outcomes. Structural equation modeling and high dimensional multivariate mediation were employed to examine the influence of pain catastrophizing (PC) on pain outcomes. Differences in pain outcomes between religious/spiritual (R/S) and atheist participants were also explored. Results: TMD participants exhibited accelerated brain aging which was associated with pain severity and interference but not placebo analgesia. Structural equation modeling revealed that PC did not mediate pain outcomes or influence placebo analgesia. However, resting-state functional connectivity between the hippocampus and cuneal cortex mediated the relationship between PC and pain interference. Although there were no significant differences in behavioral outcomes between R/S and atheist participants, differential connectivity underlying these outcomes was found, such that atheist participants exhibited greater reliance on low-level sensory input compared to R/S participants who utilized higher cognitive regions for better emotional regulation of pain. Conclusion: This work provides valuable insights into the neurobiological mechanisms underlying how brain age, PC, and R/S factors influence chronic pain and placebo-induced pain reductions. By unraveling the complexities of pain processing using a whole-person approach, this research promotes a more holistic understanding of chronic pain and contributes to the development of more effective interventions for individuals suffering from chronic pain.
    • Regulation of Epidermal Skin Immunity by a Tick Bite

      Marnin, Liron; Pedra, Joao H. F. (2023)
      Hard ticks are hematophagous arthropods of public health and veterinary importance. Following a tick bite, these arthropods take prolonged, continuous bloodmeals that facilitate pathogen transmission. Successful bloodmeals are attributed to components of the tick saliva that alter inflammation, inhibit hemostasis, and block pain and itch responses in the mammalian skin. Recent studies have reported that extracellular vesicles (EVs) in the saliva enable tick feeding and redirect skin immunity. Notably, the skin epidermis, which interfaces with the external environment, has been mostly neglected when studying the relationship between ticks and their mammalian hosts. Here, we report that Ixodes scapularis EVs enable tick feeding by affecting epidermal γδ T cells frequency and co-receptor expression at the skin site. Epidermal γδ T cells have a critical role in wound healing and interact with keratinocytes, which comprise 95% of the epidermal layer. We further coupled entomological approaches with flow cytometry, single cell RNA sequencing, and animal strains devoid of epidermal γδ T cells to demonstrate that tick EVs disrupt networks involved in keratinocyte proliferation, suggesting an effect on epithelium wound repair. Collectively, this work broadens our knowledge of ectoparasitology, vector-host interactions, and principles of immunology.
    • Characterization of Plasmodium vivax transcriptome by Single Cell RNA-sequencing in a non-human primate model

      Hazzard, Brittany; Serre, David (2023)
      Malaria is a significant cause of morbidity and mortality worldwide, responsible for 241 million clinical cases and 627,000 deaths in 2020 according to the World Health Organization. Plasmodium vivax infections, while less deadly than the more widely studied Plasmodium falciparum, is a significant risk to many people in SE Asia and South America. P. vivax infections often consist of heterogenous populations of parasites at different developmental stages and with distinct transcriptional profiles, which complicates gene expression analyses. The advent of single cell RNA sequencing (scRNA-seq) enabled disentangling this complexity and has provided robust and stage-specific characterization of Plasmodium gene expression. However, scRNA-seq information is typically derived from the end of each mRNA molecule (usually the 3’-end) and therefore fails to capture the diversity in transcript isoforms documented in bulk RNA-seq data. P. vivax infections also often contain multiple, genetically-distinct, parasites but the consequences of this polyclonality on the regulation of asexual parasites, their sexual differentiation, and the transmission to a new host remain unknown. Here, we describe the sequencing of P. vivax scRNA-seq libraries obtained from a non-human primate model using traditional Illumina sequencing and Pacific Biosciences (PacBio) chemistry to characterize full-length Plasmodium vivax transcripts from single cell parasites, as well as experimental infections with the NIH-1993-F3 and the Chesson strains of P. vivax. Our results show that many P. vivax genes are transcribed into multiple isoforms, primarily through variations in untranslated region (UTR) length or splicing, and that the expression of many isoforms is developmentally regulated. Our experimental coinfection experiments revealed limited polyclonality when the strains were inoculated one after the other, but yielded robust polyclonal infections by simultaneous infection. In contrast to our hypothesis, this polyclonality did not seem to modify the regulation of individual parasites, or their sexual commitment. Overall, this work yielded unique insights on the mechanisms regulating the establishment of polyclonal P. vivax infections, and their consequences for disease transmission, and provided a validated framework to further study of Plasmodium polyclonal infections.