Neuropsychiatric disorders arise from circuit defects established during development. However, risk-genes involved in circuit organization have not been thoroughly investigated due to the large cost of making transgenic animals for each genetic manipulation. We aimed to create a high-throughput CRISPR toolkit to investigate the effects of risk-gene modulation in the developing brain. This toolkit includes CRISPR knockout to study protein function, CRISPR knockin for protein localization, and CRISPR activation (CRISPRa) for gene dosage studies. We used in utero electroporation to introduce CRISPR agents into the developing brain and analyzed neuronal migration and axonal targeting phenotypes with fluorescent microscopy. We developed a sparse CRISPR knockout method to investigate gene function in the developing brain, validating this method with known phenotypes like Pten increased cell soma size and Dab1 neuronal migration defects. We also identified a novel axonal phenotype with Dctn2 knockout were knockout axons produced large varicosities in the corpus callosum. Similar callosal phenotypes were seen in patients with loss-of-function mutations of DCTN2. We then compared several common tags with more efficient CRISPR knockin. We find that the single-epitope HA tag has similar fluorescence intensity and higher knockin efficiency compared to the tandem-derivative 3xHA and mScarlet-I with β-Actin and IgLON4 knockin. Finally, we show that the transcriptional activator VPR frequently used with CRISPRa affects neuronal migration in a gRNA-independent manner, affecting the ability to identify phenotypes with changes in specific gene dosage. We also show that CRISPRa affects neuronal migration during development and will need to be optimized further to be used to investigate gene dosage of risk-genes. This work has provided a significant advance in the development of a CRISPR toolkit for investigating risk-genes in the developing brain and has identified important variables that should be taken into consideration when designing these experiments.

Alcohol use disorder (AUD) and Huntington Disease (HD) are neuropsychological diseases with an impact on the striatum. AUD is characterized by inflexible drinking, which is thought to be mediated by effects of chronic intermittent ethanol exposure on the dorsal striatum, the input nucleus of the basal ganglia. In our study of AUD, we used single-nuclei RNA-sequencing (snRNA-seq; n = 86,715 cells) to examine the impact of chronic intermittent ethanol exposure on the dorsal striatum in C57BL/6 male and female mice. We detected 462 differentially expressed genes at FDR < 0.05, the majority of which were mapped to spiny projection neurons (SPNs), the most prominent cell type in the striatum. Gene co-expression network analysis and functional annotation of differentially expressed genes revealed down-regulation of postsynaptic intracellular signaling cascades specifically in canonical SPNs. Inflammation-related genes were down-regulated across many striatal cell types. Gene set enrichment analyses also pointed to altered states of rare cell types, including the induction of angiogenesis-related genes in vascular cells. These data provide important clues as to the impact of ethanol on striatal biology and provide a key resource for future investigation. As a monogenic disorder, Huntington’s disease (HD) is at the forefront for gene-therapy innovation in neurodegeneration. One challenge to clinical innovation is our ability to discern detailed information about how HD and its potential therapeutics impact the brain. Single-nucleus RNA sequencing is a cutting-edge technology capable of providing this level of detail. Here, we studied cell type-specific transcriptomic effects of HD mutations in the striatum of a knock-in mouse model, HttQ111/+. Further, we examined the cellular impacts of a non-allele specific Htt-lowering antisense oligonucleotide (ASO). We characterized substantial transcriptional effects of the HD mutation in spiny projection neurons, the cell type most vulnerable to HD neurodegeneration. Surprisingly, Htt-lowering ASO treatment exacerbated many of these transcriptional changes. Understanding these cell type-specific effects of ASO treatment could aid in the interpretation of Htt-lowering toxicity that has recently been observed in human patients.

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.

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.

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.

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.

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.

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.

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.

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.