Since the advent of recommendations for placing infants in the supine position during sleep to reduce the incidence of sudden infant death syndrome, clinicians have noted an increase in the frequency of cranial asymmetry due to deformation of suture sections of the infants’ skulls as a result of constant concentrated stress in one area at the back of their head. This specific form of cranial deformation is known as positional plagiocephaly and its rate of occurrence has increased from 0.3% in 8.2% within the past 30 years. Current treatments and methodologies for preventing and correcting positional plagiocephaly such as stretching exercises, bedding pillows, and cranial molding are not optimized for effectiveness and comfort. Literature surrounding the implementation of these methodologies or devices often assesses the relative effectiveness of each treatment through statistical means, or studies complications associated with their use. There is a lack of quantified mechanical analysis for determining the effectiveness of each treatment or engineered solutions. In this study, a finite element model was created and validated to study the effect of wearing a cranial helmet, as the most effective non-surgical device for treatment of positional plagiocephaly, on reducing concentrated stress from the back of the baby’s head during sleep. The results from this model were then compared to two other finite element models with a healthy baby sleeping in supine position on a pillow, and a patient diagnosed with a severe case of positional plagiocephaly sleeping on the flat side of his head in supine position. The geometries representing the head of the babies in these models are the refined 3D laser-scanned file of a patient’s head contour at Hanger Clinic as well as the cavity inside the cranial helmet that was used for treatment of the baby. After successfully assigning section and contact properties to different regions of the models, applying proper loading and boundary conditions, and performing mesh convergence studies for each of the three models, the average Von Mises stress values of each of the 13 different suture segments of each model were summarized in tables and evaluated using mathematical and qualitative methods. The stress value data obtained from different suture regions of the model with the cranial helmet resulted in the smallest standard deviation among all three populations which supports that wearing the cranial helmet helps to reduce stress concentrations. Use of the cranial helmet during sleep also showed a significant decrease of the average Von Mises stress within the posterior fontanelle by 90% compared to the healthy baby sleeping in supine position and 73.4% compared to the deformed head sleeping on the flat surface of the head. The major limitations of this study are correlated with the simplifying assumptions and geometries in generating and validating the models. Future studies need to focus on overcoming these limitations and generating more complex models using a similar approach. The methods used in this study and the results obtained from the models can serve as a basis for future development of engineered solutions that are more effective than the existing solutions in the market and reduce the side-effects and complications associated with their use.

Friedreich's ataxia (FRDA) is the most prevalent inherited ataxia, affecting one in every 50,000 individuals in the United States. This hereditary condition is caused by an abnormal GAA trinucleotide repeat expansion within the first intron of the frataxin gene resulting in decreased levels of the frataxin protein (FXN). Insufficient cellular frataxin levels results in iron accumulation, increased reactive oxygen species production and mitochondrial dysfunction. Tissues most heavily impacted are those most dependent on oxidative phosphorylation as an energy source and include the nervous system and muscle tissue. This is evident in the clinical phenotype which includes muscle weakness, ataxia, neurodegeneration and cardiomyopathy. However, there has been a lack of data regarding the cell type specific contributions in FRDA pathogenesis. We generated a cohort of induced pluripotent stem cells (iPSCs) consisting of FRDA patient lines, CRISPR-Cas9 edited controls, carriers and non-related controls. Our preliminary data identified a hyperinflammatory microglial phenotype with extensive defects in mitochondrial function; since microglia are the primary innate immune cell of the brain, we hypothesized microglia may decrease neuronal viability which contributes to FRDA pathology. To investigate this, the iPSC cohort was utilized to generate microglia (iMGs) and neurons to better understand microglia-mediated neurodegeneration and how this contributes to pathology. An in vitro co-culture model composed of neurons, astrocytes and microglia was employed to better understand microglia-neuronal communication in FRDA. Healthy neurons co-cultured with FRDA iMG or with FRDA iMG-conditioned media demonstrated higher incidences of caspase-3 mediated apoptosis. These findings were recapitulated in vivo as xenotransplantation of FRDA microglia progenitors into a murine model resulted in reduced Purkinje cell survival in the cerebellum. Previous research has demonstrated the therapeutic potential of wildtype microglia to rescue the FRDA phenotype in the Y8GR mouse model of FRDA. To further explore the potential mechanisms behind this rescue, the delivery of mitochondria and FXN to FRDA microglia and neurons was investigated. CRISPR-Cas9 edited microglia demonstrated transfer of healthy mitochondria to FRDA microglia and neurons in an in vitro co-culture model. To investigate the transfer of frataxin protein, an FRDA iPSC line was transduced with an FXN-GFP lentivirus. Restoring FXN expression was demonstrated to rescue the FRDA microglial morphological phenotype. FXN-GFP microglia demonstrated transfer of frataxin protein to FRDA microglia suggesting the potential role of microglia as a therapeutic vehicle in FRDA. Together these findings show that FRDA microglia have a deleterious effect on neuronal viability, while healthy microglia may work as a therapeutic vehicle through the delivery of mitochondria and frataxin to FRDA cells.

Introduction: For children born with physical disabilities, the perspectives and actions of their parents prove significant to their childhood developmental outcomes clinically, educationally, socially, and with regard to community participation. The lived world and perceptions of parents who have children with disabilities however is not well investigated. This study sought to understand parents’ framing of theirs and their children’s disability experiences. Family systems together with family systems intervention models, and disability theory were used to provide structure to interview instrumentation and subsequent analysis. Child-centered and ecologic influences were also used to track the transformative processes over time that infuses parental themes. Methods: Methods for this study followed traditions of heuristic phenomenology. Open-ended parental interviews, written and spoken, together with field notes were used to explore the meanings given to disability. Analysis focused on collective descriptions and critical themes. Results: The nine parents in this study revealed four dominant themes around which their children’s lived lives were both understood and framed. Navigating normal for us; Our pride and joy; Anything but disability; Lived lives, looking back. Each is expressed in the words of parents who reared a child with disabilities into adulthood. Discussion and Recommendations: Parental disability frameworks differ from medical model frameworks and those of disability studies but share similarities with each. The parent themes provided holistic views of what these families have lived and learned. Their perspectives provide potentially vital markers and points of inquiry for interventionists and team members who work with children and families. Themes may also offer categorical means to explore well-being and child outcomes. Additionally, the themes were transformative and empowering for parents, both in the discussion of individual matters and in their narratives. All participants iterated that they welcomed having their voices invited and heard.

DNA damage response pathways are a complicated network of proteins that function to remove and/or reverse DNA damage. Following genetic insult, a signal cascade is generated, which alerts the cell to the presence of damaged DNA. Once recognized, the damage is either removed or the damaged region is excised, and the original genetic sequence is restored. However, when these pathways are defective the cell is unable to effectively mediate the DNA damage response and the damage persists unrepaired. Thus, the proteins that maintain the DNA damage response pathway are critical in preserving genomic stability. One essential DNA repair protein is the Breast Cancer Associated gene, BRCA1. BRCA1 is essential for mediating the DNA damage response, facilitating DNA damage repair, and activating key cell cycle checkpoints. Moreover, mutations in BRCA1 lead to a higher incidence of breast and ovarian cancer, highlighting the importance of BRCA1 as a tumor suppressor. In an effort to better understand how BRCA1 carried out these functions, researchers sought to identify additional BRCA1 interacting proteins. This led to the identification of several proteins including the BRCA1 Associated C-terminal Helicase, BACH1. Due to the direct interaction of BACH1 with a region of BRCA1 essential for DNA repair and tumor suppression, it was speculated that BACH1 may help support these BRCA1 function(s). In fact, initial genetic screenings confirmed that mutations in BACH1 correlated not only with hereditary breast cancer, but also with defects in DNA damage repair processes. The initial correlation between BACH1 and cancer predisposition was further confirmed when mutations in BACH1 were identified in the cancer syndrome Fanconi anemia (FA) (complementation group FA-J), thus giving BACH1 its new name FANCJ. These findings supported a previously established link between the FA and BRCA pathways and between FA and DNA repair. In particular, we demonstrated that similar to other FA/BRCA proteins, suppression of FANCJ lead to a substantial decrease in homologous recombination and enhanced both the cellular sensitivity to DNA interstrand cross-linking agents and chromosomal instability. What remained unknown was specifically how FANCJ functioned and whether these functions were dependent on its interaction with BRCA1 or other associated partners. In fact, we identified that FANCJ interacted directly with the MMR protein MLH1. Moreover, we found that the FANCJ/BRCA1 interaction was not required to correct the cellular defects in FA-J cells, but rather that the FANCJ/MLH1 interaction was required. Although both the FA/BRCA and MMR pathways undoubtedly mediate the DNA damage response, there was no evidence to suggest that these pathways were linked, until recently. Our findings not only indicate a physical link between these pathways by protein-protein interaction, but also demonstrated a functional link.

Huntington’s disease (HD) is an autosomal dominant, progressive neurodegenerative disorder. Invariably fatal, HD is caused by expansion of the CAG repeat region in exon 1 of the Huntingtin gene which creates a toxic protein with an extended polyglutamine tract 1. Silencing mutant Huntingtin messenger RNA (mRNA) is a promising therapeutic approach 2-6. The ideal silencing strategy would reduce mutant Huntingtin while leaving the wild-type mRNA intact. Unfortunately, targeting the disease causing CAG repeat expansion is difficult and risks targeting other CAG repeat containing genes. We examined an alternative strategy, targeting single nucleotide polymorphisms (SNPs) in the Huntingtin mRNA. The feasibility of this approach hinges on the presence of a few common highly heterozygous SNPs which are amenable to SNP-specific targeting. In a population of HD patients from Europe and the United states, forty-eight percent were heterozygous at a single SNP site; one isoform of this SNP is associated with HD. Seventy-five percent of patients are heterozygous at least one of three frequently heterozygous SNPs. Consequently, only five allele-specific siRNAs are required to treat three-quarters of the patients in the European and U.S. patient populations. We have designed and validated siRNAs targeting these SNPs. We also developed artificial microRNAs (miRNAs) targeting Huntingtin SNPs for delivery using recombinant adeno-associated viruses (rAAVs). Both U6 promoter driven and CMV promoter driven miRNAs can discriminate between matched and mismatched targets in cell culture but the U6 promoter driven miRNAs produce the mature miRNA at levels exceeding those of the vast majority of endogenous miRNAs. The U6 promoter driven miRNAs can produce a number of unwanted processing products, most likely due to a combination of overexpression and unintended export of the pri-miRNA from the nucleus. In contrast, CMV-promoter driven miRNAs produce predominantly a single species at levels comparable to endogenous miRNAs. Injection of recombinant self complementary AAV9 viruses carrying polymerase II driven Huntingtin SNP targeting miRNAs into the striatum results in expression of the mature miRNA sequence in the brain and has no significant effect on endogenous miRNAs. Matched, but not mismatched SNP-targeting miRNAs reduce inclusions in a knock-in mouse model of HD. These studies bring us closer to an allele-specific therapy for Huntington’s disease.

GM1 gangliosidosis is an autosomal recessive lysosomal storage disease, caused by a deficiency in the enzyme β-galactosidase. The disease affects the CNS, liver, kidney, heart and skeletal system, leading to severe neurodegeneration and death. We propose to treat this disorder using ex vivo hematopoietic stem cell therapy. The effectiveness of this therapy requires the recruitment of transduced donor cells to the CNS. This is only found to occur after mice are conditioned with total body irradiation, due to the increase in CNS cytokine production and blood brain barrier permeability that occurs. As the use of total body irradiation in pediatric patients has been linked to future developmental problems, this myeloablation approach is often avoided in younger patients in favor of a conditioning regimen using the chemotherapy drugs, busulfan and cyclophosphamide. Whether donor cells can enter the CNS when a busulfan and cyclophosphamide conditioning regimen is used has not been determined. In this study we plan to quantify the cytokine and blood-brain barrier permeability increases necessary for donor cells to be recruited to the CNS after total body irradiation. We will then investigate whether busulfan and cyclophosphamide conditioning and/or the chronic neuroinflammation present in GM1 mice can produce similar conditions and facilitate the recruitment of donor hematopoietic stem cells to the CNS. Finally we will assess whether ex vivo hematopoietic stem cell gene therapy is still an effective therapy when busulfan and cyclophosphamide are used for myeloablative conditioning.

Disruptions in cardiac development cause congenital heart disease, the most prevalent and deadly congenital malformation. Genetic and environmental factors are thought to contribute to these defects, however molecular mechanisms remain largely undefined. Recent work highlighted potential roles of chromatin- modifying enzymes in congenital heart disease pathogenesis. Histone deacetylases, a class of chromatin-modifying enzymes, have developmental importance and recognized roles in the mature heart. This thesis aimed to characterize functions of Hdac3 in cardiac development. We found loss of Hdac3 in the primary heart field causes precocious progenitor cell differentiation, resulting in hypoplastic ventricular walls, ventricular septal defect, and mid- gestational lethality. In primary heart field progenitors, Hdac3 interacts with, deacetylates, and functionally suppresses transcription factor Tbx5. Furthermore, a disease-associated Tbx5 mutation disrupts this interaction, rendering Tbx5 hyperacetylated and hyperactive. By contrast, deletion of Hdac3 in second heart field progenitors bypasses these defects, instead causing malformations in the outflow tract and semilunar valves, with lethality prior to birth. Affected semilunar valves and outflow tract vessels exhibit extracellular matrix and EndMT defects and activation of the Tgfβ1 signaling pathway. In normal second heart field development, Hdac3 represses Tgfβ1 transcription, independent of its deacetylase activity, by recruiting the PRC2 methyltransferase complex to methylate the Tgfβ1 promoter. Importantly, knockouts of Hdac3 in differentiated cardiac cells do not fully recapitulate the progenitor-specific knockout phenotypes. These results illustrate spatiotemporal roles of Hdac3, both deacetylase-dependent and deacetylase-independent, in cardiac development, suggesting that dysregulation of Hdac3 in cardiac progenitor cells could be a contributing factor in congenital heart disease pathogenesis.

KCNE1 (E1) peptide is the founding member of the KCNE family (1-5), which is a class of type I transmembrane ß-subunits. KCNE1 peptides assemble with and modulate the gating, ion conducting properties and pharmacology of a variety of voltage-gated K+ channel a-subunits, including KCNQ1 (Q1). Mutations that interfere with the function of either E1 and/or Q1 and disrupt the assembly and trafficking of KCNE1- KCNQ1 channel complexes give rise to diseases such as Romano-Ward (RW) and Jervell Lange Nielsen Syndrome (JLNS), two different forms of Long QT Syndrome (LQTS). Using enzymatic deglycosylation assays, immunofluorescence techniques and quantitative cell surface labeling, we showed that KCNE1 peptides are retained in the early stages of the secretory pathway as immaturely N-linked glycosylated proteins. KCNE1 co-assembly with KCNQ1 leads to E1 progression through the secretory pathway and glycan maturation, resulting in cell surface expression. N-linked glycosylation of some membrane proteins is critical for proper folding, co-assembly and subsequent trafficking through the biosynthetic pathway. Previous studies have shown that genetic mutations that disrupt one of the two N-linked glycosylation sites on KCNE family members lead to LQTS (T7I, KCNE1 and T8A, KCNE2) (Schulze-Bahr et al., 1997; Sesti et al., 2000a; Park et al., 2003). Having confirmed that KCNE1 proteins acquire N-linked glycans, we examined the kinetics and efficiency of N-linked glycan addition to KCNE1. We showed that KCNE1 has two distinct N-linked glycosylation sites. The N-terminal sequon is a traditional co-translational site. The internal sequon (which is only ~ 20 residues away from the N-terminal sequon) acquires N-linked glycans primarily after protein synthesis (post-translationally). Surprisingly, mutations that prevent N-glycosylation at the cotranslational site also reduce the glycosylation efficiency of post-translational glycosylation at the internal sequon, resulting in a large population of unglycosylated KCNE1 peptides that are retained in the early stages of the secretory pathway and do not reach the cell surface with their cognate K+ channel. We showed that KCNE1 post-translational N-glycosylation in the endoplasmic reticulum is a cellular mechanism that ensures E1 proteins acquire the maximal number of glycans needed for proper channel assembly and trafficking. Our findings provide a new biogenic mechanism for human disease by showing that the JLNS mutation, T7I, not only inhibits glycosylation of the N-terminal sequon, but also indirectly prevents the glycosylation of the internal sequon, giving rise to a large population of assembly incompetent hypoglycosylated KCNE1 peptides. To further investigate the two N-linked glycosylation sites on KCNE1, we generated structure-function deletion scans of KCNE1 and performed positional glycosylation scanning mutagenesis. We examined the glycosylation pattern of glycosylation mutants in an effort to define the glycosylation window important for proper KCNE1 assembly and trafficking. Our findings suggested a nine amino acid periodicity to serve as a desirable glycosylation site and a better substrate for N-glycosylation. Appendix II shows work on the characterization of the C-terminally HA-tagged KCNE1 protein, which was used throughout the experiments presented in Chapter II, Chapter III and Chapter IV. Analysis of the C-terminally HA-tagged KCNE1 protein revealed that in heterologous expression systems KCNE1 had an internal translational start site, a methionine at position 27. A proteolytic cleavage site was also identified at the arginine cluster spanning residues 32 through 38 bearing the two known Long QT mutations (R32H and R36H) (Splawski et al., 2000; Napolitano et al., 2005). My work in Professor Craig C. Mello’s lab during the first four years of my graduate study is presented in Appendix I. The highly conserved Wnt/Wingless glycoproteins regulate many aspects of animal development. Wnt signaling specifies endoderm fate by controlling the fate of EMS blastomere daughters in 4-cell stage Caenorhabditis elegans embryos. A suppressor genetic screen was performed using two temperature sensitive alleles of mom-2/Wnt to identify additional regulators of the Wnt/Wingless signaling pathway during C. elegans endoderm specification. Five intragenic suppressors and three extragenic suppressors of mom-2/Wnt embryonic lethality were identified. We cloned ifg-1, eIF4G homologue, as one of the extragenic suppressors suggesting an intriguing connection between the Wnt signaling pathway and the translational machinery.

Genomic instability, in the form of gene mutations, insertions/deletions, and gene amplifications, is one of the hallmarks in many types of cancers and other inheritable genetic disorders. Trinucleotide repeat (TNR) disorders, such as Huntington’s disease (HD) and Myotonic dystrophy (DM) can be inherited and repeats may be extended through subsequent generations. However, it is not clear how the CAG repeats expand through generations in HD. Two possible repeat expansion mechanisms include: 1) polymerase mediated repeat extension; 2) persistent TNR hairpin structure formation persisting in the genome resulting in expansion after subsequent cell division. Recent in vitro studies suggested that a family A translesion polymerase, polymerase θ (Polθ), was able to synthesize DNA larger than the template DNA. Clinical and in vivo studies showed either overexpression or knock down of Polθ caused poor survival in breast cancer patients and genomic instability. However, the role of Polθ in TNR expansion remains unelucidated. Therefore, we hypothesize that Polθ can directly cause TNR expansion during DNA synthesis. The investigation of the functional properties of Polθ during DNA replication and TNR synthesis will provide insight for the mechanism of TNR expansion through generations.

The endoplasmic reticulum (ER) is a multi-functional cellular compartment that functions in protein folding, lipid biosynthesis, and calcium homeostasis. Perturbations to ER function lead to the dysregulation of ER homeostasis, causing the accumulation of unfolded and misfolded proteins in the cell. This is a state of ER stress. ER stress elicits a cytoprotective, adaptive signaling cascade to mitigate stress, the Unfolded Protein Response (UPR). As long as the UPR can moderate stress, cells can produce the proper amount of proteins and maintain a state of homeostasis. If the UPR, however, is dysfunctional and fails to achieve this, cells will undergo apoptosis. Diabetes mellitus is a group of metabolic disorders characterized by persistent high blood glucose levels. The pathogenesis of this disease involves pancreatic β-cell dysfunction: an abnormality in the primary function of the β-cell, insulin production and secretion. Activation of the UPR is critical to pancreatic β-cell survival, where a disruption in ER stress signaling can lead to cell death and consequently diabetes. There are several models of ER stress leading to diabetes. Wolcott-Rallison syndrome, for example, occurs when there is a mutation in the gene encoding one of the master regulators of the UPR, PKR-like ER kinase (PERK). In this dissertation, we show that Wolfram Syndrome 1 (WFS1), an ER transmembrane protein, is a component of the UPR and is a downstream target of two of the master regulators of the UPR, Inositol Requiring 1 (IRE1) and PERK. WFS1 mutations lead to Wolfram syndrome, a non-autoimmune form of type 1 diabetes accompanied by optical atrophy and other neurological disorders. It has been shown that patients develop diabetes due to the selective loss of their pancreatic β-cells. Here we define the underlying molecular mechanism of β-cell loss in Wolfram syndrome, and link this cell loss to ER stress and a dysfunction in a component of the UPR, WFS1. We show that WFS1 expression is localized to the β-cell of the pancreas, it is upregulated during insulin secretion and ER stress, and its inactivation leads to chronic ER stress and apoptosis. This dissertation also reveals the previously unknown function of WFS1 in the UPR. Positive regulation of the UPR has been extensively studied, however, the precise mechanisms of negative regulation of this signaling pathway have not. Here we report that WFS1 regulates a key transcription factor of the UPR, activating transcription factor 6 (ATF6), through the ubiquitin-proteasome pathway. WFS1 expression decreases expression levels of ATF6 target genes and represses ATF6-mediated activation of the ER stress response (ERSE) promoter. WFS1 recruits and stabilizes an E3 ubiquitin ligase, HMG-CoA reductase degradation protein 1 (HRD1), on the ER membrane. The WFS1-HRD1 complex recruits ATF6 to the proteasome and enhances its ubiquitination and proteasome-mediated degradation, leading to suppression of the UPR under non-stress conditions. In response to ER stress, ATF6 is released from WFS1 and activates the UPR to mitigate ER stress. This body of work reveals a novel role for WFS1 in the UPR, and a novel mechanism for regulating ER stress signaling. These findings also indicate that hyperactivation of the UPR can lead to cellular dysfunction and death. This supports the notion that tight regulation of ER stress signaling is crucial to cell survival. This unanticipated role of WFS1 for a feedback loop of the UPR is relevant to diseases caused by chronic hyperactivation of ER stress signaling network such as pancreatic β-cell death in diabetes and neurodegeneration.