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Human Cytomegalovirus (HCMV) is the most common cause of viral congenital infection in the world. Around 5-10% of HCMV infected children are symptomatic at birth, and 50-90% of these develop severe manifestations with a 30% mortality rate. Among the asymptomatic children at birth, 10-15% develop late sequelae. The major cell entry glycoproteins of HCMV form three complexes: gC-I containing gB; gC-II containing gM & gN; and gC-III containing gH, gL, and gO (or UL128-131). These entry glycoproteins are polymorphic, producing different glycoprotein genotypes. The polymorphic nature of the glycoproteins as well as their ability to elicit neutralizing antibodies made them of interest in correlating them with the severity and outcome of the disease. This study aimed to develop a robust system to identify clusters of glycoprotein genotypes and to correlate them with disease manifestation. PCR assays of high sensitivity were used to identify all six glycoproteins. The PCR products were digested using restriction enzymes (RFLP) to identify the glycoprotein genotypes. Available laboratory strains (AD169, Towne, Davis, Toledo, and Merlin) as well as 112 clinical samples were amplified and genotyped using the assay, and their glycoprotein genotype profile was determined. A reliable sensitive assay was successfully developed to identify all glycoprotein genotypes including a novel gM assay using PCR/RFLP. The clinical samples were grouped according to disease manifestation (Group 1: congenital/postnatal patients (subgroup 1A: confirmed congenital patients & subgroup 1B: patients with either congenital or postnatal infection), Group 2: immunocompetent patients, Group 3: immunocompromised patients (subgroup 3A: immunocompromised patients with primary infection, subgroup 3B: immunocompromised patients with recurrent infection & subgroup 3C: immunocompromised patients with unconfirmed primary or recurrent infection)). Genotype gB1 was found predominantly prevalent in congenital/postnatal and immunocompromised patients, while gB3 was the most common genotype in immunocompetent patients. This result along with the phylogenetic analysis performed in this study suggest a relationship between gB genotypes and the immune response of the patients, where gB3 may be positively selected by host immune pressure. The novel gM assay genotyped the highly conserved gene (UL100) into three distinct genotypes; gM3 genotype associated with the congenital/postnatal group; which may provide an insight into understanding viral attachment and spread into the host cell. In congenital/postnatal infection, gH1 (72.7%) and gL4 (65.1%) were the most prevalent genotypes (gH1= 32/44, gL4= 28/43; P=0.000). In immunocompetent patients, mixed gH and mixed gL genotypes significantly correlated with the group, and in the immunocompromised group gH2 and mixed gL genotype were the most common genotypes (51.1% and 46.9% respectively). Glycoproteins gO, gH and gL are components of gC-III complex and gO1 was found to be the most prevalent gO genotype in all infection types (Group 1= 32.1%, Group 2= 85.7%, Group 3= 18.8%; P < 0.05). Also, in congenital/postnatal infection gN and gO were found to significantly link with each other and this is expected since both glycoproteins are highly polymorphic and are located on adjacent gene loci in HCMV genome (gN1+gO1a (P=0.000), gN3a+gO4 (P= 0.000)). The specific gN-gO linkages found here could be potential indicators for congenital/postnatal infection. In congenital/postnatal infection group, gH had significant linkages to gN and gO (gH1+gN1 (P=0.023, gH1+gO1a (P=0.013)) suggesting that interlinked selection of glycoprotein genotypes in the gC-II and gC-III complexes is involved in the development of congenital infection. High viral loads were found trending with immunocompromised patients, while low viral loads were significantly associated with mixed infected patients. This study has shown significant associations between a number of glycoproteins and congenital infection. Previously ignored glycoproteins gM and gL have been shown to be potentially of significant interest in this study and a larger study to confirm this is needed. In most cases the pattern of glycoprotein genotypes in congenital infection is more similar to that of immunocompromised than immunocompetent patients and it is possible that immune pressure is selecting for or against particular glycoprotein genotypes. The relationship between mixed infection and sample type may offer opportunities for development of prognostic biomarkers for congenital disease and further work is warranted.

There are many complex factors that will affect whether children with a unilateral congenital below-elbow deficiency (UCBED) will use a prosthetic limb to interact within their environment. Children face higher rates of prosthesis abandonment at 35-45%, compared to adults at 23-26%. Ultimately, for a child to wear and use their prosthesis, it must facilitate the effective performance of daily tasks and promote healthy social interactions. Although beginning to emerge, multiarticulate upper limb prostheses for children remain sparse despite the continued advancement of mechatronic technologies that have benefited adults with upper limb amputations. In contrast, pediatric devices typically provide a single open-close grasp (if a grasping function is available at all) and often offer non-anthropomorphic appearances, falling short of meeting the criteria essential to prosthesis adoption. Moreover, this population presents unique challenges, as they were born never having actuated a hand, and with forearm musculature that never fully developed–a stark departure from those with acquired limb absence. Due to the lack of investigation into how children with UCBED actuate their muscles coupled with the limited advancement in pediatric upper limb devices, the effective translation of dexterous prostheses remains a prominent issue. This dissertation builds the fundamental groundwork necessary for the effective translation of dexterous prosthetic hands for children with UCBED. It begins with an examination of how typically developing children use their hands to interact within their environment to inform dexterous device development (Chapter 3). Here we found that children, like adults, use a small subset of hand movements to perform object manipulation in home settings. Subsequently, a child-sized dexterous prosthetic hand was developed to serve as a dedicated research platform (Chapter 4). A thorough benchmark of this research platform was performed to validate its functional grasping ability and it was shown to be a robust device within a research environment. Prior to using this device, a cohort of children with UCBED were recruited, and an in-depth analysis of state-of-the-art prosthetic control, namely surface electromyography (sEMG) as a measure of affected muscle electrical activity, was conducted (Chapter 5). Upon investigation, participants exhibited a measurable degree of consistency and repeatability of their affected musculature as obtained through sEMG when they attempted missing hand and wrist movements. Furthermore, through tuning features, i.e., sEMG characteristics, and classification algorithms, we found a novel generalized feature set that provided increased classification to decode hand motor intent (Chapter 6). Moreover, we benchmarked the real-time performance of these children to execute hand movements, adding a translational dimension to our findings (Chapter 7). This forms a crucial foundation for understanding muscle actuation and use of advanced prostheses among children with UCBED. Through this work, we have laid the foundation to understand the capacity of children with UCBED to control their affected musculature. This begins to address the translational aspect of child-size dexterous upper limb devices and has the potential to remove barriers to device acceptance.

Background: Cytomegalovirus (CMV) is the most common congenital infection in the U.S. Approximately 0.5-0.7% of newborns are infected with CMV in utero, translating to about 22,800 infants infected each year. This dissertation focused on women of reproductive age, considered here to be 18-44 years, as this is the population who may make decisions regarding newborn CMV screening, and who could potentially transmit CMV congenitally during a pregnancy. Methods: Data from two sources were used. First, a survey was conducted at the 2018 Minnesota State Fair, where women ages 18-44 years asked about their preferences related to newborn CMV screening. Second, publicly available data from the National Health and Nutrition Examination Survey 1999-2004 were obtained, including the most recently available data on CMV seropositivity and urinary shedding in adults. Seroprevalence and prevalence of recent infections were assessed in women ages 18-44 years and stratified by racial and ethnic groups. Associations between physiological and social stressors and CMV urinary shedding were evaluated among seropositive women. Results: Women surveyed were generally supportive of newborn screening for CMV, with 86% stating they would choose to have their newborn screened. Women who also supported other newborn health activities were 30% more likely to choose newborn CMV screening than women who were not supportive of these activities. Non-Hispanic White women had lower CMV seroprevalence (48%) than non-Hispanic Black (85%) or Mexican American women (85%) but appeared to be slightly more likely to have evidence of a recurrent CMV infection (1.3% compared with 0.6% and 0.4%). When examining associations between physiologic and social stressors and CMV urinary shedding, point estimates suggested higher prevalence relative to not having the stressor, consistent with our expectation. However, for most stressors the 95% confidence intervals were extremely wide and consistent with associations ranging from much lower prevalence to much higher. Discussion: This dissertation adds evidence that women of reproductive age are supportive of newborn screening for CMV, especially women who also support other routine newborn healthcare activities. This finding is particularly timely, as universal newborn CMV screening was just implemented in Minnesota this year and is being considered in other states. A better understanding of factors related to CMV seroprevalence, timing of primary or recurrent infections, and shedding may help define risk factors for transmission among seropositive women and inform subsequent counseling among the 60% of pregnant women who are CMV positive so that the number of congenitally infected infants can be reduced.

Congenital cytomegalovirus (CMV) infection can cause severe neurological sequelae or even fetal death. We present a 17-year-old pregnant woman with fetal CMV infection, leading to voluntary termination of pregnancy. Fetopsy demonstrated a brainstem hemorrhage and focal polymicrogyria. CMV inclusions were observed in the lung, liver, thyroid, pancreas, kidneys, adrenal, placenta, and central nervous system. Intracranial hemorrhage is a rare finding in the context of congenital CMV infection, with isolated brainstem hemorrhage being an exceptional form of presentation. Polymicrogyria appears to be a more frequent finding, although its actual incidence is unknown. Future studies are needed to determine the causal association.

Pulmonary valve stenosis (PS) is the most common congenital heart defect in dogs. The condition is due to abnormal valve anatomy present at birth that leads to stenosis of the right ventricular outflow tract. The valve stenosis results in pressure overload of the right ventricle. If severe enough, the stenosis leads to right ventricular hypertrophy, arrhythmias, exercise intolerance, and right-sided heart failure. Severity of disease is typically determined based on velocity of blood flow across the lesion via echocardiography. The median survival time for dogs with severe PS is 4-5 years with medical management, while moderately affected cases have a variable prognosis. The prognosis for mild PS is generally good. The Bulldog, which is the fourth most popular breed according to AKC dog rankings, is a breed highly overrepresented in PS cases. Due to this breed predisposition PS is suspected to be inherited. Mildly affected dogs are frequently not diagnosed as traditional auscultation-based tests are insensitive in dogs with profound airway sounds and barrel-shaped chests such as the Bulldog. These subclinical cases make it incredibly difficult to successfully screen and remove affected individuals from the breeding program. Thus, hindering breeding efforts to reduce disease prevalence. Therefore, the development of a genetic screening test represents the most practical approach to identification and reduction of disease frequency in the breed. Additionally, severe and moderately affected PS cases are treated with traditional balloon valvuloplasty, which has good but variable success. The procedure has a significant cost, does not restore normal function, and there is considerable anesthetic risk for the brachycephalic breeds that are predisposed to PS. Additionally, some PS-affected dogs have concurrent coronary anomalies, which are expensive to identify and make balloon valvuloplasty contraindicated, further highlighting the need for genetic screening tests for this condition. There is limited information about the genetic cause of PS in humans with the majority of mutations involving the RAS-MAPK pathway. Although PS is commonly seen in puppies, no mutation to date has been identified, although literature suggests a possible recessive pattern of inheritance. Through the combined efforts of the clinical cardiology service at the University of California Davis, clinical cardiology service at Colorado State University, and the Translational Cardiac Genetics and Pharmacogenomics Laboratory in the University of California Davis School of Veterinary Medicine we aim to elucidate the genetic mechanisms of PS in Bulldogs. The goal of this dissertation is to compile and contribute to what is currently known about the genetics of PS in humans and animals. A retrospective study in a large referral hospital population allowed determination of the prevalence of this disease in dogs and confirmed common breed predisposition. A pedigree analysis was performed in the most affected breed, the Bulldog, to elucidate a common mode of inheritance. A genome-wide association study was performed to refine a region of interest in the canine genome that is associated with PS. Whole genome sequencing followed by variant analysis allowed identification of mutations associated with the disease. The most plausible variants underwent validation in a large population of dogs using MassArray and those that continued to segregate were followed up with immunohistochemistry in cardiac tissue. Finally, the results of this project will contribute to the understanding of heart development and congenital heart disease in dogs overall, thus guiding novel prevention and therapeutic strategies for PS.

Background:Unlike adults, children quickly outgrow any intravascular metal stents. Great interest exists in the development of stents that can either grow with or resorb in the arteries. This study aimed to develop self-expanding stents that could grow with the vasculature, examine them in a chronic animal model of a growing swine, and investigate the effects of the stent’s geometry and radial forces on the growing tissue. Methods: Computer Aided Design (CAD) and Finite Element Analysis (FEA) was used to select four self-expanding nitinol stents with a range of radial forces. Stents were designed and manufactured to meet predetermined mechanical specifications including crossing profile and fully expanded stent size of ≥20 mm. Hysteresis plots were determined for each stent. Fourteen stents were implanted in the abdominal aorta and iliac artery of four three-months old, 50 kg swine. Quantitative angiography, and both gross pathology and histopathology was assessed at 3 months (n=2) and 6 months (n=12). Results: The luminal area of all stented vessels grew more than the adjacent non-stented vessels and the average rate of diameter growth was 34%–49% and 20%–23% for stented and non-stented vessels, respectively. There was no intraluminal thrombus or significant stenosis seen in any of the arteries. Variable degrees of compression and laceration of the internal elastic lamina and/or media was observed to correlate with the initial vessel size but not with the radial force of the stent on the vessel. Similar correlation was observed with inflammatory response and with the degree of neointimal formation. No animals had major complications, issues with deployment, dissection, aneurysm formation, stent fracture, or migration during vessel growth. Conclusions: Self-expanding stents can be designed to grow with an artery to mirror and even exceed somatic growth. Although longer term testing is needed, it is possible to make custom tailor self-expanding stents to grow after arterial implantation in pediatric patients.

INTRODUCTION: Currently, patients diagnosed with Congenital Heart Disease (CHD) are living longer lifespans, leading to an increased number of these patients presenting for non-cardiac procedures/surgeries. Little research has been recently done analyzing intraoperative complications/risks for CHD patients undergoing non-cardiac surgeries. This study aims to identify common intraoperative events experienced by CHD patients undergoing non-cardiac surgeries using more recent data, while at the same time analyzing to see if there is any difference in frequency of intraoperative events experienced between different types of CHD diagnoses. METHODS: After receiving IRB approval, patients with CHD presenting for non-cardiac procedures/surgeries between the years 2008 and 2012 were pulled from Boston Children’s Hospital’s Electronic Medical records. 1,024 non-cardiac surgical encounters from 362 patients were analyzed to determine average age, average weight, patient gender, average ASA class, frequency of CHD diagnoses, ventricular function, type of non-cardiac procedure, premedication administration, type of induction and type and frequency of intraoperative events experienced. The 1,024 encounters were divided into two groups: those done in patients diagnosed with single ventricle physiology (n=79) and those done in patients diagnosed with non-single ventricle physiology (n=945). Unpaired Mann-Whitney tests were performed to determine if there was a significant difference in overall and specific intraoperative event occurrence between the single ventricle and non-single ventricle groups. RESULTS: Average age and weight at the time of these surgical encounters was 4.86 years and 20.57 Kg. A majority of the surgical encounters were done in males (59.2%). Atrial septal defect was the most common type of CHD, and most of the patients in these surgical encounters received an ASA class of 3. Intraoperative events occurred in 24.4% of the surgical encounters with cardiovascular events being the most common (44.82% of total events). Other events made up 30.49% of events experienced intraoperatively, with respiratory events making up the remaining 24.70%. There was a significant difference in the occurrence of overall events between the single ventricle and non-single ventricle group (P

Leukocyte Adhesion Defiency type III (LAD-III) is a rare congenital defect that affects the normal adhesive function of phagocytic cells of the myeloid lineage. Bone marrow from a patient with LAD-III was obtained and used to generate induced pluripotent stem cells (iPSCs). The iPSCs were determined to have normal karyotype, phenotype, differentiation capacity, cell surface markers, self-renewal capabilities and pluripotency. We hypothesize that iPSCs from this patient can be used to generate hematopoietic cells. In addition, the generated hematopoietic cells will have the same characteristics as the patient cells. We also evaluated the hematopoietic cells generated from the iPSCs, including their number and differentiation status, and their ability to migrate and adhere in response to inflammatory mediators. The main purpose of this project is to successfully model the cellular functional defect in LAD-III using iPS derived hematopoietic cells for in vitro cell culture analysis. Our results showed that we were able to generate hematopoietic progenitor cells and myeloid cells from our patient derived iPSCs. Furthermore, the differentiated myeloid cells had sustained the expected LAD-III functional defects of cellular adhesion and migration. In summary, this study was able to show the functional cellular defects in LAD-III by using patient-specific iPSCs to model the disease in vitro.

Abstract: Technological advances for treatment of congenital heart disease (CHD) have led to decreases in mortality over the past thirty years. Persistent morbidity into adolescence and adulthood necessitates regular medical follow-up and the influence of ongoing physical health issues on the teens emotional health merits clinician and researcher attention. Employing interpretive description research methodology, 22 interviews with teens aged 13-17 years were analyzed to understand how adolescents with CHD describe everyday life and relate to questions about quality of life (QOL). Although the majority of these teens viewed themselves as normal, CHD was a part of their everyday life that they situated into the foreground or background of their lives, as it suited their needs. These teens spoke of QOL issues in a concrete manner focusing on physical activity limitations and their need to fit in. Further discussion of these issues must be undertaken as adolescents with CHD transition to adulthood.

The human cytomegalovirus (HCMV) viral chemokine gene, UL146, shows a high degree of variability in clinical isolates. The UL146-produced viral chemokine, vCXCL-1, has homology to CXC chemokines and is predicted to be an immune modulator that may contribute to the pathogenesis of HCMV infections. In the analysis of clinical isolates from congenitally infected infants, we found 11 distinct vCXCL-1 clades. Although the four cysteine residues that create two disulfide bonds providing the essential structure for CXC chemokines,are conserved, the N-loop region, which is important for receptor binding and activation, was hypervariable. One clade also contained a modified glutamic acid-leucine-arginine (ELR) motif (asparagine-glycine-arginine / NGR), which regulates binding to CXCR1 and CXCR2 receptors. Based on this sequence information, we hypothesize that these proteins differentially activate neutrophils, which may have a role in HCMV pathogenesis. To address these functional differences, we produced representative vCXCL-1 proteins from each of the 11 clades using a baculovirus protein expression system. Using competition binding assays, we have examined their binding affinities to either CXCR1 or CXCR2 expressed on HEK293 cells. All vCXCL-1s bound to CXCR2 with different binding affinities. Interestingly, only three vCXCL-1s bound to CXCR1 while the others demonstrated did not. We analyzed functional differences between the vCXCL-1s in calcium mobilization, adhesion molecule induction, and chemotaxis on human peripheral blood neutrophils (PBNs). Although the binding affinities to CXCR2 and/or CXCR1 were variable, all vCXCL-1s were capable of inducing intracellular calcium mobilization in PBNs and upregulating adhesion molecules on the surface of PBNs to similar levels as human CXCL1. However, the potency of the vCXCL-1s in the chemotaxis of neutrophils varied and was affinity independent. We also examined secondary chemokine production upon vCXCL-1 treatment on neutrophil-like HL60 T2 cells using real-time PCR. The results showed CCL22 induction was affinity dependent. Taken together, these results provide insights into the potential role of vCXCL-1 in HCMV pathogenesis and how the variability in these chemokines can affect neutrophil function.