Genetic disorders like Cystic Fibrosis (CF) and X-linked Diseases (XLD) are inherited by offspring from parents who are healthy carriers of the autosomal recessive or allosomal genes. About 10-million Americans are healthy carriers of a mutant cysticfibrosis gene (predominantly F508del) and about 4% of newborns are at risk of being born with an X-linked disease. The current clinically approved mitigation plan for preventing genetic disorders in newborns from “at-risk couples” is to consider Preimplantation Genetic Testing for Monogenetic diseases (PGT-M). PGT-M involves an invasive microsurgical procedure that requires the removal of cells from 3-5day old embryos. To minimize this invasiveness, we proposed a less invasive approach to prevent genetic disorders in newborns by genotypically sorting sperm cells which may be used for fertilization events (IUI/IVF/ICSI) with specially characterized antigens on the sperm surface membrane. For the disease models being adopted in our study – CF and XLD; we utilized certain monoclonal antibodies (mab) to target the H-Y male antigen and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein which are both selectively expressed on the sperm surface. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

Lung disease is the third leading cause of death worldwide. The only curative intervention for end-stage lung disease is lung transplantation, which remains limited by the shortage of viable donor organs. Strategies to improve outcomes for patients with end-stage lung disease include: (i) ex vivo recovery of initially unusable donor lungs to a level suitable for transplantation, and (ii) repair of damaged lungs in situ to avoid the need for transplantation. Recovery of damaged lungs both ex vivo and in situ necessitates precise regulation of the lung extracellular environment, which includes biochemical, physical, and mechanical stimuli across scales. This thesis describes the development of bioengineering tools, including bioreactors and biomaterials, that leverage the lung extracellular environment across cellular, tissue, and organ scales to: (i) recover whole injured donor lungs ex vivo, (ii) assess and repair regional lung tissue injury in situ, and (iii) study the pathological cellular microenvironment in cystic fibrosis. In Chapter 1, regulation of the organ macroenvironment (ventilation, perfusion, systemic metabolism) with a homeostatic cross-circulation bioreactor enabled up to 100 hours of ex vivo lung support and recovery of injured human donor lungs. In Chapter 2, quantitative analysis of localized lung tissue properties, including lung sounds, enabled detection and assessment of pulmonary air leak, and recapitulation of lung microenvironmental features (structure, mechanics, composition) in a therapeutic biomaterial sealant enabled rapid treatment of air leaks. In Chapter 3, the first quantitative characterization of the cystic fibrosis matrisome (matrix proteome) identified pathological alterations to the microenvironment, and investigated implications for inflammation and immunity in cystic fibrosis. Collectively, these studies demonstrate that macro- and microenvironmental signals, including ventilation and perfusion mechanics, homeostatic metabolic regulation, and extracellular matrix structure and composition, can be leveraged to reveal previously unknown drivers of disease and promote recovery and repair of damaged lungs.

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The purpose of these studies was to further research on bone density in children with cystic fibrosis, particularly as it pertains to nutritional parameters and care. The first paper presented a comparison of a group of 50 children with cystic fibrosis to a group of 32 control children. There were no significant differences between the groups in any of the pertinent bone density measurements. Serum 25(0H) vitamin D was positively correlated with spine density z score in the cystic fibrosis group. The second paper, incorporating all of the information obtained from the first paper, describes an intervention study with the implementation of a fortified milk to determine the effects of additional calcium and vitamin D on bone density in the cystic fibrosis group. A follow-up bone scan was done. The fortified milk did not significantly improve bone density, but the fortified milk group did have significantly higher lung function scores on follow-up.

PURPOSE: A retrospective chart review was conducted to determine if nutritional source of feeding and/or infant age at diagnosis of cystic fibrosis had any effect on nutritional status and subsequent growth accretion. Additionally, an attempt was made to identify predictors for poor growth in patients with undiagnosed cystic fibrosis. METHODS: Data was collected from medical and clinic charts at Primary Children's Medical Center (PCMC), Salt Lake City, Utah, for subjects born between January 1, 1995 and December 31, 200 I, who were diagnosed with cystic fibrosis before 1 year of age. Thirty-one subjects met inclusion parameters. These subjects were divided into two groups: an "early" diagnosis group (N= 13) included those who were diagnosed before 9 weeks of age, and a "late" diagnosis group (N= 18) included those who were diagnosed after 9 weeks of age. "Breastfed at diagnosis" (N=7) and "not breastfed at diagnosis" (N= 17) groups were established as well, with nutritional source of feeding remaining unknown for 7 of the 31 subjects. RESULTS: Paired t-tests indicated that children who were primarily breastfed at time of diagnosis did not grow significantly more than children who were formula-fed at time of diagnosis, although regression analysis indicated that nutritional source of feeding at time of diagnosis was a significant predictor of growth later in life. This contradiction could have come about due to the small sample size. Age at diagnosis had a significant effect on growth, at diagnosis, at age 2 years, and age 6 years. Children who were diagnosed early grew taller and weighed more than the children who were diagnosed after 9 weeks of age, both at the 2-year mark and at the 6- year mark. Additionally, low blood albumin levels at diagnosis were predictive of more growth at age 2 years and 6 years. Other identified predictors of growth included gender, age at diagnosis, and whether the child had a family history of cystic fibrosis. This research highlights the crucial need for early detection and correction of malnutrition in infants and children with cystic fibrosis. It should be viewed as a pilot study, with more research needed in this area.

Cystic Fibrosis (CF) is an autosomal recessive disease that significantly affects quality of life and lifespan. There are currently no effective animal models of CF that mimic the human disease state. This prevents the development of pharmaceutical treatments for patients. Sheep have been considered for a useful animal model because of their size, life expectancy, and similarities in their anatomy and physiology. In order to generate a sheep transgenic model to study CF we have produced two Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene targeting DNA vectors containing large regions of homology to the CFTR gene in sheep. One of these targeting vectors (ΔF508Neo) contains sequences designed to delete the phenylalanine at amino acid position 508 of CFTR. Another targeting vector (G27XDTNeo) contains sequences designed to introduce an early stop codon into the CFTR gene such that no CFTR is produced. These two targeting vectors were used to transfect White Romney fibroblast cells. Donor sheep oocytes were collected for use in somatic cell nuclear transfer (scNT) with the two genetically modified cell lines. Embryos produced from scNT were transferred to recipient ewes which resulted in the birth of 11 ΔF508 heterozygous lambs and 3 G27XDT heterozygous lambs

Indiana University-Purdue University Indianapolis (IUPUI) / The Gram-negative bacterium Pseudomonas aeruginosa causes numerous acute and chronic opportunistic infections in humans. One of its most formidable weapons is a type III secretion system (T3SS), a multi-protein molecular syringe that injects powerful toxins directly into host cells. The toxins lead to cell dysfunction and, ultimately, cell death. Identification of regulatory pathways that control T3SS gene expression may lead to the discovery of novel therapeutics to treat P. aeruginosa infections. In a previous study, it was found that expression of the magnesium transporter gene mgtE inhibits T3SS gene transcription. MgtE-dependent inhibition appeared to interfere with the synthesis or function of the master T3SS transcriptional activator ExsA, although the exact mechanism was unclear. In this work, we demonstrate that mgtE expression acts through the GacAS two-component system to activate transcription of the small regulatory RNAs RsmY and RsmZ. This event ultimately leads to inhibition of exsA translation. Moreover, our data reveal that MgtE acts solely through this pathway to regulate T3SS gene transcription. Our study reveals an important mechanism that may allow P. aeruginosa to fine-tune T3SS activity in response to certain environmental stimuli. In addition, a previous study has shown that the P. aeruginosa gene algR abrogates mgtE mediated regulation of cytotoxicity. AlgR has pleiotropic effects in P. aeruginosa, including regulation of synthesis of the exopolysaccharide alginate. In the second part of my thesis, I show that algR and mgtE genetically crosstalk to inhibit ExsA driven T3SS gene transcription. This genetic interaction between algR and mgtE seems to be specifically directed towards regulation of T3SS gene expression rather than having an indiscriminate effect on multiple virulence attributes in P. aeruginosa. Additionally, we have further demonstrated that AlgR inhibits mgtE transcription. These studies suggest the presence of a T3SS inhibitor that is inhibited by both AlgR and MgtE. Future work will involve transcriptomic and proteomic analysis to identify such an inhibitor. Taken together, this study provides important insight into the molecular mechanisms of mgtE expression and function in P. aeruginosa. We have established that mgtE has pleiotropic effects on cytotoxicity in P. aeruginosa. Thus, given the role that cytotoxicity regulation plays in shaping P. aeruginosa pathogenesis and associated clinical outcomes, mgtE might be an interesting drug target, though extensive future studies are required to validate this proposition. Nevertheless, this research, provides clues for identification of novel therapeutic targets in P. aeruginosa. Hence this work, in the long run, serve to ameliorate the morbidity and mortality in patients infected with P. aeruginosa.

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A recent study by Leonard, Lebecque, Dingemanse, and Leal [1] tested the effect of Miglustat, an alpha inhibitor on the cystic fibrosis conductance regulator gene using total chloride secretion in the nasal epithelium as the key variable estimated from basal nasal potential differences. The conclusion was drawn that “There was no evidence of a treatment effect on any nasal potential difference variable.” This conclusion may not be correct because of a slight misinterpretation of their statistical results. There also is a question of whether longer exposure periods than 8 days would have produced a more pronounced effect.