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Studies centered on pulmonary drug delivery have primarily focused on new device technologies to improve the generation of aerosols and their deposition in the respiratory system. Although considerable progress has been made in this field, no significant superior treatment of respiratory diseases has been achieved in recent times. One of the principal reasons is a scarcity in knowledge concerning inhaled/aerosol drug fate post-deposition in the lungs. The first barrier encountered by inhaled drugs in the pulmonary system is the mucus layer. This plays a protective role that is believed to be largely achieved by its ability to act as a physical barrier but also hinders molecular diffusion. The impeding nature of the mucus on aerosolised drug permeation is poorly understood. Indeed, the effects of direct interactions between mucus and inhaled drugs have been largely overlooked and most of the available evidence on mucus-drug interactions are related to the gastrointestinal tract. In this doctoral thesis, the main aim was to gain a better understanding of the relationship between the physicochemical properties of aerosolised pulmonary agents and their diffusion across the pulmonary mucus layer. This knowledge would provide valuable information for the development of more effective pulmonary agents. The first objective was to develop and characterise an in vitro mucus model suitable for drug permeation studies. That was comprised of a thin mucus layer derived from pig trachea covering the semi-permeable membrane of Transwell® inserts. Rheometric measurements on pig tracheal mucus secretions after a cleaning procedure to remove blood contaminants demonstrated that the obtained mucus was non-Newtonian. Furthermore, cryogenic-scanning electron microscopy (cryo-SEM) revealed pore sizes with diameters of ∼400 ± 200 nm. The barrier properties of the mucus model were verified by measuring the diffusion of various fluorescent dyes across mucus layers coating the Transwell® inserts. This revealed that the physicochemical properties of the compounds influenced their permeation rate with lipophilic dyes preferentially binding to the mucus layer. A global metabolic profiling approach was applied to characterise the low molecular weight compounds present in pig tracheal mucus before and after the cleaning procedure. A total of 816 metabolites were common to both sets of raw and cleaned mucus samples. Approximately 50% of those were lipids, products of amino acid metabolism and carbohydrate metabolism, which was in line with the previously reported composition of human bronchial mucus. A deposition system consisting of a Penn-Century MicroSprayer® mounted into a holder glass desiccator was successfully assembled to allow aerosolisation of test molecules at the surface of the mucus layers. After optimisation of the distance between the MicroSprayer® and the Transwell® inserts, the volume of the spray and the geometrical arrangement of the Transwell® inserts underneath the spray, a reproducible dose could be delivered onto four Transwell® inserts. Ipratropium bromide, glycopyrronium bromide, salbutamol sulphate, formoterol and indacaterol maleate were chosen as model pulmonary agents to investigate the effect of drug physicochemical properties on their interactions with airway mucus using the in-vitro mucus model and aerosoliser system previously developed. The extent of drug retention within the mucus layer varied between the compounds tested. Although no significant correlation could be established due to the low number of drugs studied, logP appeared the most important physico-chemical parameter driving drug affinity for pulmonary mucus. This PhD thesis has led to the development of simple in vitro systems to investigate the effect of the mucus layer on drug fate in the lungs and has identified mucus as a possible barrier to drug diffusion, in particular, for lipophilic compounds.

The rheological and structural characteristics of gastric (MUC5AC) and duodenal (MUC2) mucin solutions, the structural basis of the adherent mucus layer in the two organs, and their interactions with polyphenols, the phytochemicals which are linked with a number of health benefits, were investigated using particle tracking microrheology and scattering techniques. We used biochemically well characterised porcine mucins as models for human mucins to characterise their viscoelasticity, structure and dynamics as a function of concentration and pH. Additionally, the mesoscopic forces that mediate the integrity of the network were investigated using reducing (dithiothreitol) and chaotropic agents (guanidinium chloride and urea). Mucins in solution were found to be flexible and three distinct viscoelastic regimes were identify ed. At neutral pH, both types of mucin were found to form flexible self-assembled semi-dilute networks above a critical concentration (c*) where the viscosity scales as c 0.53+-0.08 and c 0.53 +-0.06 for MUC5AC and MUC2 respectively. Above a second critical concentration, the entanglement concentration (Ce), the peptide backbones reptate and entangle and there is a sharp increase in viscosity, c 3.92+- 0.38 for MUC5AC and c 5.1 0+-0.08 for MUC2. At low pH, both types of mucin solution undergo a sol-gel transition, forming pH-switchable gels. The addition of tea-derived polyphenols and tea extracts to the mucin solutions has revealed the strong interaction of galloylated phenolic molecules with mucins, which eventually leads to the gelation of the solution. Cross-linking of mucins by galloylated polyphenols is thus expected to have an impact on the physicochemical environment of the stomach and small intestine; the alteration of the organisation of the mucin polymer network is expected to modulate the barrier properties of the two adherent mucus layers which will affect nutrient absorption and the viscoelastic microenvironment of intestinal bacteria.

Amphibian population declines have occurred across a diverse range of geographic regions, and of approximately 7900 described amphibian species, an estimated 40% are threatened. Various factors, including disease and habitat degradation, have been linked to population declines. The Eastern hellbender (Cryptobranchus alleganiensis alleganiensis) is classified as endangered in the state of Ohio. To increase Ohio populations, conservation programs collect eggs from the wild and thousands of hellbenders have been successfully captive-reared and reintroduced to wild sites. However, mortality rates in captive hellbenders are highly variable and causes of mortality are poorly understood. Over a five-month period (November 2019 through March 2020), a high mortality rate (14%, 50/353) was observed in a captive population of hellbenders. Gross necropsies of 44 individuals revealed 86% (38/44) had cutaneous lesions (e.g. thick mucus layers, ulcerations, or white cottony aggregates). Histopathologic examination revealed cutaneous erosion, ulceration, or necrosis in 93% (41/44) of individuals. Bacteria was seen in 77% (34/44) and filamentous hyphae were seen in 68% (30/44) of individuals. Based on the presence of severe cutaneous disease in a large number of animals, we used 16S rRNA sequencing to assess the cutaneous bacterial microbiota of 116 live hellbenders from the same cohort group-housed in multiple tanks. We found variation in skin microbial diversity based on animal weight and tank location. This suggests that animals of different body weights exhibit microbial differences that may influence susceptibility to cutaneous and systemic disease and that local conditions influence skin microbial communities in hellbenders. Future research should investigate the influence of husbandry practices on the skin microbiome of hellbenders under human care and how skin microbiome is related to the presence of cutaneous and systemic disease. Ultimately, this research can be used to identify risk factors for disease development in order to prevent morbidity and mortality in Eastern hellbenders.

Cryptosporidium and Eimeria are two coccidian parasites that infect a wide range of animals including humans for Cryptosporidium, resulting in disease and, in some cases, death of the host. Infection involves three stages; ingestion, excystation and the subsequent invasion of the epithelial cells lining the gastrointestinal or respiratory (as can be the case for Cryptosporidium) tract. The mechanisms employed by the parasites to withstand the hostile environment of the host, and to penetrate to and invade a host cell are not clearly understood. The objective of this study was to provide more information on these three steps to infection, to elucidate the mechanisms involved for Cryptosporidium and Eimeria tenella, and to establish the extent to which these features are shared by the parasites. An in vitro procedure for inducing Cryptosporidium excystation was optimised. Anaerobic reducing conditions produced the highest excystation rates, with oocysts from different species excysting most efficiently at the pH values akin to their in vivo locations in the gastrointestinal tract. Oocyst age affected the excystation process, with the number of sporozoites released declining with increasing age. A novel method for determining the viability of sporozoites was developed. This employed the use of the vital stains acridine orange and bis-benzimide using fluorescence microscopy. Sporozoite survival during incubation with different pH buffers varied with the species of parasite, exemplifying that each species is adapted for sui'vival at the pH similar to their infection site in vivo. Inhibitors of mitochondrial function had little effect on either C. parvum or E. tenella. These data are consistent with C. parvum lacking a mitochondrion, and E. tenella sporozoites not requiring the function of this organelle. C. muris was, to an extent, affected by some of the metabolic inhibitors, suggesting that this species either relies in part on a mitochondrion, or that the inhibitors were affecting other processes in the parasite. Biotinylated probes and antibodies raised against Leishmania mexicana cysteine proteinases were used to analyse the lysates of C. parvum, E. tenella and Toxoplasma gondii for the presence of proteinases. Evidence was found for the presence of both cysteine and serine proteinases in all of the coccidia, with the apparent localisation of a cysteine proteinase on the surface of E. tenella sporozoites. Stage-specific differences were observed when sera against L. mexicana cysteine proteinases were used to analyse unsporulated oocysts and sporozoites of E. tenella. A survey of a range of enzyme activities showed the presence of a number of enzymes and some apparent variations between life cycle stages. However attempts to detect sialidase in Cryptosporidium and Eimeria using fiuorogenic substrates were not successful. A system was developed to study the penetration of mucus layers. The results showed that C. parvum sporozoites and E. tenella sporozoites and merozoites were able to penetrate the layers and, through the use of specific enzyme inhibitors, that motility, cysteine and serine proteinases, polyamines and sialidase were involved in mucus penetration by the parasites. Host cell invasion by C. parvum sporozoites and E. tenella sporozoites and merozoites was investigated using Madin Darby Bovine Kidney (MDBK) cells and specific enzyme inhibitors. The results suggest that cysteine, serine, metallo- and aspartic proteinases play a role in sporozoite invasion, as do polyamines, whereas sialidase was not important. Invasion by merozoites also involved cysteine proteinases and polyamines, but sialidase did appear to have a role. The MDBK cell cycle was analysed using bromodeoxyuridine (BrdU) labelling and 4,6,-diamino-2-phenylindole (DAPI) staining and its influence on parasite invasion was investigated. Sporozoites of both E. tenella and C. parvum invaded the host cells at the highest rate when the host cells were four hours into S phase. It was also discovered that more MDBK cells from cultures infected with E. tenella were in S phase than in control cultures. Thus the studies performed provide information on how Cryptosporidium and Eimeria survive within the hosts intestinal tract and penetrate to and invade a host cell. The two parasites appear to employ some similar mechanisms to facilitate the infection process, yet there are stage-specific features of each parasite.

A layer of mucus covers the surface of the pulmonary airways. Mucus is a hydrogel mainly composed of water, mucins (glycoproteins), DNA, proteins, lipids, and cell debris, which conditions the inhaled air and protects us from airborne threats. The natural protective role of mucus is nowadays acknowledged as a major barrier to be overcome in non-invasive drug delivery. The heterogeneity of mucus components offers a wide range of potential chemical interaction sites for macromolecules, while the mesh-like architecture given to mucus by the intermolecular cross- linking of mucin molecules results in a dense network that physically, and in a size-dependent manner, hinders the diffusion of macromolecules and nanoparticles through mucus. In this thesis, native airway mucus samples of porcine and human origin have been exhaustively characterized utilizing techniques that allow its investigation while preserving its original viscoelastic properties. The rheological properties and the penetration of differently sized nanoparticles either mechanically mixed with mucus or deposited as an aerosol onto mucus layers have been investigated. Additionally, a significant research effort has been spent in the development of in vitro models aimed at mimicking the bronchial region under diverse physiological conditions, including bacterial infection. These models have been proved suitable for investigating the translocation of nanoparticle-based drug delivery systems through the airways as well as for testing the efficacy of antibiotics in the context of cystic fibrosis.

A mechanism is described whereby the superficial mucus layers of the small intestines of immune rats trap Trichinella spiralis infective larvae and, with the action of peristalsis, the superficial mucus and the trapped larvae get rolled up into globules and cleared distally. Over 50% of the larvae recovered from the anterior small intestines within a.few hours of immune rats being infected either orally or intraduodenally, are trapped in such globules, the figure for control rats being, at most, 5.5%. Following an oral infection a maximum of 2.6% of the administered larvae penetrate through to the epithelium, as against up to 50% in control rats. This protective mechanism is not associated with a detectable anaphylactic reaction but can be partially blocked with high dose corticosteroids. Using 3000 larvae per rat there is not a detectable mucus discharge from the intestine (over and above the normal discharge). It does not act against fourth stage larvae of the unrelated nematode, Nippostrongylus brasiliensis. Hyperimmune serum transfers a partial degree of protection and the protective mechanism is associated chronologically with a significant fall in anti larval IgG serum antibodies. A crude in vitro assay was developed which confirmed the ability of immune serum to mediate larval trapping as could control serum which had not been heat inactivated; the latter mechanism not being thought to be important in vivo. The in vitro assay did not demonstrate larval trapping to be mediated by immune (or control) bile, a source of IgA antibodies.