Thesis (Ph. D.)--Catholic University of America, 1927. / Bibliography: p. 158-165.

There has been an escalating interest in the medicinal use of Cannabis sativa in recent years. Cannabidiol (CBD) and ∆9-tetrahydrocannabinol (THC), the main constituents of Cannabis sativa, have well documented immunomodulatory effects in vitro and following administration of high doses to animals. However, these effects have not been clearly evident in humans following oral administration of cannabinoids, probably due to low systemic bioavailability. To note, cannabis and cannabis-containing medicines are currently used for symptomatic relief in autoimmune diseases, such as multiple sclerosis (MS), and in cases of immunodeficiency, such as in cancer patients on chemotherapy regimens. In this thesis, we aimed to elucidate the impact of enhancing the transport of orally administered cannabinoids to the intestinal lymphatic system, the major host of immune cells, on the immunomodulatory effects of cannabinoids. Oral administration of lipophilic cannabinoids with long-chain triglycerides (LCT) was investigated as a simple approach to enhance the intestinal lymphatic transport. The effect of LCT on the intraluminal processing of orally administered cannabinoids was assessed by means of in vitro lipolysis model. The results of in vitro lipolysis demonstrated that at least one-third of CBD dose would be solubilised and readily available for absorption to the enterocytes when orally administered in LCT-formulation. The association of CBD with chylomicrons (CM) in the enterocytes and subsequent intestinal lymphatic transport was estimated using an in silico model, in vitro association by artificial CM-like lipid particles, and ex vivo uptake by plasma-derived CM from rats and humans. The results of CM association studies revealed high intestinal lymphatic transport potential for CBD in rats and humans. Similar high lymphatic transport potential was also reported for THC in our laboratory. Oral co-administration of CBD and THC with LCT to rats increased the systemic exposure by 3-fold and 2.5-fold, respectively, compared to lipid-free formulations. The underlying mechanism of increased bioavailability is likely to enhanced intestinal lymphatic transport and decreased pre-systemic metabolism in the liver. The results of biodistribution experiments indicated that the intestinal lymphatic transport of CBD and THC was, indeed, enhanced following oral co-administration of lipids as denoted by the dramatic increase in the concentrations recovered in MLN and intestinal lymph. The concentrations of CBD and THC in intestinal lymph fluid were in the range of 120 and 60 µg/mL compared to 0.5 and 0.6 µg/mL in plasma, respectively. Moreover, CBD and THC showed dose-dependent immunosuppressive effect on lymphocytes isolated from rats and peripheral blood mononuclear cells (PBMC) isolated from humans as assessed by lymphocyte proliferation assay and flow cytometry analysis of inflammatory cytokines. These effects were only significant at concentrations achieved in the intestinal lymphatic system, but not in plasma, following oral co-administration of cannabinoids with LCT. CBD showed more immunosuppressive effects on lymphocyte proliferation and the expression of inflammatory cytokines comparing to THC. Also, PBMC from MS patients were more susceptible to the immunomodulatory effects of cannabinoids than PBMC from healthy volunteers and cancer patients on chemotherapy. In conclusion, oral administration of cannabinoids with lipids can enhance the intestinal micellar solubilisation and augment the systemic exposure to cannabinoids by enhancing intestinal lymphatic transport. The concentrations of lipophilic cannabinoids recovered in the intestinal lymphatic system were extremely high and exceeded the immunosuppressive threshold of CBD and THC. The increase in systemic exposure to cannabinoids in humans is of potentially high clinical importance as it could turn a barely effective dose of orally administered cannabis into highly effective one, or indeed a therapeutic dose into a toxic one. In addition, administering cannabinoids, in particular CBD, with a high-fat meal, as cannabis-containing food, or in lipid-based formulations could represent a valid therapeutic approach to improve the treatment of MS, or other T cell-mediated autoimmune disorders. However, in immunocompromised patients, administration of cannabinoids in this way may deepen the immunosuppressive effects.

Pulmonary arterial hypertension (PAH) is a progressive disease of the small pulmonary arteries, characterised by pulmonary vascular remodelling due to excessive proliferation and resistance to apoptosis of pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells (PASMCs). The increased pulmonary vascular resistance and elevated pulmonary artery pressures result in right heart failure and premature death. Germline mutations of the bone morphogenetic protein receptor-2 (bmpr2) gene, a receptor of the transforming growth factor beta (TGF-β) superfamily, account for approximately 75%-80% of the cases of heritable form of PAH (HPAH) and 20% of sporadic cases or idiopathic PAH (IPAH). IPAH patients without known bmpr2 mutations show reduced expression of BMPR2. However only ~ 20% of bmpr2-mutation carriers will develop the disease, due to an incomplete penetrance, thus the need for a ‘second hit’ including other genetic and/or environmental factors is accepted. Diagnosis of PAH occurs most frequently when patients have reached an advanced stage of disease. Although modern PAH therapies can markedly improve a patient’s symptoms and slow the rate of clinical deterioration, the mortality rate from PAH remains unacceptably high. Therefore, the development of novel therapeutic approaches is required for the treatment of this multifaceted disease. Noncoding RNAs (ncRNAs) include microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). MiRNAs are ~ 22 nucleotide long and act as negative regulators of gene ex-pression via degradation or translational inhibition of their target mRNAs. Previous studies showed extensive evidence for the role of miRNAs in the development of PAH. LncRNAs are transcribed RNA molecules greater than 200 nucleotides in length. Similar to classical mRNA, lncRNAs are translated by RNA polymerase II and are generally alternatively spliced and polyadenylated. LncRNAs are highly versatile and function to regulate gene expression by diverse mechanisms. Unlike miRNAs, which exhibit well-defined actions in negatively regulating gene expression via the 3’-UTR of mRNAs, lncRNAs play more diverse and unpredictable regulatory roles. Although a number of lncRNAs have been intensively investigated in the cancer field, studies of the role of lncRNAs in vascular diseases such as PAH are still at a very early stage. The aim of this study was to investigate the involvement of specific ncRNAs in the development of PAH using experimental animal models and cell culture. The first ncRNA we focused on was miR-143, which is up-regulated in the lung and right ventricle tissues of various animal models of PH, as well as in the lungs and PASMCs of PAH patients. We show that genetic ablation of miR-143 is protective against the development of chronic hypoxia induced PH in mice, assessed via measurement of right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling. We further report that knockdown of miR-143-3p in WT mice via anti-miR-143-3p administration prior to exposure of mice to chronic hypoxia significantly decreases certain indices of PH (RVSP) although no significant changes in RVH and pulmo-nary vascular remodelling were observed. However, a reversal study using antimiR-143-3p treatment to modulate miR-143-3p demonstrated a protective effect on RVSP, RVH, and muscularisation of pulmonary arteries in the mouse chronic hypoxia induced PH model. In vitro experiments showed that miR-143-3p overexpression promotes PASMC migration and inhibits PASMC apoptosis, while knockdown miR-143-3p elicits the opposite effect, with no effects observed on cellular proliferation. Interestingly, miR-143-3p-enriched exosomes derived from PASMCs mediated cell-to-cell communication between PASMCs and PAECs, contributing to the pro-migratory and pro-angiogenic phenotype of PAECs that underlies the pathogenesis of PAH. Previous work has shown that miR-145-5p expression is upregulated in the chronic hypoxia induced mouse model of PH, as well as in PAH patients. Genetic ablation and pharmacological inhibition (subcutaneous injection) of miR-145-5p exert a protective against the de-velopment of PAH. In order to explore the potential for alternative, more lung targeted delivery strategies, miR-145-5p expression was inhibited in WT mice using intranasal-delivered antimiR-145-5p both prior to and post exposure to chronic hypoxia. The decreased expression of miR-145-5p in lung showed no beneficial effect on the development of PH compared with control antimiRNA treated mice exposed to chronic hypoxia. Thus, miR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, while the inhibition of miR-143-3p prevented the development of experimental pulmonary hypertension. We focused on two lncRNAs in this project: Myocardin-induced Smooth Muscle Long noncoding RNA, Inducer of Differentiation (MYOSLID) and non-annotated Myolnc16, which were identified from RNA sequencing studies in human coronary artery smooth muscle cells (HCASMCs) that overexpress myocardin. MYOSLID was significantly in-creased in PASMCs from patients with IPAH compared to healthy controls and increased in circulating endothelial progenitor cells (EPCs) from bmpr2 mutant PAH patients. Exposure of PASMCs to hypoxia in vitro led to a significant upregulation in MYOSLID expres-sion. MYOSLID expression was also induced by treatment of PASMC with BMP4, TGF-β and PDGF, which are known to be triggers of PAH in vitro. Small interfering RNA (siR-NA)-mediated knockdown MYOSLID inhibited migration and induced cell apoptosis without affecting cell proliferation and upregulated several genes in the BMP pathway in-cluding bmpr1α, bmpr2, id1, and id3. Modulation of MYOSLID also affected expression of BMPR2 at the protein level. In addition, MYOSLID knockdown affected the BMP-Smad and BMP-non-Smad signalling pathways in PASMCs assessed by phosphorylation of Smad1/5/9 and ERK1/2, respectively. In PAECs, MYOSLID expression was also induced by hypoxia exposure, VEGF and FGF2 treatment. In addition, MYOSLID knockdown sig-nificantly decreased the proliferation of PAECs. Thus, MYOSLID may be a novel modulator in pulmonary vascular cell functions, likely through the BMP-Smad and –non-Smad pathways. Treatment of PASMCs with inflammatory cytokines (IL-1 and TNF-α) significantly in-duced the expression of Myolnc16 at a very early time point. Knockdown of Myolnc16 in vitro decreased the expression of il-6, and upregulated the expression of il-1 and il-8 in PASMCs. Moreover, the expression levels of chemokines (cxcl1, cxcl6 and cxcl8) were sig-nificantly decreased with Myolnc16 knockdown. In addition, Myolnc16 knockdown decreased the MAP kinase signalling pathway assessed by phosphorylation of ERK1/2 and p38 MAPK and inhibited cell migration and proliferation in PASMCs. Thus, Myolnc16 may a novel modulator of PASMCs functions through anti-inflammatory signalling pathways. In summary, in this thesis we have demonstrated how miR-143-3p plays a protective role in the development of PH both in vivo animal models and patients, as well as in vitro cell cul-ture. Moreover, we have showed the role of two novel lncRNAs in pulmonary vascular cells. These ncRNAs represent potential novel therapeutic targets for the treatment of PAH with further work addressing to investigate the target genes, and the pathways modulated by these ncRNAs during the development of PAH.

Parkinson’s disease (PD) is caused by the degeneration of dopaminergic cells in the substantia nigra (SN) and ventral tegmental area (VTA) of the midbrain. Until recently, PD research predominantly focused on motor symptoms which are synonymous with the condition. Along with the acknowledgment that PD is no longer considered a ‘motor disorder’, there has been a significant increase in research designed to increase the understating of the nature, origins and management of a range of nonmotor symptoms, which can accompany a PD diagnosis. Investigations into a frequently reported memory decline in PD, have largely neglected to account for the influence of dopamine replacement therapies on such decline and specifically, the impact of second generation, non-ergot derived, D2 agonists, ropinirole and pramipexole have yet to be explored. In a between groups comparison, 2 clinically matched subgroups of PD patients, 1 medicated with ropinirole and the other with pramipexole were administered a test of recognition memory, familiarly and recollection on two separate occasions, once in a medicated state and once after a period of withdrawal. Results revealed that when in a medicated state, the PD subgroup medicated with pramipexole exhibited a significantly poorer recollection performance than the subgroup medicated with ropinirole. Furthermore, recollection significantly improved in the subgroup medicated with pramipexole when tested after a period of withdrawal, whilst ropinirole had no effect on memory performance. These findings suggested for the first time that pramipexole may induce a recollection impairment in PD. However, with the between groups methodology employed in this study, 3 possible scenarios remain as potential explanations of these findings; a PD phenotype, characterized by a memory decline, for which pramipexole is a only a maker based on its effectiveness in alleviating tremor and depressive symptoms; an drug effect, the high binding affinity that pramipexole has for d3 subreceptors, prevalent in the hippocampus, disrupts hippocampally dependent episodic memory processes, whereas ropinirole does not by virtue of a broad spectrum binding affinity for d2/d3/d4 subreceptors; a phenotype*drug interaction, a synthesis of the other 2 scenarios, where a PPX phenotype has a memory deficit which is particularly vulnerable to further impairment induced by pramipexole. To investigate these findings further, a fully powered, randomised controlled, crossover trial is required, whereby a PD cohort usually medicated with either ropinirole or pramipexole are combined and subsequently tested on each drug. A pilot trial was conducted to show how analysis could investigate the 3 scenarios described above, to obtain recollection estimates for a power calculation and to validate a battery of neuropsychological assessments to inform the design of a fully powered trial. Furthermore, recall measures were used and their relationship with recollection assessed to identify a clinically accessible test that could be administered in a clinical environment that is simpler and quicker to complete than a recollection assessment based on the remember/know paradigm. Throughout the duration of the pilot trial, recruitment was a major challenge. To explore this, eligible PD patients who declined to participate - and their caregivers - were interviewed to explore their perceived barriers to participation in clinical trials. A thematic analysis revealed four themes (switching medication, trial accessibility, fear of the unknown and caregiver workload) and several sub themes which represent decliner’s primary concerns. The findings presented in this thesis, contribute to current understanding of how memory is affected in PD, not just by the disease pathology but by dopamine replacement therapies. Findings have implications for dopaminergic modulation of hippocampal processes literature. Potential mechanisms for the dopaminergic disruption of glutamatergic and cholinergic modulation of hippocampal processes and the adequacy of the dopamine overdose model in accounting for acute dopamine therapies and the clinical management of PD are discussed. Furthermore, a number of recommendations are made to reduce perceived barriers to recruitment when designing and managing drug trials, not just with PD patients, but other clinical populations.

In mammals light is the principal timing cue for alignment of physiology to the external environment. Illumination from the unrelenting 24-hour day-night cycle enters the biological system and is communicated to the master pacemaker, the suprachiasmatic nucleus (SCN) to drive circadian entrainment. The decoding of light by the retina and the signalling pathways to and from the SCN rely on neural excitation mechanisms, achieved through changes in membrane potential from a resting state stabilised by K2P channels. With TASK-3 being the most abundant K2P channel in the rodent SCN it is feasible this channel has a crucial role in regulating SCN neural transmission for effective circadian entrainment. This study investigates this role through the use of transgenic TASK-3 KO mice. In the first experimental chapter I demonstrate the presence of TASK-3 mRNA in the SCN and retina of wild type mice. Further, I reveal a circadian pattern in TASK-3 mRNA expression with significant midday nadir which feasibly influences resting membrane potential (RMP) supporting increased neuronal excitation reported at this time. The following three chapters explore TASK-3 conductance in behavioural output rhythms via locomotor activity studies under light-dark cycles and in constant darkness. This series of experiments highlights how TASK-3 is essential for effective adjustment to changing light and how loss of this channel reduces light-driven and endogenous activity intensity and rhythm amplitude. With light entering the circadian system exclusively via the eyes, the role of TASK-3 at the level of the retina is of upmost importance to entrainment. This is investigated in chapter 6 using pupillary light reflex as a measure of retinal sensitivity and decoding capacity. Through manipulation of intensity and wavelength specific classes of photoreceptor are studied for their contribution to this non-image forming response. These experiments show TASK-3 ablation significantly attenuates retinal sensitivity to sub-saturating light in a mechanism likely to be melanopsin-independent. Finally examination of mRNA expression of core clock genes reveals the role of TASK-3 at the level of the SCN. Here, loss of TASK-3 conductance is shown to alter daily rhythms in several key genes thereby linking the properties of this background leakage channel to the molecular clockwork. Overall these experiments demonstrate some of the roles TASK-3 conductance plays within the SCN and in output rhythms; and the requirement of this channel within the retina for effective retinal decoding across the visible spectrum over a range of light intensities.

This study has its origins in a question posed by a patient diagnosed with a psychotic illness, as to why her husband could not administer depot injection. Following local and national discussion the study aims were; - to explore the elements of risk management involved in enabling carers (supportive persons) to give depot injections - to develop a training package that may be useful for others to use should such a request be made - to establish whether enabling supportive persons to give depot injections would have an effect on the relationship between the user (recipient of the medication) and the supportive person (giver of medication) - to ascertain the views, concerns and attitudes of medical staff (prescribers) and mental health nurses (administrators of depot injections) about enabling carers/relatives (supportive persons) to give depot intramuscular injection medication. An action research study informed by empowerment theoretical perspectives and influenced by recovery philosophies was used to explore the issues about ‘supportive person’ depot administration. Methods used to collect data included case studies, interviews, observation, reflection and three validated evaluation tools. Data were analysed through thematic analysis, and alongside establishing data, relating to the study aims, additional themes i.e. stigma, disclosure, concealment and trust evolved from the data. The study has relevance for clinical practice, policy and service provision. Current government policies promote choice and collaborative working and health and social care staff are encouraged to be responsive to the views of mental health service users and carers in relation to their experiences and expectations of care. Mental health services are being asked to deliver and translate these policies into practice alongside expectations of gainful employment for service users.

In clinical trials, sirolimus (rapamycin, a macrocyclic lactone) has been shown to exhibit antitumor activity across a variety of human cancers by binding to and inhibiting the activation of the mammalian target of rapamycin (mTOR), thus preventing cell cycle progression from the G1 to S phase. Inhibitors of mTOR have received regulatory approval as immunosuppressive agents for the treatment of allograft rejection and as antitumor agents for kidney cancer (Rapamune®). In these clinical trials tumour cell proliferation was dramatically reduced without sirolimus being formulated in a drug carrier. The more challenging question is whether strategies can be developed to improve the delivery of sirolimus directly to tumour cells and maximize mTOR inhibition? The aim of the research reported herein was to develop, characterise and evaluate the anti-cancer activity of sirolimus loaded liposome formulations. Liposome-drug formulations were prepared using the thin film hydration method and were characterised using particle size analysis, atomic force icroscopy (AFM), differential scanning calorimetry (DSC) and X-ray photoelectron spectroscopy (XPS). The particle size analysis of the liposome formulations showed that the liposome-drug formulations were stable over a 6 month period of time. Further characterization of the liposome-drug formulations using XPS and DSC studies demonstrated the incorporation of the drug (sirolimus) in the liposome bilayer. In order to ascertain the anti-tumour activity of the sirolimus formulations, in-vitro studies using MTT assays were carried out on human breast cancer cell lines (MCF-7 and BT-474). The cytotoxicity studies using pure sirolimus showed anti-proliferative action at concentrations above 40 μg/mL and the formulated liposome formulations also demonstrated anti-proliferative effects when incubated with the cancer cells. Parameters such as lipid composition, incubation time and drug loading were established as important factors that play a key role in the therapeutic efficacy of the sirolimus loaded liposomes. Fluorescent images obtained from the cellular uptake and apoptosis studies also provided supporting data which demonstrated the anti-proliferative effect of the liposome formulations. In addition, sirolimus was designed to actively target breast cancer cells by conjugating transferrin on the surface of the sirolimus loaded liposomes. Both in-vitro and in-vivo studies of the conjugated sirolimus formulations demonstrated the formulation to be more effective in inducing anti-proliferative effects compared to the passive formulation (non-conjugated sirolimus loaded liposomes). Sirolimus loaded liposome anticancer activity towards prostate cancer cell lines (LNCAP and DU-145) has also been evaluated. Similar results to the breast cancer studies were obtained; specific parameters were also shown to influence the anti-proliferative efficacy of the sirolimus liposome formulations in prostate cancer cells.

Studies of pancreatic elastase were undertaken to examine substrate binding and of human leukocyte elastase to try to gain insight into the disease, pulmonary emphysema. Porcine pancreatic elastase was inhibited by various inhibitors p-toluene sulphonyl fluoride, phenylmethane sulphonyl fluoride, 1-dimethylamino-naphthalene-S-sulphonyl fluoride or chloride and p-nitrophenyl-anthranilate. The enzyme was only fully inhibited by the first two inhibitors which were then treated under alkaline conditions to convert the serine-19S, -CH20H to -CH2 to form the anhydroelastase. The enzyme was shown to be totally inactive but X-ray diffraction data to 2.SA resolution from one preparation showed no changes at the active site. Further studies were carried out to modify the serine-19S to cysteine by thiolation of the inhibited enzyme and anhydroelastase but proved unsuccessful. Binding studies were carried out with the prepared hexapeptide substrate, H.Pro-Ala-Pro-Ala-Lys-Phe.OH and tetrapeptide inhibitors, Ac-Pro-Ala-Pro-Ala.OH (1), Ac-Pro-Ala-Pro-Alaninal (2) and TFA-Pro-Ala-Pro-Alaninal (3) using anhydroelastase and native elastase. Inhibition with inhibitors (2) and (3) showed TFA-peptide-aldehyde bound tighter than Ac-peptide-aldehyde with K for (2) - 6.1 llM and for (3) - 8.6 llM. A TFA-pepiide-chloromethYlketone was also examined. It showed anomalous binding with the TFA group bound to the active site region whilst the rest of the peptide bound in parallel mode with the main chain residues 214-216. Data on substrate (1) was collected but no binding around the active site region was observed. Small crystals of human leukocyte elastase were obtained by the technique of vapour diffusion, however sealed tube X-ray experiments and using the sychroton revealed no diffraction pattern~ Moreover, studies were done on the enzyme to remove the sugar content using various glycosidases of which almond emulsin showed some conclusive results. In addition, the crystal structures of two small molecules containing the heavy atoms ruthenium and platinium were solved by conventional techniques of X-ray crystallography.

The liver is recognised as a potential target site for nanoparticle (NP) toxicity, as NPs have been observed to accumulate within this organ subsequent to exposure via injection, inhalation or instillation. The liver's unique structure has to be taken into consideration when evaluating NP toxicity, as a variety of cell types of distinct morphology and function are evident, and potentially affected by NP exposure. Of particular interest are hepatocytes, due to their abundance and importance to the maintenance of normal liver function, and macrophages due to their role in host defence. The uptake and intracellular fate of fluorescent polystyrene particles (20nm and 200nm) by hepatocytes was evaluated (with exposure times of up to 60 minutes). Within the studies conducted comparisons of the response of primary rat hepatocytes, with C3A and HepG2 hepatocyte cell lines to NP exposure were made in order to investigate whether cell lines are a relevant model of hepatocyte behaviour. It was found that the uptake of particles by the primary hepatocytes, and both cell lines was size and time dependent. Specifically, it appeared that the internalisation of 200nm particles was limited, occurred at later time points (60 minutes), with the majority of particles evident at the cell surface. Polystyrene NPs (20nm) were internalised by cells after a 10 minute exposure time, after which NPs compartmentalised either within and/or between adjacent cells. The nature of the NP ‘compartments', and therefore fate of internalised NPs was then investigated to determine if the compartments developed as a consequence of the mechanism of uptake, or due to the attempted elimination of NPs from cells. It was found that NPs were not contained within early endosomes or lysosomes. However it was apparent that polystyrene NPs were eliminated to a limited extent within the bile canaliculi of all cell types, and may accumulate within the mitochondria of cell lines after a 60 minute exposure, which warrants further investigation. The impact of the PARTICLE_RISK particle panel [consisting of ultrafine carbon black (ufCB), CB, carbon nanotubes (CNTs), C60 (carbon fullerene) QD621 (positively charged quantum dots) and QD620 (negatively charged quantum dots)] on hepatocyte function was then determined. It was consistently observed that QD621 and QD620 were able to elicit the greatest extent of toxicity, evidenced within their ability to deplete cellular GSH, induce cytotoxicity, initiate a pro-inflammatory response (indicated by an increase in IL-8 production) and decrease bile secretion, in the hepatocyte couplet, in vitro model. It was observed that the pattern of response was similar within the cell lines and primary cells. Differentiated monocytic THP-1 cells (to represent the resident liver macrophages, Kupffer cells) were exposed to the PARTICLE_RISK particle panel to obtain conditioned medium (CM) that was exposed to hepatocytes, in order to gain insight into the ability of macrophages to influence NP mediated toxicity to hepatocytes. Firstly, the response of macrophages to particle exposure was considered and it was apparent that the toxicity that was observed within hepatocytes was paralleled within the response of differentiated monocytic cells (THP-1). Accordingly, QD621 were again proven to have the greatest toxic potential, with QD620 able to induce toxicity to a more limited extent. The exposure of hepatocytes to CM potentiated the toxicity observed when cells were exposed to particles alone, so that the pattern of response was comparable, but the extent of toxicity greater, and evident at earlier time points. It was apparent that QDs were able to induce an inflammatory response (characterised by TNFα and IL-8 production) within the liver that was primarily mediated by macrophages. When considering the results from all experiments it is evident that some of the particles contained within the PARTICLE_RISK panel were more capable of eliciting toxicity within the liver, and that their toxicity can be ranked in the following order: QD621>QD620>CNT=ufCB=C60>CB.

Over recent decades the modern lifestyle dynamic has led to an increased parental reliance on commercially marketed complementary infant foods in the UK, which has been highlighted by the Diet and Nutrition Survey of Infants and Young Children. The current nutritional labelling formats for ready-to-eat complementary infant foods are a duplicate of the legislative requirements for manufacturing of ready meals, intended for the general population, the implication of this is that a number of important nutrients maybe limiting or excessive, which will affect their nutritional quality and suitability as an infant food. Furthermore nutritional databases, such as McCance and Widdowson provide limited data on the composition of these types of food products. The European Food Safety Authority has highlighted that nutrient intake data after six months of life is currently inadequate as well as insufficient and urgently needs to be addressed. Therefore the nutritional content of these food products needs to be assessed to ascertain whether or not infants are meeting dietary requirements when consuming such products. The aims of this study were to evaluate the nutritional suitability of infant food products currently available on the UK market, according to the most up to date recommendations and recent relevant legislation and to explore the microencapsulation of docasahexaenoic acid (DHA) to optimise the nutrient content of infant food products. New protocols were developed for the quantitative analysis of certain key nutrients including High Pressure Liquid Chromatography (HPLC) – Charged Aerosol Detection for essential fatty acids, HPLC and UV spectrophotometry for fat soluble vitamins A and E, competitive enzyme immunoassay (Vitakit D™) for vitamin D and Inductively Coupled Plasma Optical Emission Spectroscopy for essential elements (Ca, Cu, Fe, K, Mg, Na, P and Zn) in commercial infant foods in the UK. The estimated daily intakes of these products were compared against current dietary recommendations for infants. In addition the Ca:P ratio was also determined in a range of commercial infant foods and compared with recommendations in relation to bone health. Furthermore, the effects of commonly practiced re-heating treatments used by parents were examined to establish whether different preparation methods affected the fatty acid content of manufactured infant formula milks. Finally, through the nutritional evaluation of these infant food products, the infant’s diet was found to be low in DHA, which provided opportunities for scope and product optimisation to improve the nutritive value of infant food products. Therefore microencapsulation of DHA was explored as a potential way to improve the nutritional quality of infant food products. The nutritional evaluation of the essential fatty acid content of a 6-9 month old infant’s diet highlighted that pre-formed long chain polyunsaturated fatty acids (LCPUFA) DHA and arachidonic acid (AA) intakes (at 23.3 mg/day and 36.7 mg/day, respectively) were below recommendations set by the US, at 103.3 mg/day and 147.5 mg/day, respectively. This provides scope for product optimization to improve the nutritive value of commercial infant food products. With respect to the precursor essential fatty acids, the dietary intake of the n-6 fatty acid linoleic acid (LA) was found to be above recommendations at 3147.9 mg/day, whereas the n-3 fatty acid α-linolenic acid (ALA) was found to be below recommendations at 296.4 mg/day, which increases the LA:ALA ratio of the diet; this may have implications for allergy. As the fortified infant formula was identified as the major dietary contributor and due to the fact that unsaturated fatty acids are prone to oxidation, the impact of re-heating treatments used by parents on the fatty acid content of formula milk was investigated and a degree of statistically significant changes were observed. In relation to the transparency of the nutritional information declared on the labels by the manufacturers, infant formula milks were all within the limits of EU regulations although there was a degree of significant variation between the quantitative values analyzed in this study and the declared values on the labels. With regards to the vitamin A and E analysis, normal phase HPLC was employed for the simultaneous quantification of retinyl acetate, retinyl palmitate, α-tocopherol and γ-tocopherol; reverse phase HPLC was used for the quantification of β-carotene and UV spectrophotometry for the quantification of carotenoids from selected meat and vegetable ‘ready-to-feed’ commercial infant meals. Based on the results of the study, the estimated total daily intake for a 6-9 month old infant of vitamin A (retinol equivalents, RE) and vitamin E (α-tocopherol equivalents, ATE) from both infant food and formula milk were 1.74 mg RE/day and 10.4 mg ATE/day, respectively. These intakes exceed the recommendations set by the Department of Health (1991). The main dietary contributor was highlighted as being the fortified infant formula which highlights the importance of nutrient dense foods in situations where infant formula is reduced or compromised. The study into the essential elemental content of dairy based commercial infant food products found that the Ca:P ratio of a 7-12 month old infant’s diet was 1.49:1, which was within the recommended range of 1-2:1. However, the level of intake for each of the elements analyzed, with the exception of sodium, were found to be above the Recommended Nutrient Intake (RNI) set by the Department of Health (1991), which warrants further investigation in relation to both micronutrient interactions, and in situations where the intake of fortified infant formula milk is compromised. In addition, as this study was the first to include consumption of infant snack products, the level of total calorie intake was also assessed, which indicated that energy intakes exceed recommendations set by the Scientific Advisory Committee of Nutrition (2011) by 42%, which may have implications for obesity. This highlights that parents need to select appropriate snack products. In relation to bone health, vitamin D was also quantified in a range of commercial infant meals. The total dietary intake of vitamin D3 was determined to be 9.61 μg/day, which is 137% higher than the RNI set by the Department of Health (1991) for 7-12 month old infants. However 120% is contributed from fortified infant formula, which may raise a cause for concern over deficiency issues, in situations where infant formula is reduced or compromised or the infant is breastfed. Furthermore the National Diet and Nutrition Survey have shown evidence for an increased risk of vitamin D deficiency in all age and sex groups in the UK. Consequently, following the nutritional evaluation of commercial infant food products, an infant’s diet is not meeting recommendations for the pre-formed long chain polyunsaturated fatty acids, DHA and AA. DHA may be of more significance due to endogenous production. Therefore, two approaches have been explored for the encapsulation of DHA in the pH dependent polymer hydroxypropyl methylcellulose acetate succinate (HPMCAS). In the first approach direct spray drying was implemented for the microencapsulation of DHA/HPMCAS organic solutions, while in the second approach solid lipid nano-emulsions of DHA, produced by high pressure homogenization, were subsequently spray dried in HPMCAS aqueous solutions. The direct spray drying approach resulted in significantly higher quantities of DHA being encapsulated, at 2.09 g/100 g compared to 0.60 g/100 in the spray dried solid lipid nano-emulsions. DHA stability was increased by the direct spray drying approach and the release of DHA was analysed by a dissolution methodology. / The encapsulated powders produced by the desired method offer a source of DHA that has the potential to be incorporated into infant foods to increase their dietary DHA consumption.