Protein kinases are major drug targets for many diseases. Among these are the STE20/SPS1-related proline/alanine-rich kinase (SPAK) and the oxidative- stress-responsive kinase 1 (OSR1), which are two related serine/threonine protein kinases. Both kinases are key components of the WNK-SPAK/OSR1 signalling pathway that has emerged as a key regulator of electrolyte homeostasis, body fluid and blood pressure. Various knock-in and knock-out SPAK and OSR1 mouse models exhibited reduced blood pressure. This highlighted SPAK and OSR1 kinases as promising targets in the treatment of hypertension. Encouraged by this, this project was initiated to discover specific WNK-signalling inhibitors by targeting SPAK and OSR1 kinases as potential novel antihypertensive agents. My work led to the identification of an allosteric pocket located in the highly conserved C-terminal domains of SPAK and OSR1, which influences their kinase activities. Using in silico screening, Rafoxanide, an anti-parasitic agent, was identified as a novel allosteric inhibitor of SPAK and OSR1. Additionally, high throughput screening led to the discovery of the clinically used agent, Verteporfin, as a novel and potent WNK-signalling inhibitor. Moreover, several fragment-binders to the C-terminal domain of OSR1 kinase were identified using NMRfragment based screening. In addition, the NMR backbone assignments of the C-terminal domain of OSR1 kinase were determined and used to map the previously unknown binding site of different OSR1 and SPAK inhibitors. Collectively, these findings have significantly advanced the field of SPAK and OSR1 kinase inhibition and provided key tools that will facilitate the future discovery of other SPAK and OSR1 kinase inhibitors.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2016-04-13T16:30:14Z No. of bitstreams: 1 DIS_PRICILA_KURZ_DE_ASSUMPCAO_PARCIAL.pdf: 290160 bytes, checksum: d018575426ad9071019ec6e098c90a03 (MD5) / Made available in DSpace on 2016-04-13T16:30:14Z (GMT). No. of bitstreams: 1 DIS_PRICILA_KURZ_DE_ASSUMPCAO_PARCIAL.pdf: 290160 bytes, checksum: d018575426ad9071019ec6e098c90a03 (MD5) Previous issue date: 2016-01-12 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Objectives: To evaluate the functional capacity of obese children and adolescents compared with normal-weight individuals and investigate possible relationships with biochemical parameters in the blood. Methods: In this study were included children and adolescents, between 6 and 18 years, divided into control (eutrophic) and obese groups according to body mass index (BMI). Data were collected regarding demographic, anthropometric, waist circumference and functional capacity through the Shuttle Walk Test (SWT). In the obese group were evaluated biochemical parameters in the blood (total cholesterol, HDL, LDL, triglycerides and glucose), and the application of a physical activity questionnaire. For statistical analysis were used the chi-square test, Student's t test for independent samples and the Pearson correlation test. Results: 77 participants were included, 27 in the control group and 50 obese. There was no significant difference between the two groups regarding sample characteristics, except for body weight, BMI and waist circumference. Most obese presented results of biochemical tests within the desirable limit, though none were considered active. There was a significant reduction (p <0.001) functional capacity in obese group compared to control subjects. Positive correlations were identified SWT with age and height, and negative correlation with BMI. However, there were no correlations with the biochemical parameters analyzed. Conclusions: Obese children and adolescents have reduced physical capacity when compared to normal individuals. The SWT performance seems to have a negative association with BMI, but is not correlated with the blood biochemical parameters. / Objetivos: Avaliar a capacidade funcional de crian?as e adolescentes obesas em compara??o com indiv?duos eutr?ficos e verificar poss?veis correla??es com par?metros bioqu?micos no sangue. Materiais e m?todos: Foram inclu?das crian?as e adolescentes, entre 6 e 18 anos, divididos em grupos controle (eutr?fico) e obeso de acordo com o ?ndice de massa corporal (IMC). Coletaram-se dados referentes ?s caracter?sticas demogr?ficas, antropom?tricas, circunfer?ncia abdominal e capacidade funcional atrav?s do Shuttle Walk Test (SWT). No grupo de obesos, foram avaliados par?metros bioqu?micos no sangue (colesterol total, HDL, LDL, triglicer?deos e glicemia), al?m da aplica??o de um question?rio de atividade f?sica. Para an?lise estat?stica, utilizaram-se os testes de qui-quadrado, t de Student para amostras independentes e o teste de correla??o de Pearson. Resultados: Foram inclu?dos 77 participantes, sendo 27 no grupo controle e 50 obesos. N?o houve diferen?a significativa entre os dois grupos em rela??o ?s caracter?sticas da amostra, exceto quanto ao peso corporal, IMC e circunfer?ncia abdominal. A maioria dos obesos apresentaram resultados dos exames bioqu?micos dentro do limite desej?vel, por?m nenhum foi considerado ativo. Houve redu??o significativa (p<0,001) da capacidade funcional no grupo obeso quando comparado aos indiv?duos controles. Foram identificadas correla??es positivas do SWT com idade e altura, e correla??o negativa com o IMC. No entanto, n?o foram encontradas correla??es com os par?metros bioqu?micos analisados. Conclus?es: Crian?as e adolescentes obesos apresentam redu??o da capacidade f?sica quando comparados a indiv?duos eutr?ficos. O desempenho no SWT parece ter associa??o negativa com o IMC, por?m n?o se correlaciona com par?metros bioqu?micos no sangue.

The objective of this work was to develop suitable delivery systems for biological agents that have antimicrobial activities using biocompatible polymers, aiming to reduce their toxicity when administered. Two biological agents, colistin as an antibacterial agent and nystatin (Nys) as an antifungal agent, are the focus of this thesis as they are potent treatments for current pathogen infections, especially to the multidrug-resistant (MDR) bacteria/fungi, but have potential toxicity to human. Polymeric drug delivery systems, including prodrug, hydrogel and micelle formulations, have been developed and discussed for their potential as topical and systemic regimes. The majority of the work was focused on the effect of the covalently attachment of synthetic polymers onto the biological agents upon their antimicrobial activities and the toxicity. The conjugation between colistin and polymers was achieved successfully through either irreversible or releasable linkages. Although irreversible polymer modifications on colistin showed no antimicrobial activity (chapter 2), an acceptable antibacterial activity was observed from the polymer-colistin conjugates with a releasable linkage through either ‘grafting-to’ (chapter 3) or ‘grafting-from’ (chapter 4) approaches. On the other hand, even though the pure polymer-Nys conjugate with a releasable imine linkage cannot be obtained due to the nature of the labile imine bond, the crude conjugate showed an excellent antifungal activity and a reduced toxicity compared to the native Nys (chapter 6). Other polymeric delivery systems were also discussed in this thesis. The incorporation of colistin within a developed hydrogel delivery system as an antibacterial patch for burn infections was investigated through in vitro and in vivo studies, showing a similar antibacterial activity as the native colistin solution against MDR Gram-negative bacteria with no systemic toxicity (chapter 5). Finally, an amphiphilic polymer containing boronic acid groups on the side chains was synthesised and used to target the hydroxyl groups on Nys, expecting to build up an environmental responsive micelle through dynamic boronate ester bond (chapter 7). Although more work is still needed, this system showed a potential to improve Nys solubility.

Natural α,β-epoxyketone proteasome inhibitors produced primarily by Streptomyces species, such as TMC-86A, are potent anticancer compounds. The enzymes responsible for biosynthesis of their key α,β-epoxyketone pharmacophore had yet to be determined at the onset of this research. Elucidation of the responsible enzymes and their putative substrate would allow for stereoselective completion of a synthetically challenging epoxidation reaction, responsible for the high cost of epoxyketone pharmaceuticals produced by industry. The cytochrome P450 TmcI and the flavin-dependent TmcF enzymes from the TMC- 86A biosynthetic gene cluster in Streptomyces chromofuscus, believed to have a role in epoxyketone biosynthesis, were cloned, expressed and overproduced. Potential substrates were produced through a peptide coupling synthetic route. In vitro assays demonstrated that EpnF, a TmcF homologue, could produce α,β-epoxyketone compounds with an α-dimethyl-β-keto carboxylic acid substrate. These substrates were prone to decarboxylative degradation, therefore stable methyl ester precursors were synthesised as an alternative. Using an esterase, methyl ester precursors were hydrolysed to reproduce the previously observed epoxyketone compound in a one pot reaction with EpnF. Substrate specificity of EpnF was probed by in vitro assay using an alanine derived analogue, which was found to be a viable substrate for the enzyme. Lastly, the EpnF catalytic cycle was interrogated with a synthesised authentic standard of a proposed intermediate, which was observed to co-elute with the desired intermediate from a quenched EpnF assay using UHPLC-MS chromatography.

Chapter 1 begins with an introduction to azetidines, including a discussion of the methodologies for their synthesis, their applications, relevance in natural products and as building blocks in medicinal chemistry. It then describes the development of a new asymmetric route to 2-substituted azetidin-3-ones using Enders’ SAMP/RAMP auxiliary. A one-pot process was developed involving the metalation of SAMP hydrazones of N-Boc-azetidin-3-one, alkylation and subsequent in situ hydrolysis to give the substituted products. Various bases and reaction conditions were explored to find optimal conditions for maximal yield and enantioselectivity. A representative range of electrophiles were screened including alkyl, allyl and benzyl halides and carbonyl compounds, producing enantioselectivities of up to 85% ee. Multiple substitution on the azetidin-3-one ring was briefly explored by repetition of the alkylation/hydrolysis sequence. Derivitisation by way of Pictet-Spengler reactions was used to confirm the absolute configuration at the newly created stereocentre. Chapter 2 begins with an introduction to 1,2-diazetidines outlining methods for their synthesis, before introducing the relevance of these nitrogen spirocycles. This chapter then describes two routes for the synthesis of these novel spirocyclic 1,2- diazetidines by (i) formation of the diazetidine ring and (ii) functionalisation of a range of 3-methylene-1,2-diazetidines including differentially protected variants. The diazetidines were subjected to dichloro- and difluorocyclopropanation with the latter achieved in high yields. Additionally, reactions with tetracyanoethylene by way of highly asynchronous [2π+2π] cycloadditions proceeded in near quantitative yield. In this way, a range of novel 4,5-diazaspiro[2.3]hexane and 1,2- diazaspiro[3.3]heptane spirocycles were produced. Chapter 3 details the experimental procedure and characterisation for all the novel compounds synthesised.

The natural compound cordycepin (3’-deoxyadenosine) causes a reduction in breast cancer cell viability. Microarray analysis showed that growth related genes are down-regulated by cordycepin. Indeed, mTOR, ERK and AMPK signalling was shown to be altered by cordycepin, but the effect was too fast to be mediated by transcriptional changes. It was hypothesised that cordycepin affected signal transduction through translation. However, polysome profiling did not identify clear candidates for the effects of cordycepin on signal transduction but unveiled that cordycepin leads to translation repression on 5’ terminal oligopyrimidine (TOP) mRNAs. As TOP mRNAs are known to be regulated by mTOR signaling, this result consistently suggests mTOR signaling is inhibited by cordycepin treatment. To test if it is possible that cordycepin affects gene expression via signal transduction, we compared its effects to various signal transduction inhibitors and an activator. So far, Pictilisib, a pan-PI3K inhibitor, is the only inhibitor that mimics both the gene expression and signal transduction effects of cordycepin, indicating the PI3K-PDK1-AKT axis is affected by cordycepin. The RNAs upregulated by cordycepin were highly enriched in a group of non-coding RNAs, which are also appeared to induce during serum withdrawal. Knockdown of poly(A) polymerases induced these RNAs, indicating that they probably are degraded by the PABPN1 and poly(A) polymerase dependent nuclear RNA decay pathway. Thus the data suggest that cordycepin affects gene regulation by two distinct pathways, one affecting signal transduction and growth related mRNA expression and another affecting polyadenylation mediated decay of non-coding mRNAs.

Cordycepin (3′-deoxyadenosine) is a medicinal bioactive component of the caterpillar fungi (Cordyceps and Ophicordyceps). It is reported to have nephroprotective, antiapoptotic, anti-metastatic, hepatoprotective (Yue et al. 2013), inflammatory effects, antioxidant, anti-tumor, immunomodulatory and vasorelaxation activities. Cordycepin is well known to terminate and inhibit polyadenylation, both in vitro and in vivo. Other proposed mechanisms of action of cordycepin include activation of adenosine receptors, activation of AMP dependent kinase (AMPK) and inhibition of PARP1. The purpose of this study is to elucidate the biological and pharmacological effects of cordycepin on cancer cell lines such as MCF-7 cells. In this study I found that cordycepin reduces the cell proliferation in all examined cell lines without always exerting an effect on 4EBP phosphorylation and protein synthesis rates. Therefore, the effects on protein synthesis via inhibition of mTOR, which were previously reported, are not only the sole reason for the effect of cordycepin on cell proliferation. Knockdown of poly (A) polymerases reduces cell proliferation and survival, indicating that poly (A) polymerases are potential targets of cordycepin. I studied different adenosine analogues and found that 8 aminoadenosine, the only one that also consistently inhibits polyadenylation, also reduces levels of P-4EBP. It also inhibits the expression of specific genes indicating that the effects on polyadenylation, mTOR signalling and gene expression are linked. Also consistent with polyadenylation inhibition as the major mode of action is the fact that the effects of cordycepin on gene expression are predominantly post-transcriptional. However, knockdown of poly (A) polymerases did not have the same effects on gene expression or on polyadenylation, indicating that cordycepin may act as a dominant negative rather than as a null mutant. This is consistent with the fact that cordycepin is known to arrest a normally transient polyadenylation complex. We performed microarray analysis of cordycepin treated MCF-7 cells and found that the downregulated mRNAs were predominantly involved in transcriptional regulation, cell proliferation, cell cycle and cell migration. These data show that cordycepin is a promising new drug for cancer and indicates that the mode of action it is likely to be through the inhibition of polyadenylation.

Ng, Chun Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 134-150). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vii / Table of Contents --- p.ix / Abbreviations --- p.xii / List of Figures --- p.xv / List of Tables --- p.xviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- "Introduction to Cardiovascular Disease, Hypertension and Atherosclerosis" --- p.1 / Chapter 1.1.1 --- Cardiovascular Disease --- p.1 / Chapter 1.1.2 --- Hypertension --- p.2 / Chapter 1.1.2.1 --- Background --- p.2 / Chapter 1.1.2.2 --- Causes of Hypertension --- p.3 / Chapter 1.1.2.3 --- Current Western Management and Medication --- p.6 / Chapter 1.1.3 --- Atherosclerosis --- p.9 / Chapter 1.1.3.1 --- Background --- p.9 / Chapter 1.1.3.2 --- Pathogenesis of Atherosclerosis --- p.10 / Chapter 1.1.3.3 --- Current Western Treatment and Medication --- p.12 / Chapter 1.2 --- Selection and Introduction of Current Chinese Medicine Formula --- p.16 / Chapter 1.2.1 --- Cardiac Syndrome in Traditional Chinese Medicine --- p.16 / Chapter 1.2.2 --- Traditional Chinese Medicine as an Complementary or Alternative Medicine --- p.17 / Chapter 1.2.3 --- Selection of TCM Formula from Pharmacopoeia --- p.18 / Chapter 1.2.3.1 --- Compound Formula --- p.18 / Chapter 1.2.4 --- Introduction to Constitutional Herbal Medicine --- p.19 / Chapter 1.2.4.1 --- Danshen (Radix Salviae miltiorrhizae) --- p.19 / Chapter 1.2.4.2 --- Gegen (Radix Puerariae lobatae) --- p.20 / Chapter 1.2.4.3 --- Yanhusuo (Rhizoma Corydalis) --- p.21 / Chapter 1.2.4.4 --- Composition of the Final Formula Used in the Present Study --- p.21 / Chapter 1.2.5 --- Previous work on Danshen-Gegen Formula and its limitations --- p.22 / Chapter 1.3 --- Objectives of the Present Study --- p.25 / Chapter 1.3.1 --- Research Plan --- p.26 / Chapter Chapter 2 --- Experimental Design and General Methodology --- p.27 / Chapter 2.1 --- Source and Authentication of Raw Herbs --- p.27 / Chapter 2.2 --- Materials --- p.29 / Chapter 2.3 --- Ethical Approval --- p.31 / Chapter 2.4. --- General Methods --- p.32 / Chapter 2.4.1 --- Blood Pressure Measurement --- p.32 / Chapter 2.4.2 --- Blood Profile Measurement --- p.33 / Chapter 2.4.3 --- Vascular Reactivity Studies --- p.36 / Chapter 2.5 --- Statistical Analysis --- p.38 / Chapter Chapter 3 --- Anti-hypertensive Studies of Danshen-Gegen Formula on Rat --- p.39 / Chapter 3.1 --- Introduction --- p.39 / Chapter 3.1.1 --- In vivo Anti-Hypertensive Studies --- p.39 / Chapter 3.1.1.1 --- Spontaneously Hypertensive Rat (SHR) --- p.40 / Chapter 3.1.1.2 --- Tail-cuff Blood Pressure Measurement --- p.41 / Chapter 3.1.2 --- Detailed Underlying Mechanistic Studies --- p.42 / Chapter 3.1.2.1 --- Nitric Oxide-mediated Vasodilation --- p.42 / Chapter 3.1.2.2 --- Prostacyclin-mediated Vasodilation --- p.43 / Chapter 3.1.2.3 --- Hyperpolarization-mediated Vasodilation --- p.43 / Chapter 3.1.2.4 --- Endothelium-dependent/-independent Vasodilation --- p.46 / Chapter 3.1.3 --- Long Term Underlying Mechanistic Studies --- p.48 / Chapter 3.2 --- Methods --- p.49 / Chapter 3.2.1 --- In vivo Anti-Hypertensive Studies --- p.49 / Chapter 3.2.2 --- Detailed Underlying Mechanistic Studies --- p.51 / Chapter 3.2.3 --- Long Term Underlying Mechanistic Studies --- p.53 / Chapter 3.2.4 --- Statistical analysis --- p.56 / Chapter 3.3 --- Results --- p.58 / Chapter 3.3.1 --- In vivo Anti-Hypertensive Studies --- p.58 / Chapter 3.3.1.1 --- Preventive Effect in Hypertension --- p.58 / Chapter 3.3.1.2 --- Therapeutic Effect in Hypertension --- p.62 / Chapter 3.3.2 --- Detailed Underlying Mechanistic Studies --- p.66 / Chapter 3.3.2.1 --- DG extract-induced Vasodilation --- p.66 / Chapter 3.3.2.2 --- Endothelium-independent Vasodilation --- p.67 / Chapter 3.3.2.3 --- Nitric Oxide-mediated Vasodilation --- p.68 / Chapter 3.3.2.4 --- Prostacyclin-mediated Vasodilation --- p.69 / Chapter 3.3.2.5 --- Hyperpolarization-mediated Vasodilation --- p.70 / Chapter 3.3.3 --- Long Term Underlying Mechanistic Studies --- p.74 / Chapter 3.4 --- Discussion --- p.79 / Chapter Chapter 4 --- Anti-atherosclerosis Studies of Danshen-Gegen Formula in Rabbits --- p.89 / Chapter 4.1 --- Introduction --- p.89 / Chapter 4.1.1 --- Intima-Media Thickening --- p.89 / Chapter 4.1.2 --- Effect of High Cholesterol Diet in Rabbit --- p.90 / Chapter 4.1.3 --- Thiobarbituric Acid Reactive Substances --- p.91 / Chapter 4.2 --- Methods --- p.93 / Chapter 4.2.1 --- Pilot Study for Establishment of Experimental Protocol --- p.93 / Chapter 4.2.2 --- Effect of DG extract on Intima-media Thickening --- p.97 / Chapter 4.2.3 --- Statistical analysis --- p.99 / Chapter 4.3 --- Result --- p.100 / Chapter 4.3.1 --- Study of the Anti-atherosclerosis Effect of DG extract - First Run --- p.100 / Chapter 4.3.2 --- Study of the Anti-atherosclerosis Effect of DG extract - Second Run --- p.108 / Chapter 4.4 --- Discussion --- p.117 / Chapter Chapter 5 --- General Discussion and Conclusion --- p.122 / Chapter 5.1 --- Significance of the Study --- p.122 / Chapter 5.2 --- Limitations and Future work --- p.127 / Chapter 5.3 --- Clinical Implication of the Use of the DG Preparations for Patients with CVD --- p.132 / References --- p.134

Wong, Ying Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 73-78). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abstract --- p.II / 摘要 --- p.IV / List of Tables --- p.VI / List of Figures --- p.VII / List of Abbreviations --- p.VIII / Chapter Chapter 1 --- Introduction --- p.3 / Chapter 1.1 --- Chemical Constituents of Rhodiola Genus --- p.5 / Chapter 1.1.1 --- Phenylethyl Derivatives --- p.5 / Chapter 1.1.2 --- Phenylpropanoids --- p.8 / Chapter 1.1.3 --- Phenolic Derivatives --- p.11 / Chapter 1.1.4 --- Flavonoids --- p.14 / Chapter 1.1.4.1 --- Flavone and Flavone Glycosides --- p.15 / Chapter 1.1.4.2 --- Flavonols and Their Glycosides --- p.17 / Chapter 1.1.4.3 --- Flavan-3-ol Derivatives --- p.23 / Chapter 1.1.5 --- Monoterpenoids --- p.26 / Chapter 1.1.6 --- Triterpenes --- p.30 / Chapter 1.1.7 --- Miscellaneous Compounds --- p.32 / Chapter 1.2 --- Biological Activities of Rhodiola Genus --- p.33 / Chapter 1.2.1 --- Anti-oxidative Effect --- p.34 / Chapter 1.2.1.1 --- Protective Effect on Ischemia and Reperfusion --- p.34 / Chapter 1.2.1.2 --- Anti-Aging Effect --- p.35 / Chapter 1.2.2 --- Learning and Memory --- p.36 / Chapter 1.2.3. --- Immune Response --- p.37 / Chapter 1.2.4 --- Anti-cancer Effect --- p.38 / Chapter 1.3 --- Objective --- p.39 / Chapter Chapter 2 --- Experimental --- p.40 / Chapter 2.1 --- General Experimental Procedures --- p.40 / Chapter 2.2 --- Plant Materials --- p.40 / Chapter 2.3 --- Extraction and Isolation --- p.41 / Chapter 2.3.1 --- Isolation and Purification of the Ethyl Acetate (E.A.) Fraction --- p.41 / Chapter 2.3.2 --- Isolation and Purification of the Butanol Fraction --- p.44 / Chapter 2.4 --- Characterization of the Isolated Compounds --- p.46 / Chapter 2.4.1 --- β-Sitosterol (1) --- p.46 / Chapter 2.4.2 --- Tyrosol (2) --- p.46 / Chapter 2.4.3 --- trans-Hydroxycinnamic acid (3) --- p.47 / Chapter 2.4.4 --- Geranyl-β-glucopyranoside (4) --- p.47 / Chapter 2.4.5 --- Neryl-β-glucopyranoside (5) --- p.48 / Chapter 2.4.6 --- Hexyl β-Glucopyranoside (6) --- p.48 / Chapter 2.4.7 --- Gallic Acid (7) --- p.49 / Chapter 2.4.8 --- Epigallocatechin-3-Gallate (8) --- p.49 / Chapter 2.4.9 --- Rhodiolgin (9) --- p.50 / Chapter 2.4.10 --- lsolariciresinol-9-β-Glucopyranoside (10) --- p.51 / Chapter 2.4.11 --- Rhodiooctanoside (11) --- p.52 / Chapter 2.4.12 --- Sacranoside B (12) --- p.52 / Chapter Chapter 3 --- Results and Discussion --- p.53 / Chapter 3.1 --- Structural Determination of the Isolated Compounds --- p.53 / Chapter 3.1.1 --- Identification of β-sitosterol (1) --- p.53 / Chapter 3.1.2 --- Identification of Tyrosol (2) --- p.54 / Chapter 3.1.3 --- Identification of trans-Hydroxycinnamic Acid (3) --- p.55 / Chapter 3.1.4 --- Identification of Geranyl-jS-glucopyranoside (4) --- p.56 / Chapter 3.1.5 --- Identification of Neryl-β-glucopyranoside (5) --- p.58 / Chapter 3.1.6 --- Identification of Hexyl β-Glucopyranoside (6) --- p.59 / Chapter 3.1.7 --- Identification of Gallic Acid (7) --- p.60 / Chapter 3.1.8 --- Identification of (-)-Epigallocatechin 3-Gallate (8) --- p.61 / Chapter 3.1.9 --- Identification of Rhodiolgin (9) --- p.63 / Chapter 3.1.10 --- Identification of lsolariciresinol-9-β-glucopyranoside (10) --- p.65 / Chapter 3.1.11 --- Identification of Rhodiooctanoside (11) --- p.67 / Chapter 3.1.12 --- Identification of Sacranoside B (12) --- p.69 / Chapter Chapter 4 --- Conclusion --- p.70 / References --- p.73

Introduction: Despite the importance of pharmacokinetic (PK) information for patient management there are low numbers of paediatric PK studies and little guidance available on optimum study design and conduct. Method: Drawing on Implementation Science, a mixed-methods study was conducted, including a scoping review (SR) (PK literature: 1990-2015) and quantitative and qualitative inquiry (stakeholders: lay population, service users and health-care professionals). Aim: to explore the feasibility and acceptability of paediatric PK research. Results: The SR (203 papers) highlighted significant problems with participant recruitment, retention and sampling. Stakeholders (n=240) added insight into these phenomenon, with lack of research staff, additional blood-sampling and appointments highlighted as significant barriers to recruitment and conduct. Facilitators included sensitivity and timeliness of approach, communication, involvement of child/young person (CYP) in decision-making, engagement between research and clinical teams, reassurance of safety, pain minimisation, and avoidance/reduction of burden to the CYP and family. Dedicated research support was viewed as critical to success. Discussion: PK research was viewed as feasible and acceptable by service users and health professionals, even in the context of critical illness. Novel, evidence-based, patient-centred, recommendations for future PK study conduct and design have been generated which are applicable for those designing, approving and implementing PK research.