The active pharmaceutical ingredient (API) and excipients are vital for determining the behaviour of drug release from tablet compacts. Macro attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopic imaging can be employed for in situ studies of dissolving tablets. This thesis describes new developments that applied macro ATR-FTIR spectroscopic imaging to investigate the stability and dissolution of amorphous APIs, the effect of carriers for improving drug release, the stability of ionised drug candidates and the behaviour of multiple formulations in microfluidic devices. Solid dispersions containing an amorphous drug formulated with different polymers were investigated using ATR-FTIR spectroscopy and spectroscopic imaging. Crystallisation of the amorphous drug was detected during stability and tablet dissolution experiments. The implications of this form change inhibited dissolution of the drug into solution. ATR-FTIR spectroscopic imaging was also used in combination with ultraviolet detection to study the release of a drug formulated with selected carriers. Hydrogen bonded interactions between the drug and carrier were characterised and resulted in an increased rate of drug release. When these interactions were not present in the tablet, a slower rate of dissolution was observed. Disproportionation of an ionised drug was investigated by ATR-FTIR spectroscopic imaging and Raman mapping. During dissolution experiments in acidic solution, chemical changes of the ionised API were detected in real time that resulted in the formation of the less soluble form of the drug. Exciting results were obtained by simultaneously screening the behaviour of multiple formulations in microfluidic channels using macro ATR-FTIR spectroscopic imaging. Moreover, the precipitation of a dissolved drug that crystallised upon contact with an acidic solution was investigated. Overall, the research in this thesis has demonstrated that macro ATR-FTIR spectroscopic imaging can address the challenges of studying a range of innovative delivery systems that can ultimately lead to the development of more efficient pharmaceutical formulations.

Animal behaviour is coordinated by biological rhythms over multiple timescales. In the temporal cortex, rhythmic oscillatory activity in the 5 to 12 Hz theta frequency range supports behaviour through temporal windows of changes in excitability and circuit computation. Subcortical inputs, including those from the medial septum are thought to generate the theta oscillatory activity. However, the rules of innervation of identified types of cortical cells in different cortical regions by a diverse population of septal neurons are largely unknown. During my DPhil studies, I tested the hypothesis that medial septal GABAergic neurons which exhibit distinct activity patterns would express distinct molecular marker combinations and have distinct synaptic partners. In Chapters 1 and 2, I formulate my research questions and explain the methodology used. My main findings are presented in chapters 3 to 7. In Chapter 3, I present the strategy used to identify four populations of rhythmic medial septal neurons based on their spike-train dynamics in the mouse in vivo. In Chapter 4, I present a detailed analysis of the spike-timing and molecular parameters of a specialised population of rhythmic neuron, the Teevra cells. Teevra cells are PV+, do not change their firing rate from rest to running and fire short bursts of action potentials preferentially at the trough of both CA1 theta and slow irregular activity recorded in stratum pyramidale. In Chapter 5, I show that Teevra cells selectively innervate axo-axonic cells and CCK-immunopositive interneurons in hippocampal area CA3, bypassing CA1, CA2, and the dentate gyrus. In Chapter 6, I show that Teevra cell axons terminate in restricted septo-temporal segments of CA3 according to their rhythmicity. Finally, in Chapter 7, I present the Komal cells, which fire long bursts of action potentials at the peak of theta, increase their firing rate during running, and preferentially innervate extrahippocampal cortices. I discuss the broad relevance of my observations in Chapter 8. Overall, my results show an unexpected specialisation in the organisation of medial septal GABAergic inputs to the temporal cortex. Teevra cells fire maximally at the trough and preferentially innervate the CA3 whereas Komal cells fire maximally at the peak and target extrahippocampal cortices, thereby coordinating the timing of converging glutamatergic inputs to CA1 via disinhibition. This cortical-region-specific GABAergic innervation by physiologically distinct septal neuronal types suggests a general mechanism for the coordination of network activity, both during regular theta oscillations and irregular large amplitude waves, providing windows of computation through synchronisation of neuronal activity in the cortex.

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Von Hippel–Lindau (VHL) is the E3 ubiquitin ligase targeting hypoxia-inducible transcription factor-alpha (HIF α) for proteasomal degradation. The crucial function of VHL in response to hypoxia and cellular oxygen sensing are well established, owing to the use of genetic tools through knockout and knockdown that inactivate VHL. However, the functional consequences of specifically interrupting the interaction between VHL and HIF α remain to be elucidated. The development of a chemical probe that unambiguously blocks the VHL:HIF α interaction downstream of HIF α hydroxylation by prolyl hydroxylase domain (PHD) enzymes, would address biological questions about VHL molecular targets and functional consequences of disrupting the interaction. Here, small molecules inhibiting the VHL:HIF α interaction were shown for the first time to stabilise HIF α and elicit HIF transcriptional activity in cells. The most potent VHL inhibitor identified is VH298. VH298 is potent, cell-permeable, selective, and not toxic at the concentration required for HIF α stabilisation. Further characterisation shows that VHL inhibitor exclusively induces HIF-dependent changes in global gene and protein expression, demonstrating the specificity of the inhibitor. VHL protein level was found to increase in the presence of VHL inhibitor, which in turn promotes the degradation of HIF α in prolonged inhibition. The work herein characterises the VHL inhibitor as a chemical probe of the hypoxia signalling pathway with great potential to address biological questions regarding the roles and regulation of VHL. The VHL inhibitor is a unique tool due to its on target selectivity and specificity in inducing HIF activity, without affecting HIF-independent response, and exerts its effect further downstream than PHD hydroxylation. This work provides a foundation and cellular proof of concept for future studies evaluating therapeutic potential of VHL inhibitor in diseases, such as chronic anaemia, ischaemia, and inflammation-driven diseases.

Skin secretions of amphibians, which contain a large amount of bioactive compounds, play an essential role in their survival. For instance, some toxic or irritant secretions can protect them against predators. Due to the pharmacological and bioactive characteristics of the components in amphibian skin secretion, these secretions have been considered as one of the world’s richest source of such agents. In this study, the skin secretions of the frogs Odorrana livida, Hylarana latouchii and Phyllomedusa sauvagii were collected from the dorsal surface and immediately frozen in liquid nitrogen. Afterwards, a series of molecular techniques, including mRNA isolation, cDNA library construction, PCR, agarose gel analysis and DNA sequencing were applied. The novel peptide sequences were deduced from cloned cDNAs and then confirmed by using MALDI-TOF mass spectrometry and MS/MS fragmentation sequencing. Finally, three novel peptides were chemically-synthesised. The determination of the therapeutic bioactivities of the three novel peptides was carried out by a series of assays. QUB-1384, exhibited BK-receptor agonist-like functions which induced the contraction of rat bladder and uterus tissues. QUB-1906 is a Bowman-Birk inhibitor which could efficiently inhibit the activity of trypsin. The novel dermaseptin peptide, QUB-2949, had broad-spectrum anticancer activities against human cancer cell lines including PC-3 (ATCC-CRL-1435), U251MG (ecacc-09063001), H157 (ATCC-CRL-5802), MCF-7 (ATCC-HTB-22) and MDA-MB-435S (ATCC-HTB-129). Meanwhile, like other dermaseptin peptides, QUB-2949 showed effective growth inhibitory activity against the standard Gram-negative bacterium Escherichia coli (NCTC 10418), the standard Gram-positive bacterium Staphylococcus aureus (NCTC 10788), the pathogenic yeast Candida albicans (NCPF 1467), the drug-resistant bacterium Enterococcus faecalis (ATCC 29212), Methicillin-resistant Staphylococcus aureus (MRSA) (ATCC BAA-2094) and Pseudomonas aeruginosa (NCTC 13437). In addition, according to the results of haemolysis assay and HMEC-1 MTT assay, all the peptides exhibited low cytotoxicity. In conclusion, three novel peptides were found and assessed in this project. The multifunction and low cytotoxicity of these bioactive peptides make them promising candidates for natural drug discovery and provide new prospect for clinical treatments.

For thousands of years, habitats of amphibians have changed dramatically from the initial marine to current freshwater aquatic types. This transition provided diverse resources but presented significant challenges from the environment and from predators. As a consequence, amphibians adapted to secrete abundant noxious substances from their skins in order to survive. These substances attracted the attention of many researchers in recent times who believe that there is a great potential in these secretions to discover new pharmaceuticals from natural products. In recent years, the biochemical/pharmaceutical components including peptides, proteins and nucleic acids, derived from amphibians, have been subjected to intensive studies. Original research work on these bioactive substances such as bradykinin-related peptides and antimicrobial peptides, indicates that they have some clear clinical therapeutic implications including uses as anti-inflammatories, anti-bacterials and even anticancer agents. This project mainly focused on the isolation and activity evaluation of three novel peptides derived from three different frog species belonging to different families. In the genomic aspect of the study, three novel peptides were identified by molecular cloning and named according to their molecular masses. Two bradykinin-related peptides, named R-13-R (QUB1379) and RVA-(V1,T6)-BK (QUB1344), were identified from the skin secretions of the Chinese brown frog, Rana chensinensis and the Bamboo leaf frog, Odorrana versabilis, respectively. A novel dermaseptin peptide, named dermaseptin-Ph (QUB2536), was isolated from skin secretions of the Northern orange-legged leaf frog, Phyllomedusa hypochondrialis. The cDNAs were cloned by molecular cloning and the primary structures of their respective encoded peptide biosynthetic precursors were deduced by translation. Reverse phase high performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS/MS) techniques were applied to confirm their structures. The peptides were then synthesised by a solid phase peptide synthesis (SPPS) technique and their degrees of putity were identified by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometer analysis. The determination of the bioactivities of the three novel peptides was carried out by employment of a series of bioassays. QUB1379 was found to be a bradykinin-receptor antagonist which inhibited the function of bradykinin inducing vasodilation on rat tail artery tissues and contraction on rat ileum tissues. However, the other novel bradykinin-related peptide, QUB 1344, exhibited a BK-receptor agonist-like function which induced the contraction of rat bladder and uterus tissues. The novel dermaseptin peptide, QUB2536, had a broad-spectrum of anticancer activities against PC-3 cells (ATCC-CRL-1435), U251MG cells (ECACC-09063001), H157 cells (ATCC-CRL-5802), MCF-7 cells (ATCC-HTB-22) and MDA-MB-435S cells (ATCC-HTB-129) and effective growth inhibitory activity against Gram-negative bacteria Escherichia coli (NCTC 10418) and Pseudomonas aeruginosa (ATCC 27853); Gram-positive bacteria Staphylococcus aureus (NCTC 10788) and Enterococcus faecalis (NCTC 12697); and pathogenic yeast Candida albicans (NCYC 1467). In recent years, both agonists and antagonists have significant clinical therapeutics based on the literature published. Kinin receptors are involved in pain and other neurological reactions. Various modifications of kinin receptor antagonists agents rely on the natural BRPs discovered from the natural world. What’s more, due to the rapid development of multi-drug resistant bacteria, discovering and producing of new types of antibiotics is in urge of investigation. Hence, this project can provide new insight for the research and development of new agents. Besides, great efforts need to be made to identify and assess new substances discovered from natural source.

Amphibian skin can secrete mucus from skin glands and this mucus always contains many types of antimicrobial peptides (AMPs) which can protect the hosts from bacterial infection. AMPs have been studied for a few decades with significant results as increasing numbers have been found, explored and developed to be evaluated as clinical drugs. This is a huge source of new molecules to help humans in their fight against diseases. In Chapter 3, a phylloxin-like AMP, QUB1966 and its analogue QUB2260, were studied. The results showed that QUB1966 exhibited antimicrobial effects against Gram-positive (Staphylococcus aureus) bacteria, Gram-negative (Escherichia coli) bacteria and yeast (Candida albicans). The modification increased the positive charge of the AMP, which enhanced the interaction between the peptide and the membrane of the selected microbes. The results showed that the modified peptide, QUB2260, expressed more potent antimicrobial function than the wild-type peptide QUB1966, increasing its inhibitory effect by 32-fold against E. coli and C. albicans, and 4-fold against S. aureus. Thus, the modified peptide might provide favourable prospects for novel biomedicine design and antibiotics substitution. In Chapter 4, a bradykinin-related peptide (BRP), QUB1315 (RAA-Val1, Thr6-bradykinin), was isolated from the skin secretion of Odorrana schmackeri with the defined primary sequence of RAAVPPGFTPFR. After bioactivity studies, QUB1351 revealed a dose-dependent contractile property on rat bladder. The analogue peptide, QUB1281 (RAA-Val1, Thr6, Leu8-bradykinin), was modified based on QUB1315 by replacing a single amino acid from Phe8 to Leu8 to test the antagonistic activity. However, the results showed that the modified peptide, QUB1281, was still an agonist. QUB1281 expressed less potency on bladder contraction than the wild-type QUB1315. It was also found that Thr6 and Leu8 might contribute to agonist effects and the substitution at position 8 could affect the affinity between peptides and bradykinin receptors. In Chapter 5, two novel peptides from Odorrana schmackeri, QUB1517 and QUB2025, were found to exhibit antibacterial potency against Gram-positive (Staphylococcus aureus) bacteria, Gram-negative (Escherichia coli) bacteria and yeast (Candida albicans). Their similar antimicrobial effects against these three microbes assumed that the same sequence domain might contribute to the antimicrobial activity. However, they both showed higher inhibitory activity on Gram-positive bacteria than Gram-negative bacteria. It is expected that the novel data described in this thesis will contribute to the burgeoning database of biologically-active peptides from amphibian skin secretions and may ultimately provide a basis for the development of new classes of peptide drugs for both major diseases and for orphan indications.

The utilisation of natural products as drugs for the treatment of diseases and injuries has developed for thousands of years, but the current pharmaceutical system is predominately occupied by chemical-based drugs, while natural products are rarely applied. Since the widespread of antibiotic resistance and prevalence of chronic and malignant diseases (such as diabetes and cancers), there has been a significant rise of interest in discovering novel drug candidates with potent efficiency in treating these diseases. The amphibian skin, a special morphological and physiological organ, acts as the first line of defence against physical stimuli and lethal invasions. It is a rich source of pharmacological alternatives, some of which possess the potential to be developed in medical applications. In this study, both genomics and proteomics techniques have been used to study the frog skin secretions from the Burmeister’s leaf frog, Phyllomedusa burmeisteri; the hylid frog, Phyllomedusa sauvagei and the Chinese tiger frog, Hoplobatrachus rugulosus. More specifically, the molecular cloning strategy was performed to isolate target mRNAs, establish corresponding cDNA libraries and eventually clone unique peptide precursor-encoding cDNA sequences. Also, the mature peptides were analysed and confirmed via RP-HPLC, MALDI-TOF mass spectrometry and MS/MS fragmentation sequencing. After the peptide primary structure characterisation, synthetic replicates of each peptide were purified before applied for further secondary structural analysis and biofunctional assessments. The secondary structure conformations were determined by circular dichroism, and the functional analysis in this study includes the antimicrobial assay, MTT assay, haemolysis assay, insulinotropic assay and GLP-1 releasing assay. This study reports the isolation, identification and characterisation of one phylloseptin peptide Phylloseptin-PBu, one dermaseptin peptide Dermaseptin-PS1 and four tigerinin peptides (Tigerinin-HR1 to Tigerinin-HR4), they exhibited distinct biological activities. In Chapter 3, the detailed function and correlative mechanisms of novel phylloseptin peptide Phylloseptin-PBu were described. We showed that Phylloseptin-PBu could induce a dose-dependent insulinotropic activity in BRIN-BD11 cells, which is regulated by ATP-sensitive potassium channel depolarisation triggered extracellular calcium influx and GLP-1 receptor-initiated PKA signalling activation. In Chapter 4, the novel Dermaseptin-PS1 was demonstrated to possess anticancer capability in U251MG cells through the induction of intrinsic apoptosis at low concentrations (10-6M), but disrupting the cell membranes at high concentrations (≥10-5 M). In Chapter 5, GLP-1 release activity was observed after the treatment of peptide concentration gradient of four novel tigerinin peptides (Tigerinin-HR1 to Tigerinin-HR4) in STC-1 cells. These discoveries enriched the foundational knowledge of biological peptides from these three-frog species and might contribute to future drug development. This thesis includes six chapters, the Chapter 1 and Chapter 2 are general introduction and general materials and methods, respectively; the Chapter 3 to Chapter 5 are specific experimental chapters; and the Chapter 6 is general discussion chapter.

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