On Improving Generalization of CNN-Based Image Classification with Delineation Maps Using the CORF Push-Pull Inhibition Operator

Deployed image classification pipelines are typically dependent on the images captured in real-world environments. This means that images might be affected by different sources of perturbations (e.g. sensor noise in low-light environments). The main challenge arises by the fact that image quality directly impacts the reliability and consistency of classification tasks. This challenge has, hence, attracted wide interest within the computer vision communities. We propose a transformation step that attempts to enhance the generalization ability of CNN models in the presence of unseen noise in the test set. Concretely, the delineation maps of given images are determined using the CORF push-pull inhibition operator. Such an operation transforms an input image into a space that is more robust to noise before being processed by a CNN. We evaluated our approach on the Fashion MNIST data set with an AlexNet model. It turned out that the proposed CORF-augmented pipeline achieved comparable results on noise-free images to those of a conventional AlexNet classification model without CORF delineation maps, but it consistently achieved significantly superior performance on test images perturbed with different levels of Gaussian and uniform noise

IN SILICO METHODS FOR DRUG DESIGN AND DISCOVERY

Computer-aided drug design (CADD) methodologies are playing an ever-increasing role in drug discovery that are critical in the cost-effective identification of promising drug candidates. These computational methods are relevant in limiting the use of animal models in pharmacological research, for aiding the rational design of novel and safe drug candidates, and for repositioning marketed drugs, supporting medicinal chemists and pharmacologists during the drug discovery trajectory.Within this field of research, we launched a Research Topic in Frontiers in Chemistry in March 2019 entitled “In silico Methods for Drug Design and Discovery,” which involved two sections of the journal: Medicinal and Pharmaceutical Chemistry and Theoretical and Computational Chemistry. For the reasons mentioned, this Research Topic attracted the attention of scientists and received a large number of submitted manuscripts. Among them 27 Original Research articles, five Review articles, and two Perspective articles have been published within the Research Topic. The Original Research articles cover most of the topics in CADD, reporting advanced in silico methods in drug discovery, while the Review articles offer a point of view of some computer-driven techniques applied to drug research. Finally, the Perspective articles provide a vision of specific computational approaches with an outlook in the modern era of CADD

Marine Drug Research in China: Selected Papers from the 15-NASMD Conference

The Book covers this whole field, from the discovery of structurally new and bioactive natural products (including biomacromolecules), from marine macro-/micro-organisms, to the pharmacodynamics, pharmacokinetics, metabolisms, and mechanisms of marine-derived lead compounds, both in vitro and in vivo, along with the synthesis and/or structural optimization of marine-derived lead compounds and their structure–activity relationships. Taken together, this Special Issue reprint not only provides inspiration for the discovery of marine-derived novel bioactive compounds, but also sheds light on the further research and development of marine candidate drugs

Structural Analysis and Glycan Receptor Binding Specificities of Human Polyomaviruses

This thesis investigates the atomic structures and cell surface glycan receptor specificities of six human polyomaviruses. In healthy individuals, polyomaviruses establish usually asymptomatic chronic infections, but they may cause severe disease in immunocompromised patients. High-resolution X-ray structures of the major capsid protein VP1 alone and in complex with its specific glycan receptors allowed to analyze the molecular basis for underlying recognition events during early steps of the infection. The findings from crystallographic studies were corroborated with cell-based binding experiments using flow cytometry, interaction studies in solution by NMR spectroscopy, and infection studies in cell culture. The presented results highlight the enormous complexity of virus-glycan interactions and demonstrate that subtle differences in both the viral attachment protein and the cell-surface glycan receptor modulate binding specificities and affinities and thus, are key determinants for tissue and host tropism, viral infectivity, and pathogenesis. JC Polyomavirus (JCPyV) causes the fatal demyelinating disease Progressive Multifocal Leukoencephalopathy (PML) in immunocompromised individuals. A brain isolate of JCPyV, a genotype 1 strain, requires α2,6-linked sialic acids on the LSTc glycan for attachment to host cells, whereas a kidney isolate, a genotype 3 strain, was reported to interact with gangliosides featuring α2,3- and α2,8-linked sialic acids. Comprehensive structural and functional analyses of these two representative strains and their glycan receptor specificities show that engagement of LSTc is a prerequisite for functional receptor engagement for all seven JCPyV genotypes while the weaker-binding gangliosides are not required for infection. Interestingly, the majority of JCPyV isolates from PML patients contain distinct mutations within or in proximity to the LSTc binding site on VP1. The presented results reveal that binding of these mutant viruses to glycans is abolished or severely compromised rendering them not infectious. Thus, these viruses likely utilize a so far unknown receptor for the infectious entry and/or play alternative roles in PML pathogenesis. In order to explore potential strategies for the development of antiviral compounds against PML a fragment-based screening approach was carried out and subsequent X-ray structure analysis identified a novel compound binding site inside the hydrophobic cavity of the JCPyV VP1 pentamer. Further studies show that modifications to the five-fold pore of the VP1 pentamer result in a severe reduction of infectivity, suggesting that the pore is an important structural feature of polyomaviruses. Thus, targeting this pore may be proven to be an effective antiviral therapy. Crystal structures of VP1 from Trichodysplasia spinulosa-associated Polyomavirus (TSPyV) in complex with three different glycans reveal a sialic acid binding site that is shifted by about 18 Å from the ‘classical’ sialic acid binding sites of JCPyV and other polyomaviruses. Functional and cell-based studies confirm the importance of these novel interactions with sialic acids and suggest that glycolipids play an important role during TSPyV infection. Surprisingly, this new sialic acid binding site is also conserved in VP1 of Human Polyomavirus 12 (HPyV12), whereas the human New Jersey Polyomavirus (NJPyV) employs a third location for the recognition of an Neu5Ac-α2,3-Gal-containing receptor. A structure-based phylogenetic analysis suggests that TSPyV, HPyV12, and NJPyV share their sialic acid binding site with closely related non-human polyomaviruses providing initial clues about determinants of host specificity and evolution of these viruses. In contrast, Human Polyomavirus 6 and 7 (HPyV6 and HPyV7, respectively) carry uniquely elongated loops that cover the bulk of the outer virion surfaces, and moreover, obstruct the groove that binds sialylated glycan receptors in related viruses. Consistently, cell attachment and NMR studies further suggest that sialylated glycans are not required for cell attachment of both, HPyV6 and HPyV7. In conclusion, with its relatively high sequence homology and a conserved overall architecture, the growing Polyomaviridae family forms an excellent platform to analyze principles and molecular determinants of receptor specificity and antigenicity as well as critical factors for viral pathogenesis. A detailed understanding of the underlying molecular principles is important to establish a comprehensive toolbox, which can be used for new approaches for antiviral therapies and for the design of therapeutic gene vectors.Diese Dissertation untersucht die atomaren Strukturen von sechs menschlichen Polyomaviren und das spezifische Andocken dieser Viren an Zuckerstrukturen auf Zelloberflächen der Wirtszellen. Bei gesunden Menschen verursachen Polyomaviren meist chronische asymptomatische Infektionen, jedoch können einige dieser Viren bei immungeschwächten Patienten schwere Erkrankung auslösen. Mit Hilfe von Röntgenstrukturen der Kapsidproteine in Komplexen mit Zuckerverbindungen konnten die molekularen Grundlagen für das spezifische Andocken dieser Viren an Zellen identifiziert werden. Anschließend wurden die Erkenntnisse aus den kristallographischen Studien mit zellbasierten Bindungsexperimenten mittels Durchflusszytometrie, Interaktionsstudien mittels NMR-Spektroskopie in Lösung und Infektionsstudien in Zellkultur überprüft und weiter analysiert. Die erhaltenen Ergebnisse unterstreichen die enorme Komplexität von Interaktionen zwischen Viren und Zuckerstrukturen auf der Zelloberfläche und zeigen, dass feine Unterschiede in den viralen Proteine oder den gebundenen Zuckermolekülen eine wichtige Rolle für die Erkennungsprozess und die Bindungsaffinität spielen. Diese spezifischen und gut regulierten Wechselwirkungen zwischen Virus und Rezeptoren auf der Wirtszelle sind nicht nur für die initiale Erkennung wichtig sondern auch die viralen Infektiosität und Pathogenese. JC Polyomavirus (JCPyV) kann bei immungeschwächten Personen die tödliche Gehirnerkrankung Progressive Multifokale Leukoenzephalopathie (PML) auslösen. Ein JCPyV Stamm aus dem Gehirn bindet an α2,6-verknüpfte Sialinsäure der linearen Zuckerstruktur LSTc auf der Wirtszelle, während Untersuchungen für einen anderen JCPyV Stamm, der aus der Niere isoliert wurde, zeigten, dass auch Gangliosiden mit α2,3- oder α2,8- verknüpfter Sialinsäure gebunden werden. Eine umfassende strukturelle und funktionelle Analyse der zwei repräsentative Virusstämme zeigt hier jedoch deutlich, dass die Bindung an LSTc eine Voraussetzung für die Infektion für alle sieben JCPyV Genotypen ist, während die schwächer gebundenen Ganglioside nicht für die Infektion erforderlich sind. Interessanterweise besitzen JCPyV Isolate von PML-Patienten oft eine kleine Anzahl von konservierten Mutationen innerhalb oder in unmittelbarer Nähe der LSTc-Bindungsstelle auf dem Kapsidprotein. Die hier vorgestellten Ergebnisse zeigen, dass die Bindung an Zuckerstrukturen durch die Veränderungen in VP1 verhindert oder stark beeinträchtigt ist und daher diesen Viren nicht mehr infektiös sind. Somit verwenden diese PML-assoziierten Viren wahrscheinlich ein bisher unbekanntes Molekül auf der Zelloberfläche für den infektiösen Eintritt und/oder sie tragen in einer anderen Weise zur PML-Pathogenese bei. Ein Fragment-basiertes Screening wurde durchgeführt um mögliche Strategien für die Entwicklung von antiviralen Verbindungen gegen PML zu testen. Die Bindung von einem Fragment im Innenraum des hydrophoben VP1 Pentamers wurde mittels Strukturanalyse bestätigt. Zusätzlich konnte in einer Untersuchung gezeigt werden, dass Änderungen der fünffachen Pore der VP1-Pentamere die Virusinfektion verringern. Diese Erkenntnisse deuten darauf hin, dass die Poren ein wichtiger struktureller Bestandteil des Viruskapsids ist. Somit stellt auch die Pore oder gar der hydrophobe Innenraum des VP1 Pentamers einen möglichen Angriffsort für die Entwicklung von wirksamen antiviralen Therapien dar. Kristallstrukturen von VP1 des Trichodysplasia spinulosa-assoziierten Polyomavirus (TSPyV) im Komplex mit drei verschiedenen Zuckermolekülem zeigen eine Sialinsäure-Bindungsstelle, die um etwa 18 Å von der "klassischen" Sialinsäure-Bindungsstellen von JCPyV und anderer Polyomaviren entfernt ist. Funktionelle Untersuchungen bestätigen die Wichtigkeit dieser neuartigen Sialinsäure-Interaktion für die TSPyV Infektion und lassen zudem vermuten, dass besonders Glycolipide hierbei eine wichtige Rolle einnehmen. Diese neue Bindungsstelle ist auch im Kapsid des menschlichen Polyomavirus 12 (HPyV12) strukturell konserviert, während der menschliche New Jersey Polyomavirus (NJPyV) in einem dritten Bereich von VP1 mit einer spezifischer Zuckerstruktur wechselwirkt. Strukturbasierte phylogenetische Analysen zeigen, dass die Sialinsäure-Bindungsstellen von TSPyV, HPyV12 und NJPyV vermutlich auch in verwandten aber nicht-menschlichen Polyomaviren vorhanden sind und geben Hinweise über Determinanten der Wirtsspezifität und Evolution dieser Viren. Die humanen Polyomaviren 6 und 7 (HPyV6 und HPyV7) besitzen im Gegensatz dazu verlängerte VP1-Schleifenstrukturen, die den Großteil der Kapsidoberflächen abdecken und das Erkennen von Zuckermolekülen verhindert. Zusätzlich zeigen NMR-spektroskopische Untersuchungen, dass sialylierte Zucker nicht für das initiale Andocken von HPyV6 und HPyV7 an Zellen erforderlich sind. Zusammenfassend lässt sich sagen, das die wachsende Familie der Polyomaviren mit einer hohen Sequenzhomologie und konservierten Gesamtarchitektur eine hervorragende Plattform bildet, um Einblicke in grundlegende Prinzipien und molekularen Determinanten für Rezeptorspezifität, viraler Antigenität und Pathogenese zu erhalten. Ein detailliertes Verständnis der molekularen Grundlagen ist wichtig für neue Ansätze in der Entwicklung von antiviralen Therapien und auch therapeutische Genvektoren

Persistente organiske forbindelser og effekter på utviklings- og funksjonsprosesser i mus

Initial exposure to persistent organic pollutants (POPs) occurs by placental and lactational transfer during fetal and neonatal life and continues during the adult stage through ingestion, inhalation and dermal absorption. Many POPs have functional groups that resemble endogenous molecules rendering them with the potential for endocrine disruption during sensitive periods of organ development and function. Thus, adverse health effects may arise from exposure to POPs such as cancer and dysfunction of the endocrine, immune, reproductive, developmental or neurological systems. Mice are extensively used in research related to human health and disease as they are small animals with short generation time and high genetic similarity to humans. In addition, transgenic and knockout technology has led to the development of murine models with high phenotypic similarity to human disease. However, several differences between humans and mice are present in the absorption, distribution, metabolism, excretion, sensitivity and susceptibility to POPs. Furthermore, attention has recently shifted from assessing the toxicity of single compounds or well-defined chemical mixtures to evaluating large and environmentally relevant mixtures of POPs. The present study utilized two mouse models (CD-1 and A/J Min) and two exposure regimes (dietary or perinatal) to explore the effects of a human relevant mixture of POPs on female mammary gland development and ovarian folliculogenesis, liver morphology and function, and colorectal cancer development, intestinal microbiota and metabolome. The mixture composition was based on chemicals present in Scandinavian food products and the individual compound concentrations were adjusted to 0x (Control), 5 000x (Low) or 100 000x (High) human estimated daily intake levels for the general Scandinavian population. As shown previously, the present study also demonstrated gestational and lactational transfer of POPs from mothers to offspring. Furthermore, the results showed absorption, distribution, accumulation and persistence of POPs in murine tissues. The Low dose resulted in human relevant concentrations (for some chemicals) and can be considered at least partly human relevant in both its composition and concentration. Perinatal exposure to the mixture restricted mammary gland development and led to a premature arrest of gland growth in female CD-1 mice. In addition, it decreased ovarian follicle maturation and possibly also increased follicle atresia. Together this indicated potential endocrine disruption. Furthermore, the mixture caused persistent hepatocellular hypertrophy in CD-1 mice, but not in A/J Min mice. Thus, a strain-dependent difference in hepatic sensitivity was illustrated and, together with the induction of cytochrome P450 enzymes, indicated that the mixture may cause hepatotoxicity in sensitive strains. The two exposure regimes, dietary and perinatal, resulted in contradictory effects on colorectal carcinogenesis in A/J Min mice. Dietary exposure moderately increased cancer development while perinatal exposure reduced carcinogenesis. The increase was synergistically enhanced when combined with one injection of azoxymethane. Interestingly, perinatal POP exposure modulated the biochemical and microbial environment of the intestine possibly to reduce colorectal carcinogenesis in the predetermined cancer model. In conclusion, the human relevant mixture of POPs affected several developmental and functional processes in mice. The results of the present study can facilitate future mechanistic investigations into how human relevant chemical mixtures may affect biological development and function.Eksponering for persistente organiske forbindelser (POPs) starter i foster- og neonatalstadiet igjennom morkaken og brystmelk, og fortsetter videre igjennom matinntak, innånding og dermal absorpsjon. Mange POPs har funksjonelle grupper som etterligner endogene molekyler, noe som gir kjemikaliene et potensiale for endokrine forstyrrelser i løpet av sensitive perioder for organutvikling og funksjon. Alvorlige helseeffekter kan dermed oppstå som følge av eksponering, slik som kreft og dysfunksjon av ulike kroppsfunksjoner. Mus er mye brukt i forskning på human helse og sykdommer fordi de er små dyr med kort generasjonstid og har høy genetisk likhet til mennesker. I tillegg har transgen- og knockoutteknologi ført til utvikling av musemodeller med høy fenotypisk likhet til humane sykdommer. Likevel finnes det flere ulikheter mellom mennesker og mus, blant annet når det gjelder absorpsjon, distribusjon, metabolisme og ekskresjon av POPs. Videre har oppmerksomheten skiftet fra å studere toksisiteten av enkeltstoffer eller veldefinerte mikser av kjemikalier til å undersøke store og miljørelevante sammensetninger av POPs. I denne avhandlingen ble det brukt to musemodeller (CD-1 og A/J Min) og to eksponeringsregimer (diett og perinatal) til å undersøke effektene av en humanrelevant miks av POPs på hunnlig brystutvikling og utvikling av follikler i ovariene, levermorfologi og funksjon. Forekomst av tykktarmskreft, sammensetning av tarmmikrobiota og metabolom i tarminnhold og tarmvev ble også undersøkt. Miksen hadde en sammensetning av stoffer basert på kjemikalier som er tilstede i Skandinaviske matprodukter og konsentrasjonene av de individuelle stoffene ble justert til 0x (Kontroll), 5 000x (Lav) eller 100 000x (Høy) nivået av estimert humant daglig inntak for den generelle Skandinaviske populasjonen. I likhet med tidligere studier viste også dette studiet at POPs overføres fra mor til barn igjennom graviditet og amming. Videre viste resultatene absorpsjon, distribusjon, akkumulasjon og persistens av POPs i vev fra mus. Lav eksponeringsdose resulterte i konsentrasjoner (for noen av kjemikaliene) som var humanrelevante og kan delvis vurderes til å være humanrelevant i både sin komposisjon og konsentrasjon. Perinatal eksponering for miksen begrenset brystutviklingen og førte til en prematur stans i kjertelveksten i CD-1 hunnmus. Videre førte eksponeringen til en reduksjon i follikkelmodningen og mulig også en økning av follikkelatresi i ovariene. Til sammen indikerte dette en mulig endokrin forstyrrelse. Miksen førte også til persistent hypertrofi av leverceller i CD-1 mus, men ikke i A/J Min mus. Dette tydet på forskjeller i leversensitivitet mellom musemodeller og, sammen med en induksjon av cytokrom P450 enzymer, indikerte dette at miksen kunne føre til levertoksisitet i høysensitive mus. De to eksponeringsregimene, igjennom diett og perinatal overføring, førte til motstridende effekter på tarmkreft i A/J Min mus. Eksponering igjennom dietten førte til en moderat økning av kreft, mens perinatal eksponering reduserte karsinogenesen. Økningen ble synergistisk forsterket i kombinasjon med én injeksjon av azoxymetan. Perinatal POP-eksponering modulerte det biokjemiske og mikrobielle miljøet i tarmen, noe som mulig reduserte tarmkreftutviklingen i den forutbestemte kreftmodellen. Vi konkluderer med at den humanrelevante miksen av POPs påvirket utviklingen og funksjonen til flere kroppsfunksjoner i mus. Disse resultatene gir et godt grunnlag for videre forskning knyttet til hvordan humanrelevante blandinger av POPs kan påvirke utvikling og helse hos dyr og mennesker

Studies on natural products: resistance modifying agents, antibacterials and structure elucidation

This thesis describes research starting in 1999 on three areas of natural product science, namely bacterial resistance modifying agents, antibacterials and structure elucidation of natural products. Plants produce an array of structurally-complex and diverse chemical scaffolds and whilst there is an expanding volume of published literature on structure elucidation, there remains a need to understand why these compounds are produced and how they function in terms of biological activity. That can only be properly realised by a full and determined attempt at structure elucidation. This is an important concept as molecular structure describes and precedes function. The chirality and functional group chemistry of natural products defines the way in which a compound specifically binds to a receptor, protein or drug target. My independent research career started with studies on the ability of plant extracts and phytochemicals to modulate the activity of antibiotics that are substrates for bacterial multidrug efflux. These investigations are described in the first section, “Natural Product Resistance Modifying Agents”. Studies were, in the first instance, simple assays to look at potentiation and synergy of extracts and pure phytochemicals to potentiate the activity of antibiotics against resistant bacteria. This research evolved to study efflux inhibition, where we learnt much from the collaborations with Professors Piddock (Birmingham), Kaatz (Wayne State) and Bhakta (Birkbeck). Latterly, we were inspired by the highly imaginative and creative work of Dr Paul Stapleton (UCL), to study the plasmid transfer inhibitory effects of natural products; the rationale being that plasmids carry antibiotic-resistance genes and virulence factors. Inhibition of transfer could result in a reduction in the spread of antibiotic resistance and a reduction in pathogenicity. The second section of this thesis describes antibacterial natural products that were evaluated against clinically-relevant species of bacteria, in the main Gram-positive organisms such as Staphylococcus aureus and its methicillin- (MRSA) and multidrug-resistant variants and Mycobacterium tuberculosis, the causative agent of tuberculosis, which still continues to affect millions of people globally and for which antibiotic resistance is considerable. The papers described in this section detail the extraction of the plant and the bioassay-guided isolation of the active compounds, which were then subjected to structure elucidation, using in the majority of cases, Nuclear Magnetic Resonance (NMR) spectroscopy, High-Resolution Mass Spectrometry, and Infrared and Ultraviolet-Visible Spectroscopy. Natural products from the acylphloroglucinol, terpenoid, polyacetylene, alkaloid and sulphide classes are well represented in these publications with some of these antibacterial natural products displaying minimum inhibitory concentrations (MIC) values of less than 1 mg/L against MRSA and Mycobacterium tuberculosis strains. These activity levels approach those of existing clinically used antibiotics and this highlights the value of plant natural products as a resource for antibacterial templates. Mechanistic studies have also been conducted on selected compounds, for example the natural products from Hypericum acmosepalum were found to inhibit ATP- dependent MurE ligase, a key enzyme involved in bacterial cell wall biosynthesis. Other examples included the main component of cinnamon (Cinnamomum zeylanicum), an ancient medicinal material cited in the Bible in Exodus, which has been used in antiquity as an anti-infective substance. The main compound from this medicinal material is trans-cinnamaldehyde, a simple phenylpropanoid which has been shown to inhibit Acetyl-CoA Carboxylase, a pivotal enzyme that catalyses the first committed step in fatty acid biosynthesis in all animals, plants and bacteria. In collaboration with the marine natural product chemist Professor Vassilios Roussis, we have also been able to characterise the antibacterial activities of marine plants, particularly compounds of the diterpene class that display promising levels of antibacterial activity against MRSA and S. aureus strains. Work on the antibacterial properties of Cannabis sativa showed that some of the main cannabinoids display excellent potency towards drug-resistant variants of S. aureus and support the ancient medicinal usage of Cannabis as an anti-infective and wound healing preparation. The acylphloroglucinol class of plant natural products are also noteworthy, particularly from Hypericum and Mediterranean medicinal plant species such as Myrtle (Myrtus communis), again with MIC values reaching 1 mg/L against pathogenic bacteria. We synthesised some of these acylphloroglucinols and made analogues and not surprisingly, were unable to improve the activity as nature really is the best chemist of all. The final section describes early and continuing research into the isolation and structure elucidation of natural products from plants and microbes. The rationale for this research is manifold: training for isolation to understand the medicinal use of a plant or microbe, chemotaxonomic investigations, the ecological relevance of phytochemicals in plants that are halophytic and xerophytic and in some cases just plain academic curiosity. These studies use classical phytochemical techniques to isolate and determine the structures of the species of investigation and where possible, absolute stereochemistry is undertaken. It should be noted however that isolation can be exceptionally challenging and frustrating. This can be due to the paucity of biomass, low concentrations of compounds, complexity of the resulting natural product mixtures and finally a lack of chemical stability of the products. All of these issues need to be faced before structure determination can even be attempted. A word of caution is therefore needed to the young natural product chemist embarking on their first isolation project. However, words of encouragement are also needed: the isolation of new, chemically complex and exquisitely biologically active molecules is a beautiful endeavour and exceptionally rewarding on many levels.This thesis describes research starting in 1999 on three areas of natural product science, namely bacterial resistance modifying agents, antibacterials and structure elucidation of natural products. Plants produce an array of structurally-complex and diverse chemical scaffolds and whilst there is an expanding volume of published literature on structure elucidation, there remains a need to understand why these compounds are produced and how they function in terms of biological activity. That can only be properly realised by a full and determined attempt at structure elucidation. This is an important concept as molecular structure describes and precedes function. The chirality and functional group chemistry of natural products defines the way in which a compound specifically binds to a receptor, protein or drug target. My independent research career started with studies on the ability of plant extracts and phytochemicals to modulate the activity of antibiotics that are substrates for bacterial multidrug efflux. These investigations are described in the first section, “Natural Product Resistance Modifying Agents”. Studies were, in the first instance, simple assays to look at potentiation and synergy of extracts and pure phytochemicals to potentiate the activity of antibiotics against resistant bacteria. This research evolved to study efflux inhibition, where we learnt much from the collaborations with Professors Piddock (Birmingham), Kaatz (Wayne State) and Bhakta (Birkbeck). Latterly, we were inspired by the highly imaginative and creative work of Dr Paul Stapleton (UCL), to study the plasmid transfer inhibitory effects of natural products; the rationale being that plasmids carry antibiotic-resistance genes and virulence factors. Inhibition of transfer could result in a reduction in the spread of antibiotic resistance and a reduction in pathogenicity. The second section of this thesis describes antibacterial natural products that were evaluated against clinically-relevant species of bacteria, in the main Gram-positive organisms such as Staphylococcus aureus and its methicillin- (MRSA) and multidrug-resistant variants and Mycobacterium tuberculosis, the causative agent of tuberculosis, which still continues to affect millions of people globally and for which antibiotic resistance is considerable. The papers described in this section detail the extraction of the plant and the bioassay-guided isolation of the active compounds, which were then subjected to structure elucidation, using in the majority of cases, Nuclear Magnetic Resonance (NMR) spectroscopy, High-Resolution Mass Spectrometry, and Infrared and Ultraviolet-Visible Spectroscopy. Natural products from the acylphloroglucinol, terpenoid, polyacetylene, alkaloid and sulphide classes are well represented in these publications with some of these antibacterial natural products displaying minimum inhibitory concentrations (MIC) values of less than 1 mg/L against MRSA and Mycobacterium tuberculosis strains. These activity levels approach those of existing clinically used antibiotics and this highlights the value of plant natural products as a resource for antibacterial templates. Mechanistic studies have also been conducted on selected compounds, for example the natural products from Hypericum acmosepalum were found to inhibit ATP- dependent MurE ligase, a key enzyme involved in bacterial cell wall biosynthesis. Other examples included the main component of cinnamon (Cinnamomum zeylanicum), an ancient medicinal material cited in the Bible in Exodus, which has been used in antiquity as an anti-infective substance. The main compound from this medicinal material is trans-cinnamaldehyde, a simple phenylpropanoid which has been shown to inhibit Acetyl-CoA Carboxylase, a pivotal enzyme that catalyses the first committed step in fatty acid biosynthesis in all animals, plants and bacteria. In collaboration with the marine natural product chemist Professor Vassilios Roussis, we have also been able to characterise the antibacterial activities of marine plants, particularly compounds of the diterpene class that display promising levels of antibacterial activity against MRSA and S. aureus strains. Work on the antibacterial properties of Cannabis sativa showed that some of the main cannabinoids display excellent potency towards drug-resistant variants of S. aureus and support the ancient medicinal usage of Cannabis as an anti-infective and wound healing preparation. The acylphloroglucinol class of plant natural products are also noteworthy, particularly from Hypericum and Mediterranean medicinal plant species such as Myrtle (Myrtus communis), again with MIC values reaching 1 mg/L against pathogenic bacteria. We synthesised some of these acylphloroglucinols and made analogues and not surprisingly, were unable to improve the activity as nature really is the best chemist of all. The final section describes early and continuing research into the isolation and structure elucidation of natural products from plants and microbes. The rationale for this research is manifold: training for isolation to understand the medicinal use of a plant or microbe, chemotaxonomic investigations, the ecological relevance of phytochemicals in plants that are halophytic and xerophytic and in some cases just plain academic curiosity. These studies use classical phytochemical techniques to isolate and determine the structures of the species of investigation and where possible, absolute stereochemistry is undertaken. It should be noted however that isolation can be exceptionally challenging and frustrating. This can be due to the paucity of biomass, low concentrations of compounds, complexity of the resulting natural product mixtures and finally a lack of chemical stability of the products. All of these issues need to be faced before structure determination can even be attempted. A word of caution is therefore needed to the young natural product chemist embarking on their first isolation project. However, words of encouragement are also needed: the isolation of new, chemically complex and exquisitely biologically active molecules is a beautiful endeavour and exceptionally rewarding on many levels