A collection of twenty three selectively mono-protected di- and triamines, masked with the Boc, Fmoc or Ddiv protecting groups, were synthesised via continuous flow synthesis in a self-assembled meso-scale PTFE flow reactor. The continuous flow strategy offered direct access to the mono-protected compounds in good yields, especially in the case of the Fmoc carbamates which circumvented the use of another sacrificial protecting group. Two of the mono-Boc-protected carbamates were used as starting materials to generate N-alkylglycine monomers; synthesised via tandem mono-alkylation and Fmoc carbamation, linked by an in-line scavenging protocol using a silica-based trisamine scavenger resin. The final step of the monomer synthesis employed catalytic transfer hydrogenolysis using 20% Pd(OH)2/C and 1,4- cyclohexadiene. The three-step flow procedure gave access to two monomers, with one of them being a novel N-alkylglycine unit bearing a triethylene glycol bridge. The monomers were used as building blocks to assemble new oligo-N-alkylglycines (peptoids) via microwave-assisted solid phase synthesis. Three different types of peptoids were synthesised: (i) oligo-N-(6-aminohexyl)glycines (“standard” peptoids), (ii) oligo-N-{2-[2-(2-aminoethoxy)ethoxy]ethyl}glycines (“triethylene glycol” [TEG] peptoids) and (iii) hetero-oligomers of alternating “standard” and “TEG” monomers (“hybrid” peptoids). The peptoids were evaluated for their cellular permeability and cytotoxicity with HeLa, HEK-293 and CHO cells. All the peptoids were shown to be non-cytotoxic at 10 μM based on cell proliferation assays. In general, it was found that the cellular uptake of the hybrid peptoids outperformed their standard and TEG analogues. Flow cytometry and confocal microscopy results revealed that the hybrid nonamer had the highest cellular uptake efficiency of all the peptoids synthesised. At a concentration of 1 μM, it outperformed the second best molecular transporter (standard nonamer) by a factor of seven.

This thesis explores new synthetic routes for the formation of CDE & EFG fragments of pectenotoxin-4. Chapter 1: Introduction and Previous Work: This chapter reviews the discovery and biological activities of members of the pectenotoxin family. Two previous total syntheses are discussed, and previous work regarding the synthesis of ABC, E and FG fragments within the group is introduced. Chapter 2: Synthesis of the E Ring Fragment of Pectenotoxin-4: The synthesis of the E ring fragment is discussed. Key reactions include Negishi coupling and osmium mediated oxidative cyclisation. Chapter 3: First Generation Strategy for the Synthesis of the D ring: A simple model towards the D ring core was completed using alkyne-epoxide opening strategy. The application on a more sophisticated system was tested. Chapter 4: Second Generation Strategy for the Synthesis of the D ring: Sonogashira coupling was successfully tested as key step to unite two coupling partners; and further functionalisation towards the D ring skeleton was studied. Chapter 5: Third Generation Strategy for the Synthesis of the D ring: The new strategy including a Lewis acid assisted coupling and mercury mediated hydration of alkyne sequence was completed on a simple model. The application on a more sophisticated system was tested. Chapter 6: Synthesis of EFG Fragment of Pectenotoxin-4: Key Julia-Kocienski olefination between E ring fragment and FG ring fragment was examined. The further functionalisation of the resulting coupling product towards EFG fragment was finished. Chapter 7: Experimental: Full experimental procedures and characterisation of compounds are reported.

In 2009, based on its commitment to take action on the climate change, the aviation industry accorded a group of objectives to reduce carbon dioxide emissions. Although only 2% of all human-induced carbon dioxide (CO2) emissions are produced by the global aviation industry, the aviation industry is set to grow in the next 30 years. In order to maintain this growth without increasing its negative environmental impact, the future aircraft have to be cleaner and greener. In order to reduce carbon emissions and increase the operative efficiency, novel technologies have been developed and applied on aircraft. One of the recently introduced technologies is the flow control over the wing by employing active flow control methods. Amongst the active flow control methods, synthetic jets have emerged as a developing and promising technology. The latter have been extensively investigated since 1990 in laboratory based investigations. In spite of the fact that many experimental studies have been performed to design synthetic jet actuators for optimal flow control, due to the the vast number of operating parameters involved, and the lack of current measurement technologies, they can be impractical and highly expensive. Hence, there is a need for a systematic analysis to establish the optimal operating conditions with the highest effectiveness at the cost of minimum energy input, and the most suitable orientation of synthetic jet orifices. This would require enhanced comprehension of the inherent features of synthetic jets and their corresponding near wall effects. By using numerical simulations with a commercial CFD software (Star-CCM+), this thesis investigates some features associated with synthetic jet performance that are not fully understood, such as: • The optimal working configuration of a synthetic jet array embedded into a laminar detached boundary layer for flow separation control. • The effect of orifice orientation (inclined and skewed synthetic jets) over normal synthetic jets and their optimal working configuration in an attached laminar boundary layer.

abstract: Synthetic biology is constantly evolving as new ideas are incorporated into this increasingly flexible field. It incorporates the engineering of life with standard genetic parts and methods; new organisms with new genomes; expansion of life to include new components, capabilities, and chemistries; and even completely synthetic organisms that mimic life while being composed of non-living matter. We have introduced a new paradigm of synthetic biology that melds the methods of in vitro evolution with the goals and philosophy of synthetic biology. The Family B proteins represent the first de novo evolved natively folded proteins to be developed with increasingly powerful tools of molecular evolution. These proteins are folded and functional, composed of the 20 canonical amino acids, and in many ways resemble natural proteins. However, their evolutionary history is quite different from natural proteins, as it did not involve a cellular environment. In this study, we examine the properties of DX, one of the Family B proteins that have been evolutionarily optimized for folding stability. Described in chapter 2 is an investigation into the primitive catalytic properties of DX, which seems to have evolved a serendipitous ATPase activity in addition to its selected ATP binding activity. In chapters 3 and 4 we express the DX gene in E. coli cells and observe massive changes in cell morphology, biochemistry, and life cycle. Exposure to DX activates several defense systems in E. coli, including filamentation, cytoplasmic segregation, and reversion to a viable but non-culturable state. We examined these phenotypes in detail and present a model that accounts for how DX causes such a rearrangement of the cell. / Dissertation/Thesis / Ph.D. Biology 2011

Synthetic cannabinoids were first created in a pharmaceutical setting where scientists were studying marijuana. Researchers were trying to develop medically beneficial marijuana analogs. The compounds, however, were found to give physiological effects that were more potent than marijuana. Presently, synthetic cannabinoids have become a psychoactive drug of abuse, sold in head shops and over the Internet. New compounds are constantly being synthesized, which makes analysis of the drugs difficult. Solid phase extraction (SPE) is a well-studied method used in toxicological analysis to extract drugs and their metabolites from biological fluids. This sample preparation method is necessary to isolate the desired components of a sample for analysis by gas chromatography and mass spectrometry (GC/MS). This study sought to compare four brands of commercially available SPE cartridges using a procedure from United Chemical Technologies (UCT) for the simultaneous extraction of the three synthetic cannabinoid metabolites, JWH-018 N-(4-hydroxypentyl), JWH-122 N-(5-hydroxypentyl), and JWH-250 N-(5-hydroxypentyl), from urine. The cartridges from UCT, Thermo Scientific, Agilent Technologies, and SiliCycle were evaluated to determine how they performed throughout the SPE procedure. A recovery efficiency study was conducted to measure the amount of extracted metabolites from the urine. The responses of the quantification ion of the metabolites from an extracted urine sample were compared to a neat sample and the percent recovery was calculated. A within-run precision study was also utilized to measure the reproducibility of the cartridges, which was determined by the coefficient of variation (CV) of the different brands. The outcome of this research led to a development of a GC/MS method for detection of the three metabolites, creation of calibration curves for quantification, use of SPE for the extraction of the metabolites from urine, and the quantification of the extracted compounds to determine the efficacy and consistency of four brands of SPE cartridges. Method optimization was able to minimize the interday variations seen in the results of aliquots of the same samples. Optimal parameters include initial validation of the GC/MS method, a clean liner for the analysis of synthetic cannabinoid metabolites, using a GC column with a high temperature limit, and derivatization of the extracts before injection into the GC. While this study shows it is possible to use GC/MS for the analysis of these metabolites, LC/MS does not have the same restrictions because a liner, temperature elution, and derivatization of the analytes are not utilized. It was determined from the results of these studies that SiliCycle had the most reproducible and efficient cartridges. SiliCycle cartridges had a consistent and fast flow rate with a percent recovery efficiency within ±20% of the actual value. The results from SiliCycle were followed by cartridges from UCT, Thermo Scientific, and Agilent brands, respectively.

Less than half of patients with epithelial ovarian cancer (EOC) survive five years following diagnosis, underscoring the imperative need for improved treatment. Many patients, including those with advanced disease, initially respond to platinum agents, which constitute the backbone of therapy. However, tumors ultimately become resistant, rendering further treatment ineffective. Additionally, the poor tolerability of these agents warrants the exploration of more targeted treatments – one such strategy is exploiting synthetic lethal genetic relationships. Recent genomic sequencing efforts have revealed that as many of half of EOCs have homologous recombination (HR) alterations. HR is a critical pathway for the repair of platinum-induced ICLs, thus compromised HR is hypothesized to explain the initial response to chemotherapy in many patients. Accordingly, women whose tumors harbor mutations in the critical HR genes, BRCA1 or BRCA2 (BRCA1/2), demonstrate improved prognosis. BRCA1/2 mutations also confer exquisite sensitivity to inhibitors of the enzyme, poly(ADP-ribose) polymerase 1 (PARPis), hence loss of BRCA1/2 and PARP1 is synthetic lethal. A number of models have been proposed to explain this synthetic lethality, yet a consensus model that accounts for the diverse cellular roles of BRCA1/2 and PARP1 has yet to be established. Delineating the precise molecular underpinnings of PARPi action in BRCA1/2-deficient cells will aid clinicians in identifying the appropriate population of women with EOC likely to benefit from PARPi treatment and provide insight into resistance mechanisms that arise in these patients. Combining this approach with retrospective analysis of PARPi clinical trials will best define the proper indication for PARPi in EOC and other human cancers.

This thesis describes the application of flow chemistry to discovery and development of medicinal compound synthesis and new chemical methodologies respectively. It is divided into three distinct sections. The first section addresses a brief introduction to flow chemistry, highlighting the advantages and challenges that have been faced in the past and present and also the outlook to the future. The second section reports the integration of machine-assisted methods with batch processes to produce two medicinal compounds, a precursor to the sacubitril and OZ439 respectively. In the respect to the precursor to sacubritil, a flow-batch integrated synthesis is developed to provide the desired product in 54% yield over 7 steps from commercially available 4-iodophenyl. In particular, a tube-in-tube gas flow reactor was employed in three gas-liquid reactions without the need for installing a costly highpressure autoclave. These gas-lquid reactions were an ethylene Heck coupling reaction, an anti-Markovnikov Wacker oxidation and a rhodium-catalysed stereoselective hydrogenation respectively. In addition, a diastereoselective Reformatsky-type carbethoxyallylation using zinc metal was also highlighted in this synthesis to install an important stereocentre. A new antimalarial agent, OZ439 containing a trioxolane unit as the main structural feature, has the unique property of providing a single-dose cure for malaria in humans and has recently completed phase IIb trials. A machine-enabled process for the preparation of OZ439 was developed in 33% overall yield over 5 steps without the need of column chromatography purification. This preparation features a selective continuous hydrogrenation, Griesbaum ozonlysis and a Zn-catalysed amide reduction in the present of triethoxylsilane. The third section contains the development of two new methodologies of diazo compounds with organoboron compounds. The first methodology involves an in situ generation of transient allylic boronic species by reacting TMSCHN2 and E-vinyl boronic acids in flow, followed by subsequent trapping with a range of aldehydes (15 examples, 55-97% yield) and on a large scale (10 mmol) to provide homoallylic alcohols with high diastereoselectivity (>20:1 dr confirmed by 1H NMR). This multicomponent metal-free reaction could also be applied under batch conditions (20 further examples, 60-82% yield). The second methodology involves the preparation of an organodimetallic compound, α-trimethylsilyl benzylboronic acid pinacol esters, by reacting TMSCHN2 and phenylboronic anhydrides (21 examples, 60-91% yield), and the development of their applications as bifunctional building blocks to complex structures.

Tropical forests are extremely important ecosystems which play a substantial role in the global carbon budget and are increasingly dominated by anthropogenic disturbance through deforestation and forest degradation, contributing to emissions of greenhouse gases to the atmosphere. There is an urgent need for forest monitoring over extensive and inaccessible tropical forest which can be best accomplished using spaceborne satellite data. Currently, two key processes are extremely challenging to monitor: forest degradation and post-disturbance re-growth. The thesis work focuses on these key processes by considering change indicators derived from radar remote sensing signal that arise from changes in forest structure. The problem is tackled by exploiting spaceborne Synthetic Aperture Radar (SAR) and Interferometric SAR (InSAR) observations, which can provide forest structural information while simultaneously being able to collect data independently of cloud cover, haze and daylight conditions which is a great advantage over the tropics. The main principle of the work is that a connection can be established between the forest structure distribution in space and signal variation (spatial statistics) within backscatter and Digital Surface Models (DSMs) provided by SAR. In turn, forest structure spatial characteristics and changes are used to map forest condition (intact or degraded) or disturbance. The innovative approach focuses on looking for textural patterns (and their changes) in radar observations, then connecting these patterns to the forest state through supporting evidence from expert knowledge and auxiliary remote sensing observations (e.g. high resolution optical, aerial photography or LiDAR). These patterns are descriptors of the forest structural characteristics in a statistical sense, but are not estimates of physical properties, such as above-ground biomass or canopy height. The thesis tests and develops methods using novel remote sensing technology (e.g. single-pass spaceborne InSAR) and modern image statistical analysis methods (wavelet-based space-scale analysis). The work is developed on an experimental basis and articulated in three test cases, each addressing a particular observational setting, analytical method and thematic context. The first paper deals with textural backscatter patterns (C-band ENVISAT ASAR and L-band ALOS PALSAR) in semi-deciduous closed forest in Cameroon. Analysis concludes that intact forest and degraded forest (arising from selective logging) are significantly different based on canopy structural properties when measured by wavelet based space-scale analysis. In this case, C-band data are more effective than longer wavelength L-band data. Such a result could be explained by the lower wave penetration into the forest volume at shorter wavelength, with the mechanism driving the differences between the two forest states arising from upper canopy heterogeneity. In the second paper, wavelet based space-scale analysis is also used to provide information on upper canopy structure. A DSM derived from TanDEM-X acquired in 2014 was used to discriminate primary lowland Dipterocarp forest, secondary forest, mixed-scrub and grassland in the Sungai Wain Protection Forest (East Kalimantan, Indonesian Borneo) which was affected by the 1997/1998 El Niño Southern Oscillation (ENSO). The Jeffries- Matusita separability of wavelet spectral measures of InSAR DSMs between primary and secondary forest was in some cases comparable to results achieved by high resolution LiDAR data. The third test case introduces a temporal component, with change detection aimed at detecting forest structure changes provided by differencing TanDEM-X DSMs acquired at two dates separated by one year (2012-2013) in the Republic of Congo. The method enables cancelling out the component due to terrain elevation which is constant between the two dates, and therefore the signal related to the forest structure change is provided. Object-based change detection successfully mapped a gradient of forest volume loss (deforestation/forest degradation) and forest volume gain (post-disturbance re-growth). Results indicate that the combination of InSAR observations and wavelet based space-scale analysis is the most promising way to measure differences in forest structure arising from forest fires. Equally, the process of forest degradation due to shifting cultivation and post-disturbance re-growth can be best detected using multiple InSAR observations. From the experiments conducted, single-pass InSAR appears to be the most promising remote sensing technology to detect forest structure changes, as it provides three-dimensional information and with no temporal decorrelation. This type of information is not available in optical remote sensing and only partially available (through a 2D mapping) in SAR backscatter. It is advised that future research or operational endeavours aimed at mapping and monitoring forest degradation/regrowth should take advantage of the only currently available high resolution spaceborne single-pass InSAR mission (TanDEM-X). Moreover, the results contribute to increase knowledge related to the role of SAR and InSAR for monitoring degraded forest and tracking the process of forest degradation which is a priority but still highly challenging to detect. In the future the techniques developed in the thesis work could be used to some extent to support REDD+ initiatives.

Advances in DNA synthesis technology have led to rapid growth in the field of synthetic biology, heralding a nascent era of synthetic genomics. Sc2.0 (Saccharomyces cerevisiae version 2.0) is an international consortium with the aim of designing and constructing a fully‐synthetic eukaryotic genome. Fundamental design changes to the synthetic genome include the removal of unstable tRNA genes and their intended collation onto a “tRNA neochromosome”, with the aim of producing a more robust and stable synthetic genome structure. To maintain viability of a synthetic yeast, the tRNA neochromosome is therefore considered an important if not essential aspect of this project. The application of engineering principles is synonymous with synthetic biology, regularly employing the recursive Design‐Build‐Test cycle to improve experimental approach. This doctoral study explores the design, construction and characterisation of a tRNA neochromosome in Saccharomyces cerevisiae. A series of design principles influenced by engineering concepts were used to rationalise the complexities of de novo chromosome engineering, maximise its stability and ensure function in vivo. A methodology based on in vivo homologous recombination was then developed and shown to reliably construct the neochromosome from its constituent parts. Experimental characterisation revealed that genetic elements function as expected, and that the parental strain can tolerate the sole presence of one each of three single‐copy, essential tRNA genes (SUP61, TRT2 and TRR4), although Northern blot revealed potential precursor accumulation of the SUP61 tRNA caused by the presence of a synthetic 5’ flanking sequence. Following the addition of synthetic telomere seed sequences, pulsed‐field gel electrophoresis (PFGE) and deep sequencing revealed complex structure variations in two independent strain backgrounds. Except for these structural variations, successful neochromosome construction demonstrated the applicability of the approaches used and the remarkable ability of the yeast model to support the presence of a 17th chromosome housing an additional 275 tRNA genes. The research in this thesis has for the first time described the design, construction and characterisation of a eukaryotic neochromosome de novo. It is hoped that the findings presented will further our understanding of tRNA biology and enhance the aims of the Sc2.0 project.

Leishmaniasis is a disease caused by obligate intracellular parasites of the genus Leishmania, including 20 species that are pathogenic to humans. Female sand fly is the known vector that can transmit the disease. Visceral leishmaniasis is the severe form of the disease that affects internal organs and can be fatal with inappropriate diagnosis or treatment. Leishmania donovani is the causative agent of visceral leishmaniasis. Approximately 350 million in 89 countries are at risk of infection. Around 2 million new cases are reported annually with 500,000 of these are visceral leishmaniasis. Current drug therapies are inadequate due to their toxicity, high cost, severe adverse reaction, limited availability, and the emergence of resistance. With all these limitations, the need for new drugs is urgent. Pentavalent antimonials are the first line of treatment for leishmaniasis since the 1940s. Although amphotericin B, pentamidine and paromomycin are current drugs that treat leishmaniasis, they were discovered initially as a treatment for other pathogens. Furthermore, miltefosine the only available oral drug for leishmaniasis is an anticancer drug that found to be active against Leishmania. Therefore, we used our quantitative Leishmania donovani axenic amastigote assay and the clinically relevant infected macrophage assay to identify new antileishmanial hits from unstudied or understudied natural product sources such as mangrove endophytic fungi, Antarctic deep-sea coral, and terrestrial plants. We also used the same assays to screen synthetic compounds form multiple chemical scaffolds. Our well-established assays led to the identification of new antileishmanial hits from unstudied natural products and the discovery of new classes of molecules from synthetic compounds that possess potent activity against Leishmania donovani. Finally, we conducted an in vivo hamster study on an active hit that revealed high efficacy against Leishmania donovani in this severe model leading to promising antileishmanial drug development.