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The technique of Fluorescence Lifetime Imaging Microscopy (FLIM) has been employed to quantitatively and spatially map the fluid composition and temperature within microfluidic systems. A molecular probe with a solvent-sensitive fluorescence lifetime has been exploited to investigate and map the diffusional mixing of fluid streams under laminar flow conditions within a microfluidic device. Using FLIM, the fluid composition is mapped with high quantification and spatial resolution to assess the extent of mixing. This technique was extended to quantitatively evaluate the mixing efficiency of a range of commercial microfluidic mixers employing various mixing strategies, including the use of obstacles fabricated within the channels. A fluorescently labelled polymer has been investigated as a new probe for mapping temperature within microfluidic devices using FLIM. Time Correlated Single Photon Counting (TCSPC) measurements showed that the average fluorescence lifetime displayed by an aqueous solution of the polymer depended strongly on temperature, increasing from 3 ns to 13.5 ns between 23 and 38 oC. This effect was exploited using FLIM to provide high spatial resolution temperature mapping with sub-degree temperature resolution within microfluidic devices. A temperature-sensitive, water-soluble derivative of the rhodamine B fluorophore, effective over a wide dynamic temperature range (25 to 91 oC) has been used to map the temperature distribution during the mixing of fluid streams of different temperatures within a microchannel. In addition, this probe was employed to quantify the fluid temperature in a prototype microfluidic system for DNA amplification. FLIM has been demonstrated to provide a superior approach to the imaging within microfluidic systems over other commonly used techniques, such as fluorescence intensity and colourimetric imaging.

Continuous cover forestry (CCF), an alternative forest management approach to clearfelling, is increasingly being adopted in the UK. It aims at enhancing stand structural diversity and favouring natural regeneration and subsequent seedling growth below the existing canopy of plantation forests. One area of limited knowledge is the critical level of below-canopy light for the growth of naturally regenerating seedlings. In addition, plant growth beneath canopies is influenced by other factors (e.g. herbivory). Picea sitchensis (Bong.) Carr. (Sitka spruce) seedlings under canopies have been observed to be severely damaged by Elatobium abietinum (Walker) (green spruce aphid) attacks. The combined effects of light availability and insect attack on seedling growth are not well understood, however, this understanding is crucial in order to ensure successful management of regeneration within CCF systems. A controlled experiment, which mimicked different natural understorey light levels along with repeated artificial aphid infestation, was conducted over two years to look at seedling performance through structural and physiological (chlorophyll fluorescence) measurements. Aphid population assessments showed significant increased population density under shaded conditions. Nevertheless, aphid impacts were mainly localised in extent while the impact of light was the major component that described seedling growth. Light was the primary factor affecting the whole-plant biomass, whereas aphids had only localised effects on the total dry weight of older needles and roots, and on leader extension growth. A significant interaction between light levels and presence/absence of aphid infestation was found for main leader extension growth of the seedlings during the second year of the experiment, with lower values at low light levels under infestation. Plant biomass allocation was affected strongly by light, while aphid presence did not result in significant changes. At the leaf physiologylevel, the light environment was found to be the main driving factor affecting photosynthetic response, whilst aphid presence had only a short-term localised effect on photosynthesis. The impact of light levels and aphid presence on seedling growth were also determined at an experimental field site where plots were located across two light regimes typical of CCF conditions in upland UK coniferous forests. The comparison of the photosynthetic response of the seedlings in both the controlled and field experiment highlighted the importance of considering the temporal heterogeneity of the light environment experienced by understorey seedlings in CCF stands, while aphid and aphid x light interaction effects could not be determined due to very low aphid levels during the year. Finally, a pre-existing light model was parameterised to predict the understorey light environment required to promote successful seedling growth in CCF stands. Sensitivity and validation analyses were also performed.

The steady-state and time-resolved fluorescence of 2-aminopurine (2AP) have been used to monitor base dynamics and base stacking interactions in DNA single strands and dinucleotides, and to investigate the interactions between DNA and a polymerase, Pfu-Pol. A new class of base analogues has also been investigated using a combination of experiment and quantum chemical computation. In recent years, 2AP has been widely used as a fluorescent probe to study conformational changes and inter-bases interactions in duplex DNA, but the conformational behaviour of DNA in single strands has been far less investigated. In the present work, six 2AP–labelled single strands have been studied by steady-state and time-resolved fluorescence measurements. Single strands were found to show similar conformational heterogeneity (manifested by 4-exponential fluorescence decays) to duplex DNA, but highly stacked conformations, in which 2AP is rapidly quenched by inter-base charge transfer, are less populated in single strands, whereas imperfectly stacked (weakly quenched) conformations are more highly populated. The effect of base pairing in constraining base mobility is evident. To further investigate the influence of base stacking interaction on DNA conformation and the mechanism of inter-base quenching of 2AP, the time-resolved fluorescence of 2AP-containing dinucleotides was measured. The fluorescence decay of 2AP-containing dinucleotides in PBS buffer at room temperature is also multiexponential and the shortest lifetime varies with the identity of the natural base partner, in a manner consistent with quenching by inter-base electron transfer. When the dinucleotides are frozen to 77 K, the quenching of 2AP is almost eliminated, demonstrating the importance of thermal fluctuations of the bases in facilitating inter-base quenching at room temperature. In the frozen dinucleotides, an additional decay component with a lifetime significantly longer than unquenched 2AP is also observed, suggesting the formation of a new, delocalised, inter-base excited-state. Archaeal family-B DNA polymerases bind tightly to uracil and stall replication when they encounter this base in template strands, four bases ahead of the primer-template junction. If the polymerase progresses further towards the uracil, the 3′-5′ proof reading exonuclease becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and the introduction of mutations into the genome. Time-resolved fluorescence of 2AP has been used to investigate the role played by unwinding of primer-templates in this mechanism. 2AP-labelled primer-templates (2AP positioned next to the terminal 3′ base of the primer strand), with a misincorpated uracil at the +2 position (U+2) or +4 position (U+4) from the replication fork in the complementary template strand, were investigated in complex with the polymerase Pfu-Pol. For the U+2 primer-template, the fluorescence decay parameters show clear evidence for a decrease in the amount of double-stranded DNA on polymerase binding, manifested by marked weakening of inter-base stacking and a large transfer of population from highly stacked to poorly stacked conformations. In contrast, for the U+4 primer-template only a small perturbation to inter-base stacking is seen, together with the persistence of a high population of strongly stacked states. A new class of base analogues with selenium replacing oxygen at the 4 position of thymine and the 6 position of guanine has been investigated experimentally and computationally. These base analogues are interesting because they show a large shift (>80 nm) in their absorption spectrum compared with the natural bases, taking their absorption into the visible region, with minimal change in molecular structure. The potential of two examples of these analogues, 4-selenium thymine-3’-phosphate and 6-selenium-2’-deoxyguanosine-3-phosphate as luminescent probes has been investigated. However, they prove to have very low emission quantum yields for both fluorescence and phosphorescence. The effect of selenium-substitution on the structural and photophysical properties of the bases has been studied by various ab initio computational methods. It has been found that replacement of oxygen by selenium does not affect the ground state structure but changes the structure of the first excited-state from buckled to nearly planar. The shift in the absorption spectrum on introduction of selenium is successfully predicted by the calculations; the red-shifted absorption band of selenium-substituted thymine is due to a new electronic transition that is not present in the natural base, whereas that of selenium-substituted guanine is from red-shifting of a guanine-like transition.

Neurotensin (NT) is a tridecapeptide neurotransmitter found in the central nervous system and gastrointestinal tract. Neurotensin receptor 1 (NTS1), a high affinity receptor for NT, is a member of the GPCR superfamily and is a putative target for the treatment of conditions such as Schizophrenia, Parkinson’s Disease and drug addiction. Overexpression and purification are typically limiting steps in the high resolution structure determination of GPCRs. In this study, through the optimisation of the E.coli strain used for overexpression of rat NTS1 (NTS1) and the inclusion of phospholipids in the purification buffers to prevent delipidation, an approximate 3-fold improvement in active receptor yield was obtained relative to existing protocols. Preliminary electron microscopy (negative stain and cryo) confirmed a monodisperse receptor population. Purified NTS1 is now being produced at a sufficient level for high resolution structural studies, including 3D crystallisation and further electron microscopy studies. The existing construct for the expression of NTS1 in E.coli, termed NTS1B, was modified to contain a fusion to the genes encoding either the eCFP or eYFP fluorescent proteins. These constructs were used for the E.coli expression of NTS1 tagged with either fluorescent protein at the C-terminus. Tagged receptor was successfully expressed at levels of up to 0.29 ± 0.03 mg per l of culture. Successful purification and proteolytic removal of the MBP and TrxA-His10 fusion partners was achieved whilst retaining both fluorescence and ligand binding capability (K_d = 0.91 ± 0.17 nM). Purified, fluorescent receptor was reconstituted into brain polar lipid (BPL) liposomes in an active conformation which was both fluorescent and able to bind NT. Experimentation with alternative lipid compositions suggested that specific lipids are required in order to maintain ligand-binding activity. FRET between the eCFP- and eYFP-tagged receptors was observed in reconstituted samples. The FRET efficiency was comparable to that observed in vivo for other GPCRs, including the yeast α-factor receptor, which is believed to be dimeric. This suggests that NTS1 could also be multimeric. In contrast, no FRET was observed in detergent samples. Therefore, a functioning in vitro system has been developed which enables the study of NTS1 multimerisation in lipid bilayers and future studies will attempt to implement single molecule fluorescence techniques. In addition, fluorescent derivatives of NT were successfully synthesised and purified. Radioligand competition assays and fluorescence correlation spectroscopy (FCS) confirmed that the fluorescent peptides bound to purified NTS1 in specific competition with unlabelled NT. Surface plasmon resonance (SPR) was used to confirm the ligand binding activity of purified NTS1. A novel approach was utilised which involved the measurement of the binding of detergent-solubilised NTS1 to immobilised, N-terminally biotinylated NT on the sensor surface. The use of a rigorous control, which consisted of immobilised ‘scrambled sequence’ NT, demonstrated a specific interaction. Analysis of the kinetics revealed a multiphasic interaction with a K_d in the nanomolar range. In summary, improvements to the expression and purification of NTS1, the generation of fluorescent constructs as useful tools in the study of receptor multimerisation and the optimisation of lipid-reconstitution protocols have opened up several preliminary lines of study which show considerable potential for future research.

To date, surgery is the most common and successful way to treat cancer. Tumour identification during surgery, however, can be challenging as it relies on the surgeon’s ability to differentiate healthy from diseased tissue, based on visual appearance and palpation. Additional contrast mechanisms are needed to further improve cancer detection during surgery. In this work, I explore the possibility of improving surgical outcomes by using intra-operative fluorescence imaging technologies to identify otherwise invisible lesions. A theoretical model is developed to quantify and characterise the imaging performance of fluorescence image-guided surgery (FIGS) and to guide the development of imaging systems. This model shows excellent potential for performance characterisation of FIGS devices, particularly when small lesions are involved. The design, development and testing of FIGS devices for open and keyhole surgery are described. These devices exploit near infrared (NIR) wavelengths to achieve a superior depth penetration while minimising tissue autofluorescence. Unlike existing systems, the devices described in this work use a single miniaturised camera to simultaneously detect bright-field and fluorescence from multiple dyes. Practical tests indicate nano-molar detection of clinically approved fluorescence dyes. The proposed technology is tested in a clinical study for detecting the sentinel lymph node (SLN) in gynaecological cancers. Results using two non-specific NIR dyes show excellent SLN detection rate in real time during open surgery and laparoscopy. In addition, multi-spectral fluorescence allows independent visualisation of different lymphatic pathways, crucial for understanding the mechanisms of metastasis through the lymphatic system. The FIGS devices are also used to test novel tumour-specific markers in vivo, ex vivo and in vitro. Promising results are reported, suggesting that this imaging technology is suitable for fluorescence molecular imaging. In conclusion, I report the development and applications of a novel multi-spectral FIGS technology that can effectively improve outcomes in surgical oncology.

The field of cardiac electrophysiological imaging has advanced tremendously in the past three decades with developments in fluorescent dyes, photodetectors, optical filters, illumination sources, computers and electronics. This thesis describes several scalable multi-parametric imaging systems and their application to cardiac tissue preparations at various levels of complexity. Using off-the-shelf components, single-camera multi-parametric optical mapping systems are described for various fluorescent dye combinations and single-element photodiode-based fibre-optic detection systems are described for drug-testing applications. The instruments described take advantage of modern voltage-sensitive dyes, multi-band optical filters and powerful light-emitting-diodes, from the ultraviolet to the red. The two electrophysiological parameters focused on were transmembrane voltage and the intracellular calcium concentration. Several voltage and calcium dye combinations were established, which produce no signal cross-talk. Furthermore, second- and third-generation voltage dyes were characterized in cardiac tissue, in vitro and in vivo. The developed systems were then applied to isolated Langendorff-perfused whole-hearts, in vivo whole-hearts, thin ventricular tissue-slices and human induced pluripotent stem cell-derived cardiac tissue. The interventions applied include accurately-timed electrical and mechanical local stimulation of the whole-heart to generate ectopic beats, cardiotoxic drugs and flash-photolysis of caged-compounds. With the high-throughput demands of drug discovery and testing, further development of scalable optical electrophysiological systems may prove critical in reducing attrition and costs. And for in vivo optical mapping, development of minimally-invasive and clinically-relevant optical systems will be essential in validating existing theories based on in vitro experiments and exploring cardiac function and behaviour with the heart intact in the organism.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Platinum drugs are among the most commonly used chemotherapeutics for the treatment of testicular, head and neck, ovarian, small cell lung, and colorectal carcinomas. Although the current set of platinum chemotherapeutics has proven somewhat successful, the overall success of platinum based drugs is limited due to acquired drug resistance and a limited range of tumor types that are treatable with the current regime. The development of novel cytotoxic platinum based compounds, both trans- and polynuclear, provides for the promising treatment of clinical platinum drug resistant tumors. While the cytotoxic activity of platinum drugs provides for a hopeful outlook, the ultimate factors that affect the success of chemotherapeutics are the fine balance between cytotoxic activity and metabolic deactivation. In general, this work reports the drug design/drug action, and pharmacokinetic consequences of anticancer compounds aimed to fight mechanisms of cisplatin resistance. In the first project, we report the biological and biophysical studies aimed at understanding and improving upon the pharmacokinetic properties of chemotherapeutics; specifically, understanding their interactions with serum proteins. This work resulted in the discovery of using carboxylate ligands to modulate the reactivity of trans-platinum based compounds towards sulfur containing proteins with consequent effects on drug efficacy. In addition, we report an in depth look into the biological consequences of non-covalent platinum drug-protein interactions on drug efficacy, and introduce the use of novel Platinum-NBD fluorescent conjugates as probes for drug metabolism. In the second project we report the design, synthesis, and biological consequences of fluorescent drug derivatives based on the NBD fluorophore, for use in understanding drug action and drug metabolism. As a result of this fluorescent drug labeling, TriplatinNC, a non-covalent platinum based chemotherapeutic, was found to specifically target nucleolar DNA/RNA, due to its high charge, and inhibit ribosomal RNA production in cancer cells. The use of fluorescent derivatization also resulted in the development of a series of novel water-soluble trans-platinum complexes, with greater cytotoxicity than cisplatin. Therefore, these data resulted in the understanding of, and improvement upon the pharmacokinetic profile of platinum chemotherapeutics, as well as the development of novel fluorescent platinum conjugates with novel metabolic and cytotoxic profiles.