Multimodality Imaging of Tumour Pathophysiology and Response to Pharmacological Intervention

This thesis describes the need for imaging the tumour pathophysiological microenvironment in order to understand response to treatment. Specifically looking at tumour vascularisation in in vivo murine xenograft models of disease, response to treatment with vascular disruption is assessed via photoacoustic tomography (PAT) and magnetic resonance imaging (MRI). Photoacoustic imaging is a novel imaging modality based on the detection of ultrasound waves created by the absorption of nano-second pulsed laser energy within tissue chromophores. It has the spectral specificity of optical techniques whilst also achieving the high resolution of ultrasound. Haemoglobin is the main chromophore found in biological tissue and this modality is therefore ideally suited to imaging tumour vascularisation. Using a Fabry-Perot interferometer this thesis demonstrates for the first time the feasibility of using PAT for re-clinical research and the characterisation of typical tumour vascular features in a non-invasive non-ionising manner. Response to different concentrations of a vascular disrupting drug is then demonstrated, with novel insights in to how tumours recover from vascular damage observed. MRI of response to vascular disruption is also presented. As MRI is widely used in the clinic it can serve as a translational tool of novel imaging biomarkers, and serves to further understand the differences in response of pathologically vascularised of tumours. This thesis looks at markers associated with disruption of haemodynamics, using apparent diffusion (ADC) to elucidate onset of necrosis, increase in haemoglobin concentration (R2*) as indication of impaired flow, and arterial spin labelling (ASL) as a marker of tumour blood perfusion. This is shown in both subcutaneous and clinically relevant liver metastasis models. Taken as whole, the results from this thesis indicate that whilst understanding the response of the tumour vasculature to pharmacological intervention is complex, novel imaging techniques can provide invaluable translational information on the pathophysiology of tumours

Cervical brachalgia: Assessment by cervical CT epidurography post transforaminal injection

Adjunct cervical CT epidurography (CCTE) can be used to image impingement in patients with cervical brachalgia undergoing fluoroscopic-guided cervical transforaminal injection (TFI) of steroid/local anaesthetic where magnetic resonance imaging (MRI) is contraindicated. CCTE images of the 9 patients on whom the authors performed CCTE post TFI over 6 years from 1998 to 2003 were retrospectively reviewed. CCTE is able to provide good images of the cervical spinal canal and its contents. CCTE may be an alternative imaging method for impingement in patients with cervical brachalgia contraindicated for MRI

The potential use of single-particle electron microscopy as a tool for structure-based inhibitor design

Recent developments in electron microscopy (EM) have led to a step change in our ability to solve the structures of previously intractable systems, especially membrane proteins and large protein complexes. This has provided new opportunities in the field of structure-based drug design, with a number of high-profile publications resolving the binding sites of small molecules and peptide inhibitors. There are a number of advantages of EM over the more traditional X-ray crystallographic approach, such as resolving different conformational states and permitting the dynamics of a system to be better resolved when not constrained by a crystal lattice. There are still significant challenges to be overcome using an EM approach, not least the speed of structure determination, difficulties with low-occupancy ligands and the modest resolution that is available. However, with the anticipated developments in the field of EM, the potential of EM to become a key tool for structure-based drug design, often complementing X-ray and NMR studies, seems promising

A Convergent, Umpoled Synthesis of 2-(1-Amidoalkyl)pyridines

A convenient, one-pot, two-component synthesis of 2-(1-amidoalkyl)pyridines is reported, based upon the substitution of suitably-activated pyridine N-oxides by azlactone nucleophiles, followed by decarboxylative azlactone ring-opening. The synthesis obviates the need for precious metal catalysts to achieve a formal enolate arylation reaction, and constitutes a formally ‘umpoled’ approach to this valuable class of bioactive structures

Three-Component Synthesis of Pyridylacetic Acid Derivatives by Arylation/Decarboxylative Substitution of Meldrum’s Acids

A convenient and simple three-component synthesis of substituted pyridylacetic acid derivatives is reported. The approach centers on the dual reactivity of Meldrum’s acid derivatives, initially as nucleophiles to perform substitution on activated pyridine-N-oxides, then as electrophiles with a range of nucleophiles to trigger ring-opening and decarboxylation

LAT1 (SLC7A5) and CD98hc (SLC3A2) complex dynamics revealed by single-particle cryo-EM

Solute carriers are a large class of transporters that play key roles in normal and disease physiology. Among the solute carriers, heteromeric amino-acid transporters (HATs) are unique in their quaternary structure. LAT1–CD98hc, a HAT, transports essential amino acids and drugs across the blood–brain barrier and into cancer cells. It is therefore an important target both biologically and therapeutically. During the course of this work, cryo-EM structures of LAT1–CD98hc in the inward-facing conformation and in either the substrate-bound or apo states were reported to 3.3–3.5 Å resolution [Yan et al. (2019), Nature (London), 568, 127–130]. Here, these structures are analyzed together with our lower resolution cryo-EM structure, and multibody 3D auto-refinement against single-particle cryo-EM data was used to characterize the dynamics of the interaction of CD98hc and LAT1. It is shown that the CD98hc ectodomain and the LAT1 extracellular surface share no substantial interface. This allows the CD98hc ectodomain to have a high degree of movement within the extracellular space. The functional implications of these aspects are discussed together with the structure determination

Decomposition of spontaneous fluctuations in tumour oxygenation using BOLD MRI and independent component analysis

Solid tumours can undergo cycles of hypoxia, followed by reoxygenation, which can have significant implications for the success of anticancer therapies. A need therefore exists to develop methods to aid its detection and to further characterise its biological basis. We present here a novel method for decomposing systemic and tumour-specific contributions to fluctuations in tumour deoxyhaemoglobin concentration, based on magnetic resonance imaging measurements

Shiny app to predict agricultural tire dimensions

The main objective of this project, carried out in an industrial context, was to apply a multivariate analysis to variables related to the specifications required for the production of an agricultural tire and the dimensional test results. With the exploratory data analysis, it was possible to identify strong correlations between predictor variables and with the response variables of each test. In this project, the principal component analysis (PCA) serves to eliminate the effects of multicollinearity. The use of regression analysis was intended to predict the behavior of the agricultural tire considering the selected variables of each test. In the case of Test 1, when applying the Stepwise methods to select the variables, the model with the lowest value of Akaike Information Criterion (AIC) was achieved with the technique “Both”. However, the lowest value of AIC for Test 2 was achieved with “Backward”. Regarding the validation of assumptions, both Test 1 and Test 2 were validated. Therefore, all the quantitative variables are important, both in Test 1 and Test 2, because they are a linear combination that determines the principal components. In order to make it easier to compute predictions for future agricultural tires, an application that was developed in Shiny allows the company to know the behavior of the tire before it was produced. Using the application, it is possible to reduce the industrialization time, materials and resources, thus increasing efficiency and profits.This work has been supported by FCT – Fundação para a Ciˆencia e Tecnologia within the R&D Units Project Scope: UIDB/00319/2020

Structure-Based Identification and Characterization of Inhibitors of the Epilepsy-Associated KNa1.1 (KCNT1) Potassium Channel

Drug-resistant epileptic encephalopathies of infancy have been associated with KCNT1 gainof-function mutations, which increase the activity of KNa1.1 sodium-activated potassium channels. Pharmacological inhibition of hyperactive KNa1.1 channels by quinidine has been proposed as a stratified treatment, but mostly this has not been successful, being linked to the low potency and lack of specificity of the drug. Here we describe the use of a previously determined cryo-electron microscopy-derived KNa1.1 structure and mutational analysis to identify how quinidine binds to the channel pore and, using computational methods, screened for compounds predicated to bind to this site. We describe six compounds that inhibited KNa1.1 channels with low- and sub-micromolar potencies, likely also through binding in the intracellular pore vestibule. In hERG inhibition and cytotoxicity assays, two compounds were ineffective. These may provide starting points for the development of new pharmacophores and could become tool compounds to study this channel further