This thesis reports on the degradation of two different models of organic pollutants in two contrasting Scottish river sediments. The first type of sediment (sediment 1) was collected close to an urban area with high percentage of organic matter content (1.3%). The second type of sediment (sediment 2) was collected from a pristine area and was less organic (0.2%). As organic pollutant model, radiolabelled (14C) 2,4-Dichlorophenoxyacetic acid (Acid-ring-UL-14C) and Naphthalene (Naphthalene-1-14C) were amended to individual microcosms (50g of sediment and 50ml of river water) at different concentrations (0.5, 5.0 and 50 mg per microcosm). Degradation was assessed by emission of 14CO2 during a period of 40 and 124 days for 2,4-D and Naphthalene, respectively. 2,4-D was mostly degraded between 20-35 days, and degradation rates (5 to 750 mgd-1) were proportional to initial concentrations. Almost 25% of applied Naphthalene was degraded by 40 days, at different degradation rates (2.7 to 405 mgd-1) according to sediment type. Assessment of the impact of 2,4-D and Naphthalene on the sediment microbial community was made using Biolog, DGGE and PLFA analysis to provide for a comparative physiological, molecular and chemotaxonomic assessment, respectively. These analyses linked differences in the rates and extents of 2,4-D degradation to differences in composition of the microbial communities. To assess the impact of 2,4-D, DGGE-profiling was used and show genetic changes were more significant in sediment 2 microcosms than sediment 1. Biolog suggested differences in the microbial structure of both sediments, but PLFA demonstrates that sediment 1 treatments were more affected by 2,4-D amendments than sediment 2. The extent of Naphthalene biodegradation was restricted by bioavailability, sediment type and volatilisation.

Human leukocytes have been radiolabelled indirectly via targeting leukocytes inside the body (in vivo) and directly by radiolabelling these cells in the laboratory (in vitro), the latter being the most commonly used.

Human leukocytes have been radiolabelled indirectly via targeting leukocytes inside the body (in vivo) and directly by radiolabelling these cells in the laboratory (in vitro), the latter being the most commonly used.

No description available.

Positron emission tomography (PET) is a quantitative and non-invasive investigative tool, which permits the identification of pivotal biomarkers and their role in disease onset, transformation and progression. Quantitative detection of these disease biomarkers has qualified PET as a pioneering approach to rapid diagnosis. The tool is a powerful approach to patient diagnosis and investigation of inter- and intra- patient disease heterogeneity thereby supporting patient stratification and forging a truly personalised medicine approach. The sensitivity of PET is complemented by use of highly selective radiopharmaceuticals; this has fuelled the popularity of radiolabelled affinity peptides and proteins (APPs). Inspired by the high selectivity and affinity of biological protein-protein interactions, APP-based radiotracers are an increasingly popular class of radiotracer. Monoclonal antibodies (mAbs) are an important category of APP-based radiotracer; their prevalence in the field is attributed to their high target selectivity and affinity characteristics, which, will likely, uphold their popularity in the field. Yet, advancements in protein engineering has transformed the landscape of APP-based radiotracers, indicated by a preference for small radiolabelled APPs which exhibit rapid pharmacokinetics whilst retaining high target specificity and affinity. Their fast pharmacokinetics is matched by the half-life of 18F which has been defined as an ideal PET radionuclide. 18F APP radiolabelling hasn't yet met its full potential due to its inherent challenges; many approaches have been adopted, ranging from non-site specific radiolabelling with amine reactive prosthetic groups to site-specific methods including oxime bond formation. The radiochemistry of 18F APP radiolabelling is diverse and evolving; yet ideal goals are apparent including site-specificity, fast reaction kinetics, mild labelling conditions and applicability to automation. Automation is challenging for multi-step 18F APP radiolabelling methods, but is a key developmental step. Automation helps with conformance to good manufacturing practice (GMP) by enhancing process robustness, consistency and reliability. It also permits the radiosynthesis of clinically relevant radiotracer doses. The development of an automated generic platform, or a method that adopts a general approach, for APP radiolabelling with 18F would assist in [18F]APPs meeting their potential in PET and, importantly, aid their translation from bench-to-bedside.

Optimization of Affibody molecules would allow for high contrast imaging of cancer associated surface receptors using molecular imaging. The primary aim of the thesis was to develop Affibody-based PET imaging agents to provide the highest possible sensitivity of RTK detection in vivo. The thesis evaluates the effect of radiolabelling chemistry on biodistribution and targeting properties of Affibody molecules directed against HER2 and PDGFRβ. The thesis is based on five published papers (I-V). Paper I. The targeting properties of maleimido derivatives of DOTA and NODAGA for site-specific labelling of a recombinant HER2-binding Affibody molecule radiolabelled with 68Ga were compared in vivo. Favourable in vivo properties were seen for the Affibody molecule with the combination of 68Ga with NODAGA. Paper II. The aim was to compare the biodistribution of 68Ga- and 111In-labelled HER2-targeting Affibody molecules containing DOTA, NOTA and NODAGA at the N-terminus. This paper also demonstrated favourable in vivo properties for Affibody molecules in combination with 68Ga and NODAGA placed on the N-terminus. Paper III.  The influence of chelator positioning on the synthetic anti-HER2 affibody molecule labelled with 68Ga was investigated. The chelator DOTA was conjugated either at the N-terminus, the middle of helix-3 or at the C-terminus of the Affibody molecules. The N-terminus placement provided the highest tumour uptake and tumour-to-organ ratios. Paper IV. The aim of this study was to evaluate if the 68Ga labelled PDGFRβ-targeting Affibody would provide an imaging agent suitable for PDGFRβ visualization using PET. The 68Ga labelled conjugate provided high-contrast imaging of PDGFRβ-expressing tumours in vivo using microPET as early as 2h after injection. Paper V. This paper investigated if the replacement of IHPEM with IPEM as a linker molecule for radioiodination of Affibody molecules would reduce renal retention of radioactivity. Results showed that the use of the more lipophilic linker IPEM reduced the renal radioactivity retention for radioiodinated Affibody molecules. In conclusion, this thesis clearly demonstrates that the labelling strategy is of great importance with a substantial influence on the targeting properties of Affibody molecules and should be taken under serious considerations when developing new imaging agents.

Chapter One introduces molecular irnaging and the modalities available for oncological irnaging. The radioisotopes and imaging agents for Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) are discussed together with the bifunctional chelator approach for radio labelling of biomolecules. Finally, the chemistry and radioisotopes of copper are described, and copper bis(thiosemicarbazonato) complexes introduced in the context of PET irnaging. Chapter Two describes the synthesis and characterisation of novel carboxylate- and maleirnide- functionalised bis(thiosemicarbazonates) and their conjugation to biologically active molecules. Radiolabelling of a chelator-bombesin conjugate demonstrated site-specific labelling at room temperature and preliminary in vitro and in vivo studies confirmed its potential as an imaging agent. Bioconjugation to a model protein and subsequent radiolabelling was also investigated. Chapter Three introduces molecular irnaging of hypoxia with a focus on CuATSM. An overview of the currently accepted mechanism of hypoxia selectivity is presented. The emphasis is placed on the relationship between oxygenation status, uptake and retention which display cell- and tumour- line dependency. Chapter Four presents the synthesis of copper bis(thiosemicarbazonates), radiolabelled either at the metal (64CU) or at the ligand e8F or 123n for mechanistic studies. The physicochemical characteristics of the copper complexes were measured and the complexes evaluated for their in vitro hypoxia selectivity. Chapter Five describes in vitro and in vivo studies of the orthogonally radiolabelled complexes, inclusive of control experiments with [64Cu]CuATS~, the radiolabelled proligand and [64CU]CU2+ salts. In vitro cellular assays, as well as in vivo biodistribution studies including dynamic PET and SPECT were performed. Stability studies contrasting the in vitro and in vivo behaviour were carried out. The collective data suggest that the currently proposed redox trapping mechanism might not provide a full understanding of the factors governing biodistribution and tumour uptake. Chapter Six contains full experimental details for the work described in this thesis.

A method for purification and radiolabelling phosphoglucose isomerase was devised in order to develop a sensitive quantitative radioimmunoassay for the detection of the enzyme irrespective of its catalytic activity. For four genetic variants of PGI no difference in the molecular specific activity was observed. In one variant (PGI-Denton), liver and heart tissue extracts, and in mature erythrocytes (as compared to normal erythrocytes), a decreased molecular specific activity was observed which initially may imply that these samples contain cross-reactive material which is not catalytically active.

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of: Biophysics and Physical Chemistry Consultant: Doc. Ing. Alice Lázníčková, CSc. Student: Pavla Kosová Title of thesis: Radiolabeling of PCTA-Lys3-Bombesine and determination of stability of the radiolabeled product The dissertation deal with study of radiolabeling of new receptor-specific peptide - bombesin, which was modified with chelator PCTA, PCTA-Lys3-bombesin, with radionuclide 177 Lu, and determination of labelled product quality and its stability. For quality control we used method high-efficiency liquid chromatography (HPLC) and thin-layer chromatography (ITLC) on ITLC-SG. HPLC offer clear proof about pollution and possible dissociative outputs. Comparison of both methods for determination of purity of labelled peptide showed that HPLC is more accurate than TLC method.

Positron Emission Tomography (PET) has great value in research and clinical applications from oncology to neurodegenerative disorders. However, there is a barrier in translating biological knowledge into new PET applications due in part to the lack of efficient, widely applicable methods for labelling compounds with PET radioisotopes. Herein, a generic approach to radiolabelling is presented which is direct, broadly applicable and potentially adaptable to either of the two most commonly used PET radioisotopes, 11C and 18F. This approach employs the advantages of solid phase synthesis to achieve selective release of only the desired radiolabelled product from a solid support in a single step, simplifying purification and hence improving synthetic efficiency. Polystyrene resin was functionalised with a 1,2-diol group; this allowed the covalent attachment of compounds bearing boronic acid groups via formation of a boronate ester linkage. A Suzuki-Miyaura reaction with methyl iodide was used to cleave a model compound from the resin in 61% conversion after five minutes. This reaction was adapted to develop a fully automated radiosynthesis with [11C]- methyl iodide which generated a radiolabelled model compound in 2 – 7% non-decay-corrected radiochemical yield. This provided proof of concept for the simultaneous cleavage of compounds from the resin and radiolabelling with 11C. A boronic acid precursor of the known radiotracer [11C]-M-MTEB was attached to the resin and successfully radiolabelled with 11C in 2.4% non-decay-corrected radiochemical yield and 96 – 100% radiochemical purity under the same conditions. Furthermore, the potential adaptability of this solid phase approach to 18F radiolabelling was demonstrated by treatment of the resin-bound small molecules and peptides with potassium bifluoride, which released the compounds rapidly as trifluoroborate salts.