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In many countries the current plan for the management of intermediate and high level radioactive wastes is to dispose of the radioactive materials underground in a Geological Disposal Facility (GDF) to prevent release of radioactivity to the environment. In the UK, the repository for intermediate level waste may be backfilled with cementitious material and it is clear that grout and cement will be used during many disposal concepts. Upon saturation, the cement will react creating a region of hyperalkaline geochemical conditions extending away from the GDF, within which, significant changes in radionuclide behaviour are expected. Therefore, this thesis utilises a range of experimental and analytical techniques to try to gain a mechanistic understanding of the behaviour of some key radionuclides (U(VI), Np(V) and Eu(III) as an analogue for Cm(III)/Am(III)) in a range of high pH systems of direct relevance to any cementitious GDF. U(VI) interaction with calcite (calcium carbonate, a common component in high pH cements and the natural environment) surfaces was studied in the 'old' (Ca(OH)2 solution; pH 10.5) and 'young' (Na+, K+, Ca2+; pH 13.3) leachates. In the 'old' leachate, luminescence spectroscopy, batch experiments and kinetic modelling suggested that at low concentrations (smaller or equal to 0.42 µM) a Ca2UO2(CO3)3-like surface complex formed. At higher concentrations, batch experiments, extended X-ray absorption fine structure spectroscopy and luminescence suggested that a surface mediated precipitation mechanism was controlling U(VI) concentrations. Further TEM analysis confirmed that a calcium uranate (CaUO4) solid phase was forming on the calcite surfaces. In the 'young' leachate, batch experiments showed that U(VI) had little affinity for the calcite surface, with no statistically relevant removal from solution observed over a 18 month period. Small angle X-ray diffraction data demonstrated that the U(VI) was probably present in the form of U(VI) intrinsic colloids. Np(V) solubility and sorption to calcite under hyperalkaline conditions were studied using batch, X-ray absorption spectroscopy, and geochemical modelling techniques. It was determined that Np(V) solubility in 'old' cement leachates was consistent with the literature. However, in 'young' cement leachates, an unidentified calcium containing phase was controlling solubility. It was demonstrated that sorption to calcite in 'old' leachates was controlled by the formation of a >CO3NpO2 surface complex, whereas, in the 'young' leachates interaction with the calcite surface was controlled by a precipitation mechanism. Eu(III) sorption to a potential GDF backfill material, Nirex Reference Vault Backfill (NRVB) cement, was studied. The kinetics of removal were rapid with 98.5% Eu(III) removal within 24 hours. Ultrafiltration experiments indicated that all Eu(III) remaining in solution was associated with NRVB derived colloids. Additional experiments using ethylenediaminetetraacetic acid (EDTA) as a competing ligand show that removal from solution was significantly reduced at high concentrations (>0.01 M). These EDTA experiments also indicated some irreversibility in the systems, possibly caused by incorporation into the C-S-H or calcite structures.

Deep geological disposal is a method of managing high level, long-­‐lived nuclear waste. It is a concept that many countries are exploring for the possibility of managing nuclear waste generated from power production. For deep geological disposal to be viable then areas where problems may surface have to be explored. Bentonite clay has been proposed as the material to be used for the backfill of the repositories. Its swelling properties ensure that it will expand to plug the bore holes that will be made for the waste, its impermeable nature restricts contact between groundwater and the waste package and its stability on a geological timescale all make it desirable as a backfill material. This project looks at the role that colloids formed from the bentonite clay could have in facilitating radionuclide transport away from a nuclear waste repository. Several radionuclides (Eu(III), U(VI), Th(IV) and Am(III)) have been considered in this research, and information from these studies will be used in the BELBaR project’s outputs, which will eventually support a disposal safety case. Ternary systems of 152Eu(III), bulk bentonite and EDTA ([Eu] = 7.9 x 10-­‐10 M; pH = 6.0 – 7.0) have been studied. Without EDTA, there was slow uptake in a two-­‐stage process, with initial rapid sorption of Eu(III) (96%), followed by slower uptake of a smaller fraction (3.0 % over a period of 1 month). The reversibility of Eu(III) binding was tested by allowing Eu(III) to sorb to bentonite for 1 – 322 days. EDTA was added to the pre-­‐equilibrated Eu bentonite systems at 0.01 M. A dissociation rate constant of approximately 4.3 x 10-­‐8 s-­‐1 (values in the range 2.2 x 10-­‐8 – 1.0 x 10-­‐7 s-­‐1) for pre-­‐equilibration times ≥ 7 days was measured. Eventually, the amount of Eu(III) remaining bound to the bentonite was within error of that when EDTA was also present prior to contact (4.5 % ± 0.6). Eu interactions with colloidal bentonite were studied, and the dissociation rate constant measured by a resin competition method. A dissociation rate of 8.8 x 10-­‐7 s-­‐1 and a range of 7.7 x 10-­‐7 – 9.5 x 10-­‐7 s-­‐1 were measured. For both bulk and colloidal bentonite slow dissociation was observed for Eu(III), but there was no evidence for ‘irreversible’ binding. The interactions of 232U(VI) with bentonite colloids ([U] = 5.43 x 10-­‐10 M; pH = 8.8 ± 0.2) have been studied using a resin ion exchange competition technique. The reversibility of the interaction was studied by allowing U(VI) to sorb to bentonite colloids for periods from 1 – 35 days. A fraction of the U(VI) was removed from the solution instantaneously (28-­‐50 %), and after 3 days, the amount of U(VI) remaining on the bentonite colloids was 17-­‐ 25%. With time, the amount of U(VI) retained by the bentonite colloid is reduced further, with a first order dissociation rate constant of 5.6 x 10-­‐7 s-­‐1. Whilst the dissociating fraction was small (24% (+34; -­‐12 %)), complete dissociation was not observed. Although slow dissociation was observed for U(VI), there was no convincing evidence for ‘irreversible binding’ of the radionuclide by the colloid. The interactions of 228Th(IV) ([Th] = 3.79 x 10-­‐12 M; pH = 8.8 ± 0.2) and 241Am(III) ([Am] = 3.27 x 10-­‐9 M; pH = 8.8 ± 0.2), with bentonite colloids have been studied using an ion exchange competition technique. Th(IV) was not fully associated with the bentonite colloids, and filtration showed that the uptake after 1 week was 78.3% (± 2.7%). Am(III) was weakly associated to the bentonite colloids, the uptake after 1 week was 20.1 % (± 5.2 %). Cellulose phosphate was added to the radionuclide/bentonite colloid systems (1 g for Th(IV), 0.2 g for Am(III)), an amount that was sufficient to retain the radionuclide when no bentonite colloids are present. A fraction of the Th(IV) is initially removed by the Cellphos (75-­‐93 %), and after 7 days the amount of Th(IV) remaining on the colloids is 1 -­‐ 3 %. Over the time of the experiment, the amount of Th(IV) retained by the bentonite colloid appears to remain level and the amount bound to the bentonite colloid at the end of the experiment is 2.1 % ± 0.88 % which is within experimental error of the steady state equilibrium of the system. A fraction (48-­‐94 %) of the Am(III) is also initially removed by the Cellphos, after 7 days the8amount of Am(III) remaining on the colloids is 1.2 – 9.3 %. However, after 35 days of contact time with the cellulose phosphate it appears that Am(III) is released back into the system, preventing dissociation rates from being calculated in this case. Studies of the association of Eu(III) to the clay colloids and its subsequent dissociation in this thesis follow similar trends to those described elsewhere in the literature (Missana et al. (2008), Bouby et al. (2011)). The Eu/bentonite colloid dissociation rate calculated here (8.8 x 10-­‐7 s-­‐1 (± 9.1 x 10-­‐7 s-­‐1)) is within error of the dissociation rates for trivalent ions estimated by Wold (2010) (Am(III) 5.6 x 10-­‐7 s-­‐1 Cm(III) 1.7 x 10-­‐6 s-­‐1). The U(VI) studies in this thesis show a dissociation rate of 5.6 x 10-­‐7 s-­‐1 (± 4.2 × 10-­‐7) which is within error of the U(VI) dissociation rate estimated by Wold (2010) (8.3 X 10-­‐7 s-­‐1). Reliable dissociation rates could not be obtained from the Am(III) and the Th(IV) studies in this thesis, other studies (e.g. Bouby et al. (2011) showed signs of irreversible binding of Th(IV) to bentonite colloids, however, no irreversible binding was observed in this thesis. Am(III) did not appear to be a close analogue of Eu(III) in these systems. All of the isotopes studied in this thesis showed no evidence of irreversible binding to bentonite or bentonite colloids. As such, the role that bentonite colloids will have in the facilitated transport of radioisotopes away from a repository is likely to have only a limited impact, at most, on the environmental safety case.

The investigation of an abdominal mass in a child is a common problem in the radiology department of the Red Cross Children's Hospital. The majority of these masses involve the urinary tract. The commonest neoplasm is a Wilms' tumour of the kidney. Against a pathological and clinical background, the investigation of Wilms' tumour by diagnostic imaging is presented. The imaging modalities currently utilised are the intravenous urogram (IVU), ultrasound (US), computed tomography (CT) and magnetic resonance (MR). Using the material available in the last decade, the principles, techniques and imaging characteristics of these modalities are investigated and compared. These results are reflected against those reported in the medical literature. This literature is not yet extensive as the current technology has only been available for the last six to seven years. The IVU has in the past been the main imaging modality and we still use it extensively. Its strengths and weaknesses are discussed. In the last five years US has taken its place as the primary method of diagnostic imaging. We have found that with our increasing experience that this is justified. The use of US and IVU in a practiced hand is a powerful diagnostic combination. CT as a primary investigation is not readily available at our institution. We have used it for comparative purposes in about 20% of our recent cases. CT has not added greatly to our initial diagnostic impression. However, it has been most useful for follow up of metastasis and for assessing the normality of the lungs before ceasing chemotherapy. Our experience with MRI is limited and confined to unusual presentations in the last year. Other modalities such as arteriography and nuclear medicine have special indications which are to be discussed. The remaining tumours of the upper urinary tracts are all rare, but are reported and the literature researched. In the lower urinary tract the main pelvic lesion is a rhabdomyosarcoma. The comparative advantages of the IVU, US, CT and MRI are also noted. In the pelvis, US has also become the primary imaging modality, and is replacing contrast medium cystography. However, examples of the latter are included as it still has a place, particularly in the less sophisticated institutes. CT and MRI, when available, have imaging advantages in the pelvis and are becoming the methods of choice for follow up. The main objective of this document has been to investigate the available imaging techniques, but, against this overall theme, the clinical care of the child is most important. With this in mind the treatment protocols that are used at our hospital are noted in the appendices to the thesis.

Affibody molecules are a promising class of scaffold-based targeting proteins for radionuclide-based imaging and therapy of cancer. This thesis work is based on 5 original research articles (papers I-V), which focus on optimization of molecular design of HER2-binding Affibody variants for high contrast imaging of this predictive biomarker as well as development of Affibody molecules suitable for radionuclide-based targeted therapies.  Papers I and II were dedicated to evaluation of the influence of the macrocyclic chelator DOTA positioning at N-terminus, in the middle of helix-3 and at C terminus of a synthetic Affibody molecule, ZHER2:S1. These synthetic variants were labelled with different radionuclides i.e. 111In and 68Ga to study also the effect of different labels on their biodistribution properties. In paper III a 2-helix variant, Z342min, was developed using native ligation cyclization to cross-link helices one and two resulting in a stable 2-helix scaffold and characterized in vivo. This study was performed with the aim to obtain structure-properties relationship for development of smaller Affibody molecules.   Papers IV and V were devoted to development of therapeutic strategies. In paper IV, a series of peptide based chelators was investigated for labelling of Affibody molecules with 188Re to provide low renal retention. In paper V, a pretargeting approach using peptide nucleic acid was investigated. These studies were performed with the aim to overcome the high renal retention of Affibody molecules when labelled with residualizing therapeutic radionuclides. Otherwise, the particle emitting radiometals could damage the kidneys more than the tumours. The results obtained for anti-HER2 Affibody molecules summarized in this thesis might be of importance for the development of other scaffold protein based targeting agents.

Affibody molecules are promising candidates for targeted radionuclide-based imaging and therapy applications. Optimisation of targeting properties would permit the in vivo visualization of cancer-specific surface receptors with high contrast. In therapy, this may increase the ratio of radioactivity uptake between tumour and normal tissues.  This thesis work is based on 5 original research articles (papers I-V) and focuses on optimisation of targeting properties of anti-HER2 affibody molecules by optimising the labelling chemistry. Paper I and II report the comparative evaluation of the anti-HER2 ZHER2:2395 affibody molecule site specifically labelled with 111In (suitable for SPECT imaging) and 68Ga (suitable for PET imaging) using the thiol reactive derivatives of DOTA and NODAGA as chelators. The incorporation of different macrocyclic chelators and labelling with different radionuclides modified the biodistribution properties of affibody molecules. This indicates that the labelling strategy may have a profound effect on the targeting properties of radiotracers and must be carefully optimized. Paper III reports the study of the mechanism of renal reabsorption of anti-HER2 ZHER2:2395 affibody molecule. An unknown receptor (not HER2) is suspected to be responsible for the high reabsorption of ZHER2:2395 molecules in the kidneys. Paper IV reports the optimization and development of in vivo targeting properties of 188Re-labelled anti-HER2 affibody molecules. By using an array of peptide based chelators, it was found that substitution of one amino acid by another or changing its position can have a dramatic effect on the biodistribution properties of 188Re-labelled affibody molecules. This permitted the selection of –GGGC chelator whichdemonstrated the lowest retention of radioactivity in kidneys compared to other variants and showed excellent tumour targeting properties. Paper V reports the preclinical evaluation of 188Re-ZHER2:V2 as a potential candidate for targeted radionuclide therapy of HER2-expressing tumours. In vivo experiments in mice along with dosimetry assessment in both murine and human models revealed that future human radiotherapy studies using 188Re-ZHER2:V2 may be feasible. It would be reasonable to believe that the results of optimisation of anti-HER2 affibody molecules summarized in this thesis can be of importance for the development of other scaffold protein-based targeting agents.

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Familial amyloidosis with polyneuropathy (FAP) is a neuropathic form of heredofamilial systemic amyloidosis. Clusters of patients have been reported predominantly from Portugal, Japan and Sweden. The present study examines the involvement of the heart in individuals with the Swedish variety of FAP. During long-term ECG recording in 16 patients, a high frequency of disturbances of sinus node function and atrioventricular conduction were observed. Long-term ECG may help considerably in the evaluation of symptoms attributable to disturbances of heart rhythm in FAP. A retrospective survey of 20 patients with FAP treated with a pacemaker showed that the indication for pacing was advanced atrioventricular block (12 cases), dysfunction of the sinus node (5 cases), and atrial fibrillation with a slow ventricular response (3 cases). All patients experienced the effective relief of symptoms attributable to a slow ventricular rate. The long-term prognosis, however, seemed unaffected by this treatment. Histopathological examination of the sinoatrial (9 cases) and atrioventricular (6 cases) parts of the conduction system showed marked amyloid infiltration in all cases, which may explain the high occurrence of disturbances of cardiac rhythm and conduction. Twelve patients were examined by two-dimensional echocardiography and changes of varying degrees, such as highly refractile myocardial echoes (12 cases) and thickened ventricular walls (8 cases) and valves (7 cases), could be observed. Technetium-99m-pyrophosphate scintigraphy of the same patients revealed abnormal myocardial uptake of the isotope only in four. Echocardiography thus seems to be superior to scintigraphy for non-invasive detection of cardiac involvement in FAP. Myocardial samples from regions producing highly refractile myocardial echoes were obtained at in vitro échocardiographie examination of hearts from FAP autopsy cases. Histological examination showed that the highly refractile echoes corresponded to more or less sharply delineated nodules, containing amyloid and collagen in various amounts. / digitalisering@umu

<p>Targeted radionuclide therapy (TRT) holds great promise for the treatment of cancer. In TRT, radioactive nuclides are delivered specifically to tumours by molecules that recognise and bind to structures overexpressed by, or specific to, cancer cells. Human epidermal growth factor receptor like protein 2 (HER-2) is an oncogene product overexpressed in e.g. urological, breast, or ovarian cancers that have been correlated to poor prognosis and resistance to hormonal therapy. There is also evidence that tumour cells retain their HER-2 overexpression in metastases.</p><p>Trastuzumab and pertuzumab are two humanised monoclonal antibodies targeting different parts of HER-2. This thesis describes the radiolabelling of these antibodies for use in TRT and diagnostics. The thesis also investigates possible methods for modifying uptake and retention of radioactivity delivered with antibodies binding to HER-2. Modification of the cellular retention of ¹²⁵I by using polyhedral boron anion based linker molecules (DABI and NBI) is investigated, and it is shown that linking ¹²⁵I to trastuzumab using DABI increases cellular accumulation of radioactivity by 33%. It is also shown that trastuzumab can be efficiently coupled to the positron emitter ⁷⁶Br by using NBI. Furthermore, it is shown that cellular uptake of ¹²⁵I can be modified by stimulating EGFR (HER-1) with EGF.</p><p>When labelled with the alpha emitter ²¹¹At, trastuzumab could specifically kill cells in vitro. This cell killing effect could be prevented by saturating the receptors of the target cells with non-radiolabelled trastuzumab.</p><p>Pertuzumab was radiolabelled with the low energy beta emitter ¹⁷⁷Lu without losing affinity or immunocompetence. [¹⁷⁷Lu]pertuzumab was specific to HER-2 in vitro and in vivo. This targeting conjugate was shown to increase median time to tumour progression in mice bearing xenografts of the radioresistant SKOV-3 cell line. </p><p>In conclusion, antibodies against HER-2, especially pertuzumab radiolabelled with ¹⁷⁷Lu, show promise as TRT agents.</p>