Affibody molecules are small (7 kDa) affinity proteins of non-immunoglobulin origin that have been generated to specifically interact with a large number of clinically important molecular targets. In this thesis, Affibody molecules have been employed as tracers for radionuclide molecular imaging of HER2- and IGF-1R-expressing tumors, paper I-IV, and for surface knock-down of EGFR, paper V. In paper I, a tag with the amino acid sequence HEHEHE was fused to the N-terminus of a HER2-specific Affibody molecule, (ZHER2), and was shown to enable facile IMAC purification and efficient tri-carbonyl 99mTc-labeling. In vivo evaluation of radioactivity uptake in different organs showed an improved biodistribution, including a 10-fold lower radioactivity uptake in liver, compared to the same construct with a H6-tag. In paper II, it was further shown that an N-terminally placed HEHEHE-tag on ZHER2 provided lower unspecific uptake of radioactivity in liver compared to its H6-tagged counterpart even when radiolabeling was at the C-terminus using alternative chemistries to attach 99mTc, 111In or 125I. In paper III, the H6-tag’s composition and position was varied with regards to charge, hydrophobicity and its C- or N-terminal placement on ZHER2. Among the ten variants investigated, it was found that an N-terminal HEHEHE-tag provided the most favorable overall biodistribution profile and that introduction of hydrophobic and positively charged amino acids provoked liver uptake of radioactivity. In paper IV, the HEHEHE-tag was shown to enable IMAC purification and tri-carbonyl 99mTc-labeling of an IGF-1R-specific Affibody molecule and improved its overall biodistribution when compared to the same construct with a H6-tag. In paper V, the aim was to develop an intracellular receptor-entrapment system to reduce the surface levels of EGFR. An EGFR-specific Affibody molecule was expressed as a fusion to different mutants of an intracellular transport protein in SKOV-3 cells, resulting in a collection of cell lines with 50%, 60%, 80% and 96% reduced surface level of EGFR. Analysis of the proliferation rate of these cell lines showed that a modest reduction (15%) in proliferation occurs between 60% and 80% reduction of the surface level of EGFR. / <p>QC 20130129</p>

Affibody molecules have lately shown great potential as tools for in vivo molecular imaging. These small, 3-helical bundles, with their highly stable protein scaffold, are well suited for the often harsh conditions of radiolabeling. Their small size allows for rapid clearance from the blood circulation which permits the collection of images already within hours after injection. This thesis includes four papers aimed at engineering different variants of a HER2-binding Affibody molecule to enable effective  and  flexible  radiolabeling  and  enhancing  the  molecular  imaging  in  terms  of  imaging contrast and resolution. In paper I an Affibody molecule was engineered to function as a multifunctional platform for site-specific labeling with different nuclides for radionuclide imaging. This was done using only natural amino  acids,  thereby  allowing  for  both  synthetic  and  recombinant  production.  By  grafting  the amino acid sequence -GSECG to the C-terminal of our model-protein, a HER2-binding Affibody molecule, we enabled site specific labeling with both trivalent radiometals and with  99m Tc. Maleim-ide-DOTA was conjugated to the cysteine residue for labeling with  111 In, while the peptide sequence was able to chelate  99m Tc directly. This approach can also be used for site-specific labeling with other probes available for thiol-chemistry, and is applicable also to other protein scaffolds. In paper II we investigated the impact of size and affinity of radiolabeled Affibody molecules on tumor targeting and image contrast. Two HER2-targeting Affibody molecules, a two-helix (~5 kDa) and a three-helix (~7 kDa) counterpart, were synthetically produced, labeled with  111 In via chelation by  DOTA  and  directly  compared  in  terms  of  biodistribution  and  targeting  properties.  Results showed  that  the  smaller  variant  can  provide  higher  contrast  images,  at  the  cost  of  lower  tumor uptake,  in  high-expressing  HER2-tumors.  However,  neither  the  tumor  uptake  nor  the  contrast of the two-helix variant is sufficient to compete with the three-helix molecule in tumors with low expression of HER2. In paper III and IV we were aiming to find methods to improve the labeling of Affibody molecules with  18 F for PET imaging. Current methods are either complex, time-consuming or generate heavily lipophilic conjugates. This results in low yields of radiolabeled tracer, low specific activity left for imaging, undesirable biodistribution or a combination thereof. In paper III we demonstrate a swift and efficient 2-step, 1-pot method for labeling HER2-binding Affibody molecules by the formation of aluminum  18 F-fluoride (Al 18 F) and its chelation by NOTA, all in 30 min. The results show that the  18 F-NOTA-approach is a very promising method of labeling Affibody molecules with  18 F and further investigation of this scheme is highly motivated. In the last paper we pursued the possibility of decreasing the high kidney retention that is common among small radiotracers with residual-izing radiometabolites. In this work  18 F-4-fluorobenzaldehyde (FBA) was conjugated to a synthetic HER2-targeting Affibody molecule via oxime ligation. However, to avoid elevated liver retention, as seen in previous studies with this kind of label, a hydrophilic triglutamyl spacer between the aminooxy moiety and the N-terminal was introduced. A comparison of the two constructs (with and without the triglutamyl spacer) showed a clear reduction of retention in both kidney and liver in NMRI mice at 2 h p.i. when the spacer was included. In the light of these promising results, further studies including tumor-bearing mice, are in preparation. / <p>QC 20130203</p>

The Manatee River Watershed (Manatee County, FL) has experienced heavy anthropogenic development over the last 100 years and was relatively pristine previous to this development. The population growth within the watershed has surpassed the national trends and has doubled in the last 30 years. The heavy anthropogenic development has led to depletion in natural resources, nutrient loading, coastal erosion, and increased pollution. This study constructs records of sedimentological processes to compare the pre-development records to the past 100 years of anthropogenic development. The first portion of this study identifies specific changes in sedimentation attributed to anthropogenic activity in the Manatee River. Anthropogenic development has increased the input of terrigenous material into the river by as much as an order of magnitude (0.3-3.0 g/cm2/yr) over three periods; 1) the predevelopment period (1900-1941), 2) the agricultural development period (1941-1970's), and 3) the urban development period (1970's-2010). The second portion of this study examines records of heavy metal (As, Cu, Pb) enrichment in the Manatee River. There are areas in the Manatee River that currently have, or recently have had, concentrations of heavy metals above the EPA regional screening levels. Throughout all of the Manatee River sediment cores there has been a continuous increase in the concentration of arsenic (0.32-20.91 ppm), lead (0.35-35.79 ppm) and copper (1.49-49.55 ppm) from 1900-2010. The third portion of this study utilizes calcareous tests from benthic foraminifera (Ammonia beccarii) in the longest sediment core to determine the Mg/Ca, 18O/ 16O, and 13C/ 12C ratios as proxies for river water temperature, salinity and nutrient content. These proxies allow for the assessment of changes in rates and range of river water parameters from the pre-anthropogenic to the anthropogenic periods. A Manatee River temperature record, precipitation/evaporation record and nutrification record have been constructed for the last 450 years (1550-2009 CE). These records are necessary to inform and enhance future coastal resource management practices.

Enhanced soil loss from the watershed is a major environmental issue. Increased soil loss from a watershed can potentially increase sediment delivery and loading to aquatic ecosystems such as rivers and estuaries. An increase in sediment delivery and loading to freshwater and transitional marine ecosystems can impact water quality and supply specifically by: (1) reducing water clarity, (2) transporting nutrient and pollutant laden sediments and (3) reducing the storage capacity of reservoirs. To address these broader environmental impacts of increased sediment delivery and loading to aquatic ecosystems it is imperative that potential source areas of sediments available for transport are identified in the watershed. It is also important that sediment source areas are linked to sediment transport and delivery to aquatic ecosystems. This study attempted to establish a link between soil loss from watersheds and sediment delivery in two estuaries on the island of Puerto Rico. The two estuarine systems used in this investigation were the Rio Espiritu Santo (RES) riverine-estuarine system and the Jobos Bay (JB) Estuary. Soil loss from both study watersheds was estimated using RUSLE. Sediment cores and surface grab samples were collected from both estuaries. In addition, soil samples were collected from the two watersheds. Gamma analyses were performed in order to measure activity concentrations of 137Cs and excess 210Pb in sediment cores, surface and soil samples. 137Cs inventories were computed for each core collected from both watersheds. Also, grain-size and LOI were performed on the sediments to describe the sedimentological characteristics of collected sediment cores, surface samples and soil samples. A conceptual framework was developed and implemented for linking sediment production, availability (supply), transport and delivery to study estuaries. Results from the RUSLE model indicated that soil loss within both watersheds were low with patchy instances of erosional hotspots. These results did not provide any information on sediment supply or insights into the hydrologic connectivity of the study watersheds. 137Cs inventories computed from the RES watershed indicated that sediment cores located further upstream had the highest inventories. With reference to the JB Estuary, statistical analysis showed that location had an effect on distribution of Cs in surface samples within the bay. Sedimentological characteristics varied between cores. The implementation of the conceptual model in both study watersheds allowed for the identification of potential source areas of sediments that were available for transport and delivery to adjacent aquatic systems. This investigation revealed that to link soil loss to sediment delivery it is essential that key processes and variables (rainfall, soils, LULC and geomorphology) must be included in the analysis. This conceptual model may be a valuable tool for monitoring and managing soil loss within the watershed and consequently, addressing problems of increased sediment delivery to aquatic and transitional marine ecosystems such as estuaries.

As increasing emphasis is placed upon the role surface processes play in regulating tectonic behaviour, the need for accurate measurements of denudation rate has become paramount. The quantity and quality of denudation rate studies has grown with the advent of cosmogenic radionuclide techniques, capable of recording denudation rates over timescales of 100 to 1000000 years. This study seeks to utilise cosmogenic 10Be concentrations measured in alluvial sediments in order to further develop this method and to investigate rates of basin-wide denudation in the San Bernardino Mountains, an active orogen associated with the San Andreas Fault system. The theory which underpins measurements of basin-wide denudation rates with cosmogenic radionuclide analysis is evaluated in light of recent understanding of production mechanisms. Field testing of the assumptions required by the basinwide denudation rate model highlights the importance of sampling thoroughly mixed sediments. Denudation rates ranging over three orders of magnitude are measured by applying the cosmogenic radionuclide technique in thirty-seven basins throughout the San Bernardino Mountains. Results show a relationship between denudation rate and slope which provides quantification of the threshold slope angle in high relief granitic environments and suggests tectonic activity is the first order control of denudation rates in these mountains. Mean annual precipitation is shown to exert no significant influence over the rates measured in the San Bernardino Mountains. Questions concerning denudation rates recorded over differing timespans are addressed using the cosmogenic technique, (U-Th)/He thermochronometry, incision into dated horizons and modern day sediment flux data. This comparison reveals that a decrease in rates with distance from the San Andreas Fault has been consistent throughout the lifespan of the San Bernardino Mountains and provides further evidence that a tectonic mechanism is driving denudation in this region. The relevance of both spatial and temporal scale in geomorphic studies is considered in light of these results, highlighting the need for a greater appreciation of their role in the interpretation of basin-wide denudation rates.

This thesis focuses on the evaluation of biomarkers for radio-immunodiagnostics and radio-immunotherapy and on radiosensitization strategies after HSP90 inhibition, as a step towards more personalized cancer medicine. There is a need to develop new tracers that target cancer-specific biomarkers to improve diagnostic imaging, as well as to combine treatment strategies to potentiate synergistic effects. Special focus has been on the cell surface molecule CD44 and its oncogenic variants, which were found to exhibit unique expression patterns in head and neck squamous cell carcinoma (HNSCC). The variant CD44v6 seems to be a promising target, because it is overexpressed in this cancer type and is associated with radioresistance. Two new radioconjugates that target CD44v6, namely, the Fab fragment AbD15179 and the bivalent fragment AbD19384, were investigated with regard to specificity, biodistribution and imaging performance. Both conjugates were able to efficiently target CD44v6-positive tumors in vitro and in vivo. PET imaging of CD44v6 with 124I-AbD19384 revealed many advantages compared with the clinical standard 18F-FDG. Furthermore, the efficacy of the novel HSP90 inhibitor AT13387 and its potential use in combination with radiation treatment were evaluated. AT13387 proved to be a potent new cancer drug with favorable pharmacokinetics. Synergistic combination effects at clinically relevant drug and radiation doses are promising for both radiation dose reduction and minimization of side effects, or for an improved therapeutic response. The AT13387 investigation indicated that CD44v6 is not dependent on the molecular chaperone HSP90, and therefore, radio-immunotargeting of CD44v6 in combination with the HSP90 inhibitor AT13387 might potentiate treatment outcomes. However, EGFR expression levels did correlate with HSP90 inhibition, and therefore, molecular imaging of EGFR-positive tumors may be used to assess the treatment response to HSP90 inhibitors. In conclusion, these results demonstrate how tumor targeting with radiolabeled vectors and chemotherapeutic compounds can provide more specific and sensitive diagnostic tools and treatment options, which can lead to customized treatment decisions and a functional diagnosis that provides more precise and safer drug prescribing, as well as a more effective treatment for each patient.

iThemba LABS is a particle accelerator facility housing a radionuclide production facility that uses a 66 MeV proton beam to produce radionuclides for medical and industrial use. Ionising radiation is produced by a variety of sources at Themba LABS. Ionising is a health hazard. High doses can cause acute radiation syndrome, i.e. "radiation sickness". Lower doses cannot cause acute symptom, but carry a risk of radiation-related cancer. Ionising radiation is also detrimental to materials, and can damage polymers and lubricants e.g. Shielding is used to reduce radiation levels to values that should be safe for the intended level of human occupancy. Shield performance is vital to human health and the life expectancy of polymers and lubricants, so that quality management in shield design is very important. However, until 2003, there has been no formal system at iThemba LABS to evaluate and improve all radiation shield designs and layouts from a radiation protection perspective. This study deals with evaluating and redesigning radiation shielding in the radionuclide production facility of iThemba LABS. There are several designs and layouts in the radionuclide production building of iThemba LABS that lead to unnecessary exposure of personnel to ionising radiation. The shielding in these areas are sub-standard. Performance criteria for radiation shields are developed. Inadequate radiation shields are identified. The identified inadequate shields are: the processing hotcells, the target store room and the hotcells in the radiopharmaceutical dispensing laboratory, Point-Kernel radiation shielding calculations are done to specify materials and material thickness that will adequately protect workers against ionising radiation. / Thesis (MSc. ARST) North-West University, Mafikeng Campus, 2004

Background: In an ongoing study conducted at Karolinska Institutet &amp; Karolinska University Hospital, Positron Emission Tomography (PET)/Computed Tomography (CT) scans are performed on patients with tibia fractures and deformations treated with Taylor Spatial Frames (TSFs) in order to monitor their bone remodeling progress. Each patients receive an administration of approximately 2 MBq/kg bodyweight of Na18F associated with PET scans on two sessions, six and twelve weeks after the attachment of the TSF. These PET/CT scans provide information about the progress of the healing bone and can be used to estimate the optimal time point for de-attachment of the TSF. The Standardized Uptake Value (SUV) is used as a measure of the rate of bone remodeling for these patients, however, there is a need for verification of this practice by a method independent of the PET scanner. Furthermore, information regarding the biodistribution of the Na18F throughout the body of these patients and the effects of the TSF on the CT scan X‑rays is required. Additionally, an investigation of alternative methods that have the potential to provide similar information with a lower absorbed dose to the patients is desirable. Materials and methods: Thermoluminescent Dosimeters (TLDs) were attached on the skin at the position of the heart, urinary bladder, femurs, fracture, and the contralateral tibia of twelve patients during the first one hour and five minutes after the administration of the Na18F. Additional TLD measurements were performed during the CT scan of two of these patients. From the PET scan images, SUVs at the fracture site of these patients were collected. An investigation of the possibility of exploiting the “naturally” occurring bone seeking radionuclide Strontium-90 (90Sr) in the human body to gain information about the fracture site was undertaken. Using a 90Sr source, three different detection techniques were evaluated and a practical methodology for in vivo measurements on the tibia fracture patients was developed. As it was concluded that TLD based measurements were the most suitable technique for this purpose, and it was tested on five patients with tibia fractures. Results: From the collected TLD data, it was concluded that for these patients the urinary bladder is the organ receiving the greatest amount of absorbed dose and the organ most affected as the administered activity exceeds 2 MBq/kg. On average, a three times higher surface dose was measured on the tibia fracture compared to the un-fractured tibia. A linear relationship between the surface dose and SUVmax was shown. A strong positive correlation between the activity concentration at the fracture site and the amount of injected activity was found, and it was demonstrated that this also affects the SUVs. For patients who were administered different amounts of Na18F for the two PET scans, maximum activity concentrationwas less affected than mean activity concentration. It was concluded that TSF’s effect on the scatter of the X-rays to organs higher up in the body is negligible. Regarding “naturally” occurring 90Sr in the human body, no higher activity at the fractured tibia compared to the non‑fractured tibia could be found. Conclusions: This project assessed the accumulation of Na18F in the fracture site of patients treated with TSF by a method independent of the PET scanner. The methodology of using SUVs as an indicator for bone remodeling was verified. It was shown that the uptake of Na18F by the fracture site is strongly correlated to the amount of injected activity. The importance of considering the amount of injected activity when evaluating and comparing SUVs was highlighted. In vivo measurements using LiF:Mn TLDs did not indicate any quantifiable higher concentration of 90Sr at the fracture in the tibia bone.

No description available.

The growing understanding of tumor biology and the identification of tumor specificgenetic and molecular alterations, such as the overexpression of human epidermal growthfactor receptor 2 (HER2), opens up for personalization of patient management using targeted therapies. However, this puts stringent demands on the diagnostic tools usedto identify patients that are likely to respond to a particular treatment. Radionuclide molecular imaging is a promising non-invasive method to visualize and characterize the expression of such targets. This thesis, based on five papers, is focused on the development of radiolabeled Affibody molecules for imaging of HER2-expression in malignant tumors. Affibody molecules, which represent a rather novel class of affinity proteins developed by combinatorial protein engineering of the protein A derived Z-domain, display manyfeatures that make them promising tracers for molecular imaging applications. The aim of the work presented here was to further develop the tracer format for improved in vivo properties and flexibility in the choice of radionuclide. In paper I, the development of an assay that enables quantitative studies of the internalization rate and cellular processing of high affinity Affibody molecules is described. The assay was applied to a HER2-binding Affibody variant that was efficiently retained by HER2-expressing cells, although characterized by a slow internalization rate. This may have implications for the choice of label for Affibody molecules since high affinity to the target may be equally, or more, important for good imaging quality than residualizing properties of the radiolabel. In paper II, a HER2-binding Affibody molecule and the monoclonal antibody trastuzumab were labeled with positron emitting 124I, for a head-to-head in vivocomparison of the two tracer formats. The effects of tracer size and presence of an Fc region on the biodistribution profile were investigated. In paper III, a HER2-binding Affibody molecule was site-specifically labeled with radiocobalt and evaluated in vitro and in vivo.A head-to-head in vivo comparison with the well-studied 111In-labeled counterpart was performed, revealing promising potential for the cobalt-labeled molecule as a PET-tracerfor visualization of HER2. Paper IV describes the in vitro and in vivo evaluation of a panel of Affibody molecules with different C-terminal peptide-based chelators for the coordination of 99mTc. Even small changes in the C-terminal sequence had appreciable impact on the biodistribution of the Affibody molecules and by optimizing the design of the chelator, the kidney uptake of 99mTc could be significantly reduced. Finally, in paper V we describe the development of a HER2-targeting Affibody variant equipped with a Sel-tag for site-specific labeling with the short-lived positron emitter 11C. This novel Affibody tracer could be used to image HER2-expressing tumors in vivo within one hour after injection. Taken together, Affibody molecules show great promise as targeting tracers for radionuclide molecular imaging of HER2. Careful design and optimization of the tracer protein is important and can be used to improve the biodistribution and targeting properties of Affibody molecules. / QC 20110922