Developing nanoparticulate delivery systems that will allow easy movement and localisation of a drug to the target tissue and provide more controlled release of the drug in vivo is a challenge for researchers in nanomedicine. The aim of this study was to evaluate the biodistribution of two nano-delivery systems namely, poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles containing samarium-153 oxide ([153Sm]Sm2O3) as radiotracer and solid lipid nanoparticles (SLNs) containing technetium-99m-methylene diphosphonate (99mTc-MDP), after oral and intravenous administration to rats to prove that orally administered nanoparticles indeed alter the biodistribution of a drug as compared to the drug on its own. Stable samarium-152 oxide ([152Sm]Sm2O3) was encapsulated in polymeric PLGA nanoparticles. These were then activated in a nuclear reactor to produce radioactive [153Sm]Sm2O3 loaded-PLGA nanoparticles. Both the stable nanoparticles as well as the fully decayed activated nanoparticles, were characterized for size, Zeta potential and morphology using dynamic light scattering and scanning electron microscopy (SEM) or transmission electron microscopy (TEM), respectively. SLNs were a form of delivery system which was used to encapsulate the radiotracer, 99mTc-MDP. 99mTc-MDP SLNs were characterized before and after encapsulation for size and Zeta potential. Both nanoparticle compounds were orally and intravenously (IV) administered to rats in order to trace their uptake and biodistribution through imaging and ex vivo biodistribution studies. The PLGA nanoparticles containing [153Sm]Sm2O3 were spherical in morphology and smaller than 500 nm, therefore meeting the objective of producing radiolabelled nanoparticles smaller than 500 nm. Various parameters were optimized to obtain an average particle size ranging between 250 and 300 nm, with an average polydispersity index (PDI) ≤ 0.3 after spray drying. The particles had a Zeta potential ranging between 5 and 20 mV. The Sm2O3-PLGA nanoparticles had an average size of 281 ± 6.3 nm and a PDI average of 0.22. The orally administered [153Sm]Sm2O3-PLGA nanoparticles were deposited in various organs which includes bone with a total of 0.3% of the Injected Dose (ID) per gram vs the control of [153Sm]Sm2O3which showed no uptake in any organs except the GI-tract. The IV injected [153Sm]Sm2O3-PLGA nanoparticles exhibit the highest localisation of nanoparticles in the spleen (8.63%ID/g) and liver (3.07%ID/g). The 99mTc-MDP-labelled SLN were spherical and smaller than 500 nm. Optimization of the MDP-loaded SLN emulsions yielded a slightly higher PDI of ≥0.5 and a size range between 150 and 450 nm. The Zeta potential was between -30 and -2 mV. The MDP-loaded SLN had an average size of 256 ± 5.27 and an average PDI of 0.245.The orally administered 99mTc-MDP SLN had the highest localisation of nanoparticles in the kidneys (8.50%ID/g) and stomach (8.04%ID/g) while the control, 99mTc-MDP had no uptake in any organs except the GI-tract. The IV injected 99mTc-MDP SLN also exhibited a high localisation of particles in the kidneys (3.87%ID/g) followed by bone (2.66%ID/g). Both the IV and oral 99mTc-MDP SLN reported significantly low deposition values in the heart, liver and spleen. Based on the imaging and the biodistribution studies, it can be concluded that there was a significant transfer of the orally administrated radiolabelled nanoparticles from the stomach to other organs vs the controls. Furthermore, this biodistribution of the nano carriers warrants surface modification and optimisation of the nanoparticles to avoid higher particle localisation in the stomach. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Previous studies have shown that the formulation of an active pharmaceutical ingredient (API) entrapped in the Pheroid® (Pheroid for simplification) delivery system enhances absorption of the API, suppresses its metabolism, and may contribute to an increase in the quantity of the API present at the site of action. Higher drug levels at the active site should particularly increase the effectiveness of a drug with a narrow therapeutic index and reduce the incidence of the resistance that may otherwise arise if the sub-therapeutic levels of the API are in contact with the site of interest. Two approaches were followed in this study. First, the radioactive tracer molecule 99mTechnetium methylene diphosphonate (99mTc MDP) was used. Intravenously injected 99mTc MDP is an extremely effective bone-seeking radiopharmaceutical used in the diagnosis of bone disorders such as bone metastases in patients. However, if entrapped inside a Pheroid vesicle, it will locate to that site, usually an organ, where the Pheroid vesicles may tend to accumulate. Experiments conducted with 99mTc MDP alone or with Pheroid will therefore establish how efficiently Pheroid vesicles localize and will also indicate the preferred site of localization inside a body. The process would involve the oral administration of 99mTc MDP either alone or with Pheroid, involving an animal model. It would also involve tracking localization to particular organs, blood or other sites. The second approach requires the use of chloroquine (CQ) labeled with carbon-14 (14C-CQ,) to compare absorption of the drug both with and without the Pheroid system. The intention was to compare oral absorption and bio-distribution of 14C-CQ administered either alone or entrapped in the Pheroid system. It was also possible to establish whether the Pheroid affects the biological half-lives of the CQ and residence times of CQ in the different organs of the body. Absorption of free 99mTc MDP (orally adminsistered) through the intestinal tract is negligible but it was anticipated that increased absorption will be observed when 99mTc MDP was entrapped in the Pheroid system. In the 99mTc MDP study, different routes of administration of 99mTc MDP, as well as 99mTc MDP entrapped and not entrapped in the Pheroid system, were investigated. The Sprague Dawley rat was used as animal model. Rats were divided into three groups of four rats each for the first part of the study. In the first group, only 99mTc MDP was injected intravenously in order to establish natural distribution of the 99mTc MDP. For the second group, 99mTc MDP was administered orally in order to establish whether there was any absorption through the intestinal tract. In the third group, the 99mTc MDP was entrapped in Pheroid vesicles and this formulation was administered orally in order to establish whether the Pheroid system enhanced oral absorption. The animals were sacrificed four hours after administration and organs were harvested and were counted for radioactivity to determine the percentage of injected/administrated dose in each organ. After oral administration, the Pheroid system was found to have facilitated absorption of 99mTc MDP through the intestinal tract into the blood. 99mTc MDP concentrations in the femur, although lower, were still comparable with that observed after intravenous administration of 99mTc MDP in the absence of Pheroid. Thus, overall, excellent absorption of the Pheroid entrapped 99mTc MDP through the intestinal tract was seen in contrast to little or zero absorption of the compound in the reference formulations. The half-life of the radio-labelled compound in the blood was prolonged after oral administration owing to the Pheroid. To investigate the bio-distribution of radioactive chloroquine (14C-CQ) Sprague Dawley rats were divided into two groups of four rats each. In the first group, 14C-CQ in deionised (DI) water was administered orally, and in the second group 14C-CQ entrapped in Pheroid vesicles was administered, also orally. The animals were sacrificed one, two and four hours after administration and subjected to comprehensive macroscopic inspection. All the organs were harvested and radioactivity was determined with liquid scintillation after applicable sample preparation. The Pheroid system produced much higher organ and blood concentrations of 14C-CQ and enhanced residence times within the organs and blood in comparison with that of 14C-CQ administered alone. Commercial applications of these results are possible, as a number of radiopharmaceutical products can presently be administered only intravenously. The added potential of these new Pheroid formulations could be of significance in the treatment of malaria, as chloroquine is inexpensive and widely available. Another point of interest is that the use of these formulations may enable micromolar drug concentrations to be achieved using drug dosage regimes that usually produce only nanomolar levels. However, safety aspects would have to be carefully monitored. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

Developing nanoparticulate delivery systems that will allow easy movement and localisation of a drug to the target tissue and provide more controlled release of the drug in vivo is a challenge for researchers in nanomedicine. The aim of this study was to evaluate the biodistribution of two nano-delivery systems namely, poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles containing samarium-153 oxide ([153Sm]Sm2O3) as radiotracer and solid lipid nanoparticles (SLNs) containing technetium-99m-methylene diphosphonate (99mTc-MDP), after oral and intravenous administration to rats to prove that orally administered nanoparticles indeed alter the biodistribution of a drug as compared to the drug on its own. Stable samarium-152 oxide ([152Sm]Sm2O3) was encapsulated in polymeric PLGA nanoparticles. These were then activated in a nuclear reactor to produce radioactive [153Sm]Sm2O3 loaded-PLGA nanoparticles. Both the stable nanoparticles as well as the fully decayed activated nanoparticles, were characterized for size, Zeta potential and morphology using dynamic light scattering and scanning electron microscopy (SEM) or transmission electron microscopy (TEM), respectively. SLNs were a form of delivery system which was used to encapsulate the radiotracer, 99mTc-MDP. 99mTc-MDP SLNs were characterized before and after encapsulation for size and Zeta potential. Both nanoparticle compounds were orally and intravenously (IV) administered to rats in order to trace their uptake and biodistribution through imaging and ex vivo biodistribution studies. The PLGA nanoparticles containing [153Sm]Sm2O3 were spherical in morphology and smaller than 500 nm, therefore meeting the objective of producing radiolabelled nanoparticles smaller than 500 nm. Various parameters were optimized to obtain an average particle size ranging between 250 and 300 nm, with an average polydispersity index (PDI) ≤ 0.3 after spray drying. The particles had a Zeta potential ranging between 5 and 20 mV. The Sm2O3-PLGA nanoparticles had an average size of 281 ± 6.3 nm and a PDI average of 0.22. The orally administered [153Sm]Sm2O3-PLGA nanoparticles were deposited in various organs which includes bone with a total of 0.3% of the Injected Dose (ID) per gram vs the control of [153Sm]Sm2O3which showed no uptake in any organs except the GI-tract. The IV injected [153Sm]Sm2O3-PLGA nanoparticles exhibit the highest localisation of nanoparticles in the spleen (8.63%ID/g) and liver (3.07%ID/g). The 99mTc-MDP-labelled SLN were spherical and smaller than 500 nm. Optimization of the MDP-loaded SLN emulsions yielded a slightly higher PDI of ≥0.5 and a size range between 150 and 450 nm. The Zeta potential was between -30 and -2 mV. The MDP-loaded SLN had an average size of 256 ± 5.27 and an average PDI of 0.245.The orally administered 99mTc-MDP SLN had the highest localisation of nanoparticles in the kidneys (8.50%ID/g) and stomach (8.04%ID/g) while the control, 99mTc-MDP had no uptake in any organs except the GI-tract. The IV injected 99mTc-MDP SLN also exhibited a high localisation of particles in the kidneys (3.87%ID/g) followed by bone (2.66%ID/g). Both the IV and oral 99mTc-MDP SLN reported significantly low deposition values in the heart, liver and spleen. Based on the imaging and the biodistribution studies, it can be concluded that there was a significant transfer of the orally administrated radiolabelled nanoparticles from the stomach to other organs vs the controls. Furthermore, this biodistribution of the nano carriers warrants surface modification and optimisation of the nanoparticles to avoid higher particle localisation in the stomach. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Previous studies have shown that the formulation of an active pharmaceutical ingredient (API) entrapped in the Pheroid® (Pheroid for simplification) delivery system enhances absorption of the API, suppresses its metabolism, and may contribute to an increase in the quantity of the API present at the site of action. Higher drug levels at the active site should particularly increase the effectiveness of a drug with a narrow therapeutic index and reduce the incidence of the resistance that may otherwise arise if the sub-therapeutic levels of the API are in contact with the site of interest. Two approaches were followed in this study. First, the radioactive tracer molecule 99mTechnetium methylene diphosphonate (99mTc MDP) was used. Intravenously injected 99mTc MDP is an extremely effective bone-seeking radiopharmaceutical used in the diagnosis of bone disorders such as bone metastases in patients. However, if entrapped inside a Pheroid vesicle, it will locate to that site, usually an organ, where the Pheroid vesicles may tend to accumulate. Experiments conducted with 99mTc MDP alone or with Pheroid will therefore establish how efficiently Pheroid vesicles localize and will also indicate the preferred site of localization inside a body. The process would involve the oral administration of 99mTc MDP either alone or with Pheroid, involving an animal model. It would also involve tracking localization to particular organs, blood or other sites. The second approach requires the use of chloroquine (CQ) labeled with carbon-14 (14C-CQ,) to compare absorption of the drug both with and without the Pheroid system. The intention was to compare oral absorption and bio-distribution of 14C-CQ administered either alone or entrapped in the Pheroid system. It was also possible to establish whether the Pheroid affects the biological half-lives of the CQ and residence times of CQ in the different organs of the body. Absorption of free 99mTc MDP (orally adminsistered) through the intestinal tract is negligible but it was anticipated that increased absorption will be observed when 99mTc MDP was entrapped in the Pheroid system. In the 99mTc MDP study, different routes of administration of 99mTc MDP, as well as 99mTc MDP entrapped and not entrapped in the Pheroid system, were investigated. The Sprague Dawley rat was used as animal model. Rats were divided into three groups of four rats each for the first part of the study. In the first group, only 99mTc MDP was injected intravenously in order to establish natural distribution of the 99mTc MDP. For the second group, 99mTc MDP was administered orally in order to establish whether there was any absorption through the intestinal tract. In the third group, the 99mTc MDP was entrapped in Pheroid vesicles and this formulation was administered orally in order to establish whether the Pheroid system enhanced oral absorption. The animals were sacrificed four hours after administration and organs were harvested and were counted for radioactivity to determine the percentage of injected/administrated dose in each organ. After oral administration, the Pheroid system was found to have facilitated absorption of 99mTc MDP through the intestinal tract into the blood. 99mTc MDP concentrations in the femur, although lower, were still comparable with that observed after intravenous administration of 99mTc MDP in the absence of Pheroid. Thus, overall, excellent absorption of the Pheroid entrapped 99mTc MDP through the intestinal tract was seen in contrast to little or zero absorption of the compound in the reference formulations. The half-life of the radio-labelled compound in the blood was prolonged after oral administration owing to the Pheroid. To investigate the bio-distribution of radioactive chloroquine (14C-CQ) Sprague Dawley rats were divided into two groups of four rats each. In the first group, 14C-CQ in deionised (DI) water was administered orally, and in the second group 14C-CQ entrapped in Pheroid vesicles was administered, also orally. The animals were sacrificed one, two and four hours after administration and subjected to comprehensive macroscopic inspection. All the organs were harvested and radioactivity was determined with liquid scintillation after applicable sample preparation. The Pheroid system produced much higher organ and blood concentrations of 14C-CQ and enhanced residence times within the organs and blood in comparison with that of 14C-CQ administered alone. Commercial applications of these results are possible, as a number of radiopharmaceutical products can presently be administered only intravenously. The added potential of these new Pheroid formulations could be of significance in the treatment of malaria, as chloroquine is inexpensive and widely available. Another point of interest is that the use of these formulations may enable micromolar drug concentrations to be achieved using drug dosage regimes that usually produce only nanomolar levels. However, safety aspects would have to be carefully monitored. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

Two novel synthetic routes to formation of gold-magnetite nanoparticles have been designed. Treatment of preformed magnetite nanoparticles with ultrasound in aqueous media with dissolved tetrachloroauric acid resulted in the formation of gold-magnetite nanocomposite materials. The other route involved irradiation of preformed magnetite nanoparticles by UV light in aqueous media with dissolved tetrachloroauric acid. This method resulted in the formation of gold-magnetite nanocomposite materials. These materials maintained the morphology of the original magnetite particles. The morphology of the gold particles could be controlled by adjusting experimental parameters, like addition of small amounts of solvent modifiers such as methanol, diethylene glycol, and oleic acid as well as variation of the concentration of the tetrachloroauric acid solution and time of the reaction. The nanocomposite materials were magnetic and exhibited optical properties similar to gold nanoparticles. Since we were not able to directly synthesize core shell gold magnetite nanoparticles, TiO2 was used as a bridging material. TiO2 nanoparticles with embedded magnetite were suspended in aqueous HAuCl4 and irradiated with ultraviolet light to photodeposit gold. The degree of gold coating and the wavelength of absorbance could be controlled by adjusting concentration of HAuCl4. Absorbance maxima were between 540-590 nm. Particles exhibited superparamagnetic properties (blocking temperature ~170 K) whether or not coated with gold. These particles have potential applications as drug delivery agents, magnetic imaging contrast agents, and magnetically separatable photocatalysts with unique surface properties. Another goal was to synthesize and characterize indium doped magnetite nanoparticles for application as radiotracers for in vivo fate studies. The labeled particles will be useful for determination of pharmacological behavior in biological systems. Indium doped magnetite particles with varying size and surface chemistry were synthesized with wet chemical techniques. The synthesized nanoparticles were characterized in terms of the size and shape with the help of TEM, the elemental composition by ICP and EDS, the crystal structure by XRD and magnetic properties by SQUID measurements. It was found that the indium loading could be controlled even though the magnetic properties were similar to undoped magnetite.

Le mélanome cutané est un cancer très invasif, dont l'évolution est rapidement fatale au stade métastatique du fait d'une absence de thérapies réellement efficaces. Face à ce constat, une stratégie de multimodalité a été évaluée au moyen d'un vecteur spécifique des cellules de mélanome et présentant la double potentialité diagnostique (imagerie TEP) et thérapeutique (radiothérapie interne vectorisée (RIV)) en fonction du radioélément introduit. Dans ce but, des analogues iodés et fluorés d'ICF01012, un vecteur ciblant la mélanine surexprimée dans un grand nombre de mélanomes, ont été synthétisés puis radiomarqués (iode-125 d'une part et fluor-18 d'autre part). Les profils pharmacocinétiques des radiotraceurs ont par la suite été étudiés in vivo sur modèle murin de mélanome, par imagerie scintigraphique γ (125I) et par imagerie TEP (18F). A l'issue de ces études, le traceur 4 a été sélectionné comme molécule leader de cette série, en raison d'un tropisme tumoral spécifique, élevé et durable, associé à élimination rapide des tissus non-cibles. Ce composé a ensuite été radiomarqué à l'iode-131 pour une évaluation en RIV sur le même modèle murin. Le traitement avec [131I]4 a induit une inhibition significative de la croissance tumorale et une augmentation significative de la médiane de survie. Au vu des résultats prometteurs obtenus avec le traceur 4, des études de métabolisme sur le même modèle murin ont été effectuées. En conclusion, en termes de chimie, radiochimie, stabilités in vitro et in vivo, TEP et RIV, le traceur 4 a validé notre concept de multimodalité. A terme, ce composé pourrait être transféré pour des études cliniques afin : de sélectionner les patients présentant des lésions de mélanome pigmentées (18F/TEP) et donc susceptibles de répondre au protocole de RIV ; de traiter ces patients (131I/RIV) ; de suivre la réponse au traitement (18F/TEP). En parallèle a été développé le premier groupement prosthétique iodé et fluoré multimodal, le 4-fluoro-3-iodobenzoate de tétrafluorophényle 108, pour le marquage de vecteurs d'intérêt (peptides, protéines, nanoparticules…). Le composé 108 et les précurseurs de radiomarquages correspondants ont été synthétisés en peu d'étapes, avec de bons rendements. Les radiomarquages ont ensuite été mis au point, et ont permis d'obtenir [125I]108 et [18F]108 en des temps relativement courts, avec d'excellents rendements et puretés radiochimiques. Le groupement prosthétique a ensuite été couplé avec succès à un ligand organique et deux peptides. En conclusion, nous avons démontré que le composé multimodal 108 peut être utilisé pour la radiofluoration et la radioiodation de vecteurs d'intérêt, autorisant des applications à la fois diagnostiques (TEP/18F) et thérapeutiques (RIV/131I) pour la prise en charge de nombreux cancers. / Melanoma is the most serious form of skin cancer with a poor prognosis for patients with metastatic disease. Our project deals with a multimodal approach, using a single fluorinated and iodinated melanintargeting compound, and offering both imaging (PET/18F) and therapeutic (targeted radionuclide therapy (TRT)/131I) applications, depending on the radioisotope introduced. Furthermore, using PET imaging, our strategy allows a selection of TRT-responded patients (i.e. bearing pigmented metastases) as well as a monitoring of treatment response. In previous works, the iodinated quinoxaline-carboxamide compound ICF01012 was evaluated in a TRT protocol, using melanoma-bearing mice models. It demonstrated efficacy, with significant inhibition of tumoural growth and improvement of the median survival. Based on these results, iodinated and fluorinated analogs of ICF01012 were synthesized, for multimodality purposes. All new compounds were then radiolabelled with iodine-125 and fluorine-18 (fully automated radiosyntheses), with good radiochemical yields and excellent radiochemical purities. For pharmacokinetic profile studies on melanoma-bearing mice, [125I]radiotracers were evaluated by γ-scintigraphy and [18F]radiotracers by PET. Compound 4 emerged as the lead tracer, with a specific and long-lasting tumoural uptake and a fast clearance from non-specific tissues, leading to highly contrasted images. The tracer 4 was then radiolabelled with iodine-131, with excellent radiochemical yield and purity, to perform a TRT assay on the same melanoma model. Treatment with [131I]4 significantly inhibited tumoural growth and lung metastasis occurrence. Moreover, it significantly improved the median survival. As tracer 4 demonstrated promising results in PET imaging and TRT of melanoma, its metabolism was investigated with [125I]4 and [18F]4: radiotracers were found unchanged in melanin-containing tissues (tumour and eyes), while a fast breakdown was observed in excretion organs and fluids (four metabolites were identified). In conclusion, in terms of chemistry, radiochemistry, in vitro and in vivo stability, PET imaging and TRT, compound 4 validated our multimodality concept. We also developed the first bimodal fluorinated and iodinated prosthetic group, the 2,3,5,6-tetrafluorophenyl 4-fluoro-3-iodobenzoate (108), as a suitable acylating agent for the labelling of a large variety of compounds. In this approach, this new compound allows applications in diagnosis (PET imaging/18F) and therapy (TRT/131I). Compound 108 and its corresponding precursors for radiolabelling were synthesized in very few steps, with good yields. [125I]108 was then prepared in one-step starting from a perfluorostannane precursor, and purified by F-SPE cartridge to avoid time-consuming HPLC. As for [18F]108, it was produced by a fully automated three steps, two-pots radiosynthesis process. [125I]108 and [18F]108 were both obtained in a short time, with excellent radiochemical yields and purities. These prosthetic groups were then successfully used to radiolabel small organic ligand N,N-diethylethylenediamine and peptides NDP-MSH and PEG3[c(RGDyK)]2, in mild conditions, with good yields. In conclusion, we demonstrated that compound 108 could be a promising acylating bimodal prosthetic group for radiofluorination and radioiodination of small organic molecules, peptides, proteins, antibodies as well as nanoparticles.

<p>Over the past 200 years, the Earth's atmospheric carbon dioxide (CO₂) concentration has increased by more than 35%, and climate experts predict that CO₂ levels may double by the end of this century. Understanding the mechanisms of resource management in plants is fundamental for predicting how plants will respond to the increase in atmospheric CO₂. Plant productivity sustains life on Earth and is a principal component of the planet's system that regulates atmospheric CO₂ concentration. As such, one of the central goals of plant science is to understand the regulatory mechanisms of plant growth in a changing environment. Short-lived positron-emitting radiotracer techniques provide time-dependent data that are critical for developing models of metabolite transport and resource distribution in plants and their microenvironments. To better understand the effects of environmental changes on resource transport and allocation in plants, we have developed a system for real-time measurements of metabolite transport in plants using short-lived positron-emitting radiotracers. This thesis project includes the design, construction, and demonstration of the capabilities of this system for performing real-time measurements of metabolite transport in plants.</p><p>The short-lived radiotracer system described in this dissertation takes advantage of the combined capabilities and close proximity of two research facilities at Duke University: the Triangle Universities Nuclear Laboratory (TUNL) and the Duke University Phytotron, which are separated by approximately 100 meters. The short-lived positron-emitting radioisotopes are generated using the 10-MV tandem Van de Graaff accelerator located in the main TUNL building, which provides the capability of producing short-lived positron-emitting isotopes such as carbon-11 (¹¹C; 20 minute half-life), nitrogen-13 (¹³N; 10 minute half-life), fluorine-18 (¹⁸F; 110 minute half-life), and oxygen-15 (¹⁵O; 2 minute half-life). The radioisotopes may be introduced to plants as biologically active molecules such as ¹¹CO₂, ¹³NO₃^-, ¹⁸F^--[H₂O], and H₂<sup>15<\sup>O. Plants for these studies are grown in controlled-environment chambers at the Phytotron. The chambers offer an array of control for temperature, humidity, atmospheric CO₂ concentration, and light intensity. Additionally, the Phytotron houses one large reach-in growth chamber that is dedicated to this project for radioisotope labeling measurements.</p><p>There are several important properties of short-lived positron-emitting radiotracers that make them well suited for use in investigating metabolite transport in plants. First, because the molecular mass of a radioisotope-tagged compound is only minutely different from the corresponding stable compound, radiotracer substances should be metabolized and transported in plants the same as their non-radioactive counterparts. Second, because the relatively high energy gamma rays emitted from electron-positron annihilation are attenuated very little by plant tissue, the real-time distribution of a radiotracer can be measured <em>in vivo</em> in plants. Finally, the short radioactive half-lives of these isotopes allow for repeat measurements on the same plant in a short period of time. For example, in studies of short-term environmental changes on plant metabolite dynamics, a single plant can be labeled multiple times to measure its responses to different environmental conditions. Also, different short-lived radiotracers can be applied to the same plant over a short period of time to investigate the transport and allocation of various metabolites.</p><p>This newly developed system provides the capabilities for production of ¹¹CO₂ at TUNL, transfer of the ¹¹CO₂ gas from the target area at TUNL to a radiation-shielded cryogenic trap at the Phytotron, labeling of photoassimilates with ¹¹C, and <em>in vivo</em> gamma-ray detection for real-time measurements of the radiotracer distribution in small plants. The experimental techniques and instrumentation that enabled the quantitative biological studies reported in this thesis were developed through a series of experiments made at TUNL and the Phytotron. Collimated single detectors and coincidence counting techniques were used to monitor the radiotracer distribution on a coarse spatial scale. Additionally, a prototype Versatile Imager for Positron Emitting Radiotracers (VIPER) was built to provide the capability of measuring radiotracer distributions in plants with high spatial resolution (~2.5 mm). This device enables detailed quantification of real-time metabolite dynamics on fine spatial scales.</p><p>The full capabilities of this radiotracer system were utilized in an investigation of the effects of elevated atmospheric CO₂ concentration and root nutrient availability on the transport and allocation of recently fixed carbon, including that released from the roots via exudation or respiration, in two grass species. The ¹¹CO₂ gas was introduced to a leaf on the plants grown at either ambient or elevated atmospheric CO₂. Two sequential measurements were performed per day on each plant: a control nutrient solution labeling immediately followed by labeling with a 10-fold increase or decrease in nutrient concentration. The real-time distribution of ¹¹C-labeled photoassimilate was measured <em>in vivo</em> throughout the plant and root environment. This measurement resulted in the <em>first</em> observation of a rapid plant response to short-term changes in nutrient availability via correlated changes in the photoassimilate allocation to root exudates. Our data indicated that root exudation was consistently enhanced at lower nutrient concentrations. Also, we found that elevated atmospheric CO₂ increased the velocity of photoassimilate transport throughout the plant, enhanced root exudation in an annual crop grass, and reduced root exudation in a perennial native grass.</p> / Dissertation

Les mécanismes physiopathologiques liés aux maladies neurodégénératives restent encore largement méconnus. Deux processus semblent être particulièrement en cause dans les phénomènes de neurodégénérescence : la neurotoxicité par afflux massif de calcium due à une activation excessive des récepteurs NMDA (GluN) et la neurotoxicité par déstabilisation du cytosquelette du neurone par le biais de la phosphorylation anormale de la protéine tau. L'imagerie moléculaire par le biais de la tomographie par émission de positons (TEP) et de radiotraceurs, en étudiant les mécanismes moléculaires in vivo, permet de détecter et quantifier ces phénomènes. Ce travail a eu pour objet d'étudier un dérivé de la mémantine, un antagoniste des GluN se fixant sur un site intra-canal accessible uniquement lorsque ces récepteurs sont activés ce qui en fait donc un vecteur d'imagerie intéressant pour étudier leur activation. Nous avons mis au point la synthèse d'un nouveau radiotraceur dérivé de la mémantine : la [18F]-FNM (Fluoroéthylnormémantine). Il s'agit d'une synthèse par substitution nucléophile d'un groupement tosylate par du [18F], suivie d'une hydrolyse acide. Cette synthèse est reproductible avec un rendement de 10%, son activité spécifique est > 355 GBq/µmol. Chez le rat, le traceur passe la barrière hémato-encéphalique et sa distribution cérébrale est bien corrélée avec la localisation des GluN (r=0.622, p<0.0001). Sa cinétique de fixation (40 minutes) est compatible avec son utilisation en TEP. En ce qui concerne les tauopathies, la protéine tau stabilise l'organisation microtubulaire. Lors d'une phosphorylation anormale, l'interaction avec les microtubules diminue et les protéines tau vont s'accumuler en formant des Paires de Filaments en Hélice (PHF). Nous avons optimisé la radiosynthèse de l' [18F]-AV1451 ciblant les PHF. Notre rendement de synthèse est de 30% et l'activité spécifique du traceur > 10 GBq/µmol. Nous avons réalisé des autoradiographies sur des coupes de cerveaux atteints de tauopathie et nous avons constaté la capacité du traceur à différencier les coupes saines des coupes malades. La production de cet outil dans notre centre va nous permettre d'étudier la présence de PHF chez le marmouset, un primate particulièrement intéressant dans l'étude du vieillissement. Nous avons donc réalisé la synthèse de deux radiotraceurs innovants : la [18F]-FNM et le [18F]-AV1451, les synthèses sont reproductibles et les rendements compatibles avec des productions de doses en recherche pré-clinique et clinique. / The pathophysiological mechanisms associated with neurodegenerative diseases remain largely unknown. Two processes appear to be particularly involved in the phenomena of neurodegeneration: neurotoxicity induced by massive influx of calcium caused by excessive activation of NMDA receptors (GluN) and neurotoxicity by destabilization of neuron cytoskeleton through abnormal protein tau phosphorylation. Molecular imaging through positron emission tomography (PET) and radiotracers, by studying the molecular mechanisms in vivo, allows to detect and quantify these phenomena. This work was intended to study a memantine derivative, a GluN antagonist. We chose to develop a ligand that selectively binds to the ion channel in the open and active state which therefore makes it an interesting vector to study their overactivation. We have developed the synthesis of a new memantine analogue radiotracer: the [18F]-FNM (Fluoroéthylnormémantine). This is a synthesis by nucleophilic substitution of a tosylate with [18F], followed by acid hydrolysis. This synthesis is reproducible with a yield of 10%, its specific activity was> 355 GBq / µmol. In rats, the tracer cross the blood-brain barrier and brain distribution correlates well with the location of GluN (r = 0.622, p <0.0001). The binding kinetics (40 minutes) is compatible with its use in PET. Regarding tauopathies, the tau protein stabilizes microtubule organization. During abnormal phosphorylation, interaction with microtubules and tau proteins decreases and tau will accumulate to form Paired helical Filament (PHF). We optimized the radiosynthesis of [18F] AV1451 targeting 3 tau PHF. Our yield of synthesis is 30% and the specific activity of the tracer> 10 GBq / µmol. We made autoradiography on brains sections and have shown tracer ability to differentiate healthy and pathological slices. This tool will allow us to study the presence of PHF in marmosets, a particularly interesting primate in the study of aging. So we performed the synthesis of two innovative radiotracers: the [18F]-FNM and [18F]-AV1451, syntheses are reproducible and yields compatible with doses manufacturing in pre-clinical and clinical research.

Diabetes is a rapidly growing disease with 415 million affected adults worldwide. The pancreatic endocrine cells, most importantly the insulin producing β-cells, play an important role in regulating blood glucose homeostasis. Type 1 diabetes (T1D) is characterized by the inability of the pancreas to secrete sufficient amounts of insulin due to autoimmune destruction of insulin producing β-cells. Type 2 diabetes (T2D) on the other hand is characterized by defects in insulin secretion and insulin sensitivity. Alterations in the β-cell mass (BCM) and/or function play a major role in the development and progression of the disease. Understanding BCM dynamics in disease models is therefore a key aspect for better interpretation of research results. In this thesis, we have used optical projection tomography (OPT) as a tool to evaluate a non-invasive imaging modality for β-cell scoring and to study disease dynamics in frequently used animal models for T1D and T2D. The possibility to monitor BCM in vivo would radically improve our competence in studying the pathogenesis of diabetes and in therapeutic interventions. Single photon emission computed tomography (SPECT) is a widely used technique that has become a promising approach to monitor changes in BCM in vivo. A key issue for using this approach is to evaluate the β-cell specificity and read out of the utilized radiotracers. This is most commonly performed by conventional stereological approaches, which rely on the extrapolation of 2D data. We developed a protocol for SPECT-OPT multimodal imaging that enables rapid and accurate cross evaluation of SPECT based assessments of BCM. While histological determination of islet spatial distribution was challenging, SPECT and OPT revealed similar distribution patterns of the radiotracer 111In-exendin-3 and insulin positive β-cell volumes respectively between different pancreatic lobes, both visually and quantitatively. We propose SPECT-OPT multimodal imaging as an accurate and better approach for validating the performance of β-cell radiotracers. The leptin deficient ob/ob mouse is a widely used model for studies of metabolic disturbances leading to T2D, including obesity and insulin resistance. By OPT imaging we created the first 3D-spatial and quantitative account of BCM distribution in this model. We observed a previously unreported degree of cystic lesions in hypertrophic islets, that were occupied by red blood cells (RBCs) and/or fibrin mesh. We propose that these lesions are formed by a mechanism involving the extravasation of RBCs/plasma due to increased blood flow and islet vessel instability. Further, our data indicate that the primary lobular compartments of the ob/ob pancreas have different potentials for expanding their β-cell population. Unawareness of these characteristics of β-cell expansion in ob/ob mice presented in this study may significantly influence ex vivo and in vivo assessments of this model in studies of β-cell adaptation and function. The tomographic data, on which this study was based, will be made publically available as a resource to the research community for the planning and interpretation of research involving this model. There are limited studies on early metabolic and functional changes of BCM in the settings of T1D. In order to assess initial metabolic alterations in BCM before the onset of diabetes, we characterized congenic diabetes prone Bio-breeding (BB) DR.lyp/lyp rats, a widely used model for T1D diabetes. We observed lower acute insulin response, reduced islet blood flow and a significant reduction in the BCM of small and medium sized islets at a very early stage (40 days), i.e. before insulitis and development of diabetes. Underlying changes in islet function may be a previously unrecognized factor of importance in the development of T1D.

Le diabète est une maladie chronique qui concerne plus de 380 millions de personnes dans le monde. Le diabète de type 1, qui représente 10 à 15% des cas, et le diabète de type 2, qui représente 85 à 90% des formes de diabète, ont pour caractéristique commune une diminution anatomique et/ou fonctionnelle de la masse de cellules bêta (MCB). L’estimation de la MCB des patients diabétiques présente un intérêt majeur pour l’amélioration du suivi de leur pathologie et une meilleure adaptation des traitements. Les tests métaboliques, qui sont actuellement les seuls outils disponibles pour évaluer la fonctionnalité des cellules β, présentent l’inconvénient d’être souvent lourds à mettre en place, et de ce fait, sont non utilisables en routine ou peu informatifs. L’imagerie, plus simple et plus sensible, représente une alternative attractive pour la mesure non invasive de la MCB d’autant plus qu’elle pourrait permettre à la fois une évaluation anatomique et fonctionnelle. Les outils actuellement en cours de développement visent principalement une imagerie anatomique de la MCB et s’orientent d’avantage vers le suivi longitudinal des patients que vers une quantification précise de la MCB dans un but purement diagnostique compte tenu des contraintes de sensibilité et de résolution nécessaires pour imager les cellules β. A l’heure actuelle, il n’existe pas encore d’outil idéal. Dans ce contexte, l’objectif de mon travail de thèse était le développement de fragments d’anticorps ciblant ZnT8 pour l’imagerie fonctionnelle des cellules β pancréatiques en imagerie nucléaire. Le transporteur du zinc ZnT8 est une cible intéressante pour l’imagerie des cellules β dans la mesure où il est exprimé presque exclusivement par ces dernières. De plus, ZnT8 étant situé à la surface des vésicules de sécrétion et à la surface de la cellule lors de l’exocytose de l’insuline, son ciblage devrait donc permettre de rendre compte de la fonctionnalité des cellules β. Deux F(ab’)2, puis un Fab, radiomarqués ont d’abord été évalués in vivo chez la souris. Leurs poids moléculaire élevés ne permettaient pas une cinétique sanguine favorable et ralentissaient probablement leur passage à travers un endothélium vasculaire sain les empêchant ainsi d’accéder rapidement à leur cible. Des sdAbs, qui constituent une alternative intéressante aux F(ab’)2 et aux Fab compte tenu de leur poids moléculaire plus faible, ont ensuite été sélectionnés par phage display. Pour l’instant, seule une partie de ces sdAbs a pu être produite et le meilleur candidat n’a pas pu être suffisamment purifié pour permettre son radiomarquage. / Diabetes is a chronic disease that affects 380 million people worldwide. Anatomical and/or functional diminution of beta-cell mass (BCM) is a common feature of type 1 diabetes which represents 10 to 15% of cases and type 2 diabetes which accounts for 80 to 90% of cases. Patients’ BCM estimation is of great interest for patients’ follow-up improvement and therapy adjustment. Metabolic tests which are actually the only tools available for beta cells function evaluation have the disadvantage to be cumbersome processes inapplicable for clinical routine or uninformative. Imaging, which is easier and more sensitive, is an attractive alternative for non-invasive BCM measurement since it could allow anatomical and functional evaluation. Tools currently under development mainly focused on anatomical imaging and are directed toward longitudinal follow-up of patients rather than exact BCM quantification for a merely diagnostic purpose because of sensitivity and resolution constraints necessary for beta cells imaging. Currently, there is no existing ideal tool. In this context, my PhD research was to develop antibody fragments targeting ZnT8 for pancreatic beta cells functional nuclear imaging. Zinc transporter ZnT8 is an attractive target for beta cells imaging since it is expressed almost exclusively by these cells. Moreover, ZnT8 is located on insulin containing vesicles surface and on cell surface when insulin secretion is stimulated. Its targeting should reflect beta cells functionality. Two radiolabeled F(ab’)2 then a Fab were first tested in vivo in mice. Their high molecular weight did not allow a suitable blood kinetic and probably slowed down their passage across healthy vascular endothelium preventing them from reaching quickly their target. SdAbs, which are an attractive alternative to F(ab’)2 and Fab because of their low molecular weight, were then selected by phage display. At the moment, only a part of these sdAbs have been produced and the best candidate could not be purified enough to allow its radiolabeling.