Positron emission tomography (PET) requires imaging times that last several minutes long. Therefore, when imaging areas that are prone to respiratory motion, blurring effects are often observed. This blurring can impair our ability to use these images for diagnostics purposes as well for treatment planning. While there are methods that are used to account for this effect, they often rely on adjustments to the imaging protocols in the form of longer scan times or subjecting the patient to higher doses of radiation. This dissertation explores an alternative approach that leverages state-of-the-art deep learning techniques to align the PET signal acquired at different points of the breathing motion. This method does not require adjustments to standard clinical protocols; and therefore, is more efficient and/or safer than the most widely adopted approach. To help validate this method, Monte Carlo (MC) simulations were conducted to emulate the PET imaging process, which represent the focus of our first experiment. The next experiment was the development and testing of our motion correction method. A clinical four-ring PET imaging system was modelled using GATE (v. 9.0). To validate the simulations, PET images were acquired of a cylindrical phantom, point source, and image quality phantom with the modeled system and the experimental procedures were also simulated. The simulations were compared against the measurements in terms of their count rates and sensitivity as well as their image uniformity, resolution, recovery coefficients, coefficients of variation, contrast, and background variability. When compared to the measured data, the number of true detections in the MC simulations was within 5%. The scatter fraction was found to be (31.1 ± 1.1)% and (29.8 ± 0.8)% in the measured and simulated scans, respectively. Analyzing the measured and simulated sinograms, the sensitivities were found to be 10.0 cps/kBq and 9.5 cps/kBq, respectively. The fraction of random coincidences were 19% in the measured data and 25% in the simulation. When calculating the image uniformity within the axial slices, the measured image exhibited a uniformity of (0.015 ± 0.005), while the simulated image had a uniformity of (0.029 ± 0.011). In the axial direction, the uniformity was measured to be (0.024 ± 0.006) and (0.040 ± 0.015) for the measured and simulated data, respectively. Comparing the image resolution, an average percentage difference of 2.9% was found between the measurements and simulations. The recovery coefficients calculated in both the measured and simulated images were found to be within the EARL ranges, except for that of the simulation of the smallest sphere. The coefficients of variation for the measured and simulated images were found to be 12% and 13%, respectively. Lastly, the background variability was consistent between the measurements and simulations, while the average percentage difference in the sphere contrasts was found to be 8.8%. The code used to run the GATE simulations and evaluate the described metrics has been made available (https://github.com/teaghan/PET_MonteCarlo). Next, to correct for breathing motion in PET imaging, an interpretable and unsupervised deep learning technique, FlowNet-PET, was constructed. The network was trained to predict the optical flow between two PET frames from different breathing amplitude ranges. As a result, the trained model groups different retrospectively-gated PET images together into a motion-corrected single bin, providing a final image with similar counting statistics as a non-gated image, but without the blurring effects that were initially observed. As a proof-of-concept, FlowNet-PET was applied to anthropomorphic digital phantom data, which provided the possibility to design robust metrics to quantify the corrections. When comparing the predicted optical flows to the ground truths, the median absolute error was found to be smaller than the pixel and slice widths, even for the phantom with a diaphragm movement of 21 mm. The improvements were illustrated by comparing against images without motion and computing the intersection over union (IoU) of the tumors as well as the enclosed activity and coefficient of variation (CoV) within the no-motion tumor volume before and after the corrections were applied. The average relative improvements provided by the network were 54%, 90%, and 76% for the IoU, total activity, and CoV, respectively. The results were then compared against the conventional retrospective phase binning approach. FlowNet-PET achieved similar results as retrospective binning, but only required one sixth of the scan duration. The code and data used for training and analysis has been made publicly available (https://github.com/teaghan/FlowNet_PET). The encouraging results provided by our motion correction method present the opportunity for many possible future applications. For instance, this method can be transferred to clinical patient PET images or applied to alternative imaging modalities that would benefit from similar motion corrections. When applied to clinical PET images, FlowNet-PET would provide the capability of acquiring high quality images without the requirement for either longer scan times or subjecting the patients to higher doses of radiation. Accordingly, the imaging process would likely become more efficient and/or safer, which would be appreciated by both the health care institutions and their patients. / Graduate

To address the unmet clinical need to eradicate treatment-resistant CD133+ cancer stems within tumors, a CSC-targeted radionuclide therapy (TRT) and companion diagnostic imaging probes were developed utilizing CD133-targeting antibodies and antibody fragments. In Chapter 1, background research providing context for the work in this Thesis is presented. In Chapter 2, a CD133-targeting antibody, RW03IgG, underwent radiolabeling with lutetium-177 to synthesize [177Lu]Lu-DOTA-RW03IgG for CD133-TRT. The CD133-TRT was evaluated for pharmacokinetics and treatment of a CD133 expressing human colorectal tumor bearing mouse model. Biodistribution studies on [177Lu]Lu-DOTA-RW03IgG demonstrated notable uptake in the colorectal tumors and off-target organ uptake consistent with previously reported antibody-based TRTs. Confirmation that tumor uptake was mediated by antibody-antigen binding was verified through co-injection with an excess dose of unlabeled RW03IgG. A dose-escalation therapy trial using [177Lu]Lu-DOTA-RW03IgG for treatment of the colorectal cancer mouse model revealed a dose-dependent reduction in tumor growth rate at well-tolerated doses. The decrease in tumor growth rate observed due to [177Lu]Lu-DOTA-RW03IgG treatment, along with an improvement in overall mouse survival, demonstrate the therapeutic efficacy of CD133-TRT. Additionally, histopathological and immunohistochemical (IHC) analyses indicated low off-target organ toxicity and significant anti-tumor effects. These findings suggested the potential for enhanced overall survival benefits through multiple doses. However, results on multiple-dosed CD133-TRT on the tumor growth rate and overall mouse survival were inconclusive. In Chapter 3, an orthotopic patient-derived glioblastoma (GBM) mouse model was developed that replicates anatomical pharmacokinetic challenges and CSC populations observed in patient tumors. Stereotactic engraftment of the patient GBM cells was optimized to reproducibly deliver tumor cells to the thalamus and growth was monitored using bioluminescence imaging. Ex vivo analysis confirmed various key characteristics of patient GBM, including CD133 expression, hypercellularity, and invasiveness. Biodistribution studies on [177Lu]Lu-DOTA-RW03IgG using the PDX GBM mouse model indicate antibody-antigen driven tumor uptake, determined through co-injection an excess dose of unlabeled RW03IgG. Ex vivo autoradiography supported the biodistribution results and showed elevated uptake of [177Lu]Lu-DOTA-RW03IgG in tumor relative to non-tumor bearing brain tissue. Chapters 4 and 5 centered on the development and evaluation of companion diagnostic CD133-targeted immunoPET probes. Chapter 4 specifically explored probes derived from the full antibody, RW03IgG. The probes were synthesized by conjugating RW03IgG with DFO-NCS to produce DFO-RW03IgG at different chelator-to-antibody ratios. The various DFO-RW03IgG conjugates were then radiolabeled with zirconium-89 to obtain [89Zr]-DFO-RW03IgG. Biodistribution studies and PET imaging revealed promising tumor uptake of [89Zr]-DFO-RW03IgG, and it was observed that higher chelator-to-antibody ratios led to increased accumulation in off-target organs. Chapter 5 investigated a probe derived from an scFv-Fc fragment of RW03, [89Zr]-DFO-RW03scFv-Fc. Biodistribution studies and PET images of colorectal tumor-bearing mice administered [89Zr]-DFO-RW03scFv-Fc showed favorable tumor uptake and low off-target organ accumulation. In Chapter 6, a probe for CD133-Photoacoustic Imaging (PAI) was synthesized through conjugation of RW03IgG with IR-783, an organic dye recognized for its favorable photoacoustic properties. Challenges were encountered in isolating the product, (IR-783)-RW03IgG, at high degrees of labeling (DOL) due to product aggregation. In vitro binding assays indicated that (IR-783)-RW03IgG (DOL = 1) maintained a comparable binding affinity to native RW03IgG. In vivo, colorectal tumors in mice administered (IR-783)-RW03IgG (DOL = 1) did not exhibit significant contrast from the background tissue, and the tumor PA signal did not differ significantly compared to tumors in mice administered an IR-783 labeled isotype IgG. The results suggest that a higher concentration of dye is needed within colorectal tumors for effective tumor visualization than what was provided by IR-783-RW03IgG. Chapter 7 investigated the use of Imaging Mass Cytometry (IMC) to simultaneously visualize [177Lu]Lu-DOTA-RW03IgG and multiple tumor biomarkers in tissue specimens collected from colorectal tumor xenograft mice treated with CD133-TRT. IMC showed undetectable concentrations of hafnium-177 (the decay product of lutetium-177) in tumors treated with CD133-TRT. However, lutetium-176 and lutetium-175, sourced from the carrier-added [177Lu]LuCl3 used in the synthesis of [177Lu]Lu-DOTA-RW03IgG, were present at levels sufficient for IMC visualization. The distribution of lutetium-176, representing [177Lu]Lu-DOTA-RW03IgG, within tumors, was imaged concomitantly with CD133, DNA damage markers, and several additional biomarkers that describe elements of the tumor microenvironment. These collective results endorse IMC as a useful tool to assess the distribution of TRT within tumors and uncover changes to the microenvironment in response to treatment. / Thesis / Doctor of Philosophy (PhD) / Targeted radionuclide therapy (TRT) and molecular imaging strategies were developed to aid in the elimination of the rare and particularly resilient Cancer Stem Cell (CSC) population in tumors. A fully human monoclonal antibody and antibody fragments targeting CD133, a molecular biomarker for CSCs, provided the means to deliver radioactive isotopes for therapy and imaging to CD133+ cells in tumors. The therapeutic efficacy of CD133-TRT for treatment of a colorectal cell line-derived xenograft mouse model was promising, and the treatment showed uptake in orthotopic patient derived glioblastoma tumors engrafted in mice. ImmunoPET probes targeting CD133 were optimized and successfully delineated CD133 expressing tumors from background tissue, warranting further evaluation using patient-representative cancer models. A non-invasive CD133-targeting Photoacoustic Imaging (PAI) probe was synthesized through conjugation of the CD133-targeting antibody to an organic dye, IR-783, although further probe optimization is required to provide tumor contrast. Tumor specimens from mice treated with CD133-TRT were assessed by Imaging Mass Cytometry (IMC), which revealed detectable concentrations of carrier isotopes from the therapy in the tumors, implicating the discovery of a powerful new tool for multiplexed single-cell level resolution imaging for cellular-scale analysis of targeted radionuclide therapy. The CSC-therapy and select molecular imaging probes generated in this Thesis warrant further evaluation using patient-representative mouse models of cancer.

The heterogeneity intrinsic to the ventromedial prefrontal cortex (vmPFC) is evidenced in both its anatomy and implicated function: vmPFC subregions have roles in positive affect, negative affect and autonomic/endocrine regulation. Whether different subregions serve fundamentally different functions, or whether they perform similar computations on different inputs, remains unclear. Nevertheless, the role of the vmPFC in psychopathology is widely appreciated - in mood and anxiety disorders, over-activity within constituent regions of the vmPFC is consistently implicated in symptomatology, together with its normalisation following successful treatment. However, the precise locus of change varies between studies. The work presented in this thesis investigates the causal contributions of over-activity within two key subregions of the vmPFC - the subgenual anterior cingulate cortex (sgACC, area 25) and perigenual anterior cingulate cortex (pgACC, area 32) - in discrete dimensions of behaviour and physiology affected in psychiatric disorders. Specifically, the impact of over-activity is assessed on (i) baseline physiological function; (ii) the regulation of anticipatory, motivational and consummatory aspects of reward-related behaviour; and (iii) negative affect including fear learning, stress recovery and the intolerance of uncertainty. To provide further insight into the mechanism of action of antidepressants, the efficacy of selected treatments is tested on changes induced by over-activity of these regions. Beyond the direct relevance of the results presented here to psychiatric disorders and their treatment, the thesis aims to emphasise the importance of broader themes associated with the measurement and quantification of emotion in preclinical animal studies. First, a multi-faceted approach is utilised enabling quantification of both the autonomic and behavioural aspects of emotion. In so doing, the experiments maintain relevance to studies which assess these correlates in isolation, both in humans (which typically measure subjective responses and physiology) and in rodents (which frequently assess behaviour in isolation). The assessment of more than one dimension of emotion confers these studies with improved power to detect maladaptive changes. Second, the experiments described were conducted in the marmoset, a new-world primate. The extensive anatomical homology between marmoset and human prefrontal cortex facilitates the forward-translation of functional results. In combination with the appropriate assays, this renders marmosets as an invaluable species to study the causal contributions of vmPFC subregions to symptoms of psychiatric disorders. I believe that the results of these experiments provide important insights into the causal role primate vmPFC has in relation to the behavioural and physiological aspects of psychiatric symptomatology. Most importantly, I hope that they serve as the foundation for future work to further elucidate the neuropathological processes underlying mental disorders.

Les pathologies modifiant l’intégrité de la matrice cartilagineuse (arthrose, arthrite, pathologies des disques intervertébraux, chondrosarcome…) sont caractérisées par un diagnostic tardif dû à l’absence d’un marqueur spécifique du cartilage. Notre unité de recherche développe depuis quelques années un radiotraceur à base de 99mTc capable de se lier aux protéoglycanes, un des constituants essentiels du cartilage. Son utilisation en tomographie par émission monophotonique (TEMP) a permis de mettre en évidence sa capacité à cibler spécifiquement les protéoglycanes du cartilage. Les travaux présentés dans cette thèse s’inscrivent dans la continuité de cette étude. De nouveaux radiotraceurs à base de cuivre-64 ont été synthétisés avec pour objectif de réaliser une imagerie fonctionnelle et quantifiable du cartilage en tomographie par émission de positons (TEP). La structure de base de ces radiotraceurs combine une partie vectrice ciblant spécifiquement les protéoglycanes du cartilage (fonction ammonium quaternaire) et une partie chélatante affine de l’ion cuivrique (polyazamacrocycle). Nos études préliminaires montrent que ces radiotraceurs pourraient permettre un diagnostic précoce et un suivi longitudinal des pathologies du cartilage. / Pathologies altering the integrity of the cartilaginous matrix (osteoarthritis, arthritis, intervertebral discs diseases, chondrosarcoma…) are characterized by a delayed diagnosis due to the absence of a specific marker of cartilage. Since several years, our research unit developed a radiotracer based on 99mTc able of binding proteoglycans, one of the essential constituents of the cartilage. Its use in single photon emission tomography (SPECT) had enabled to demonstrate its ability to specifically target the cartilage proteoglycans. Works reported in this thesis are a follow-up to this study. New radiotracers based on copper-64 were synthesized with the aim to achieve functional and quantifiable cartilage imaging in positron emission tomography (PET). The basic structure of these radiotracers combines a carrier function specifically targeting cartilage proteoglycan (quaternary ammonium function) and a chelating moiety for cupric ion (polyazamacrocycle). Our preliminary studies show that these radiotracers could allow early diagnosis and a longitudinal follow-up of cartilage diseases.

Introduction Since its development by Al Wolf and colleagues in the 1970s1, [11C]cyanide has been a useful synthon for a wide variety of reactions, most notably those producing [1-11C]-labeled amino acids2. However, despite its position as rote gas-phase product, the catalytic synthesis is difficult to optimize and often only perfunctorily dis-cussed in the radiochemical literature. Recently, [11C]CN– has been used in the synthesis of indole-3-[1-11C]acetic acid ([11C]IAA), the principal phytohormone responsible for a wide variety of growth and development functions in plants3. The University of Wisconsin has expertise in cyclotron production and radiochemistry of 11C and previous experience in the PET imaging of plants4,5. In this abstract, we present work on optimizing [11C]CN– production for the synthesis of [11C]IAA and the PET imaging of auxin transport in living plants. Material and Methods [11C]CH4 was produced by irradiating 270 psi of 90% N2, 10% H2 with 30 µA of 16.1 MeV protons from a GE PETtrace cyclotron. After irradiation, the [11C]CH4 was converted to [11C]CN– by passing through a quartz tube containing 3.0 g of Pt wire and powder between quartz wool frits inside a 800–1000 ˚C Carbolite tube furnace. The constituents and flow rate of the [11C]CH4 carrier gas were varied in an effort to optimize the oven\'s catalytic production of [11C]CN– from CH4 and NH3. The following conditions were investigated: i. Directly flowing irradiated target gas versus trapping, purging and releasing [11C]CH4 from a −178 ˚C HayeSep D column in He through the Pt furnace. ii. Varying the amount of anhydrous NH3 (99.995%) mixed with the [11C]CH4 carrier gas prior to the Pt furnace. Amounts varied from zero to 35 % of gas flow. iii. Varying the purity of the added NH3 gas with the addition of a hydride gas purifier (Entegris model 35KF), reducing O2 and H2O impurities to < 12 ppb. iv. Varying the flow rate of He gas carrying trapped, purged and released [11C]CH4. After flowing through the Pt furnace, the gas stream was bubbled through 300 µL of DMSO containing IAA precursor gramine (1 mg), then passed through a 60×5 cm column containing ascarite to absorb [11C]CO2, followed by a −178˚C Porapak Q column to trap [11C]CH4 and [11C]CO. After bubbling, the DMSO/gramine vial was heated to 140 ˚C to react the gramine with [11C]CN–, forming the intermediate indole-3-[1-11C]acetonitrile ([11C]IAN), which was subsequently purified by solid phase extraction (SPE). The reaction mixture was diluted into 20 mL water and loaded onto a Waters Sep-Pak light C18 cartridge, followed by rinsing with 5 mL of 0.1% HCl : acetonitrile (99 : 1) and 10 mL of the same mixture in ratio 95 : 5, and finally eluted with 0.5 mL of diethyl ether. The ether was subsequently evaporated under argon flow, followed by the hydrolysis of [11C]IAN to [11C]IAA with the addition of 300 µL 1 M NaOH and heating to 140 ˚C for 5 minutes. After hydrolysis, the solution was neutralized with 300 µL 1 M HCl and purified using preparative high-performance liquid chromatography (HPLC) using a Phenomenex Luna C18 (10μ, 250×10mm) column with a mobile phase acetonitrile : 0.1% formic acid in H2O (35 : 65) at flow rate of 3 mL/min. The [11C]IAA peak, eluting at 12 minutes, was collected and rotary evaporated to dryness, then again after the addition of 5 mL acetonitrile, followed by its reconstitution in 50 µL of water. Analytical HPLC was performed on the [11C]IAA before and after this evaporation procedure using a Phenomenex Kinetex C18 (2.6μ, 75× 4.6 mm) column with a linear gradient elution over 20 minutes of 10 : 90–30 : 70 (acetonitrile : 0.1% formic acid) at a 1 mL/min flow rate, eluting at 7.6 minutes. The transport of [11C]IAA was monitored following administration through the severed petiole of rapid cycling Brassica oleracea (rcBo) using a Siemens microPET P4 scanner. Transport was compared following administration to the first true leaf versus the final fully formed leaf in plants with and without exposure to the polar auxin transport inhibitor naphthylphthalamic acid (NPA). Results and Conclusion Optimization of the [11C]CN– gas phase chemistry was performed using two key metrics for measuring conversion yield. First is the fraction of total produced radioactivity that trapped in the DMSO/gramine solution (denoted %DMSO), and second, the fraction of DMSO/gramine-trapped activity that was able to react with gramine to form [11C]IAN (denoted %CN–). Under certain conditions, the former of these metrics experienced significant losses due to unconverted [11C]CH4 or through combustion, forming [11C]CO2 or [11C]CO. The latter metric experienced losses due to production of incomplete oxidation products of the CH4-NH3 reaction, such as methylamine. Total [11C]CH4 to [11C]CN– con-version yields is reported by the product of the two metrics. It was initially hypothesized that the irradiation of a 90% N2, 10% H2 target gas would produce sufficient in-target-hot-atom-produced NH3 to convert [11C]CH4 to [11C]CN– in the Pt furnace. However, conversion yields were found to be low and highly variable, with 13 ± 8 % trapping in DMSO/gramine, 9 ± 9 % of which reacted as CN– (n = 15). While in disagreement with previous reports1, this is likely as a result the batch irradiation conditions resulting ammonia losses in the target chamber and along the tubing walls. Yields and reproducibility were improved when combining the target gas with a stream of anhydrous NH3 gas flow with conversion yields reported in TABLE 1. However, these yields remained undesirably low, potentially as a result of the 10% H2 carrier gas having an adverse effect on the oxidative conversion of [11C]CH4 to [11C]CN–. To remedy this, the irradiated target gas was trapped, purged, released in He and combined with NH3 gas before flowing through the Pt furnace. Initial experiments using 99.995% anhydrous NH3 gas resulted in very poor (< 0.1%) [11C]CN– yields as a result of nearly quantitative combustion forming [11C]CO2. Installation of a hydride gas purifier to reduce O2 and H2O impurities in NH3 improved yields for CH4 in He, but did not significantly affect those from [11C]CH4 in N2/H2 target gas. In disagreement with previous reports2, conversion yields were found to be highly sensitive to overall carrier gas flow rate, with lower flow rates giving the best yields, as shown in TABLE 1. Optimization experiments are continuing. The total decay-corrected yield for the 1 hour synthesis of [11C]IAA in 50 µL of water is 2.3 ± 0.7 %, based on the total produced [11C]CH4 with a specific activity ranging from 1–100 GBq/µmol. The principal radiochemical impurity was determined to be indole-3-carboxylic acid. The SPE procedure isolating the [11C]IAN intermediate product was optimized to minimize this impurity in the final sample. After a rapid distribution of the administered [11C]IAA through the cut petiole and throughout the rcBO plant, upward vascular transport of auxin and downward polar auxin transport was visualized through time-activity curves (TACs) of regions of interest along the shoot. Comparison of these TACS with and without exposure to NPA yields insight into the fundamental physiological process of polar auxin transport in plants. In conclusion, the Pt-catalyzed oxidative conversion of [11C]CH4 and NH3 to [11C]CN– is a challenging process to optimize and highly sensitive to carrier gas composition and flow rate. Optimization for our experimental conditions yielded several results which disagreed with previous reports. [11C]IAA produced using [11C]CN– is well suited for PET imaging of polar auxin transport in living plants.

Der Programmed Death-Ligand 1 (PD-L1) wird von verschiedenen Tumorentitäten überexprimiert und hemmt durch die Bindung an das auf T-Zellen vorkommende Programmed Cell Death Protein (PD-1) die Immunantwort. Immuncheckpoint-Inhibitoren sind in der Lage, diese Blockade aufzubrechen und die Immunantwort zu reaktivieren. Auf eine solche Monotherapie sprechen aber nur etwa 30 % der Patienten an, sodass klinischer Bedarf an nichtinvasiven, bildgebenden Diagnosemethoden besteht. Eine Möglichkeit stellen Radioliganden für die nichtinvasive Bildgebung mittels Positronen-Emissions-Tomographie (PET) oder Einzelphotonen-Emissionscomputertomographie (SPECT) dar, welche einen Therapieerfolg prognostizieren könnten. Die vorliegende Dissertationsschrift beschäftigt sich mit der Entwicklung und Synthese einer neuen Klasse von PD-L1-adressierenden Radioliganden sowie deren biologischer Evaluierung in Zellen (in vitro) und in Kleintieren (in vivo). Nach der Identifikation von drei hochaffinen PD-L1-Inhibitoren in der Patentliteratur, wurden zunächst diese als Referenzverbindungen in einer neuen, konvergenten Synthesestrategie dargestellt. Diese ermöglichte nicht nur eine höhere Gesamtausbeute der Substanzen, sondern erlaubte auch dank des modularen Aufbaus den einfachen Zugang zu einer großen Substanzbibliothek an PD-L1-Radioliganden. Mithilfe einer Kokristallstruktur aus PD-L1 und einem Inhibitor wurden Modifikationen des Inhibitors für eine Transformation in einen Radioliganden geplant. Zur Erhöhung der Hydrophilie der Substanzen wurden löslichkeitsvermittelnde Gruppen (Sulfon-/Phosphonsäuren) in dem Molekülteil eingeführt, welcher in der „Solvent-Exposed-Region“ des Proteins lokalisiert ist. Funktionalisierung des Bindungsmotivs mit einem Alkin erlaubte die Einführung eines Linker-Chelator-Konstruktes mittels kupferkatalysierter Azid-Alkin-Cycloaddition (CuAAC) zur Komplexierung des Radiometalls. In der ersten Generation an PD-L1-Radioliganden wurden zwei Bindungsmotive und drei verschiedenen Linker erprobt, sodass nach Konjugation mit dem DOTA-Chelator sechs PD-L1-Radioliganden zur Verfügung standen. Nach erfolgreicher Radiomarkierung mit 64Cu, 68Ga und 177Lu erfolgten mit den 64Cu-markierten Radioliganden Bindungsaffinitätsbestimmungen in einem Sättigungsbindungsassay (Zugabe von 2,5 % BSA im Medium) an transduzierten PC3 PD-L1-Zellen. Dabei erwies sich [64Cu]Cu-62 mit Dimethylbiphenyl als Bindungsmotiv und Piperazinpropyl als Linker mit einem KD-Wert von 60 nM als der vielversprechendste Radiotracer, sodass dieses Bindungsmotiv als Grundlage für die folgenden Radioliganden dienen sollte. Nach bestätigter In-vitro-Stabilität der Radioliganden in verschiedenen Medien (PBS, Humanserum, gegenüber Lebermikrosomen), wurden für alle sechs Verbindungen aus der ersten Generation µPET-Aufnahmen in tumortragenden Mäusen durchgeführt. Die Aufnahmen zeigten eine geringe Akkumulation im PD-L1-positiven Tumor, eine hepatobiliäre Ausscheidung trotz niedriger log D7,4-Werten von –2,73 bis –3,50 und für kleine Moleküle ungewöhnlich lange Zirkulationszeiten. Diese konnten mit Gelelektrophorese und Größenausschlusschromatographie auf eine ausgeprägte Albuminbindung der Radioliganden zurückgeführt werden. In Echtzeit-Radioliganden-Bindungsversuchen mittels LigandTracer® konnten für die meisten Radioliganden einstellige, nanomolare KD-Werte erzielt werden. In Abwesenheit von Albumin zeigen diese Bindungsaffinitäten mit hoher Wahrscheinlichkeit die reine Bindung an PD-L1. Um die Tumoraufnahme zu erhöhen, wurden für die PD-L1-Radioliganden der zweiten Generation die funktionelle Gruppe am Pyridinring und das Halogen am zentralen Aromaten ausgetauscht. Trotz höherer KD-Werte in Sättigungsbindungsversuchen zeigten die µPET-Aufnahmen eine erhöhte Tumoraufnahme für das Brom- ([64Cu]Cu-115) und Iodderivat ([64Cu]Cu-116), vermutlich aufgrund höherer Bmax-Werte. Um eine Transchelatierung zu vermeiden, wurde für die PD-L1-Radioliganden der dritten Generation DOTA gegen NODA-GA als Chelator ausgetauscht. Außerdem wurde auf eine Verbesserung des pharmakokinetischen Profils abgezielt: Mittels Einführung von weiteren Sulfonsäuren und deren partiellen Austausch durch Phosphonsäuren sollte zum einen die Albuminbindung verringert und zum anderen die renale Ausscheidung begünstigt werden. Die sechs Radioliganden mit bis zu vier Sulfon-/Phosphonsäuren zeigten, abhängig von der Art und Anzahl der löslichkeitsvermittelnden Gruppe, stark unterschiedliche pharmakokinetische Profile. Dabei überzeugte der Radioligand [64Cu]Cu-148 mit einem KD-Wert von 94 nM im Sättigungsbindungsversuch und einem log D7,4-Wert von –3,80 insbesondere in der µPET-Bildgebung: Größtenteils renale Ausscheidung, moderate Tumoraufnahme (SUVmax = 3,5) und ausgeprägtes Signal-zu-Hintergrund-Verhältnis zum PD-L1-negativen Tumor. Die vierte Generation sollte das wünschenswerte pharmakokinetische Profil des Radioliganden [64Cu]Cu-148 mit den höheren Bmax-Werten der Radioliganden der zweiten Generation kombinieren. Die µPET-Aufnahmen zeigten für alle vier synthetisierten Radioliganden sehr geringe Tumoraufnahmen, dafür ein unerwartetes pharmakokinetisches Profil mit hoher Leberaufnahme – trotz niedriger log D7,4-Werte – und eine Akkumulation in Knochen/Knochenmark und Gelenken. Es wird vermutet, dass es trotz des räumlichen Abstandes der beiden Phosphonsäuren zu einer Ca2+-vermittelten Knochenanreicherung und/oder einer Bindung an anderen biologischen Strukturen wie Makrophagen kommt. Dank des modularen Aufbaus und der Einführung einer Alkin-Funktionalität wird über 18F-prosthetische Gruppen ein einfacher Zugang zu 18F-Radioliganden mittels CuAACReaktionen eröffnet, für welche bereits 19F-Referenzverbindungen und Markierungsvorläufer synthetisiert wurden. Nach weiteren Optimierungen des pharmakokinetischen Profils und damit kürzeren Zirkulationszeiten soll dies zukünftig den Zugang zu vielversprechenden 18F-Radiotracern ohne Chelator-Linker-Einheit ermöglichen.

The expression of monocarboxylate transporters (MCTs) is linked to pathophysiological changes in diseases, including cancer, such that MCTs could potentially serve as diagnostic markers or therapeutic targets. We recently developed [18F]FACH as a radiotracer for non-invasive molecular imaging of MCTs by positron emission tomography (PET). The aim of this study was to evaluate further the specificity, metabolic stability, and pharmacokinetics of [18F]FACH in healthy mice and piglets. We measured the [18F]FACH plasma protein binding fractions in mice and piglets and the specific binding in cryosections of murine kidney and lung. The biodistribution of [18F]FACH was evaluated by tissue sampling ex vivo and by dynamic PET/MRI in vivo, with and without pre-treatment by the MCT inhibitor α-CCA-Na or the reference compound, FACH-Na. Additionally, we performed compartmental modelling of the PET signal in kidney cortex and liver. Saturation binding studies in kidney cortex cryosections indicated a KD of 118 ± 12 nM and Bmax of 6.0 pmol/mg wet weight. The specificity of [18F]FACH uptake in the kidney cortex was confirmed in vivo by reductions in AUC0-60min after pre-treatment with α-CCA-Na in mice (-47%) and in piglets (-66%). [18F]FACH was metabolically stable in mouse, but polar radio-metabolites were present in plasma and tissues of piglets. The [18F]FACH binding potential (BPND) in the kidney cortex was approximately 1.3 in mice. The MCT1 specificity of [18F]FACH uptake was confirmed by displacement studies in 4T1 cells. [18F]FACH has suitable properties for the detection of the MCTs in kidney, and thus has potential as a molecular imaging tool for MCT-related pathologies, which should next be assessed in relevant disease models.

No description available.

Le 1,4,7-triazacyclononane (tacn) fait partie de la famille des polyazamacrocycles et a la faculté, lorsqu’il est N-fonctionnalisé par des groupements coordinants adéquats, de former des complexes de coordination stables thermodynamiquement et inertes cinétiquement avec les métaux de transition. C’est pourquoi les dérivés du tacn trouvent de nombreuses applications dans de nombreux domaines, par exemple celui de l’imagerie médicale. Notre équipe a développé des ligands N-fonctionnalisés par des groupements picolinate et méthylpyridine pour des applications en imagerie TEP et IRM.Le premier travail de cette thèse porte sur l’élaboration de nouvelles sondes bimodales TEP-imagerie optique et IRM-imagerie optique à motifs issus de tacn-picolinate décrits au laboratoire.Une seconde partie est axée sur la synthèse de nouveaux chélates du Cu(II) à base de tacn portant des bras méthylthiazole pour une potentielle application en imagerie TEP. Les complexes de Cu(II) correspondant ont été étudiés par différentes méthodes analytiques et spectroscopiques afin de juger leurs stabilités thermodynamiques et leurs inerties cinétiques.Les chélatants répondant aux critères imposés par le milieu biologique ont été sélectionnés pour une application en imagerie TEP dans le cadre du cancer de la prostate. Des analogues bifonctionnels ont ainsi été élaborés afin de les incorporer dans des radiopharmaceutiques radiomarqués au cuivre-64. / The 1,4,7-triazacyclononane (tacn) is part of the family of polyazamacrocycles, and has the faculty, when appropriately N-functionalized with coordination pendants, to form thermodynamically stable and kinetically inert coordination complexes with transition metals. Thanks to their properties tacn derivatives can be found in numerous applications such as medical imaging. Our group has developed some ligands N-functionalized with picolinate and picolyl pendants for an application in PET imaging and MRI.The first part of this thesis is focused on the development of a new class of bimodal PET-optical imaging and MRI-optical imaging probes based on tacn-picolinate units described in the laboratory.The second part deals with the synthesis of new Cu(II) chelators based on tacn-methylthiazolyl units for a potential application in PET imaging. The corresponding Cu(II) complexes were studied by different analytical and spectroscopic methods in order to evaluate their thermodynamic stabilities and kinetic inertness.The chelators that fullfiled all the criterias imposed by the biological media were selected for an applicationPET imaging of prostate cancer. Some bifunctional analogues were elaborated so as to incorporate them in radiopharmaceuticals radiolabeled with copper-64.

L’impulsivité décisionnelle est une des multiples dimensions de l’impulsivité. Elle est définie comme la préférence d’une petite récompense immédiate à une grande récompense différée, et dérive du fait que la valeur que l’on attribue à une récompense diminue avec le temps. Quand la dévaluation temporelle est excessive, l’impulsivité de choix devient un symptôme présent dans de nombreuses pathologies. Le striatum, structure cérébrale profonde composée de trois sous-territoires, le noyau caudé, le striatum ventral et le putamen, est un élément clé dans le traitement de l’impulsivité décisionnelle. En effet, il constitue la principale cible de la dopamine, connue pour être un modulateur de l’impulsivité. En lien avec le cortex, le striatum antérieur est impliqué dans les processus de prise de décision, de motivation liée à la récompense, et de sélection de l’action. Nous avons étudié le rôle spécifique des territoires du striatum dans l’impulsivité de choix chez le singe réalisant une tâche de dévaluation temporelle. En utilisant une approche comportementale et d’imagerie, nous avons montré que le noyau caudé était impliqué dans l’impulsivité de choix induite par le Pramipexole, un agoniste dopaminergique des récepteurs D2/3. Dans une seconde étude, nous avons montré que le Méthylphénidate, un inhibiteur de recapture de la dopamine utilisé comme traitement du trouble du déficit attentionnel/hyperactivité, diminuait l’impulsivité décisionnelle en passant par une action sur le striatum ventral. En résumé, ces résultats confirment le rôle de la dopamine dans l’impulsivité décisionnelle et montrent les rôles spécifiques du noyau caudé et du striatum ventral dans les processus décisionnels liés à la dévaluation temporelle / Temporal discounting is the process by which future rewards are subjectively devalued by the decision maker. Impulsive choices, defined as the tendency to prefer small immediate rewards over larger but delayed ones, derive from a high temporal discounting. This particular dimension of impulsivity is a trait of personality, but also a symptom in many neuropsychiatric disorders. It has been shown many times that impulsive choices can be modulated by dopaminergic agents. The dopamine targets the striatum, a cerebral structure linked to cortex, subdivided into three territories – the Ventral Striatum (VS), the Caudate Nucleus (CdN) and the Putamen - and involved in motivation, goal directed behaviors, decision making and action selection. All these functions are involved in impulsive behaviors, although the specific role of each territory in impulsivity remains unknown. Here, using pharmacologic and imaging approaches, we aimed to study the role of the anterior striatum in impulsive choices using the delay discounting task in non-human primates. First, we showed that the CdN supports impulsive choices triggered by Pramipexole, a D2/3 agonist suspected to produce impulsive control disorders. In a second study, we used Methylphenidate, a blocker of dopamine transporter used in attention deficit hyperactivity disorder, and we demonstrated that the VS supports Methylphenidate therapeutic effects on impulsive choices expressed in temporal discounting task. Together, our results show that dopaminergic modulation plays a specific role on the CdN and the VS in the processes of impulsive choices, and suggest a dual action between ventral and dorsal striatal territories