Charles University Fakulty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Candidate: Bc. Barbora Parýzková Supervisor: Mgr. Pavel Bárta, Ph.D. Title of diploma thesis: Radiolabelled receptor-specific peptides for cancer diagnosis and therapy. The presented diploma thesis is based on literature review and it deals with receptor- specific radioactive labeled peptides and their usage in the cancer diagnostic and treatment. It focuses on general knowledge about radiopharmaceuticals, further on the descriptions of either diagnostic or therapeutic radionuclides as well as on currently used chelating agents. The particular attention is paid to peptides like somatostatin and cholecystokinin and their usability in Nuclear Medicine for the imaging and treatment of certain malignancies. The other stated peptides in this diploma thesis are bombesin, vasoactive intestinal peptide and alpha melanocyte stimulating hormone. Keywords: receptor-specific peptides, radiolabelling, radionuclides for diagnosis and therapy, radiotherapy, radiodiagnostics, octreotide.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-06-17T11:42:15Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Pos_defesa_Final_corrigida_Biblioteca.pdf: 3212146 bytes, checksum: 9c78ac9cc8cdb05df32aea81cd5d38d3 (MD5) / Made available in DSpace on 2016-06-17T11:42:15Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Pos_defesa_Final_corrigida_Biblioteca.pdf: 3212146 bytes, checksum: 9c78ac9cc8cdb05df32aea81cd5d38d3 (MD5) Previous issue date: 2015-12-09 / A presença de diferentes substâncias genotóxicas, como os produtos do decaimento do 210Pb, um elemento amplamente encontrado no cigarro, pode causar danos genéticos e contribuir para evolução de neoplasias malignas, o que evidencia a necessidade de monitoração constante dos fumantes. Bioindicadores, a exemplo de micronúcleos (MN’s) são largamente empregados nos estudos das ações de agentes genotóxicos em seres humanos. Normalmente, em grupos formados por fumantes, são utilizadas amostras da mucosa bucal para avaliação da frequência de micronúcleos. No entanto, raros são os estudos que procuram utilizar como matriz amostras de urina para essas determinações. O presente trabalho avaliou a frequência de micronúcleo em células esfoliativas na urina, bem como os teores de Chumbo-210 em urina coletada durante 24 horas de indivíduos fumantes e não fumantes, com o objetivo de avaliar a utilização desta matriz como indicadora da suscetibilidade a danos genéticos em voluntários fumantes. Foram coletadas amostras de urina de 30 voluntários (15 não fumantes e 15 fumantes). Para as análises de micronúcleo em urina foram obtidas células esfoliativas através de uma série de centrifugações e lavagens com NaCl a 0,9%. A fixação e coloração empregadas foram metanol/ácido acético e Giemsa, respectivamente. Para determinar as concentrações de 210Pb, utilizou-se a técnica da resina trocadora de íons. Nesse caso, o 210Pb foi precipitado na forma de PbCrO4. O detector Canberra Tennelec S5E foi utilizado para determinar o 210Pb. As concentrações de 210Pb na urina dos fumantes variaram de 49,7 a 158,65 mBq.L-1, enquanto os não fumantes apresentaram valores abaixo de 45 mBq (LD). Nesta pesquisa, os resultados relacionados ao 210Pb sugerem que fatores como a idade, tempo de tabagismo e quantidade de cigarros não exerceram influência estatisticamente significante sobre os valores de excreção do 210Pb, entretanto o hábito tabagista contribui para o aumento das concentrações encontradas na urina. Foi encontrada uma frequência média para micronúcleo em não fumantes de 0,0036 ± 0,001 e, em fumantes foi de 0,0068 ± 0,002. Os resultados indicam um aumento expressivo da frequência de micronúcleos em células esfoliativas de indivíduos fumantes em relação aos não fumantes, sendo este aumento estatisticamente significativo. A análise estatística dos componentes principais (ACP) sugere que a quantidade de cigarros está relacionada com os micronúcleos, no entanto, não houve correlação estatisticamente significante com as concentrações de Chumbo-210. Os resultados obtidos sugerem que a urina pode ser utilizada como matriz biológica para a avaliação da suscetibilidade a danos genéticos ocasionados pelos compostos presentes no cigarro, comportando-se como uma ferramenta eficiente para a monitoração humana. / The presence of different genotoxic substances in cigarette smoke can cause genetic damage and contribute to development of malignancies, which highlights the need for constant monitoring of smokers. Biological indicators, such as the frequency of micronuclei (MN) are widely used to study the action of genotoxic agents in human beings. Usually in groups formed by smokers, the buccal mucosa samples are used to assess the frequency of micronuclei. However, few studies that seeks to use as a template urine samples for these determinations. The aim of this study was to evaluate the frequency of micronuclei in exfoliated cells as well as Lead-210 levels in 24-hour urine from smokers and nonsmokers, in order to evaluate the use of this matrix as an indicator of susceptibility to genetic damage in smokers volunteers. Because it is an array easy to obtain and allow an analysis of various elements were collected urine samples from 30 volunteers (15 smokers and 15 non-smokers). For the analysis of micronucleus, exfoliative cells from urine were obtained by a series of centrifugations and washings with NaCl 0.9%. Carnoy I fixation and Giemsa staining were employed, respectively. To determine the 210Pb, we used the technique of ion exchange resin. In this case, the 210Pb was precipitated in the form of PbCrO4. 210Pb beta counts were conducted in a Canberra Tennelec S5E detector with a detection limit of 45 mBq. The results indicated that the urine is an excellent matrix for assessing the degree of exposure to lead-210, especially for smokers, the group of most interest to monitoring. The non-smokers present values above 45mBq and Smokers have shown higher values for 210Pb in urine that ranged from 49.7 to 158.65 mBq.L-1. In this research, the results related to this radionuclide suggest that factors such as age, smoking time and number of cigarettes have little influence on the 210Pb excretion values, though the tobacco habit contributes to the concentrations found in the urine. The average rate for micronucleus found in nonsmokers was 0.0036 ± 0.001 and 0.0068 ± 0.002 in the smokers. The results showed a significant increase in micronuclei frequency in exfoliated cells of smokers compared to nonsmokers, which is statistically significant increase. Statistical analysis also suggest that the number of cigarettes are related to the micronuclei, however there is no statistical correlation with the concentrations of Lead-210. The results suggest that urine can be used with biological matrix for assessing genetic susceptibility to damage caused by the present compounds on the cigarette, behaving as an efficient tool for human monitoring.

La présente étude s’inscrit dans le contexte post-Fukushima et avait pour objectif la mise en place d’une méthode d’analyse pour identifier et quantifier, rapidement, des radionucléides émetteurs alpha et bêta, en situation post-accidentelle, dans les échantillons de l’environnement. La première étape de l’étude a permis de dresser la liste des radio-isotopes émetteurs alpha et bêta susceptibles d’être rejetés dans l’environnement suite à un accident nucléaire. Dans une deuxième étape, un protocole d’analyse rapide a été développé pour 17 radio-isotopes de U, Th, Pu, Am, Np et Sr. L’objectif était d’automatiser l’étape de séparation radiochimique et de la coupler à la mesure. La séparation est réalisée en employant des colonnes contenant les résines d’extraction, TEVA®, TRU et Sr. La mesure est effectuée par ICP-MS (spectrométrie de masse à plasma induit) et dans certains cas par compteur proportionnel à gaz pour quantifier le radiostrontium (89Sr et 90Sr). D’excellentes performances analytiques ont été obtenues, lors de la mesure hors-ligne, pour des échantillons d’eaux (robinet, rivière et mer) et solides (sols et filtres d’aérosols), après une minéralisation par four à micro-ondes ou par fusion alcaline suivie d’une co-précipitation avec Ca3(PO4)2. Ces performances sont caractérisées par des rendements de récupération compris entre 70 % et 100 % et des écarts types entre 5 % et 10 %. Le protocole de séparation a été ensuite automatisé et transposé en mode couplage en ligne avec l’ICP-MS. Les paramètres opératoires ont été optimisés avec un plan d’expériences, défini et exploité avec le logiciel Minitab®. Après optimisation du protocole, l’automatisation et le couplage de la séparation permettent de quantifier rapidement, en 1,5 h par échantillon, U, Th, Pu, Am, Np et Sr et de gagner un facteur 20 sur les limites de détection des radio-isotopes artificiels. / The present study follows the Fukushima power plant accident and aimed to develop an analytical method to achieve, during an emergency situation, a rapid identification and quantification of alpha and beta emitters in environmental samples. The first step of this study allowed us to list the alpha and beta emitters which can be released in the environment in case of a nuclear accident. The second step aimed towards the development of a rapid analysis method to quantify 17 radionuclides of U, Th, Pu, Am, Np and Sr. The main objective was the automation of the radiochemical separation step and its coupling for the measurement. The separation is performed using columns containing extraction resins, namely TEVA®, TRU and Sr. The measurement is performed using an ICP-MS (inductively coupled plasma mass spectrometry) and in some cases a gas proportional counter to quantify radiostrontium (89Sr and 90Sr). Excellent figures of merit were obtained, off line, with water (tap, river and sea water) and solid matrices (soil and aerosol filters), after a micro-wave digestion or an alkaline fusion dissolution followed by a Ca3(PO4)2 coprecipitation. The proposed analytical strategy showed yields between 70 % and 100 % and standard deviations between 5 % and 10 %. The newly developed separation method was then automated and coupled on-line to ICP-MS. The operating parameters were optimized using a design of experiments and the results were processed with Minitab®. The optimized automated separation coupled on-line to the ICP-MS allows the rapid quantification, in 1.5 h per sample, of U, Th, Pu, Am, Np and Sr with detection limits gain as high as 20 times for artificial radionuclides.

Le devenir des radionucléides naturels et anthropogéniques dans l’environnement demeure une préoccupation majeure des sociétés modernes nucléarisées. Parmi les compartiments environnementaux possiblement impactés, l’hydrosphère est ubiquitaire et peut transporter des composés ou éléments sur de très longues distances. L’évènement récent de Fukushima a démontré que le milieu marin pouvait être également directement impacté ce qui amène à des questionnements tant scientifiques que sociétaux. De nombreuses études ont déjà montré que les radionucléides présents dans l’eau de mer peuvent être fortement accumulés par les organismes marins mais pour autant leur spéciation est inconnue. Or cette connaissance est essentielle afin de maîtriser les mécanismes de transfert entre l’hydrosphère et la biosphère et d'évaluer in fine l’impact global sur l’Homme. Dans ce travail, nous avons choisi de déterminer expérimentalement la spéciation de trois actinides dans l’eau de mer dopée : l’uranium(VI), le neptunium(V) et l’américium(III) (via l’analogue europium(III)) en couplant des calculs de spéciation avec les outils spectroscopiques dont la Spectroscopie Laser Résolue en Temps (SLRT) et la Spectroscopie d'Absorption des Rayons X (EXAFS). Puis nous avons étudié les processus d’accumulation sur l’éponge A. cavernicola, choisi ici car considérée comme un biomoniteur de pollution pour les métaux lourds. L’accumulation de l’europium(III), de l’américium(III) et de l’uranium(VI) dans A. cavernicola a donc été investiguée à l'échelle de traces et ultra traces. Pour l'europium, les techniques d'imagerie X et électronique ont permis de localiser l'élément accumulé et d'en préciser la spéciation. / The fate of natural and anthropogenic radionuclides in the environment remains a major concern in our modern nuclearized societies. Among the environmental compartments, the hydrosphere is ubiquitous and can transport compounds or elements over very long distances. The recent event of Fukushima demonstrated that the marine environment could be directly affected and this raises both scientific and societal questions. Moreover, some studies have already shown that radionuclides present in seawater can be strongly accumulated by marine organisms although their speciation is most of the time unknown. Yet this knowledge is essential to better understand the transfer mechanisms from the hydrosphere to the biosphere and to evaluate their global impact on humans. In this work, we chose to experimentally determine the speciation of three actinides in doped seawater: uranium(VI), neptunium(V) and americium(III) (and the chemical surrogate europium(III)) by coupling speciation modeling with spectroscopic tools among which Time-Resolved Laser-Induced Fluorescence (TRLIF) and X-ray Absorption Spectroscopy (XAS). Then, we have studied the accumulation process in the sponge A. cavernicola, chosen here because it is considered as a biomonitor of heavy metal pollution. The accumulation of europium(III), americium(III) and uranium(VI) in A. cavernicola were investigated at trace and ultra-trace levels. Besides, for europium, X-ray and electronic imaging permit to localize the accumulated element in the sponge and to specify its speciation.

Quantitative soil ingestion studies employing a mass balance tracer approach have been used to determine soil ingestion rate for use in human health risk assessments (HHRAs). Past studies have focused on soil ingestion in populations living in urban/suburban environments and the results have been highly variable. Moreover, there is a paucity of reliable quantitative soil ingestion data to support human health risk assessments of other lifestyles that may be predisposed to ingesting soil, such as indigenous populations following traditional lifestyles. Thus, the primary objective of the research was to determine if populations following lifestyles typical of traditional land use practices in rural or wilderness areas ingest more soil than populations living in urban or suburban environments. Further, the research investigated the use of alternative mass balance tracers, specifically isotopes of the 238U and 232Th decay series, to reduce soil ingestion estimate variability. Mass balance tracer methods were developed and validated in a pilot canine study, and methods using isotope tracers were adapted to permit quantification of sediment ingestion in the benthic fish Moxostoma macrolepidotum (Shorthead Redhorse Sucker). A pilot human soil ingestion study of 7 subjects from an Aboriginal community in British Columbia was conducted over a 3-week period. The mean soil ingestion rate calculated using the daily means of the 4 elemental tracers with the lowest food-to-soil ratios (i.e., Al, Ce, La, Si) was observed to be approximately 74 mg d-1 (standard deviation 91 mg d-1), The median soil ingestion rate was 60 mg d-1, and the 90th percentile was 196 mg d-1. These soil ingestion rate estimates are higher than those currently recommended for HHRAs of adults, and higher than those obtained in most previous studies of adults. However, the estimates are much lower than the earlier qualitative assessments for subsistence lifestyles (i.e., 330-400 mg d-1). The study results also demonstrated that isotopes of the 238U and 232Th decay series radionuclide are not reliable mass balance tracers for estimating soil ingestion in humans; however, they may be useful for quantifying soil and sediment ingestion in wildlife.

Thesis advisor: Jeremy D. Shakun / Glaciers in the tropics have undergone significant retreat in the past several decades, but the magnitude of this retreat in the long-term context of the Holocene has mostly been qualitatively assessed. This study produces a quantitative reconstruction of Holocene glacier extent relative to today from the Quelccaya Ice Cap, Peru, and the Rwenzori Mountains of east Africa. I use measurements of in situ 14C and 10Be from bedrock that was recently exposed by glacier retreat to constrain possible bedrock exposure and erosion histories at each site. The results are strikingly similar in both areas, and suggest that ice was generally smaller than today during the first half of the Holocene and larger than today for most, if not all, of the last several millennia. These findings give evidence toward a coherent Holocene expansion of glaciers across the tropics, and suggest that recent retreat is unusual in a multi-millennial context. / Thesis (MS) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Earth and Environmental Sciences.

The paper describes the Rossendorf Beamline (ROBL) built by the Forschungszentrum Rossendorf at the ESRF. ROBL comprises two different and independently operating experimental stations: a radiochemistry laboratory for X-ray absorption spectroscopy of non-sealed radioactive samples and a general purpose materials research station for X-ray diffraction and reflectometry mainly of thin films and interfaces modified by ion beam techniques. The radiochemistry set-up is worldwide an unique installation at a modern synchrotron radiation source.

Die Entwicklung von Komplexbildnern für Kupferradionuklide im Hinblick auf eine nuklearmedizinische Anwendung ist ein intensiv bearbeitetes und in rascher Entwicklung begriffenes Forschungsgebiet. Dabei spielen verschiedene Faktoren – wie die günstigen kernphysikalischen Eigenschaften der verfügbaren Kupferradionuklide (Halbwertszeit, Umwandlungsart und -energien), die synthetische Zugänglichkeit und Modifizierbarkeit der Liganden sowie Markierungsausbeute und In-vivo-Stabilität der radioaktiven Kupferkomplexe – eine entscheidende Rolle. Liganden auf der Basis des 3,7-Diazabicyclo[3.3.1]nonans (Bispidine) besitzen für derartige radiopharmazeutische Anwendungen ein hohes Potential. Sie bilden mit Cu2+ sehr stabile Koordinationsverbindungen, wobei die Komplexstabilität im Bereich makrocyclischer Chelatoren liegt (logK ~ 20). Ferner weisen Bispidin-Derivate eine schnelle Komplexbildungskinetik mit Cu2+ auf. Damit sind radioaktiv markierte Bispidin-Liganden prinzipiell für einen Einsatz in der Nuklearmedizin (64Cu: Diagnostik, 67Cu: Therapie) geeignet und zwar insbesondere dann, wenn das Ligand-Grundgerüst mit zielsuchenden Biomolekülen, wie spezifischen Peptiden, Antikörpern oder Nukleinsäurebausteinen, funktionalisiert werden kann. Im Rahmen der vorliegenden Arbeit wurden neuartige sechszähnige Bispidin-Derivate mit Pyridyl-, Imidazolyl- und/oder Benzimidazolyl-Donoreinheiten mit Hilfe einer doppelten Mannich-Kondensation synthetisiert sowie koordinationschemisch und spektroskopisch charakterisiert. • Die Darstellung dieser Liganden erfolgte durch eine systematische Variation der Donoreinheiten an den C2/C4-Kohlenstoffatomen sowie den tertiären Aminostickstoffatomen N3/N7. Hierbei wurde insbesondere untersucht, wie sich der Einbau der unterschiedlichen Donorgruppen an den verschiedenen Positionen des Grundgerüsts auf das Löslichkeits- und Komplexbildungsverhalten auswirkt. Auf der Basis NMR-spektroskopischer Untersuchungen konnte gezeigt werden, dass die Einführung von Imidazolyl-Substituenten in N3- und N7 Position ausschließlich zu Isomeren führt, die eine trans-Stellung der Donoreinheiten an den C2/C4-Positionen aufweisen. Demgegenüber ist es möglich, durch den Einbau von Methylimidazolyl-Einheiten an C2 und C4, das im Hinblick auf die Komplexbildung mit Cu2+ gewünschte cis-Isomer zu isolieren. Diese Ergebnisse konnten anhand von Röntgenkristallstrukturanalysen der entsprechenden CuII-Komplexverbindungen bestätigt werden. • Mit Hilfe der Cyclovoltammetrie wurden CuII/CuI-Redoxpotentiale für ausgewählte CuII Bispidinkomplexe bestimmt. Da eine starke Korrelation dieser Redoxpotentiale mit den jeweiligen Stabilitätskonstanten besteht, können auf diese Weise Vorhersagen zur Komplexstabilität getroffen werden. Dabei konnte gezeigt werden, dass Bispidin-Liganden mit Pyridyl-Gruppen an N3/N7 sowie Methylimidazolyl- bzw. Pyridyl-Einheiten an C2/C4 sehr stabile Komplexe mit Cu2+ bilden. Hinsichtlich der synthetischen Zugänglichkeit ergeben sich jedoch für den Pyridyl-substituierten Liganden A Vorteile. Deshalb wurden Untersuchungen zur Biofunktionalisierung an diesem Derivat erprobt. Durch eine selektive Reduktion der Carbonylgruppe in 9-Stellung wurde der entsprechende Monoalkohol (B) als anti-Epimer dargestellt. Dieser Reaktionsschritt implizierte sowohl eine erhöhte chemische Stabilität des Liganden als auch eine gesteigerte Komplexstabilität des korrespondierenden CuII Komplexes. Die Hydrolyse der Methylestergruppen in Anwesenheit von Cäsiumhydroxid führte in hoher Ausbeute zur Verbindung C, welche an C1 und C5 über kupplungsfähige Carboxylgruppen verfügt. • Die Röntgenkristallstrukturen der CuII-Komplexe der Derivate B und C zeigen eine verzerrt oktaedrische Geometrie, wobei eine sechsfache Koordination mit dem Zentralatom vorzufinden ist. Dieser Aspekt führt zu einer nahezu vollständigen Abschirmung des Zentralatoms. Stabilitätsuntersuchungen auf der Basis der UV-Vis-Spektroskopie in Gegenwart des Konkurrenzliganden Glutathion ergaben, dass diese CuII-Komplexe selbst bei einem 100fachen Überschuss an Glutathion über mindestens 24 Stunden stabil sind. • Es wurde eine Reihe von Untersuchungen zur Kupplung von Linkereinheiten und Biomolekülen am Beispiel des Bispidin-Derivates C vollzogen. Im Rahmen der Arbeiten gelang es, Spacereinheiten wie γ-Aminobuttersäure, Ethylendiamin oder auch Maleinimid-Derivate an das Bispidin-Grundgerüst zu kuppeln. Die Funktionalisierung des Rückgrats mit Target-spezifischen Strukturen erfolgte anhand der Peptidsequenzen Neurotensin(8-13) und Bombesin(7-14). Dabei konnte demonstriert werden, dass eine Erhöhung der Ausbeuten an Peptidkonjugat ausgehend von Spacer-funktionalisierten Liganden erzielt werden kann. • Die radioaktive Markierung der Bispidin-Liganden mit den Radionukliden 64/67Cu verläuft unter physiologischen Bedingungen innerhalb weniger Minuten in hohen radiochemischen Ausbeuten (≥ 99%). In-vitro-Stabilitätsuntersuchungen (Rattenplasma, Challenge-Experimente mit Superoxid-Dismutase) an 64Cu-markierten Bispidin-Liganden und entsprechenden Peptidkonjugaten belegen, dass das Radionuklid über einen Zeitraum von 24 Stunden stabil gebunden bleibt. • Im Rahmen von In-vivo-Studien erfolgten Bioverteilungsuntersuchungen sowie Kleintier-PET-Aufnahmen an verschiedenen Bispidin-Derivaten. Das Bioverteilungsmuster der radiomarkierten Ligand- und Peptidkonjugat-Komplexe in männlichen Wistar-Ratten zeigte eine schnelle renale Ausscheidung der Verbindungen. Metabolitenanalysen bestätigten eine hohe In-vivo-Stabilität der 64Cu Bispidin-Komplexe. PET-Studien an PC3-Tumor-Mäusen unter Einsatz des 64Cu markierten Bispidin-Bombesin-Derivates belegen eine gute Tumoranreicherung dieser Verbindung sowie eine klare Visualisierung des Tumors.

Adsorption of radionuclides in granite pores and micropores ABSTRACT This graduation thesis deals with laboratory determination of adsorption isotherms parameters. Granite from the central moldanubian pluton, site Panské Dubenky, Czech Republic, was chosen to the experiment. The place is one of the candidate sites to build a deep nuclear waste disposal. A batch experiment was performed in two modes, differing in the way of addition of radioactive nuclide 90 Sr. From this experiment, distribution coefficients for a linear isotherm or parameters for Langmuir isotherm were obtained. Beside this, a through-diffusion experiment was performed. The objective of this experiment was to identify whether some of the radionuclides used (137 Cs, 90 Sr, 125 I) is able to penetrate through the pores of a granite slice barrier between two solutions of different concentrations. During the period of the experiment no radionuclide was detected reliably.

n/a / Thesis / Doctor of Philosophy (PhD)