Development and in vitro testing of liposomal gadolinium-formulations for neutron capture therapy of glioblastoma multiforme

For the improvement of current neutron capture therapy, several liposomal formulations of neutron capture agent gadolinium were developed and tested in a glioma cell model. Formulations were analyzed regarding physicochemical and biological parameters, such as size, zeta potential, uptake into cancer cells and performance under neutron irradiation. The neutron and photon dose derived from intracellular as well as extracellular Gd was calculated via Monte Carlo simulations and set in correlation with the reduction of cell survival after irradiation. To investigate the suitability of Gd as a radiosensitizer for photon radiation, cells were also irradiated with synchrotron radiation in addition to clinically used photons generated by linear accelerator.rnIrradiation with neutrons led to significantly lower survival for Gd-liposome-treated F98 and LN229 cells, compared to irradiated control cells and cells treated with non-liposomal Gd-DTPA. Correlation between Gd-content and -dose and respective cell survival displayed proportional relationship for most of the applied formulations. Photon irradiation experiments showed the proof-of-principle for the radiosensitizer approach, although the photon spectra currently used have to be optimized for higher efficiency of the radiosensitizer. In conclusion, the newly developed Gd-liposomes show great potential for the improvement of radiation treatment options for highly malignant glioblastoma.rn

Spettroscopia di assorbimento di raggi X: apparato per misure in condizioni di illuminazione ottica differenziale come primo passo verso una misura risolta in tempo della struttura locale di molecole metallo-organiche

Il continuo sviluppo negli ultimi anni di diverse declinazioni della spettroscopia d'assorbimento a raggi X (XAS) con radiazione di sincrotrone ha permesso la determinazione della struttura locale di campioni di ogni tipo, dagli elementi puri, ai più moderni materiali, indagando e approfondendo la conoscenza di quei meccanismi che conferiscono a questi ultimi delle proprietà innovative e, a volte, rivoluzionarie. Il vantaggio di questa tecnica è quello di poter ottenere informazioni sulla struttura del campione soprattutto a livello locale, rendendo relativamente agevole l'analisi di sistemi senza ordine a lungo raggio, quali per esempio i film molecolari. Nell'elaborato verrà preliminarmente illustrata la fenomenologia della XAS e l’interpretazione teorica dell'origine della struttura fine. Saranno successivamente descritte le innovative tecniche di misura che permettono di studiare i cambiamenti della struttura locale indotti dall'illuminazione con luce visibile, inclusi gli esperimenti di tipo pump probe. Un capitolo della tesi è interamente dedicato alla descrizione dei campioni studiati, di cui sono stati analizzati alcuni dati acquisiti in condizioni statiche. Quest'analisi è stata compiuta sfruttando anche dei cammini di multiplo scattering dedicando particolare attenzione alla trattazione del fattore di Debye Waller. Nella parte principale della tesi verranno descritti la progettazione ed il test di un apparato sperimentale per l'acquisizione di spettri differenziali da utilizzare alla beamline BM08 dell'European Synchrotron Radiation Facility di Grenoble. Saranno presentate principalmente le modifiche apportate al software d'acquisizione della linea e la progettazione di un sistema ottico d'eccitazione da montare nella camera sperimentale. Nella fase di studio dell'ottica è stato creato in LabView un simulatore basato sul metodo Monte Carlo, capace di prevedere il comportamento del sistema di lenti.

Cinetica e termodinamica della trasformazione metallo-idruro in nanoparticelle Mg-Ti.

L'utilizzo dell'idrogeno come vettore energetico è uno dei temi, riguardanti la sostenibilità energetica, di maggior rilievo degli ultimi anni. Tuttavia ad oggi è ancora in corso la ricerca di un sistema che ne permetta un immagazzinamento efficiente. Il MgH2 costituisce un valido candidato per la produzione di sistemi per lo stoccaggio di idrogeno allo stato solido. In questa tesi, per migliorare le proprietà cinetiche e termodinamiche di cui questo sistema, sono stati sintetizzati dei campioni nanostrutturati composti da Mg-Ti attraverso la tecnica Inert Gas Condensation. I campioni così ottenuti sono stati analizzati dal punto di vista morfologico e composizionale, mediante la microscopia elettronica a scansione, la microanalisi e la diffrazione di raggi X. Tali analisi hanno mostrato che le dimensioni delle nanoparticelle sono comprese tra i 10-30 nm e che la tecnica IGC permette una distribuzione uniforme del titanio all'interno della matrice Mg. Le misure di caratterizzazione per l'assorbimento reversibile di idrogeno sono state effettuate attraverso il metodo volumetrico, Sievert. I campioni sono stati analizzati a varie temperature (473K-573K). Cineticamente la presenza di titanio ha provocato un aumento della velocità delle cinetiche sia per i processi di desorbimento che per quelli di assorbimento ed ha contribuito ad una diminuzione consistente delle energie di attivazione di entrambi i processi rispetto a quelle note in letteratura per il composto MgH2. Dal punto di vista termodinamico, sia le pressioni di equilibrio ottenute dalle analisi PCT a diverse temperature, che l'entalpia e l'entropia di formazione risultano essere in accordo con i valori conosciuti per il sistema MgH2. All'interno di questo lavoro di tesi è inoltre presentata un'analisi preliminare di un campione analizzato con la tecnica Synchrotron Radiation-Powder X Ray Diffraction in situ, presso la facility MAX-lab (Svezia), all’interno dell’azione COST, MP1103 per la ricerca di sistemi per lo stoccaggio di idrogeno allo stato solido.

Studio della struttura locale di droganti in quantum dots mediante spettroscopia di assorbimento di raggi X

La capacità della spettroscopia di assorbimento di riuscire a determinare la struttura locale di campioni di ogni tipo e concentrazione, dagli elementi puri ai più moderni materiali nanostrutturati, rende lo studio dei meccanismi di incorporazione di droganti in matrici di semiconduttori il campo che meglio ne esprime tutto il potenziale. Inoltre la possibilità di ottenere informazioni sulla struttura locale di un particolare elemento in traccia posto in sistemi senza ordine a lungo raggio risulta, ovviamente, nello studio dei semiconduttori di grandissimo interesse. Tuttavia, la complessità di determinate strutture, generate dalla incorporazione di elementi eterovalenti che ne modificano la simmetria, può far si che all’analisi sperimentale si debbano affiancare dei metodi avanzati ab-initio. Questi approcci garantiscono, attraverso la simulazione o di strutture atomiche o dello stesso spettro XAS, di ottenere una più completa e precisa interpretazione dei dati sperimentali. Nella fase preliminare di questo elaborato si illustrerà la fenomenologia della spettroscopia di assorbimento e i fondamenti teorici che stanno alla base dell’analisi della struttura fine di soglia. Si introdurranno contemporaneamente le tecniche sperimentali con cui si realizzano le misure di spettri di assorbimento su una beamline che sfrutta sorgente di radiazione di sincrotrone facendo riferimento agli strumenti montati sulla linea LISA (o BM08) presso l’European Synchrotron Radiation Facility di Grenoble su cui si sono realizzati gli esperimenti di questo lavoro. Successivamente si realizzerà una rassegna di alcuni esperimenti simbolo della analisi della struttura locale di droganti in semiconduttori mediante XAFS, andando ad approfondire i metodi sperimentali associati. Nella parte principale della tesi verranno descritti alcuni tipi di analisi avanzate effettuate su Colloidal Quantum Dots a base di solfuro di piombo drogati con antimonio. Tali sistemi, particolarmente interessanti per potenziali applicazioni in campo optoelettrico, sono stati analizzati mediante misure di fluorescenza ottenute sulla beamline LISA. La fase di analisi ha visto la progettazione di una suite di programmi in C++ per realizzare simulazioni di uno spettro XAS teorico completo basato su strutture ottenute (anche esse) da metodi ab-initio.

Evidence and structural mechanism for late lung alveolarization

According to the current view, the formation of new alveolar septa from preexisting ones ceases due to the reduction of a double- to a single-layered capillaries network inside the alveolar septa (microvasculature maturation postnatal days 14-21 in rats). We challenged this view by measuring stereologically the appearance of new alveolar septa and by studying the alveolar capillary network in three-dimensional (3-D) visualizations obtained by high-resolution synchrotron radiation X-ray tomographic microscopy. We observed that new septa are formed at least until young adulthood (rats, days 4-60) and that roughly half of the new septa are lifted off of mature septa containing single-layered capillary networks. At the basis of newly forming septa, we detected a local duplication of the capillary network. We conclude that new alveoli may be formed in principle at any time and at any location inside the lung parenchyma and that lung development continues into young adulthood. We define two phases during developmental alveolarization. Phase one (days 4-21), lifting off of new septa from immature preexisting septa, and phase two (day 14 through young adulthood), formation of septa from mature preexisting septa. Clinically, our results ask for precautions using drugs influencing structural lung development during both phases of alveolarization.

Visualization and stereological characterization of individual rat lung acini by high-resolution X-ray tomographic microscopy

The small trees of gas-exchanging pulmonary airways which are fed by the most distal purely conducting airways are called acini and represent the functional gas-exchanging units. The three-dimensional architecture of the acini has a strong influence on ventilation and particle deposition. Due to the difficulty to identify individual acini on microscopic lung sections the knowledge about the number of acini and their biological parameters like volume, surface area, and number of alveoli per acinus are limited. We developed a method to extract individual acini from lungs imaged by high-resolution synchrotron radiation based X-ray tomographic microscopy and estimated their volume, surface area and number of alveoli. Rat acini were isolated by semiautomatically closing the airways at the transition from conducting to gas-exchanging airways. We estimated a mean internal acinar volume of 1.148mm(3), a mean acinar surface area of 73.9mm(2), and a mean of 8470 alveoli per acinus. Assuming that the acini are similarly sized throughout different regions of the lung, we calculated that a rat lung contains 5470±833 acini. We conclude that our novel approach is well suited for the fast and reliable characterization of a large number of individual acini in healthy, diseased, or transgenic lungs of different species including humans.

Neonatal steroids induce a down-regulation of tenascin-C and elastin and cause a deceleration of the first phase and an acceleration of the second phase of lung alveolarization

Pre- and postnatal corticosteroids are often used in perinatal medicine to improve pulmonary function in preterm infants. To mimic this clinical situation, newborn rats were treated systemically with dexamethasone (Dex), 0.1-0.01 mg/kg/day on days P1-P4. We hypothesized that postnatal Dex may have an impact on alveolarization by interfering with extracellular matrix proteins and cellular differentiation. Morphological alterations were observed on 3D images obtained by high-resolution synchrotron radiation X-ray tomographic microscopy. Alveolarization was quantified stereologically by estimating the formation of new septa between days P4 and P60. The parenchymal expression of tenascin-C (TNC), smooth muscle actin (SMA), and elastin was measured by immunofluorescence and gene expression for TNC by qRT-PCR. After Dex treatment, the first phase of alveolarization was significantly delayed between days P6 and P10, whereas the second phase was accelerated. Elastin and SMA expressions were delayed by Dex treatment, whereas TNC expression was delayed and prolonged. A short course of neonatal steroids impairs the first phase of alveolarization, most likely by altering the TNC and elastin expression. Due to an overshooting catch-up during the second phase of alveolarization, the differences disappear when the animals reach adulthood.

Sprouting and intussusceptive angiogenesis in postpneumonectomy lung growth: mechanisms of alveolar neovascularization

In most rodents and some other mammals, the removal of one lung results in compensatory growth associated with dramatic angiogenesis and complete restoration of lung capacity. One pivotal mechanism in neoalveolarization is neovascularization, because without angiogenesis new alveoli can not be formed. The aim of this study is to image and analyze three-dimensionally the different patterns of neovascularization seen following pneumonectomy in mice on a sub-micron-scale. C57/BL6 mice underwent a left-sided pneumonectomy. Lungs were harvested at various timepoints after pneumonectomy. Volume analysis by microCT revealed a striking increase of 143 percent in the cardiac lobe 14 days after pneumonectomy. Analysis of microvascular corrosion casting demonstrated spatially heterogenous vascular densitities which were in line with the perivascular and subpleural compensatory growth pattern observed in anti-PCNA-stained lung sections. Within these regions an expansion of the vascular plexus with increased pillar formations and sprouting angiogenesis, originating both from pre-existing bronchial and pulmonary vessels was observed. Also, type II pneumocytes and alveolar macrophages were seen to participate actively in alveolar neo-angiogenesis after pneumonectomy. 3D-visualizations obtained by high-resolution synchrotron radiation X-ray tomographic microscopy showed the appearance of double-layered vessels and bud-like alveolar baskets as have already been described in normal lung development. Scanning electron microscopy data of microvascular architecture also revealed a replication of perialveolar vessel networks through septum formation as already seen in developmental alveolarization. In addition, the appearance of pillar formations and duplications on alveolar entrance ring vessels in mature alveoli are indicative of vascular remodeling. These findings indicate that sprouting and intussusceptive angiogenesis are pivotal mechanisms in adult lung alveolarization after pneumonectomy. Various forms of developmental neoalveolarization may also be considered to contribute in compensatory lung regeneration.

Characterization of the 9L gliosarcoma implanted in the Fischer rat: an orthotopic model for a grade IV brain tumor.

Among rodent models for brain tumors, the 9L gliosarcoma is one of the most widely used. Our 9L-European Synchrotron Radiation Facility (ESRF) model was developed from cells acquired at the Brookhaven National Laboratory (NY, USA) in 1997 and implanted in the right caudate nucleus of syngeneic Fisher rats. It has been largely used by the user community of the ESRF during the last decade, for imaging, radiotherapy, and chemotherapy, including innovative treatments based on particular irradiation techniques and/or use of new drugs. This work presents a detailed study of its characteristics, assessed by magnetic resonance imaging (MRI), histology, immunohistochemistry, and cytogenetic analysis. The data used for this work were from rats sampled in six experiments carried out over a 3-year period in our lab (total number of rats = 142). The 9L-ESRF tumors were induced by a stereotactic inoculation of 10(4) 9L cells in the right caudate nucleus of the brain. The assessment of vascular parameters was performed by MRI (blood volume fraction and vascular size index) and by immunostaining of vessels (rat endothelial cell antigen-1 and type IV collagen). Immunohistochemistry and regular histology were used to describe features such as tumor cell infiltration, necrosis area, nuclear pleomorphism, cellularity, mitotic characteristics, leukocytic infiltration, proliferation, and inflammation. Moreover, for each of the six experiments, the survival of the animals was assessed and related to the tumor growth observed by MRI or histology. Additionally, the cytogenetic status of the 9L cells used at ESRF lab was investigated by comparative genomics hybridization analysis. Finally, the response of the 9L-ESRF tumor to radiotherapy was estimated by plotting the survival curves after irradiation. The median survival time of 9L-ESRF tumor-bearing rats was highly reproducible (19-20 days). The 9L-ESRF tumors presented a quasi-exponential growth, were highly vascularized with a high cellular density and a high proliferative index, accompanied by signs of inflammatory responses. We also report an infiltrative pattern which is poorly observed on conventional 9 L tumor. The 9L-ESRF cells presented some cytogenetic specificities such as altered regions including CDK4, CDKN2A, CDKN2B, and MDM2 genes. Finally, the lifespan of 9L-ESRF tumor-bearing rats was enhanced up to 28, 35, and 45 days for single doses of 10, 20, and 2 × 20 Gy, respectively. First, this report describes an animal model that is used worldwide. Second, we describe few features typical of our model if compared to other 9L models worldwide. Altogether, the 9L-ESRF tumor model presents characteristics close to the human high-grade gliomas such as high proliferative capability, high vascularization and a high infiltrative pattern. Its response to radiotherapy demonstrates its potential as a tool for innovative radiotherapy protocols.

Nanometer-size anisotropy of injection-molded polymer micro-cantilever arrays

Understanding and controlling the structural anisotropies of injection-molded polymers is vital for designing products such as cantilever-based sensors. Such micro-cantilevers are considered as cost-effective alternatives to single-crystalline silicon-based sensors. In order to achieve similar sensing characteristics,structure and morphology have to be controlled by means of processing parameters including mold temperature and injection speed. Synchrotron radiation-based scanning small- (SAXS) and wide-angle x-ray scattering techniques were used to quantify crystallinity and anisotropy in polymer micro-cantilevers with micrometer resolution in real space. SAXS measurements confirmed the lamellar nature of the injection-molded semi-crystalline micro-cantilevers. The homogenous cantilever material exhibits a lamellar periodicity increasing with mold temperature but not with injection speed. We demonstrate that micro-cantilevers made of semi-crystalline polymers such as polyvinylidenefluoride, polyoxymethylene, and polypropylene show the expected strong degree of anisotropy along the injection direction.

Efficient estimation of the total number of acini in adult rat lung

Pulmonary airways are subdivided into conducting and gas-exchanging airways. An acinus is defined as the small tree of gas-exchanging airways, which is fed by the most distal purely conducting airway. Until now a dissector of five consecutive sections or airway casts were used to count acini. We developed a faster method to estimate the number of acini in young adult rats. Right middle lung lobes were critical point dried or paraffin embedded after heavy metal staining and imaged by X-ray micro-CT or synchrotron radiation-based X-rays tomographic microscopy. The entrances of the acini were counted in three-dimensional (3D) stacks of images by scrolling through them and using morphological criteria (airway wall thickness and appearance of alveoli). Segmentation stopper were placed at the acinar entrances for 3D visualizations of the conducting airways. We observed that acinar airways start at various generations and that one transitional bronchiole may serve more than one acinus. A mean of 5612 (±547) acini per lung and a mean airspace volume of 0.907 (±0.108) μL per acinus were estimated. In 60-day-old rats neither the number of acini nor the mean acinar volume did correlate with the body weight or the lung volume.

Combining Monte Carlo methods with coherent wave optics for the simulation of phase-sensitive X-ray imaging

Phase-sensitive X-ray imaging shows a high sensitivity towards electron density variations, making it well suited for imaging of soft tissue matter. However, there are still open questions about the details of the image formation process. Here, a framework for numerical simulations of phase-sensitive X-ray imaging is presented, which takes both particle- and wave-like properties of X-rays into consideration. A split approach is presented where we combine a Monte Carlo method (MC) based sample part with a wave optics simulation based propagation part, leading to a framework that takes both particle- and wave-like properties into account. The framework can be adapted to different phase-sensitive imaging methods and has been validated through comparisons with experiments for grating interferometry and propagation-based imaging. The validation of the framework shows that the combination of wave optics and MC has been successfully implemented and yields good agreement between measurements and simulations. This demonstrates that the physical processes relevant for developing a deeper understanding of scattering in the context of phase-sensitive imaging are modelled in a sufficiently accurate manner. The framework can be used for the simulation of phase-sensitive X-ray imaging, for instance for the simulation of grating interferometry or propagation-based imaging.

Tomographic measurements of O. umbonatus and N. truempyi during the PETM and ETM-2 at ODP Sites 208-1262 and 208-1263

Predicting the impact of ongoing anthropogenic CO2 emissions on calcifying marine organisms is complex, owing to the synergy between direct changes (acidification) and indirect changes through climate change (e.g., warming, changes in ocean circulation, and deoxygenation). Laboratory experiments, particularly on longer-lived organisms, tend to be too short to reveal the potential of organisms to acclimatize, adapt, or evolve and usually do not incorporate multiple stressors. We studied two examples of rapid carbon release in the geological record, Eocene Thermal Maximum 2 (~53.2 Ma) and the Paleocene Eocene Thermal Maximum (PETM, ~55.5 Ma), the best analogs over the last 65 Ma for future ocean acidification related to high atmospheric CO2 levels. We use benthic foraminifers, which suffered severe extinction during the PETM, as a model group. Using synchrotron radiation X-ray tomographic microscopy, we reconstruct the calcification response of survivor species and find, contrary to expectations, that calcification significantly increased during the PETM. In contrast, there was no significant response to the smaller Eocene Thermal Maximum 2, which was associated with a minor change in diversity only. These observations suggest that there is a response threshold for extinction and calcification response, while highlighting the utility of the geological record in helping constrain the sensitivity of biotic response to environmental change.

Composition of suspended matter and bottom sediments from the North Dvina mouth area

Grain-size, mineral and chemical compositions of suspended particulate matter (SPM) from waters of the Severnaya (North) Dvina River mouth area during the spring flood in May 2004 is studied. Data published on composition of riverine SPM in the White Sea basin are very poor. The spring flood period when more than half of annual runoff is supplied from the river to the sea in during short time is understood more poorly. The paper considers comparison results of the grain size compositions of SPM and bottom sediments. Data of laser and hydraulic techniques of grain size analysis are compared. Short-period variations of SPM concentration and composition representing two diurnal peaks of the tide level are studied. It is found that SPM is mainly transferred during the spring flood as mineral aggregates up to 40 µm diameter. Sandy-silty fraction of riverine SPM settles in delta branches and channels, and bulk of clay-size material is supplied to the sea. Mineral and chemical compositions of SPM from the North Dvina River are determined by supply of material from the drainage basin. This material is subjected to intense mechanic separation during transfer to the sea. Key regularities of formation of mineral composition of SPM during the flood time are revealed. Effect of SPM grain size composition on distribution of minerals and chemical elements in study in the dynamic system of the river mouth area are characterized.

Gas hydrate structures and C1-C5 hydrocarbons at individual sites in the Gulf of Mexico

Gas hydrate samples from various locations in the Gulf of Mexico (GOM) differ considerably in their microstructure. Distinct microstructure characteristics coincide with discrete crystallographic structures, gas compositions and calculated thermodynamic stabilities. The crystallographic structures were established by X-ray diffraction, using both conventional X-ray sources and high-energy synchrotron radiation. The microstructures were examined by cryo-stage Field-Emission Scanning Electron Microscopy (FE-SEM). Good sample preservation was warranted by the low ice fractions shown from quantitative phase analyses. Gas hydrate structure II samples from the Green Canyon in the northern GOM had methane concentrations of 70-80% and up to 30% of C2-C5 of measured hydrocarbons. Hydrocarbons in the crystallographic structure I hydrate from the Chapopote asphalt volcano in the southern GOM was comprised of more than 98% methane. Fairly different microstructures were identified for those different hydrates: Pores measuring 200-400 nm in diameter were present in structure I gas hydrate samples; no such pores but dense crystal surfaces instead were discovered in structure II gas hydrate. The stability of the hydrate samples is discussed regarding gas composition, crystallographic structure and microstructure. Electron microscopic observations showed evidence of gas hydrate and liquid oil co-occurrence on a micrometer scale. That demonstrates that oil has direct contact to gas hydrates when it diffuses through a hydrate matrix.