Dosimetry studies for radiation therapy with photons and radiobiology using low-energy protons.

Tesis llevada a cabo para conseguir el grado de Doctor por la Universidad de Sevilla--2017-09-15This thesis work is based on the knowledge about the interaction of radiation with matter to develop new methods with the aim of applying them to the radiation therapy and radiobiology, specifically to dose measurements in radiation therapy, dose measurements with low energy proton beams and DNA damages caused by these protons on cancer cells. Thus, it presents two main parts: one regarding the presentation of a novel prototype based on silicon detector technology for treatment verification in radiotherapy, and the other, the instrumentation and the methodology for low energy proton beam characterization, dosimetry, with a special focus on the response of radiochromic films to protons, and finally the application on biological samples to study the DNA damage produced by protons. The first part of this work presents a novel system with improved performances with respect to a previous system which was filed for a patent in September 13th 2011 at the OEMP - “Oficina Espa˜nola de Patentes y Marcas”- Ministry of Industry, Tourism and Commerce, under reference number P201101009. This work was also published in specialized reviews and thesis works. The characterization of the response in terms of absorbed dose of this novel detector was presented Here, this study was repeated because of some modifications introduced in the electronics and in one of the detector’s cables, which was modified due to a breakdown. The application of this system for complex treatment verification providing a 2D dose map reconstruction in the axial plane is presented for the first time in this thesis work. The second part of the work presents the installation of instrumentation for dosimetry and radiobiology studies using proton beams produced by the 3 MV Tandem accelerator at Centro Nacional de Acceleradores, Sevilla. The original work within this part can be divided into three main points: i) the elaboration of a protocol which allows to obtain a beam profile with low current and an homogeneous profile over the whole sample surface; ii) dosimetry with low energy protons using ionization chambers and radiochromic films; iii) study of the dosimetry at the Bragg peak. Last, the application of these studies to the irradiation of cell cultures is presented and preliminary results of the DNA damage produced by protons are shown.Peer reviewe

Analgesia induced by the epigenetic drug, L-acetylcarnitine, outlasts the end of treatment in mouse models of chronic inflammatory and neuropathic pain

Background: L-acetylcarnitine, a drug marketed for the treatment of chronic pain, causes analgesia by epigenetically up-regulating type-2 metabotropic glutamate (mGlu2) receptors in the spinal cord. Because the epigenetic mechanisms are typically long-lasting, we hypothesized that analgesia could outlast the duration of L-acetylcarnitine treatment in models of inflammatory and neuropathic pain. Results: A seven-day treatment with L-acetylcarnitine ( 100 mg/kg, once a day, i.p.) produced an antiallodynic effect in the complete Freund adjuvant mouse model of chronic inflammatory pain. L-Acetylcarnitine-induced analgesia persisted for at least 14 days after drug withdrawal. In contrast, the analgesic effect of pregabalin, amitryptiline, ceftriaxone, and N-acetylcysteine disappeared seven days after drug withdrawal. L-acetylcarnitine treatment enhanced mGlu2/3 receptor protein levels in the dorsal region of the spinal cord. This effect also persisted for two weeks after drug withdrawal and was associated with increased levels of acetylated histone H3 bound to the Grm2 gene promoter in the dorsal root ganglia. A long-lasting analgesic effect of L-acetylcarnitine was also observed in mice subjected to chronic constriction injury of the sciatic nerve. In these animals, a 14-day treatment with pregabalin, amitryptiline, tramadol, or L-acetylcarnitine produced a significant antiallodynic effect, with pregabalin displaying the greatest efficacy. In mice treated with pregabalin, tramadol or L-acetylcarnitine the analgesic effect was still visible 15 days after the end of drug treatment. However, only in mice treated with L-acetylcarnitine analgesia persisted 37 days after drug withdrawal. This effect was associated with an increase in mGlu2/3 receptor protein levels in the dorsal horns of the spinal cord. Conclusions: Our findings suggest that L-acetylcarnitine has the unique property to cause a long-lasting analgesic effect that might reduce relapses in patients suffering from chronic pain

First record of plastic debris in the stomach of Mediterranean lanternfishes

This study highlights for the first time the presence of plastic debris in the stomachs of Mediterranean lanternfishes (Myctophidae): Electrona risso, Diaphus metopoclampus, Hygophum benoiti and Myctophum punctatum. Samples were collected in the central Mediterranean Sea between 2010 and 2014. Plastics ingested belonged to small microplastics (0.2 - 2 mm), large microplastics (2 - 5 mm) and mesoplastics (5 - 25 mm), having mainly clear colors. Their frequency of occurrence in stomachs was equal to 2.7%, but it increases to 5.8% if only migratory species are considered. The higher number of plastics was found in E. risso and H. benoiti (5 in both species). The plastic ingestion may represent a risk for vertical migrant lanternfishes due to the increase in buoyancy. Ecotoxicological aspects linked to the potential effects of contaminants on lanternfish biology and to the transfer of pollutants throughout the marine trophic web up to top predators should be deepened

Prvi zapis o plastičnim krhotinama u želucu mediteranskih riba žaboglavki (Myctophidae)

This study highlights for the first time the presence of plastic debris in the stomachs of Mediterranean lanternfishes (Myctophidae): Electrona risso, Diaphus metopoclampus, Hygophum benoiti and Myctophum punctatum. Samples were collected in the central Mediterranean Sea between 2010 and 2014. Plastics ingested belonged to small microplastics (0.2 - 2 mm), large microplastics (2 - 5 mm) and mesoplastics (5 - 25 mm), having mainly clear colors. Their frequency of occurrence in stomachs was equal to 2.7%, but it increases to 5.8% if only migratory species are considered. The higher number of plastics was found in E. risso and H. benoiti (5 in both species). The plastic ingestion may represent a risk for vertical migrant lanternfishes due to the increase in buoyancy. Ecotoxicological aspects linked to the potential effects of contaminants on lanternfish biology and to the transfer of pollutants throughout the marine trophic web up to top predators should be deepened.Ova studija ističe po prvi put prisutnost plastičnih otpadaka u želucima mediteranskih žaboglavki. (Myctophidae): Electrona risso, Diaphus metopoclampus, Hygophum benoiti i Myctophum punctatum. Uzorci su prikupljeni u središnjem Mediteranu u periodu između 2010. i 2014. godine. Progu tane čestice plastike pripadaju maloj mikroplastici (0,2-2 mm), velikoj mikroplastici (2-5 mm) i mezoplastici (5-25 mm), te su uglavnom jasnih boja. Njihova učestalost pojavljivanja u probavilima iznosila je 2.7%, dok je kod migratornih vrsta iznosila 5.8%. Veći broj čestica plastike pronađen je kod vrsta E. risso i H. benoiti (5 po vrsti). Gutanje plastike može predstavljati rizik za vertikalne migratore iz porodice žaboglavki zbog povećanja uzgona. Potrebno je produbiti istraživanja ekotoksikoloških aspekata povezanih s mogućim učincima zagađivala na biologiju žaboglavki, kao i onih povezanih s prijenosom zagađivala kroz trofičku mrežu u moru sve do glavnih grabežljivaca

Feasibility Study of a Proton Irradiation Facility for Radiobiological Measurements at an 18 MeV Cyclotron

A feasibility study of an experimental setup for the irradiation of biological samples at the cyclotron facility installed at the National Centre of Accelerators (Seville, Spain) is presented. This cyclotron, which counts on an external beam line for interdisciplinary research purposes, produces an 18 MeV proton beam, which is suitable for the irradiation of mono-layer cultures for the measurement of proton cell damages and Relative Biological Effectiveness (RBE) at energies below the beam nominal value. Measurements of this kind are of interest for proton therapy, since the variation of proton RBE at the distal edge of the Bragg curve may have implications in clinical proton therapy treatments. In the following, the characteristics of the beam line and the solutions implemented for the irradiation of biological samples are described. When dealing with the irradiation of cell cultures, low beam intensities and broad homogeneous irradiation fields are required, in order to assure that all the cells receive the same dose with a suitable dose rate. At the cyclotron, these constraints have been achieved by completely defocusing the beam, intercepting the beam path with tungsten scattering foils and varying the exit-window-to-sample distance. The properties of the proton beam thus obtained have been analysed and compared with Monte Carlo simulations. The results of this comparison, as well as the experimental measurement of the lateral dose profiles expected at the position of samples are presented. Meaningful dose rates of about 2–3 Gy/min have been obtained. Homogeneous lateral dose profiles, with maximum deviations of 5%, have been measured at a distance of approximately 50 cm in air from the exit window, placing a tungsten scattering foil of 200 μm in the beam path

Rapamycin and Interleukin-10 Treatment Induces T Regulatory Type 1 Cells That Mediate Antigen-Specific Transplantation Tolerance

Islet transplantation is a cure for type 1 diabetes, but its potential is limited by the need for constant immunosuppression. One solution to this problem is the induction of transplantation tolerance mediated by T regulatory cells. T regulatory type 1 (Tr1) cells are characterized by their production of high levels of interleukin (IL)-10, which is crucial for their differentiation and suppressive function. We investigated the effects of IL-10 administered in combination with rapamycin on the induction of Tr1 cells that could mediate a state of tolerance in diabetic mice after pancreatic islet transplantation. The efficacy of this treatment was compared with IL-10 alone and standard immunosuppression. Stable long-term tolerance that was not reversible by alloantigen rechallenge was achieved only in mice treated with rapamycin plus IL-10. Tr1 cells that produced high levels of IL-10 and suppressed T-cell proliferation were isolated from splenocytes of rapamycin plus IL-10–treated mice after treatment withdrawal. In rapamycin plus IL-10–treated mice, endogenous IL-10 mediated an active state of tolerance, as was observed when the blockade of IL-10 activity rapidly induced graft rejection >100 days after transplantation. CD4+ T-cells from rapamycin plus IL-10–treated mice transferred antigen-specific tolerance in mice that received new transplants. Thus rapamycin plus IL-10 not only prevented allograft rejection but also induced Tr1 cells that mediated stable antigen-specific, long-term tolerance in vivo

Role of HLA-G and extracellular vesicles in renal cancer stem cell-induced inhibition of dendritic cell differentiation

BACKGROUND: Tumor immune-escape has been related to the ability of cancer cells to inhibit T cell activation and dendritic cell (DC) differentiation. We previously identified a tumor initiating population, expressing the mesenchymal marker CD105, which fulfills the criteria for definition as cancer stem cells (CD105(+) CSCs) able to release extracellular vesicles (EVs) that favor tumor progression and metastases. The aim of the present study was to compare the ability of renal CSCs and derived EVs to modulate the behavior of monocyte-derived DCs with a non-tumor initiating renal cancer cell population (CD105(-) TCs) and their EVs. METHODS: Maturation of monocyte-derived DCs was studied in presence of CD105(+) CSCs and CD105(-) TCs and their derived EVs. DC differentiation experiments were evaluated by cytofluorimetric analysis. T cell proliferation and ELISA assays were performed. Monocytes and T cells were purified from peripheral blood mononuclear cells obtained from healthy donors. RESULTS: The results obtained demonstrate that both CD105(+) CSCs and CD105(-) TCs impaired the differentiation process of DCs from monocytes. However, the immune-modulatory effect of CD105(+) CSCs was significantly greater than that of CD105(-) TCs. EVs derived from CD105(+) CSCs and in less extent, those derived from CD105(-) TCs retained the ability to impair monocyte maturation and T cell activation. The mechanism has been mainly related to the expression of HLA-G by tumor cells and to its release in a form associated to EVs. HLA-G blockade significantly reduced the inhibitory effect of EVs on DC differentiation. CONCLUSIONS: In conclusion, the results of the present study indicate that renal cancer cells and in particular CSCs and derived EVs impair maturation of DCs and T cell immune response by a mechanism involving HLA-G. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12885-015-2025-z) contains supplementary material, which is available to authorized users

Novel dual single sided silicon strip detector chip for radiotherapy verification

A novel dual single sided silicon strip detector (SSSSD) chip was designed to meet clinical requirements in radiotherapy verification. An available design from Micron Semiconductor Ltd. (BB7, 500 µ m thick) was the base of a two-dimensional detector adapted into a special configuration with the aim of uniforming and minimizing foreing materials around the active area (64 × 64 mm2). With this purpose, two independent BB7 SSSSDs were mounted in a perpendicular configuration, separated by a 500 µ m kapton dielectric film with the same dimensions as the silicon wafers, thus minimizing air gaps in between. This new configuration, called the dual SSSSD chip design, was mounted on kapton printed circuit board (PCB). Both silicon wafers were divided into 32 strips, 2 mm width each. The aim of developing this detector was to allow 2D dose measurements, improve spatial resolution and perform radiotherapy treatment verification faster than with a previous prototype. Characteristics and performance of the novel detector are presented

Transcriptomic profiling of the development of the inflammatory response in human monocytes in vitro.

Monocytes/macrophages are key players in all phases of physiological and pathological inflammation. To understanding the regulation of macrophage functional differentiation during inflammation, we designed an in vitro model that recapitulates the different phases of the reaction (recruitment, initiation, development, and resolution), based on human primary blood monocytes exposed to sequential changes in microenvironmental conditions. All reaction phases were profiled by transcriptomic microarray analysis. Distinct clusters of genes were identified that are differentially regulated through the different phases of inflammation. The gene sets defined by GSEA analysis revealed that the inflammatory phase was enriched in inflammatory pathways, while the resolution phase comprised pathways related to metabolism and gene rearrangement. By comparing gene clusters differentially expressed in monocytes vs. M1 and vs. M2 macrophages extracted from an in-house created meta-database, it was shown that cells in the model resemble M1 during the inflammatory phase and M2 during resolution. The validation of inflammatory and transcriptional factors by qPCR and ELISA confirmed the transcriptomic profiles in the different phases of inflammation. The accurate description of the development of the human inflammatory reaction provided by this in vitro kinetic model can help in identifying regulatory mechanisms in physiological conditions and during pathological derangement

Transcriptional profiling of human bronchial epithelial cell BEAS-2B exposed to diesel and biomass ultrafine particles

Background: Emissions from diesel vehicles and biomass burning are the principal sources of primary ultrafine particles (UFP). The exposure to UFP has been associated to cardiovascular and pulmonary diseases, including lung cancer. Although many aspects of the toxicology of ambient particulate matter (PM) have been unraveled, the molecular mechanisms activated in human cells by the exposure to UFP are still poorly understood. Here, we present an RNA-seq time-course experiment (five time point after single dose exposure) used to investigate the differential and temporal changes induced in the gene expression of human bronchial epithelial cells (BEAS-2B) by the exposure to UFP generated from diesel and biomass combustion. A combination of different bioinformatics tools (EdgeR, next-maSigPro and reactome FI app-Cytoscape and prioritization strategies) facilitated the analyses the temporal transcriptional pattern, functional gene set enrichment and gene networks related to cellular response to UFP particles.Results: The bioinformatics analysis of transcriptional data reveals that the two different UFP induce, since the earliest time points, different transcriptional dynamics resulting in the activation of specific genes. The functional enrichment of differentially expressed genes indicates that the exposure to diesel UFP induces the activation of genes involved in TNFa signaling via NF-kB and inflammatory response, and hypoxia. Conversely, the exposure to ultrafine particles from biomass determines less distinct modifications of the gene expression profiles. Diesel UFP exposure induces the secretion of biomarkers associated to inflammation (CCXL2, EPGN, GREM1, IL1A, IL1B, IL6, IL24, EREG, VEGF) and transcription factors (as NFE2L2, MAFF, HES1, FOSL1, TGIF1) relevant for cardiovascular and lung disease. By means of network reconstruction, four genes (STAT3, HIF1a, NFKB1, KRAS) have emerged as major regulators of transcriptional response of bronchial epithelial cells exposed to diesel exhaust.Conclusions: Overall, this work highlights modifications of the transcriptional landscape in human bronchial cells exposed to UFP and sheds new lights on possible mechanisms by means of which UFP acts as a carcinogen and harmful factor for human health