Download Title of the Project Cellular and molecular mechanisms of ozone

Title of the Project
Cellular and molecular mechanisms of ozone therapy: an in vitro study on mesenchymal stem cells
Project duration
Two-year
Project Managers
Dr. Gabriele Tabaracci, San Rocco Clinic, Montichiari (BS), Italy
Prof. Manuela Malatesta, Department of Neurological and Movement Sciences, University of Verona, Italy
Keywords
ozone, cell culture, stem cells, confocal microscopy, transmission electron microscopy, microarray
Project summary
Oxygen-ozone therapy is a modestly invasive procedure based on the regeneration capabilities of low ozone
concentrations, and used in medicine as an alternative/adjuvant treatment for many diseases. Despite its
relatively wide use, the cellular and molecular mechanisms accounting for the positive effects of ozone
exposure are still largely unexplored.
In the present project, the effects of low ozone concentrations will be investigated on human adipose-derived
adult stem cells by combining light and electron microscopy, histochemistry, flow cytometry, and molecular
biology. These mesenchymal stem cells are present in the stromal-vascular fraction of fat tissue, and can be
induced to differentiate in vitro into meso-, ecto- and endodermal cell lineages and also be reprogrammed to
induced pluripotent stem cells, suitable for tissue regeneration/reconstruction.
Pilot studies performed in collaboration between the Project Manager and the Partner already gave interesting
data and essential technical hints; moreover, the exclusive and complementary expertise of the two teams,
and the availability of permissions and facilities in their laboratories ensure the immediate feasibility of the
planned research.
The results of this project will provide a robust scientific background to clinicians practicing ozone therapy, and
will respond to the present need of “a greater quantity of publications and research”, as recently underlined by
the International Scientific Committee of Ozone therapy. The information provided will also contribute to the
progress of regenerative medicine and tissue engineering. In addition, our results will be useful for regulatory
authorities to define a scientifically-founded framework to evaluate the biological effects of ozone exposure on
in vitro systems and, more specifically, on stem cells. Finally, understanding the biological outcomes of ozone
treatment will also contribute to increase public awareness of the benefits and limit of ozone therapy.
Detailed description of the project
Oxygen-ozone therapy is a modestly invasive procedure based on the regeneration capabilities of low ozone
concentrations, and used in medicine as an alternative/adjuvant treatment for different diseases, among
which arthritis, heart and vascular diseases, asthma, emphysema, multiple sclerosis (reviews in 1-3). Despite its
relatively wide use, the cellular and molecular mechanisms accounting for the positive effects of ozone
treatment are still largely unexplored (for a scientific overview of ozone therapy, see 4).
Molecular evidence shed light on some biological mechanisms responsible for the dose-dependent effects of
ozone treatment (5); however, structural and functional data on the effects of low ozone concentrations on
cell dynamics and on organelle structure and function are still lacking. A recent pilot study, co-financed by the
Verona University Unit and the Poliambulatorio San Rocco and performed on a human epithelial cell line, gave
interesting information on the positive effects of mild ozonisation on cytoskeletal proteins, mitochondrial
function and nuclear activity (6,7). Dott. Gabriele Tabaracci will present, as an invited speaker, the results of
this study at the National Congress of the Italian Federation of Oxygen Ozone Therapy (FIO) (Rome, June 5-7,
2015), and at the International Meeting of the Madrid Declaration on Ozone Therapy (Madrid, June 12, 2015).
In the frame of the same research, recent data on human neuronal cell lines demonstrated interesting
structural and functional effects of mild ozonisation on multiple metabolic pathways, highlighting that the
effects of ozone depends both on concentration and the cell type (unpublished data).
Based on these promising results, in the present project we aim at investigating the effects of low ozone
concentrations on human adipose-derived adult stem (hADAS) cells by combining light and electron
microscopy, histochemistry, flow cytometry, and molecular biology including high throughput Next Generation
Sequencing (NGS). In particular, the use of technologies for high-throughput sequencing of the transcriptome
will provide an innovative approach to clarify the molecular pathways accounting for the action mechanisms of
ozone identifying new markers that could represent targets for ozone therapy. Even microRNA (miRNA) could
be involved in the action mechanism of ozone therapy since they can induce epigenetic changes in gene or
protein expression, which persist over time and play important regulatory roles in a variety of biological
processes (8).
The use of in vitro systems will ensure controlled and easily reproducible experimental conditions. hADAS are
mesenchymal stem cells present in the stromal-vascular fraction of fat tissue; they have the capability to
differentiate in vitro into meso-, ecto- and endodermal cell lineages and can also be reprogrammed to induce
pluripotent stem cells more efficiently than other cell types (recent review in 9). For the present research, the
cells will be grown in appropriate media to obtain differentiation into adipoblastic, osteoblastic, chondroblastic
and neuronal lineages.
The results of the present project will contribute to fill the current lack of information on the mechanisms of
action of ozone on these important cell types, especially involved in tissue regenerative/reconstructive
processes (recent reviews in 9-12). This basic information will provide a robust scientific background to
clinicians practicing ozone therapy and will help to set up specific therapeutic protocols for different tissues
characterised by peculiar metabolic and cytokinetic features. The information provided by our study will also
be seminal for regenerative medicine and tissue engineering. In addition, our results will also provide
regulatory authorities a scientifically-founded framework to evaluate the biological effects of ozone exposure
on in vitro systems and, more specifically, on stem cells. Finally, understanding the biological outcomes of
ozone treatment will also contribute to increase public awareness of ozone therapy benefits and limits.
The research work of the present project will be subdivided into 4 Work Packages (WP).
WP1: Cell culture and exposure to ozone
WP2: Evaluation of ozone effects by microscopy techniques
WP3: Evaluation of ozone effects by biomolecular techniques
WP4: Data analysis, bioinformatics and dissemination of results
WP1: Cell culture and exposure to ozone
In the laboratory of the Verona University Unit, the isolation, expansion, characterization, and differentiation
of ADAS cells derived from liposuction aspirates are usually performed (e.g. 13-16).
Ozone exposure will be tested on both undifferentiated and differentiated ADAS cells in order to evaluate the
effects on the proliferation and differentiation potential as well as on the structural and functional features of
differentiated cells. The cells will be exposed to oxygen-ozone gas mixtures with different ozone
concentrations according to the techniques described in (17, 18). Our previous results indicate that ozone
concentrations ranging from 1 to 16 µg/ml are able to stimulate cellular activities without cell damage:
therefore, we will start our experiments under these conditions, using cells exposed to either air (untreated
samples) or O2 as controls.
Both ozone-exposed and control cells will be analysed for several structural, cytokinetic and molecular
parameters at increasing times after treatment; some cell samples will be let to proliferate for several passages
after ozone exposure in order to investigate possible effects on the cell senescence process.
WP2: Evaluation of ozone effects by microscopy techniques
To evaluate the structural and functional effects of ozone exposure on cells, the samples will be processed for
cytochemistry and immunocytochemistry at fluorescence microscopy, for flow cytometry, and for morphology
and cytochemistry at transmission electron microscopy (TEM). Experiments of correlative microscopy will also
be performed: by this advanced approach, the location of fluorescent markers may be detected at the high
resolution of TEM, by the light-induced oxidation of diaminobenzidine (photoconversion, 19).
The effects of ozone administration on cell proliferation and death or on specific subcellular organelles will be
investigated by cytochemical and immunocytochemical tests. For fluorescence immunocytochemistry, cells
grown on coverslips will be fixed with paraformaldehyde and then stored in 70% ethanol at -20°C, until use.
Cell proliferation will be monitored by dual-parameter flow cytometry of DNA content and bromodeoxyuridine
(BrdU) incorporation, to estimate the percentage of cells in G1, S, and G2-M phase of the cell cycle. In addition,
the presence of necrotic cell death in ozone-exposed samples will be estimated by the classical trypan-blue
exclusion test, while the possible occurrence of regulated forms of cell death (apoptosis or parthanatos) will be
investigated by flow cytometry after dual-fluorescence staining of unfixed cells with FITC-labeled Annexin V
and propidium iodide, or by other well-established histochemical methods (TUNEL assay or the
immunofluorescence detection of specific protein such as cleaved PARP-1, AIF, activated caspases). Some
markers of autophagy (p62, LC3-II, Beclin 1, ATG5, 7, and 12, Lamp 2, 7) will be revealed by
immunofluorescence. Particular attention will be given to the accumulation of lipid droplets in adipocytes,
which will be analysed by using histochemical and morphometrical approaches (20,21).
Since ozone is known to increase cell membrane negative charges (7) and to promote polymerization of
cytoskeletal proteins (22,23), cell adhesion capability will be analysed by counting adhering cells at increasing
times after seeding. In addition, cytoskeletal components will be investigated by means of cytochemistry (e.g.,
actin labelling by phalloidin) and fluorescence immunocytochemistry (antibodies directed against e.g. tubulin,
vimentin, desmin, neurofilaments).
The possible ozone-induced oxidative stress in cells (review in 5) will be monitored by enzyme histochemistry
(24) to detect reactive oxygen species (ROS) and, in particular, nitric oxide (NO).
For the morphological analysis at TEM, cells will be fixed with glutaraldehyde-osmium and embedded in epoxy
resins; for ultrastructural cytochemistry and immunocytochemistry, the cells will be fixed with
paraformaldehyde and embedded in LR White acrylic resin.
The possible variations in the transcription rate of cells exposed to ozone will be evaluated at TEM after
bromouridine (BrU) incorporation: cell cultures will be pulse-labelled with BrU, fixed and gold-immunolabelled;
the transcription rate will be estimated by counting the colloidal gold grains on an adequate number of nuclear
sections and by applying the appropriate statistical analyses. Variations in the RNA transcription and
processing will also be studied by immunolabelling DNA/RNA hybrids, RNA polymerases I and II, and the
protein complexes involved in transcription, elongation, splicing and cleavage (e.g., hnRNPs, snRNPs, SC-35,
fibrillarin), with particular attention to nuclear factors thought to be activated by ozone exposure (e.g. NFkB,
Nrf2, NFAT, AP-1, HIF-1a).
The subcellular organization of the cells will be observed at TEM to detect structural changes of cytoplasmic
organelles (e.g., endoplasmic reticulum, Golgi apparatus, mitochondria, lysosomes, peroxisomes) suggestive of
functional alterations. In particular, due to their high sensitivity to cell damage, mitochondria will be
investigated by ultrastructural morphology and morphometry. In parallel, the effect of ozone exposure on the
different organelles will also be estimated at light microscopy, using organelle-specific dyes (such as the
fluorescent Mito-tracker or ER-tracker) or after immunolabelling of organelle-specific proteins (such as Golgin
for the Golgi apparatus or the mitochondrial HSP 70, which is known to be affected by ozone exposure:
7,25,26). The effect of the ozone-induced oxidative stress on mitochondria will also be assessed by
ultrastructural cytochemistry and immunocytochemistry: to estimate mitochondrial competence the activity of
two key enzymes of the mitochondrial respiratory chain, i.e. cytochrome oxidase (COX) and succinic
dehydogenase (SDH) (both known to be altered by ozone: 27,28) will be studied; in addition, the possible
occurrence of mtDNA oxidation will be investigated by a reliable immunogold procedure for identifying
oxidised bases, such as 8-hydroxyguanine.
More generally, the presence and intracellular distribution of some antioxidant enzymes (e.g. SODs, GPx, GSTs,
CAT, HSPs) will be analysed by fluorescence and/or immunoelectron microscopy.
Scanning electron microscopy will be applied to investigate cell shape and surface modifications as well as cellcell interactions.
WP3: Evaluation of ozone effects by biomolecular techniques
To identify potential markers for ozone as well as molecular pathways linked to its mechanism of action, we
will sequence the coding transcriptome (mRNA sequencing) as well as all the miRNA (microRNA sequencing) in
undifferentiated and differentiated ADAS cells both at baseline and after ozone exposure. This will allow to
identify genes whose expression is modulated by the ozone treatment also in relation to the differentiation
state, and to identify sets of miRNAs modulated by ozone exposure, which could explain changes in gene
expression. Finally, based on the transcriptomic data, pathway analyses will be performed to identify the
molecular signalling linked to the effect of the ozone exposure.
The RNeasy Mini kit (Qiagen) will be used for RNA extraction and purification since it is designed for
purification of total RNA, including miRNA and other small RNA molecules, even from small amounts of
cultured cells. The RNA isolated will be quantified by a spectrophotometer (ND1000 UV-Vis, NanoDrop), and its
integrity will be evaluated by an Agilent Bioanalyser 2100 (Agilent Technologies). The isolated RNA (containing
both mRNA and miRNAs) will be used to perform gene expression profiling of the transcriptome and miRNome.
By using mRNA and microRNA sequencing, we will investigate the candidate genes and candidate miRNAs
involved in ozone mechanisms, and generate new candidates above looking at pathways modulation. mRNA
and microRNA sequencing will be performed with MiSeq platform (Illumina, Inc.). RNA will be retro-transcribed
to cDNA sense and 3’ ends will be adenylated. The following steps will be ligation to the adapters, amplification,
and validation of the libraries. Finally the libraries will be uploaded on the platform.
The genes and miRNAs that will be found modulated with the highest Fold changes will be validated by
quantitative Real Time PCR by using Taqman probe system on an Applied Biosystem Real Time Instrument.
Besides mRNA and microRNA sequencing analyses, investigations on protein expression will be also carried out
in association to microscopy studies.
Briefly, some antioxidant enzymes (e.g. SODs, GPx, GSTs, CAT, HSPs) as well as specific proteins involved in the
mechanisms of transcription and maturation will be quantified by Western blot analysis, with special attention
to nuclear factors activated by ozone exposure (e.g. NFkB, Nrf2, NFAT, AP-1, HIF-1a). Moreover, any proteins
of interest will be analysed following sequencing results.
Measure of the total free radical molecules (reactive oxygen species and reactive nitrogen species) will be
carried out fluorometrically.
WP4: Data analysis, bioinformatics and dissemination of results
For statistical evaluation, after verifying the hypothesis of identical distribution among cell samples, the data
will be pooled according to the experimental sets, to calculate the mean ± standard error of the mean (SE)
values. Statistical comparisons will be performed by parametric or non-parametric tests, as appropriate.
For transcriptomic profiling, quality control and statistical/bioinformatic analyses will be performed using
appropriate packages in R. Data analyses to get lists of genes or of miRNAs differentially modulated according
to the condition; Partek Genomic Suite 6.0 Software will be used. Pathway analyses as well as gene ontology
will be done using the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool, by
Ariadne Pathway Studio software and by Ingenuity Pathway Software. Target prediction analyses to identify
putative genes and pathways modulated by the identified miRNAs will be performed by using several miRNAs
database including TarBase, miRBase, and microRNA.org .
Short reports will be presented and discussed by the two research Units during periodical meetings. The
original results obtained will be presented at national and international meetings and conferences, and
published in peer-reviewed scientific journals (open access journals will be preferred to maximise the
dissemination of results). Results will be also presented at training courses and meetings addressed to
clinicians practicing ozone therapy.
References
1. Re L, Mawsouf MN, Menéndez S, et al. Ozone therapy: clinical and basic evidence of its therapeutic potential.
Arch Med Res 2008;39:17-26
2. Elvis AM, Ekta JS. Ozone therapy: A clinical review. J Nat Sc Biol Med 2011;2:66-70
3. Bocci V. How a calculated oxidative stress can yield multiple therapeutic effects. Free Radic Res
2012;46:1068-75
4. ISCO3 - International Scientific Committee of Ozonetherapy (Madrid, 2012). Ozone therapy and its scientific
foundations. (www.isco3.org)
5. Sagai M, Bocci V. Mechanisms of action involved in ozone therapy: is healing induced via a mild oxidative
stress? Med Gas Res 2011;1:29
6. Tabaracci G, Covi V, Malatesta M, et al. Exposure to low ozone concentrations improves adhesion and
growth of epithelial cells in vitro. European Ozone Congress, Zürich, Switzerland, October 3-5, 2014
7. Costanzo M, Cisterna B, Vella A, et al. Low ozone concentrations stimulate cytoskeletal organization,
mitochondrial
activity
and
nuclear
transcription.
Eur
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Histochem
2015;59:2515.
doi:10.4081/ejh.2015.2515
8. Rao P, Benito E, Fischer A. MicroRNAs as biomarkers for CNS disease. Front Mol Neurosci 2013;6:39
9. Ong WK, Sugii S. Adipose-derived stem cells: fatty potentials for therapy. Int J Biochem Cell Biol
2013;45:1083-6
10. Rigotti G, Marchi A, Sbarbati A. Adipose-derived mesenchymal stem cells: past, present, and future.
Aesthetic Plast Surg 2009;33:271-3
11. Salibian AA, Widgerow AD, Abrouk M, et al. Stem cells in plastic surgery: a review of current clinical and
translational applications. Arch Plast Surg 2013;40:666-75
12. Tsuji W, Rubin JP, Marra KG. Adipose-derived stem cells: Implications in tissue regeneration. World J Stem
Cells 2014;6:312-21
13. Rigotti G, Marchi A, Galiè M, et al. Clinical treatment of radiotherapy tissue damage by lipoaspirate
transplant: a healing process mediated by adipose-derived adult stem cells. Plast Reconstr Surg.
2007;119:1409-22
14. Galiè M, Pignatti M, Scambi I, et al. Comparison of different centrifugation protocols for the best yield of
adipose-derived stromal cells from lipoaspirates. Plast Reconstr Surg. 2008;122:233-4e.
15. Anghileri E, Marconi S, Pignatelli A, et al. Neuronal differentiation potential of human adipose-derived
mesenchymal stem cells. Stem Cells Dev. 2008 ;17:909-16
16. Conti G, Benati D, Bernardi P, et al. The post-adipocytic phase of the adipose cell cycle. Tissue Cell.
2014;46:520-6.
17. Larini A, Bianchi L, Bocci V. The ozone tolerance: I) Enhancement of antioxidant enzymes is ozone dosedependent in Jurkat cells. Free Radic Res 2003;37:1163-8
18. Costanzo M, Cisterna B, Covi V, et al. An easy and inexpensive method to expose adhering cultured cells to
ozonisation. Microscopie 2015;12(1):46-52 (www.pagepress.org/microscopie)
19. Malatesta M, Pellicciari C, Cisterna B, et al. Tracing nanoparticles and photosensitizing molecules at
transmission electron microscopy by diaminobenzidine photo-oxidation. Micron 2014;59C:44-51
20. Rizzatti V, Boschi F, Pedrotti M, et al. Lipid droplets characterization in adipocyte differentiated 3T3-L1
cells: size and optical density distribution. Eur J Histochem 2013;57:e24
21. Boschi F, Rizzatti V, Zamboni M, et al. Lipid droplets fusion in adipocyte differentiated 3T3-L1 cells: a Monte
Carlo simulation. Exp Cell Res 2014;321:201-8
22. Taulet N, Delorme-Walker VD, DerMardirossian C. Reactive oxygen species regulate protrusion efficiency
by controlling actin dynamics. PLoS ONE 2012;7:e41342
23. Muliyil S, Narasimha M. Mitochondrial ROS regulates cytoskeletal and mitochondrial remodeling to tune
cell and tissue dynamics in a model for wound healing. Developmental Cell 2014;28:239-52
24. Freitas I, Griffini P, Bertone V, et al. In situ detection of reactive oxygen species and nitric oxide production
in normal and pathological tissues: improvement by differential interference contrast. Exp Gerontol
2002;37:591-602
25. Bocci V, Aldinucci C, Mosci F, et al. Ozonation of human blood induces a remarkable upregulation of heme
oxygenase-1 and heat stress protein-70. Mediators Inflamm 2007;2007:26785
26. Bauer AK, Rondini EA, Hummel KA, et al. Identification of candidate genes downstream of TLR4 signaling
after ozone exposure in mice: a role for Heat-Shock Protein 70. Environ Health Perspect 2011;119:1091–
7
27. Madej P, Plewka A, Madej JA, et al. Ozone therapy in induced endotoxemic shock. II. The effect of ozone
therapy upon selected histochemical reactions in organs of rats in endotoxemic shock. Inflammation
2007;30:69-86
28. Lintas G, Molinari F, Simonetti V, et al. Time and time-frequency analysis of near-infrared signals for the
assessment of ozone autohemotherapy long-term effects in multiple sclerosis. Conf Proc IEEE Eng Med
Biol Soc 2013;2013:6171-4
Innovation and novelty
In recent years, the use of ozone as an effective therapeutic agent has become well established and known. At
present, more than forty national and international associations bring together the clinicians practicing this
therapy, promote the publication of indexed specialized journals, and organize thematic training courses and
meetings. However, the widespread application of ozone therapy is still prevented by the lack of regulation by
health authorities and by the unfounded doubts about ozone toxicity, although the concentrations of ozone
used in the clinical practice are considerably lower than the recognized toxic levels. At present, in fact, the
studies on the effects of ozonization at the cellular and molecular level are scarce, compared to the increasing
evidence of the positive effects of ozone administration on patients.
Elucidating the cellular mechanisms that account for the regeneration capabilities of therapeutic ozone
concentrations will respond to the present need of “a greater quantity of publications and research”, as
underlined by the ISCO3 - International Scientific Committee of Ozone therapy (www.isco3.org).
The results obtained in this project will be presented and discussed in national and international meetings and
conferences, and will be published in international scientific journals. It is worth noting that the involvement in
the present project of a renowned ozone-therapist (Dr. G. Tabaracci), physician and member of scientific
associations, will ensure the immediate and direct diffusion of the results among the professionals in this field.
In this study, the biological bases of ozone effects will be investigated in hADAS cells, and this will contribute to
the necessary background for designing cell- or tissue-targeted therapies as well as novel strategies to promote
regeneration/activation of specific tissues.
Team skills and competencies
Manuela Malatesta is a biologist whose main research interests concern cell biology: hypometabolism, ageing
and in vitro senescence, and skeletal muscle atrophy/dystrophy have been investigated under physiological,
pathological or experimental conditions in vivo and in vitro. Recently, she also investigated on in vitro systems
the effects of nanoparticle administration, photodynamic therapy and ozone exposure. Morphological and
immunonohistochemical methods at transmission electron microscopy were mainly used, often in association
with conventional and confocal fluorescence microscopy, biochemistry, cytometry and NMR. Co-author of 127
publications in international peer-reviewed journals (H-index: 21), she collaborates as a reviewer with many
indexed journals and is a member of the board of the Società Italiana di Scienze Microscopiche (since 2008),
having been a member of the board and Secretary of the Società Italiana di Istochimica (2010-2013). She is
Editor in chief of Microscopie (www.pagepress.org/microscopie) and Assistant Editor of the European Journal
of Histochemistry (www.ejh.it).
Gabriele Tabaracci is a physician specialised in Orthopaedics and Traumatology; he has been practicing ozone
therapy for about 20 years, and yearly organizes training courses on ozone therapy addressed to clinicians. He
is a member of the Scientific Committee of the Italian Federation of Oxygen Ozone Therapy (FIO) and attended
as invited speaker several national and international congresses.
The Project manager and the Partner supervisor with their team-mates will contribute to perform the
proposed project through their exclusive and complementary expertise. The research team in Verona has the
necessary permissions to use hADAS cells for scientific purposes. The two teams already collaborate in
concerted investigation, and the entire instrumental and technical facilities are available to perform the
planned research.
Research impact
Ozone therapy is a therapeutic approach that is increasingly used all over the world; however, to be widely
accepted by people and especially by national authorities and health institutions, the clinical practice needs to
be supported by a robust scientific evidence. In fact, the scientific literature on subject is still scarce especially
as for the cellular and molecular aspects. Therefore, the results of this project will significantly contribute to
identify the action mechanisms of ozone therapy as well as to improve knowledge of the effects of ozone on
cell dynamics and organelles’ structure and function.
In this project, hADAS cells have been selected as a model system due to their involvement in regeneration:
they have a direct role in reconstructing the connective matrix and in promoting angiogenesis, while
supporting epithelial, muscle and even nerve regeneration. It is therefore likely to hypothesize that this stem
cell population may be a primary target of ozone therapy, so that the results of our in vitro investigation could
translationally be referred to the conditions in vivo. In our system, the possibility to differentiate hADAS cells
into different cell lineages would also allow to elucidate whether ozone administration may induce peculiar
tissue-specific cytological/cytokinetic effects: this will provide a solid background for designing targeted
protocols of ozone treatment for tissue regeneration and engineering. The histochemical and molecular tests
we propose will also be useful for regulatory authorities to define a scientifically-founded protocol for
evaluating the biological effects of ozone exposure using in vitro cell systems. An application to patent the
process may be also envisaged.
GANTT Chart
WP 1: Cell culture and exposure to ozone
WP 2: Evaluation of ozone effects by microscopic techniques
WP 3: Evaluation of ozone effects by biomolecular techniques
WP 4: Data analysis and dissemination of results
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Research equipment
At the San Rocco Clinic and the Department of Neurological and Movement Sciences the following
instruments and technical facilities are already available to perform the planned research.
OZO2 Futura (Alnitec s.r.l.) ozone generator
CO2 incubators
Laminar flow cabins
Inverted microscope equipped for digital photomicrography
Centrifuges
Basic equipments for traditional morphological and histochemical techniques by light and electron
microscopy
Microtomes and ultramicrotomes for sample sectioning
Bright field, phase contrast and DIC microscopes
Fluorescence microscopes equipped for digital photomicrography and image processing
Leica TCS-SP confocal microscope system, equipped for laser excitation in the spectral regions of UV,
blue, green and red
Philips Morgagni transmission electron microscope equipped with a Megaview II camera for digital
image acquisition
Partec PAS III flow cytometer equipped with 100W mercury lamp
MiSeq platform (Illumina, Inc.)
GeneAtlas Affymetrix Microarray Technology for transcriptomic analyses
StepOne Real Time PCR
PCR systems
Nanodrop spectrophotometer
Agilent Bioanalyser 2100
Mini-Protean electrophoresis system
Computers for data collecting and analysis
Softwares specific for image analysis (AnalySIS, Image-Pro Plus, PhotoShop), statistics (SPSS), and
molecular analysis (Ariadne Pathway Studio and Ingenuity Pathway Software)