102 results on '"Duchi R"'
Search Results
2. Bortezomib, C1-Inhibitor and Plasma Exchange Do Not Prolong the Survival of Multi-Transgenic GalT-KO Pig Kidney Xenografts in Baboons
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Le Bas-Bernardet, S., Tillou, X., Branchereau, J., Dilek, N., Poirier, N., Châtelais, M., Charreau, B., Minault, D., Hervouet, J., Renaudin, K., Crossan, C., Scobie, L., Takeuchi, Y., Diswall, M., Breimer, M.E., Klar, N., Daha, M.R., Simioni, P., Robson, S.C., Nottle, M.B., Salvaris, E.J., Cowan, P.J., d’Apice, A.J.F., Sachs, D.H., Yamada, K., Lagutina, I., Duchi, R., Perota, A., Lazzari, G., Galli, C., Cozzi, E., Soulillou, J.-P., Vanhove, B., and Blancho, G.
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- 2015
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3. Long-term surface ozone variability at Mt. Cimone WMO/GAW global station (2165 m a.s.l., Italy)
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Cristofanelli, P., Scheel, H.-E., Steinbacher, M., Saliba, M., Azzopardi, F., Ellul, R., Fröhlich, M., Tositti, L., Brattich, E., Maione, M., Calzolari, F., Duchi, R., Landi, T.C., Marinoni, A., and Bonasoni, P.
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- 2015
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4. New atmospheric composition observations in the Karakorum region: Influence of local emissions and large-scale circulation during a summer field campaign
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Putero, D., Cristofanelli, P., Laj, P., Marinoni, A., Villani, P., Broquet, A., Alborghetti, M., Bonafè, U., Calzolari, F., Duchi, R., Landi, T.C., Verza, G.P., Vuillermoz, E., and Bonasoni, P.
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- 2014
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5. Transport of Stratospheric Air Masses to the Nepal Climate Observatory–Pyramid (Himalaya; 5079 m MSL) : A Synoptic-Scale Investigation
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Bracci, A., Cristofanelli, P., Sprenger, M., Bonafè, U., Calzolari, F., Duchi, R., Laj, P., Marinoni, A., Roccato, F., Vuillermoz, E., and Bonasoni, P.
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- 2012
6. Systemic and Renal Response of GAL-KO Pigs To Human Angiotensin II.: Abstract# D2856
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Casiraghi, F., Todeschini, M., Duchi, R., Gaspari, F., Carrara, F., Ferrari, S., Cannata, A., Lagutina, I., Perota, A., Remuzzi, G., Lazzari, G., Galli, C., and Perico, N.
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- 2014
7. Stratospheric intrusion index (SI2) from baseline measurement data
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Cristofanelli, P., Calzolari, F., Bonafè, U., Duchi, R., Marinoni, A., Roccato, F., Tositti, L., and Bonasoni, P.
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- 2009
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8. Improved secretory function of pancreatic islets from InsGLP1M3R piglets
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Perota, A, Mourad, Nizar, Lagutina, I, Duchi, R, Lazzari, G, Galli, C, Gianello, Pierre, 15th Congress of the International Xenotransplantation Association (IXA 2019), UCL - SSS/IREC/CHEX - Pôle de chirgurgie expérimentale et transplantation, and UCL - (SLuc) Service de chirurgie et transplantation abdominale
- Subjects
endocrine system - Abstract
Background & aims: Clinical pig islet transplantation for the treatment of type I diabetes is still hindered by obvious immunological considerations and less investigated physiological incompatibilities. Cellular encapsulation, donor genetic engineering and host immunomodulation can help improve islet survival in a xenotransplantation context but adequate insulin output from transplanted islets remains the ultimate goal to be achieved for islet transplantation to be clinically efficient. We have previously shown that adenovirus-driven expression of a cassette carrying a dipeptidyl peptidase-resistant form of glucagon-like-peptide-1 (GLP1) and a constitutively activated form of type 3 muscarinic receptor (M3R) in isolated neonatal and adult pig islets significantly increases their secretory response to in vitro glucose stimulation that is otherwise 4-10 times lower than human islets. Our aim in the current study was to replicate our previous results in a transgenic pig model with beta-cell specific expression of our GLP1M3R cassette, to characterize in vivo and in vitro islet function of these pigs and to verify whether their offspring exhibit the same improved insulin secretion as founder animals. Material & methods: Cloned transgenic pigs were produced using Talens technology and validated by TLA sequencing analyses. Pancreata were collected from 14-day old piglets. Insulin secretion in response to different stimuli was evaluated during dynamic islet perifusion experiments. After reaching adult age, selected animals were subjected to IVGTT to study in vivo islet function. Transgenic animals were then bred and islet function of their offspring was studied as for founder animals. Results: Transgenic cloned pig founders (male and female) expressing the GLP1M3R cassette at the beta cell level were successfully obtained. Islets isolated from these piglets showed a 5.5 to 7.5-fold increase of their insulin output upon stimulation with 15 mM glucose. They maintained regulated insulin secretion as basal unstimulated secretion was not significantly increased: stimulation indices of 3.7 to 7.5 compared to 2.6 for control wild type islets. In vivo islet function during IVGTT was evaluated in two animals that both showed a 2 to 3-fold increase of insulin secretion compared to controls. Finally, we examined secretory function of islets isolated from piglets born from transgenic cloned parents. In vitro perifusion experiments showed increased stimulated insulin secretion from transgenic piglets compared to their wild type siblings. Conclusion & perspectives: We obtained the first lines of genetically modified pigs exhibiting enhanced function of their pancreatic islets. Specific expression of modified GLP1 and activated M3R at the beta-cell level enhanced insulin secretion both in vivo and in vitro without affecting viability or fertility and was transmitted to the descendants of cloned animals while maintaining its positive effect on beta-cell function.
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- 2019
9. Improved secretory function of pancreatic islets from InsGLP1M3R piglets
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UCL - SSS/IREC/CHEX - Pôle de chirgurgie expérimentale et transplantation, UCL - (SLuc) Service de chirurgie et transplantation abdominale, Perota, A, Mourad, Nizar, Lagutina, I, Duchi, R, Lazzari, G, Galli, C, Gianello, Pierre, 15th Congress of the International Xenotransplantation Association (IXA 2019), UCL - SSS/IREC/CHEX - Pôle de chirgurgie expérimentale et transplantation, UCL - (SLuc) Service de chirurgie et transplantation abdominale, Perota, A, Mourad, Nizar, Lagutina, I, Duchi, R, Lazzari, G, Galli, C, Gianello, Pierre, and 15th Congress of the International Xenotransplantation Association (IXA 2019)
- Abstract
Background & aims: Clinical pig islet transplantation for the treatment of type I diabetes is still hindered by obvious immunological considerations and less investigated physiological incompatibilities. Cellular encapsulation, donor genetic engineering and host immunomodulation can help improve islet survival in a xenotransplantation context but adequate insulin output from transplanted islets remains the ultimate goal to be achieved for islet transplantation to be clinically efficient. We have previously shown that adenovirus-driven expression of a cassette carrying a dipeptidyl peptidase-resistant form of glucagon-like-peptide-1 (GLP1) and a constitutively activated form of type 3 muscarinic receptor (M3R) in isolated neonatal and adult pig islets significantly increases their secretory response to in vitro glucose stimulation that is otherwise 4-10 times lower than human islets. Our aim in the current study was to replicate our previous results in a transgenic pig model with beta-cell specific expression of our GLP1M3R cassette, to characterize in vivo and in vitro islet function of these pigs and to verify whether their offspring exhibit the same improved insulin secretion as founder animals. Material & methods: Cloned transgenic pigs were produced using Talens technology and validated by TLA sequencing analyses. Pancreata were collected from 14-day old piglets. Insulin secretion in response to different stimuli was evaluated during dynamic islet perifusion experiments. After reaching adult age, selected animals were subjected to IVGTT to study in vivo islet function. Transgenic animals were then bred and islet function of their offspring was studied as for founder animals. Results: Transgenic cloned pig founders (male and female) expressing the GLP1M3R cassette at the beta cell level were successfully obtained. Islets isolated from these piglets showed a 5.5 to 7.5-fold increase of their insulin output upon stimulation with 15 mM glucose. They maintained regul
- Published
- 2019
10. SURF1 knockout cloned pigs: Early onset of a severe lethal phenotype
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Quadalti, C., primary, Brunetti, D., additional, Lagutina, I., additional, Duchi, R., additional, Perota, A., additional, Lazzari, G., additional, Cerutti, R., additional, Di Meo, I., additional, Johnson, M., additional, Bottani, E., additional, Crociara, P., additional, Corona, C., additional, Grifoni, S., additional, Tiranti, V., additional, Fernandez-Vizarra, E., additional, Robinson, A.J., additional, Viscomi, C., additional, Casalone, C., additional, Zeviani, M., additional, and Galli, C., additional
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- 2018
- Full Text
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11. Seasonal variation of ozone and black carbon observed at Paknajol, an urban area in the Kathmandu Valley, Nepal
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Putero, D., Cristofanelli, P., Marinoni, A., Adhikary, B., Duchi, R., Das Shrestha, S., Pietro Verza, G., Landi, T., Calzolari, F., Busetto, M., Agrillo, G., Biancofiore, F., Di Carlo, P., Panday, A., Rupakheti, M., and Bonasoni, P.
- Abstract
The Kathmandu Valley in the Himalayan foothills, considered as one of the global "hot spots" for what concerns air pollution, is currently facing severe air quality problems due to rapid urbanization processes, dramatic land use changes, socioeconomic transformation and high population growth. In this work, we present the first full year (February 2013 - February 2014) analysis of simultaneous measurements of two short-lived climate forcers/pollutants (SLCF/P), i.e. ozone (O3) and equivalent black carbon (BC), and aerosol number concentration at Paknajol (27°43'4'' N, 85°18'32'' E, 1380 m a.s.l.), in the city center of Kathmandu. These observations were carried out in the framework of the SusKat-ABC (A Sustainable Atmosphere for the Kathmandu Valley - Atmospheric Brown Cloud) campaign in Nepal. The diurnal behavior of BC and aerosol number concentration indicated that local pollution sources represent the major contribution to air pollution in this city. In addition to photochemistry, the planetary boundary layer (PBL) dynamic plays an important role in determining O3 variability, as suggested by the analysis of seasonal changes of the diurnal cycles and the correlation with meteorological parameters and aerosol properties. Especially during pre-monsoon, high values of O3 were observed during the afternoon/evening. This could be related to mixing and entrainment processes between upper residual layers and the PBL. During this season, the high O3 appeared well related to the impact of major open vegetation fires occurring in Nepal. On a synoptic-scale perspective, westerly and regional atmospheric circulations appeared to be especially conducive for the occurrence of the high BC and O3 values. The very high values of the SLCF/P, detected during the whole measurement period, indicated persisting adverse air quality conditions, dangerous for the health of over 3 million residents of the Kathmandu Valley, and the environment. Consequently, all of this information may be useful for implementing control measures to mitigate the occurrence of acute pollution levels in the Kathmandu Valley and the surrounding area.
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- 2016
12. Study of short-lived climate forcers atmospheric variability at Kathmandu and at the WMO/GAW Global Station 'Nepal Climate Observatory-Pyramid' (5079 m a.s.l.) in the Himalayas
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Putero, D., Cristofanelli, P., Adhikary, B., Marinoni, A., Duchi, R., Calzolari, F., Landi, T., Verza, G., Alborghetti, M., Vuillermoz, E., Rupakheti, M., Lawrence, M., and Bonasoni, P.
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- 2014
13. Non-identical PERV profile observed in cloned pigs
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UCL - SSS/IREC/CHEX - Pôle de chirgurgie expérimentale et transplantation, UCL - (SLuc) Service de chirurgie et transplantation abdominale, Crossan, Claire, Cruikshank, V., Perota, A., Duchi, R., Lagutina, I., Lazzari, G., Mourad, Nizar, Takeuchi, Y., Galli, Cesare, Gianello, Pierre, Scobie, Linda, 2015 IPITA-IXA-CTS Joint Congress, UCL - SSS/IREC/CHEX - Pôle de chirgurgie expérimentale et transplantation, UCL - (SLuc) Service de chirurgie et transplantation abdominale, Crossan, Claire, Cruikshank, V., Perota, A., Duchi, R., Lagutina, I., Lazzari, G., Mourad, Nizar, Takeuchi, Y., Galli, Cesare, Gianello, Pierre, Scobie, Linda, and 2015 IPITA-IXA-CTS Joint Congress
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- 2015
14. Developmental potential of bovine androgenetic and parthenogenetic embryos: a comparative study
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LAGUTINA I, LAZZARI G, DUCHI R, GALLI, CESARE, LAGUTINA I, LAZZARI G, DUCHI R, and GALLI C.
- Subjects
Cell Nucleus ,Male ,Blastomeres ,Parthenogenesis ,Fertilization in Vitro ,Haploidy ,Embryo Transfer ,Diploidy ,Embryonic and Fetal Development ,Oocytes ,Animals ,Cattle ,Female ,Sperm Injections, Intracytoplasmic - Abstract
In this study, we compared the developmental capacity of bovine haploid and diploid androgenetic and parthenogenetic embryos obtained by different methods. Androgenetic embryos were produced by piezo-intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF) of enucleated oocytes with or without subsequent pronuclear transfer from one haploid zygote to another. Parthenogenetic embryos were obtained by activation of matured oocytes by ionomycin combined with cycloheximide or 6-dimethylaminopurine (DMAP) treatment. Only few cleaved androgenetic haploid embryos were able to compact (2.7%) and to form blastocysts (1.8%), while significantly more haploid parthenogenotes underwent compaction (24-37%) and a minority developed to blastocysts at different rates, depending on the activation procedure (cycloheximide 3%, 6-DMAP 14.5%). By contrast, development to blastocyst of diploid androgenotes, cloned androgenetic embryos, and parthenogenotes (31%, 39%, and 43%, respectively) was similar to IVF control embryos (35%). Cell number on Day 7 was higher for IVF blastocysts and decreased in consecutive order in diploid androgenotes, diploid parthenogenotes, and haploid uniparental embryos. Following transfer of diploid androgenetic embryos, a pregnancy was established and maintained up to Day 28.
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- 2003
15. Seasonal variation of ozone and black carbon observed at Paknajol, an urban site in the Kathmandu Valley, Nepal
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Putero, D., primary, Cristofanelli, P., additional, Marinoni, A., additional, Adhikary, B., additional, Duchi, R., additional, Shrestha, S. D., additional, Verza, G. P., additional, Landi, T. C., additional, Calzolari, F., additional, Busetto, M., additional, Agrillo, G., additional, Biancofiore, F., additional, Di Carlo, P., additional, Panday, A. K., additional, Rupakheti, M., additional, and Bonasoni, P., additional
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- 2015
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16. Targeting of a porcine EGFP line mediated by ZFNs to establish cloned red fluorescent primary cell lines suitable for Cre-mediated RMCE
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Perota, A, Lagutina, I, Duchi, R, Turini, P, Crotti, G, Colleoni, S, Lazzari, G, Lucchini, Franco, Galli, C., Lucchini, Franco (ORCID:0000-0003-0280-7062), Perota, A, Lagutina, I, Duchi, R, Turini, P, Crotti, G, Colleoni, S, Lazzari, G, Lucchini, Franco, Galli, C., and Lucchini, Franco (ORCID:0000-0003-0280-7062)
- Abstract
Recently site specific nucleases (ZFN, Tal Effector and CRISPR) emerged as powerful tools for gene modification of different cells types and EGFP-specific ZFNs were successfully used in rat (Geurtz et al. 2010) and in pig (Watanabe et al. 2010; Whyte et al. 2010). Previously (Brunetti et al. 2008, Clon. Stem. Cells) we generated an EGFP transgenic porcine line (Verro2GFP) characterized by a single integration of pCAGGS-EGFP cassette, high ubiquitous EGFP expression, mendelian transgene transmission and expression in F1. The aim of this work was to modify a transcriptionally active GFP-locus into one suitable for Cre-mediated RMCE, using EGFP-specific ZFNs. Homology arms for promoter-less targeting vector were derived from pCAGGS-EGFP vector (promoter fragment = Left-Homology-Arm = LHA; polyA sequence = Right-Homology-Arm = RHA). Cloning floxed (lox2272/lox5171) DsRedT4 reporter gene between LHA and RHA sequences, we generated the targeting/RMCE vector (pB50 30 DsRed4-PL) and its positive control (C?) for PCR set-up (100–1,000 plasmid copies). Verro2GFP fibroblasts cultured in DMEM ? M199(1:1) ? 10 %FCS, bFGF in 5 %CO2, 5 %O2, were transfected using Nucleofector (V-024 program). In ZFNs-mediated gene targeting, 2 lg of each ZFNs coding vectors (Sigma-CompoZr) and 2 lg of pB50 30 RedT4-PL/NotI vector were used to 00nucleofect00 1x106 Verro2GFP fibroblasts. Part of transfected cells were plated (3,000 cells/plate) in 10 Petri dishes ([ = 100 mm) and cultured without selection till well growing colonies appeared. Four RFP?/GFPcolonies were identified using epifluorescence, re-plated in two 150 mm dishes/colony and PCR verified. Finally 3 colonies were used in zona-free SCNT to produce reconstructed embryos, and 159 D6 blastocysts/compacted morulae were transferred into two synchronized sows that both became pregnant. One pregnancy is ongoing, another was interrupted at D58 and 7 fetuses (61.14 g ± 17.98 g) were used to generate RFP primary cell lines, finally verified b
- Published
- 2014
17. Targeted RMCE-live piglets generated by SCNT following sequential double site-specific gene modifications of a porcine EGFP line
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Perota, A, Lagutina, I, Duchi, R, Quadalti, C, Turini, P, Crotti, G, Colleoni, S, Lazzari, G, Peverali, F, Lucchini, Franco, Galli, C., Lucchini, Franco (ORCID:0000-0003-0280-7062), Perota, A, Lagutina, I, Duchi, R, Quadalti, C, Turini, P, Crotti, G, Colleoni, S, Lazzari, G, Peverali, F, Lucchini, Franco, Galli, C., and Lucchini, Franco (ORCID:0000-0003-0280-7062)
- Abstract
Site-specific nucleases (ZFN, Tal effector nucleases and CRISPRs) boosted the genome editing of different species, and EGFP-specific ZFNs were successfully used in rat (Geurtz et al. 2010) and in pig (Watanabe et al. 2010, Whyte et al. 2010). Previously (Brunetti et al., 2008) we generated an EGFP transgenic porcine line (V2G) characterized by a single integration of pCAGGS-EGFP cassette, high ubiquitous EGFP expression, Mendelian transgene transmission and expression in F1. The aim of this work was to generate live cloned piglets using modified cells after two site-specific modifications (ZFNs and RMCE) on the transcriptionally active GFP-locus (V2G), avoiding any cell rejuvenation by SCNT. Homology arms for promoterless targeting vector were derived from pCAGGSEGFP vector (promoter fragment = Left-Homology-Arm = LHA; polyA sequence = Right-Homology-Arm = RHA). Cloning floxed (lox2272/lox5171) puromycin and hygromycin resistance coding sequences between LHA and RHA sequences, and we generated the targeting/RMCE vectors (pB50 30 Puro-PL and pB50 30 Hygro-PL) and theirs positive controls (C+) for PCR set-up (100–1,000 plasmid copies). V2G fibroblasts cultured in DMEM + M199(1:1) +10 %FCS, bFGF in 5 %CO2, 5 %O2, were transfected using Nucleofector (V-024 program). In ZFN-mediated gene targeting, 2 lg of each ZFNs coding vectors (Sigma-CompoZr ) and 2 lg of pB50 30 Puro-PL/KpnI vector, were used to ‘nucleofect’ 0.9 9 106 Verro2GFP fibroblasts. Transfected cells were cultured under puromycin selection (2 lg/ml) for 8 days, and 0.45 9 106 cells were used for the Cre-mediated cassette exchange (1 lg pB50 30 Hygro-PL/KpnI + 2 lg pCAG:CreEGFP). Transfected cells were plated in 10 Petri dishes (Ø = 150 mm) and cultured under hygromycin selection (200 lg/ml) for 12 days, picked up and expanded in 24 multi-well plates for SCNT. Thirty-one colonies were PCR screened for hygromycin and 16 (51.6 %) colonies were positive for Cre-mediated targeted insertion. Four colonies were use
- Published
- 2014
18. Modeling amyotrophic lateral sclerosis in hSOD1 transgenic swine
- Author
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Chieppa, Mn, Perota, A, Corona, C, Grindatto, A, Lagutina, I, Vallino Costassa, E, Lazzari, G, Colleoni, S, Duchi, R, Lucchini, Franco, Caramelli, M, Bendotti, C, Galli, C, Casalone, C., Lucchini, Franco (ORCID:0000-0003-0280-7062), Chieppa, Mn, Perota, A, Corona, C, Grindatto, A, Lagutina, I, Vallino Costassa, E, Lazzari, G, Colleoni, S, Duchi, R, Lucchini, Franco, Caramelli, M, Bendotti, C, Galli, C, Casalone, C., and Lucchini, Franco (ORCID:0000-0003-0280-7062)
- Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that occurs in two clinically indistinguishable forms: sporadic (SALS) and familial (FALS), the latter linked to several gene mutations, mostly inheritable in a dominant manner. Nearly 20% of FALS forms are linked to mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Research on ALS relies on transgenic models and particularly on mice carrying a glycine-to-alanine conversion at the 93rd codon (G93A) of the hSOD1 gene. Although G93A transgenic mice have been widely employed in clinical trials and basic research, doubts have been recently raised from numerous reliable sources about their suitability to faithfully reproduce human disease. Besides, the scientific community has already foreseen swine as an attractive and alternative model to nonhuman primates for modeling human diseases due to closer anatomical, physiological and biochemical features of swine rather than rodents to humans. On this basis, we have produced the first swine ALS model by in vitro transfection of cultured somatic cells combined with somatic cell nuclear transfer (SCNT). To achieve this goal we developed a SOD1(G93A) (superoxide dismutase 1 mutated in Gly93-Ala) vector, capable of promoting a high and stable transgene expression in primary porcine adult male fibroblasts (PAF). After transfection, clonal selection and transgene expression level assessment, selected SOD1(G93A) PAF colonies were used as nuclei donors in SCNT procedures. SOD1(G93A) embryos were transferred in recipient sows, and pregnancies developed to term. A total of 5 piglets survived artificial hand raising and weaning and developed normally, reaching adulthood. Preliminary analysis revealed transgene integration and hSOD1(G93A) expression in swine tissues and 360° phenotypical characterization is ongoing. We believe that our SOD1(G93A) swine would provide an essential bridge between the fundamental work done in rodent models and the reality of trea
- Published
- 2014
19. Transport of short-lived climate forcers/pollutants (SLCF/P) to the Himalayas during the South Asian summer monsoon onset
- Author
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Cristofanelli, P, primary, Putero, D, additional, Adhikary, B, additional, Landi, T C, additional, Marinoni, A, additional, Duchi, R, additional, Calzolari, F, additional, Laj, P, additional, Stocchi, P, additional, Verza, G, additional, Vuillermoz, E, additional, Kang, S, additional, Ming, J, additional, and Bonasoni, P, additional
- Published
- 2014
- Full Text
- View/download PDF
20. Influence of open vegetation fires on black carbon and ozone variability in the southern Himalayas (NCO-P, 5079 m a.s.l.)
- Author
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Putero, D., primary, Landi, T.C., additional, Cristofanelli, P., additional, Marinoni, A., additional, Laj, P., additional, Duchi, R., additional, Calzolari, F., additional, Verza, G.P., additional, and Bonasoni, P., additional
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- 2014
- Full Text
- View/download PDF
21. Successful double genetic modification of porcine EGFP primary cell line using ZFN and Cre-mediated cassette exchange
- Author
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Perota, A, Lagutina, I, Duchi, R, Turini, P, Crotti, G, Colleoni, S, Lazzari, G, Lucchini, Franco (ORCID:0000-0003-0280-7062), Galli, C., Perota, A, Lagutina, I, Duchi, R, Turini, P, Crotti, G, Colleoni, S, Lazzari, G, Lucchini, Franco (ORCID:0000-0003-0280-7062), and Galli, C.
- Abstract
We previously generated EGFP transgenic porcine line (Verro2GFP) characterized by a single integration of pCAGGSEGFP cassette, high ubiquitous EGFP expression, mendelian transgene transmission and expression in F1 (Brunetti et al. 2008). Recently Zinc Finger Nucleases (ZFN) and TALENs emerged as powerful tools for gene modification of different cells types and Recombinase Mediated Cassette Exchange (RMCE) was widely tested in different species. Moreover EGFP-specific ZFNs were successfully used in rat (Geurtz et al. 2010) and in pig (Watanabe et al. 2010, Whyte et al. 2010). The purposes of our work were: a) to insert a vector suitable for RMCE into a transcriptionally active locus and b) to assess the possibility to obtain good quality cell colonies to be used in Somatic Cell Nuclear Transfer (SCNT) even after two sequential transgenic modifications (ZFN and RMCE), considering the finite life span of Verro2GFP primary fibroblasts. EGFP-specific ZFNs were purchased from Sigma (CompoZr®) and the promoter-less targeting vectors were created using a fragment of the promoter (Left-Homology-Arm = LHA) and the polyA sequence (Right-Homology-Arm = RHA) of pCAGGSEGFP expression vector. Cloning floxed (lox2272/lox5171) reporter genes (PuromycinR, HygromycinR ) between these homology sequences (LHA and RHA), we generated 2 targeting/exchanging vectors (pB53Puro-PL, pB53Hygro-PL) and their positive controls (C+). PCR on C+ was set up to obtain minimal sensitivity of 100–1000 plasmid copies. Verro2GFP fibroblasts (primary and colonies) cultured in DMEM + M199 (1:1) + FBS(10 %), 5 % CO2,5%O2, were transfected using Nucleofector (V-24 program) and selected starting at 48 h after transfection. Colonies were picked up at 8th selection day and expanded in 24-well plates for PCR screening, RMCE and/or SCNT. In ZFNs-mediated gene targeting, 2 μg of each ZFNs coding vectors (pZFN1 and pZFN2) and 2 μg of pB53Puro-PL/ KpnI vector were used to “nucleofect” primary Verro2GFP fibroblasts (3
- Published
- 2013
22. Amyotrophic lateral sclerosis (ALS) swine models: Production and preliminary characterization
- Author
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Grindatto, A, Perota, A, Chieppa, Mn, Costassa, Ev, Colleoni, S, Lo Faro, M, Duchi, R, Palmitessa, C, Lagutina, I, Tortarolo, M, Lazzari, G, Lucchini, Franco (ORCID:0000-0003-0280-7062), Bendotti, C, Corona, C, Galli, C, Casalone, C., Grindatto, A, Perota, A, Chieppa, Mn, Costassa, Ev, Colleoni, S, Lo Faro, M, Duchi, R, Palmitessa, C, Lagutina, I, Tortarolo, M, Lazzari, G, Lucchini, Franco (ORCID:0000-0003-0280-7062), Bendotti, C, Corona, C, Galli, C, and Casalone, C.
- Abstract
Introduction. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that occurs in two forms: sporadic and familial, the latter linked to mutations in the SOD1 gene. As employment of transgenic SOD1 rodent models in ALS research didn’t result in an improvement of patient prognosis, another model, more closely related to human species, is strongly demanded by the scientific community. On this basis, our group produced, by Genetic Engineering and SCNT, transgenic blastocysts and swine carrying the hSOD1G93A mutation, which is the most frequently studied in rodents, since it reproduces patients phenotype progression. Materials and Methods. SCNT blastocysts on fifth day of development were transplanted by midventral laparatomy to the synchronized sows uterus. A cesarean delivery was performed at the 114th day of gestation. In order to achieve a preliminary characterization of our swine model, tissue banking was performed on stillborn piglets and on animals that died soon after birth. Immunocytochemistry on ear biopsy fibroblasts and western blot on homogenized snap-shot frozen tissues (rabbit policlonal antibody 07–403 Millipore, concentration 1:200 and 1:1000 respectively) were performed. To assess SOD1G93A deposition pattern Immunohistochemistry (rabbit policlonal antibody GTX 100659; 1:250) and Immunofluorescence (GTX 100659; 1:250 and NeuN MAB377; 1:1000) were employed on FFPE tissues. Genomic SOD1G93A swine DNA digested by SalI+BglII (10 U/μg DNA) was hybridized with SOD-DIG probes (20 ng/ml) to assess transgene integrations number by Southern Blot. Results and Conclusions. The transfer of 638 embryos to eight recipient sows resulted in four pregnancies and in the birth of 16 vital and 12 stillborn piglets (mean blastocyst development to term efficacy, 8.78%). Five animals developed normally while the remaining piglets died due to events commonly reported in commercial herds. The transgenic protein expression was confirmed by both immunocytochemi
- Published
- 2013
23. Cellular and molecular deviations in bovine in vitro-produced embryos are related to the large offspring syndrome
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Lazzari, G., Wrenzycki, C., Herrmann, D., Duchi, R., Kruip, T., Niemann, H., and Galli, C.
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In vitro production ,Embryo ,ID-Lelystad, Instituut voor Dierhouderij en Diergezondheid ,embryonic structures ,ID Lelystad, Institute for Animal Science and Health ,Large offspring syndrome ,Gene expression - Abstract
The large offspring syndrome (LOS) is observed in bovine and ovine offspring following transfer of in vitro-produced (IVP) or cloned embryos and is characterized by a multitude of pathologic changes, of which extended gestation length and increased birthweight are predominant features. In the present study, we used bovine blastocysts to analyze cellular parameters, i.e., the number of cells in Day 7 blastocysts and the size of Day 12 elongating blastocysts, and molecular parameters, i.e., the relative abundance of developmentally important genes: glucose transporter (Glut) 1, Glut-2, Glut-3, Glut-4, heat shock protein (Hsp) 70.1, Cu/Zn-superoxide dismutase (SOD), histone H4.1, basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) I receptor (R), and IGFII-R. Some blastocysts were produced by in vitro maturation and fertilization followed by in vitro culture in synthetic oviduct fluid medium supplemented with BSA or human serum or by in vivo culture in the sheep oviduct. Other blastocysts were derived in vivo from the uterine horns of superovulated donors. The findings made in the early embryos were related to a representative number of calves obtained from each production system and from artificial insemination (AI). In vitro culture of bovine embryos in the presence of high concentrations of serum or BSA significantly increased the number of cells in Day 7 blastocysts, the size of blastocysts on Day 12, and the relative abundance of the transcripts for Hsp70.1, Cu/Zn-SOD, Glut-3, Glut-4, bFGF, and IGFI-R when compared with embryos from the in vivo production groups. Birthweights of calves derived from IVP embryos were significantly higher than those of calves derived from sheep oviduct culture, superovulation, or AI. The results support the hypothesis that persistence of early deviations in development is causally involved in the incidence of LOS, in particular in increased birthweights. The cellular and molecular parameters analyzed in this study can be considered early markers of LOS in cattle.
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- 2002
24. Generation of pre-implantation pig SCNT embryos harboring the amyotrophic lateral sclerosis related hSOD1G93A gene
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Chieppa, Mn, Perota, A, Lagutina, I, Costassa, Ev, Grindatto, A, Palmitessa, C, Corbellini, D, Tortarolo, M, Colleoni, S, Duchi, R, Lazzari, G, Corona, C, Lucchini, Franco (ORCID:0000-0003-0280-7062), Bendotti, C, Galli, C, Casalone, C., Chieppa, Mn, Perota, A, Lagutina, I, Costassa, Ev, Grindatto, A, Palmitessa, C, Corbellini, D, Tortarolo, M, Colleoni, S, Duchi, R, Lazzari, G, Corona, C, Lucchini, Franco (ORCID:0000-0003-0280-7062), Bendotti, C, Galli, C, and Casalone, C.
- Abstract
Introduction. ALS is a fatal neurodegenerative disease that occurs in two forms: sporadic and familial, the latter linked to mutations in the SOD1 gene. Pathogenic hypotheses mainly rely on transgenic rodent models however doubts have been raised about their suitability to faithfully reproduce the human disease. Therefore, a more suitable model is strongly demanded to provide a better tool to study the disease and to facilitate the preclinical findings extrapolation. Swine model plays an emerging role in biomedical research as its anatomical, physiological and biochemical features are more closely related to human species. On this basis, our group produced, by Genetic Engineering and SCNT, transgenic swine blastocysts carrying the hSOD1G93A mutation, which is the most frequently studied in rodents, since it reproduces the ALS patients phenotype progression. Material and Methods. Using the Multisite Gateway System (Invitrogen) we obtained the “EntryClone” pENTRL1L2- hSODG93A, containing the cDNA coding the hSOD1G93A gene whose open reading frame was confirmed by sequencing, and the “DestinationVector” pMGOrfA5¢3’MARpuro5171. The exchange reaction between the “DestinationVector” and the “EntryClone” was mediated by LR Clonase-Invitrogen, and was used to transform chemically competent E.coli cells (OneShotMatch1-Invitrogen). The resulting “ExpressionVector” pMG5¢3’MARPuro-hSODG93A was purified and analyzed. The same construct was successfully used to develop an ubiquitous EGFP expression vector that maintains high expression level through the next generation of pigs. This vector carries the pCAGGS hybrid promoter (CMV-IE enhancer + chicken β-actin promoter) inserted between two insulators (5′MAR of chicken lysozyme gene) to prevent positional or copy number silencing effects. 5 µg of linearized vector were used to transfect 1 × 106 Pig Adult Fibroblast (PAF) cultured in DMEM/ M199[1:1] + 10%FCS+1% β-FGF by Nucleofector (Amaxa Biosystem). Transgene expression was detect
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- 2012
25. High throughput production of multi-transgenic pig for xenotranplantation research
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Galli, C, Perota, A, Lagutina, I, Duchi, R, Colleoni, S, Lucchini, Franco, Lazzari, G., Lucchini, Franco (ORCID:0000-0003-0280-7062), Galli, C, Perota, A, Lagutina, I, Duchi, R, Colleoni, S, Lucchini, Franco, Lazzari, G., and Lucchini, Franco (ORCID:0000-0003-0280-7062)
- Abstract
Due to the increasing demand of organ for transplantation and the shortage of human donors several areas of research, including regenerative medicine and xenotransplantation, are currently explored to provide different clinical solutions to a varieties of pathological conditions. While regenerative medicine will relay on stem cell auto-regeneration and/or cell transplantation in partially compromised tissues and organs, xenotransplantation would provide ready to use tissues or organs for more severe pathologies or organ failures. Genetic engineering of porcine genome lies at the heart of xenotransplantation research since the pig is currently considered, on the risk/benefit ratio, the most appropriate species. The stepsinvolved in the creation of candidate animals for xenotransplantation research include the selection of a somatic cell line (usually fibroblasts) that is engineered using current technologies, either for the inactivation or for the insertion of candidate genes, followed by somatic cell nuclear transfer (SCNT) procedure to generate live animals. The founder animals obtained can be suitable directly for xenotransplantation and/or their cells can be further engineered. In our laboratory we have been working primarily with a male GAL-KO miniature pig cell line (provided by D. Sachs, MGH, Boston USA) with the aim of overexpressing under an ubiquitous promoter (pCGACGS) several transgenes controlling complement mediated lysis and inflammation (hCD55, hCD39), and coagulation (hEPCR, hTM). To speed up the process we cotransfected by nucleofection two transgenes at the same time, one of which carried the selectable marker (hCD55HygromycinR ? hCD39; hEPCRPuromycinR ? hTM). Forty-eight hours later the cells were subjected to drug selection for 15 days to isolate resistant cell clones that were expanded in duplicates for SCNT and phenotypic characterisation by Western blot and immunocitochemistry to assess the presence of the protein and the uniform expression of
- Published
- 2012
26. Primary versus secondary contributions to particle number concentrations in the European boundary layer
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Reddington, C. L., Carslaw, K. S., Spracklen, D. V., Frontoso, M. G., Collins, L., Merikanto, J., Minikin, A., Hamburger, T., Coe, H., Kulmala, M., Aalto, P., Flentje, H., Plass-Duelmer, C., Birmili, W., Wiedensohler, A., Wehner, B., Tuch, T., Sonntag, A., O'Dowd, C. D., Jennings, S. G., Dupuy, R., Baltensperger, U., Weingartner, E., Hansson, Hans-Christen, Tunved, Peter, Laj, P., Sellegri, K., Boulon, J., Putaud, J. -P, Gruening, C., Swietlicki, E., Roldin, P., Henzing, J. S., Moerman, M., Mihalopoulos, N., Kouvarakis, G., Zdimal, V., Zikova, N., Marinoni, A., Bonasoni, P., Duchi, R., Reddington, C. L., Carslaw, K. S., Spracklen, D. V., Frontoso, M. G., Collins, L., Merikanto, J., Minikin, A., Hamburger, T., Coe, H., Kulmala, M., Aalto, P., Flentje, H., Plass-Duelmer, C., Birmili, W., Wiedensohler, A., Wehner, B., Tuch, T., Sonntag, A., O'Dowd, C. D., Jennings, S. G., Dupuy, R., Baltensperger, U., Weingartner, E., Hansson, Hans-Christen, Tunved, Peter, Laj, P., Sellegri, K., Boulon, J., Putaud, J. -P, Gruening, C., Swietlicki, E., Roldin, P., Henzing, J. S., Moerman, M., Mihalopoulos, N., Kouvarakis, G., Zdimal, V., Zikova, N., Marinoni, A., Bonasoni, P., and Duchi, R.
- Abstract
It is important to understand the relative contribution of primary and secondary particles to regional and global aerosol so that models can attribute aerosol radiative forcing to different sources. In large-scale models, there is considerable uncertainty associated with treatments of particle formation (nucleation) in the boundary layer (BL) and in the size distribution of emitted primary particles, leading to uncertainties in predicted cloud condensation nuclei (CCN) concentrations. Here we quantify how primary particle emissions and secondary particle formation influence size-resolved particle number concentrations in the BL using a global aerosol microphysics model and aircraft and ground site observations made during the May 2008 campaign of the European Integrated Project on Aerosol Cloud Climate Air Quality Interactions (EUCAARI). We tested four different parameterisations for BL nucleation and two assumptions for the emission size distribution of anthropogenic and wildfire carbonaceous particles. When we emit carbonaceous particles at small sizes (as recommended by the Aerosol Inter-comparison project, AEROCOM), the spatial distributions of campaign-mean number concentrations of particles with diameter >50 nm (N(50)) and >100 nm (N(100)) were well captured by the model (R(2)>= 0.8) and the normalised mean bias (NMB) was also small (-18% for N(50) and -1% for N(100)). Emission of carbonaceous particles at larger sizes, which we consider to be more realistic for low spatial resolution global models, results in equally good correlation but larger bias (R(2)>= 0.8, NMB = -52% and -29%), which could be partly but not entirely compensated by BL nucleation. Within the uncertainty of the observations and accounting for the uncertainty in the size of emitted primary particles, BL nucleation makes a statistically significant contribution to CCN-sized particles at less than a quarter of the ground sites. Our results show that a major source of uncertainty i, authorCount :41
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- 2011
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27. Production and characterization of Gal-/- minipigs over-expressing hCD55
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Brunetti, D, Perota, A, Lagutina, I, Chatelais, M, Charreau, B, Duchi, R, Lazzari, G, Lucchini, Franco, Galli, C., Lucchini, Franco (ORCID:0000-0003-0280-7062), Brunetti, D, Perota, A, Lagutina, I, Chatelais, M, Charreau, B, Duchi, R, Lazzari, G, Lucchini, Franco, Galli, C., and Lucchini, Franco (ORCID:0000-0003-0280-7062)
- Published
- 2010
28. Double transgenic Gal(-/-) piglets over-expressing hCD39
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Brunetti, D, Perota, A, Lagutina, I, Chatelais, M, Charreau, B, Duchi, R, Cozzi, E, Lazzari, G, Lucchini, Franco, Anegon, I, Sachs, Dh, Galli, Cesare, Lucchini, Franco (ORCID:0000-0003-0280-7062), Brunetti, D, Perota, A, Lagutina, I, Chatelais, M, Charreau, B, Duchi, R, Cozzi, E, Lazzari, G, Lucchini, Franco, Anegon, I, Sachs, Dh, Galli, Cesare, and Lucchini, Franco (ORCID:0000-0003-0280-7062)
- Abstract
Over-expression of human CD39 in transgenic pigs is a potential strategy to bypass acute vascular rejection in xenotransplantation. The aim of this work is the production of transgenic cloned pigs using a Gal -/- CD55/CD39 cell line. A neonatal pig Gal -/- fibroblast line cultured in DMEM/M199 1:1 + 10% FCS + 5 ng/ml bFGF was co-transfected by nucleofection with two ubiquitous expression vectors, the first carrying hCD55 under Elongation Factor promoter and a HygroR cassette; the second carrying hCD39 under pCAGGS promoter and a 3¢ MAR region. After nucleofection, cells were plated in Petri dishes and selected with Hygromycin B for 8 days. Drug resistant colonies were isolated and expanded for transgene expression analysis. We used immunohistochemistry (IHC) to detect the expression of the proteins. For hCD55 we used IA10 and for hCD39 BU61. Cells co-expressing CD55–CD39 were serum starved for 24 h before being fused to enucleated oocytes. Following electric activation, embryos were grown in vitro up to compact morula/blastocyst and all (n=144) were transplanted in two synchronized sows. PAEC and fibroblasts derived from delivered piglets were analysed with FACS, using the following antibodies: BRIC110, IH4, 2G2, and MEM-118 for hCD55 and TU66 for hCD39 detection respectively. Using a double transgenic CD55/CD39 Gal -/- colony in a Somatic Cell Nuclear Transfer (SCNT) experiment we have obtained 35.4% compacted morula/blastocyst development. One of two sows resulted in a pregnancy. At day 117 of gestation, this sow was induced to farrowing and delivered two stillborn piglets that were probably too immature and died from respiratory failure. Nevertheless, IHC analysis performed on PAEC and fibroblasts derived from these piglets showed strong expression of CD55– CD39, as in the original colony. FACS analysis confirmed robust human CD39 expression but showed a very low level of hCD55 expression. Two Gal -/- CD55/CD39 piglets were obtained. Over-expression of hCD39 seem
- Published
- 2009
29. Influence of biomass burning and anthropogenic emissions on ozone, carbon monoxide and black carbon at the Mt. Cimone GAW-WMO global station (Italy, 2165 m a.s.l.)
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Cristofanelli, P., primary, Fierli, F., additional, Marinoni, A., additional, Calzolari, F., additional, Duchi, R., additional, Burkhart, J., additional, Stohl, A., additional, Maione, M., additional, Arduini, J., additional, and Bonasoni, P., additional
- Published
- 2013
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30. Primary versus secondary contributions to particle number concentrations in the European boundary layer
- Author
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Reddington, C. L., primary, Carslaw, K. S., additional, Spracklen, D. V., additional, Frontoso, M. G., additional, Collins, L., additional, Merikanto, J., additional, Minikin, A., additional, Hamburger, T., additional, Coe, H., additional, Kulmala, M., additional, Aalto, P., additional, Flentje, H., additional, Plass-Dülmer, C., additional, Birmili, W., additional, Wiedensohler, A., additional, Wehner, B., additional, Tuch, T., additional, Sonntag, A., additional, O'Dowd, C. D., additional, Jennings, S. G., additional, Dupuy, R., additional, Baltensperger, U., additional, Weingartner, E., additional, Hansson, H.-C., additional, Tunved, P., additional, Laj, P., additional, Sellegri, K., additional, Boulon, J., additional, Putaud, J.-P., additional, Gruening, C., additional, Swietlicki, E., additional, Roldin, P., additional, Henzing, J. S., additional, Moerman, M., additional, Mihalopoulos, N., additional, Kouvarakis, G., additional, Ždímal, V., additional, Zíková, N., additional, Marinoni, A., additional, Bonasoni, P., additional, and Duchi, R., additional
- Published
- 2011
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31. Embryonic genotype and inbreeding affect preimplantation development in cattle
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Lazzari, G, primary, Colleoni, S, additional, Duchi, R, additional, Galli, A, additional, Houghton, F D, additional, and Galli, C, additional
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- 2011
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32. Size-resolved aerosol chemical composition over the Italian Peninsula during typical summer and winter conditions
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Carbone, C., primary, Decesari, S., additional, Mircea, M., additional, Giulianelli, L., additional, Finessi, E., additional, Rinaldi, M., additional, Fuzzi, S., additional, Marinoni, A., additional, Duchi, R., additional, Perrino, C., additional, Sargolini, T., additional, Vardè, M., additional, Sprovieri, F., additional, Gobbi, G.P., additional, Angelini, F., additional, and Facchini, M.C., additional
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- 2010
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33. Aerosol mass and black carbon concentrations, a two year record at NCO-P (5079 m, Southern Himalayas)
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Marinoni, A., primary, Cristofanelli, P., additional, Laj, P., additional, Duchi, R., additional, Calzolari, F., additional, Decesari, S., additional, Sellegri, K., additional, Vuillermoz, E., additional, Verza, G. P., additional, Villani, P., additional, and Bonasoni, P., additional
- Published
- 2010
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34. Atmospheric Brown Clouds in the Himalayas: first two years of continuous observations at the Nepal Climate Observatory-Pyramid (5079 m)
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Bonasoni, P., primary, Laj, P., additional, Marinoni, A., additional, Sprenger, M., additional, Angelini, F., additional, Arduini, J., additional, Bonafè, U., additional, Calzolari, F., additional, Colombo, T., additional, Decesari, S., additional, Di Biagio, C., additional, di Sarra, A. G., additional, Evangelisti, F., additional, Duchi, R., additional, Facchini, MC., additional, Fuzzi, S., additional, Gobbi, G. P., additional, Maione, M., additional, Panday, A., additional, Roccato, F., additional, Sellegri, K., additional, Venzac, H., additional, Verza, GP., additional, Villani, P., additional, Vuillermoz, E., additional, and Cristofanelli, P., additional
- Published
- 2010
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35. Estimated impact of black carbon deposition during pre-monsoon season from Nepal Climate Observatory – Pyramid data and snow albedo changes over Himalayan glaciers
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Yasunari, T. J., primary, Bonasoni, P., additional, Laj, P., additional, Fujita, K., additional, Vuillermoz, E., additional, Marinoni, A., additional, Cristofanelli, P., additional, Duchi, R., additional, Tartari, G., additional, and Lau, K.-M., additional
- Published
- 2010
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36. Tropospheric ozone variations at the Nepal Climate Observatory-Pyramid (Himalayas, 5079 m a.s.l.) and influence of deep stratospheric intrusion events
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Cristofanelli, P., primary, Bracci, A., additional, Sprenger, M., additional, Marinoni, A., additional, Bonafè, U., additional, Calzolari, F., additional, Duchi, R., additional, Laj, P., additional, Pichon, J.M., additional, Roccato, F., additional, Venzac, H., additional, Vuillermoz, E., additional, and Bonasoni, P., additional
- Published
- 2010
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37. Chemical composition of PM<sub>10</sub> and PM<sub>1</sub> at the high-altitude Himalayan station Nepal Climate Observatory-Pyramid (NCO-P) (5079 m a.s.l.)
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Decesari, S., primary, Facchini, M. C., additional, Carbone, C., additional, Giulianelli, L., additional, Rinaldi, M., additional, Finessi, E., additional, Fuzzi, S., additional, Marinoni, A., additional, Cristofanelli, P., additional, Duchi, R., additional, Bonasoni, P., additional, Vuillermoz, E., additional, Cozic, J., additional, Jaffrezo, J. L., additional, and Laj, P., additional
- Published
- 2010
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38. Significant variations of trace gas composition and aerosol properties at Mt. Cimone during air mass transport from North Africa – contributions from wildfire emissions and mineral dust
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Cristofanelli, P., primary, Marinoni, A., additional, Arduini, J., additional, Bonafè, U., additional, Calzolari, F., additional, Colombo, T., additional, Decesari, S., additional, Duchi, R., additional, Facchini, M. C., additional, Fierli, F., additional, Finessi, E., additional, Maione, M., additional, Chiari, M., additional, Calzolai, G., additional, Messina, P., additional, Orlandi, E., additional, Roccato, F., additional, and Bonasoni, P., additional
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- 2009
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39. Stratospheric intrusion index (SI2) from baseline measurement data
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Cristofanelli, P., primary, Calzolari, F., additional, Bonafè, U., additional, Duchi, R., additional, Marinoni, A., additional, Roccato, F., additional, Tositti, L., additional, and Bonasoni, P., additional
- Published
- 2008
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40. Establishment, Differentiation, Electroporation, Viral Transduction, and Nuclear Transfer of Bovine and Porcine Mesenchymal Stem Cells
- Author
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Colleoni, S., primary, Donofrio, G., additional, Lagutina, I., additional, Duchi, R., additional, Galli, C., additional, and Lazzari, G., additional
- Published
- 2005
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41. Influence of biomass burning and anthropogenic emissions on ozone carbon monoxide and black carbon concentrations at the Mt. Cimone GAW-WMO global station (Italy, 2165ma.s.l.).
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Cristofanelli, P., Fierli, F., Marinoni, A., Duchi, R., Burkhart, J., Stohl, A., Maione, M., Arduini, J., and Bonasoni, P.
- Abstract
This work investigates the variability of ozone (O
3 ), carbon monoxide (CO) and equivalent black carbon (BC) concentrations at the Italian Climate Observatory "O. Vittori" (ICO-OV), part of the Mt. Cimone global GAW-WMO station (Italy). For this purpose, ICO-OV observations carried out in the period January 2007-June 2009, have been analysed and correlated with the output of the FLEXPART Lagrangian dispersion model to specifically evaluate the influence of biomass burning (BB) and anthropogenic emissions younger than 20 days. During the investigation period, the average O3 , CO and BC concentrations at ICO-OV were 54 ± 3 ppbv, 122 ± 7 ppbv and 213 ± 34 ngm-3 (mean ± expanded uncertainty with p < 95%), with clear seasonal cycles characterized by summer maxima and winter minima for O3 and BC and spring maximum and summer minimum for CO. According to FLEXPART output, BB impact is maximized during the warm months from July to September but appeared to have a significant contribution to the observed tracer concentrations only during specific transport events. We characterised in detail five major events with respect to transport scales (i.e. global, regional and local), source regions and O3 , CO and BC variations. For these events, very large variability of enhancement ratios O3 /CO (from -0.22 to 0.71) and BC/CO (from 2.69 to 29.83 ngm-3 ppbv-1 ) were observed. CO related with anthropogenic emissions (COant ) contributed to 17.4% of the mean CO value observed at ICO-OV, with the warm months appearing particularly affected by transport events of air-masses rich in anthropogenic pollution. The proportion of tracer variability that is described by FLEXPART COant peaked to 37% (in May-September) for CO, 19% (in May-September) for O3 and 32% (in January-April) for BC. During May-September, the analysis of the correlation among CO, O3 and BC as a function of the COant indicated that ICO-OV was influenced by air-masses rich in anthropogenic pollution transported from the regional to the global scale. On the other side, CO and O3 were negatively correlated during October-December, when FLEXPART does not show significant presence of recent anthropogenic emissions and only a few observations are characterized by enhanced BC. Such behaviour may be attributed to an ensemble of processes concurrent in enhancing O3 with low CO (upper troposphere/lower stratosphere intrusions) and O3 titration by NO in polluted air-masses along with lower photochemical activity. An intermediate situation occurs in January-April when CO and O3 were almost uncorrelated and BC enhancements were associated to relatively old (10 days) anthropogenic emissions. [ABSTRACT FROM AUTHOR]- Published
- 2012
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42. Continuous observations of synoptic-scale dust transport at the Nepal Climate Observatory-Pyramid (5079 m.a.s.l.) in the Himalayas.
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Duchi, R., Cristofanelli, P., Marinoni, A., Laj, P., Marcq, S., Villani, P., Sellegri, K., Angelini, F., Calzolari, F., Gobbi, G. P., Verza, G. P., Vuillermoz, E., Sapkota, A., and Bonasoni, P.
- Abstract
This study presents two years of continuous observations of physical aerosol properties at the GAW-WMO global station "Nepal Climate Observatory -- Pyramid" (NCO-P, 27°57' N, 86° 48'E), sited at 5079 m a.s.l. in the high Himalayan Khumbu Valley (Nepal). Measurements of aerosol number size distribution, aerosol optical depth (AOD) and single scattering albedo (SSA) are analysed from March 2006 to February 2008. By studying the temporal variations of coarse (1 µm < D
p ≤10 µm) particle number concentration, 53 mineral Dust Transport Events (DTEs) are identified, accounting for 22.2% of the analysed data-set. Such events occurred prevalently during pre-monsoon (for 30.6% of the period) and winter (22.1%) seasons. However, uncommon cases of mineral dust transport are observed even during the monsoon season. The main sources of mineral dust reaching NCO-P are identified in the arid regions not far from the measurement site, i.e. from Tibetan Plateau, and Lot-Thar deserts, which account for 52% of the dust transport days. Moreover, a non-negligible contribution can be attributed to the Arabian Peninsula (17%) and the Indo-Gangetic Plains (16%), as indicated by three dimensional (3-D) back-trajectory analyses performed with LAGRANTO model. The observed DTEs lead to significant enhancements in the coarse aerosol number concentration (+513%) and coarse aerosol mass (+655%), as compared with average values observed in "dust-free" conditions (0.05 ± 0.11 cm-3 and 3.4 ± 3.7 µg m-3 , respectively). During DTEs, SSA is higher (0.84-0.89) than on "dust-free" days (0.75-0.83), confirming the importance of this class of events as a driver of the radiative features of the regional Himalayan climate. Considering the dust events, a significant seasonal AOD increase (+37.5%) is observed in the post-monsoon, whereas lower increase (less than +11.1%) characterises the pre-monsoon and winter seasons confirming the influence of synoptic-scale mineral dust transports on the aerosol optical properties observed at NCO-P. [ABSTRACT FROM AUTHOR]- Published
- 2011
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43. Chemical composition of PM10 and PM1 at the high-altitude Himalayan station Nepal Climate Observatory-Pyramid (NCO-P) (5079ma.s.l.).
- Author
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Decesari, S., Facchini, M. C., Carbone, C., Giulianelli, L., Rinaldi, M., Finessi, E., Fuzzi, S., Marinoni, A., Cristofanelli, P., Duchi, R., Bonasoni, P., Vuillermoz, E., Cozic, J., Jaffrezo, J. L., and Laj, P.
- Subjects
PARTICULATE matter ,AIR quality ,ATMOSPHERIC aerosols ,ATMOSPHERIC chemistry ,SULFATES - Abstract
We report chemical composition data for PM
10 and PM1 from the Nepal Climate Observatory-Pyramid (NCO-P), the world's highest aerosol observatory, located at 5079m a.s.l. at the foothills of Mt. Everest. Despite its high altitude, the average PM10 mass apportioned by the chemical analyses is of the order of 6 μgm-3 (i.e., 10 μg/scm), with almost a half of this mass accounted for by organic matter, elemental carbon (EC) and inorganic ions, the rest being mineral dust. Organic matter, in particular, accounted for by 2.0 μgm-3 (i.e., 3.6 μg/scm) on a yearly basis, and it is by far the major PM10 component beside mineral oxides. Non-negligible concentrations of EC were also observed (0.36 μg/scm), confirming that light-absorbing aerosol produced from combustion sources can be efficiently transported up the altitudes of Himalayan glaciers. The concentrations of carbonaceous and ionic aerosols follow a common time trend with a maximum in the premonsoon season, a minimum during the monsoon and a slow recovery during the postmonsoon and dry seasons, which is the same phenomenology observed for other Nepalese Himalayan sites in previous studies. Such seasonal cycle can be explained by the seasonal variations of dry and moist convection and of wet scavenging processes characterizing the climate of north Indian subcontinent. We document the effect of orographic transport of carbonaceous and sulphate particles upslope the Himalayas, showing that the valley breeze circulation, which is almost permanently active during the out-of-monsoon season, greatly impacts the chemical composition of PM10 and PM1 in the high Himalayas and provides an efficient mechanism for bringing anthropogenic aerosols into the Asian upper troposphere (>5000m a.s.l.). The concentrations of mineral dust are impacted to a smaller extent by valley breezes and follow a unique seasonal cycle which suggest multiple source areas in central and south-west Asia. Our findings, based on two years of observations of the aerosol chemical composition, provide clear evidence that the southern side of the high Himalayas is impacted by transport of anthropogenic aerosols which constitute the Asian brown cloud. [ABSTRACT FROM AUTHOR]- Published
- 2010
- Full Text
- View/download PDF
44. Preliminary estimation of black carbon deposition from Nepal Climate Observatory-Pyramid data and its possible impact on snow albedo changes over Himalayan glaciers during the pre-monsoon season.
- Author
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Yasunari, T. J., Bonasoni, P., Laj, P., Fujita, K., Vuillermoz, E., Marinoni, A., Cristofanelli, P., Duchi, R., Tartari, G., and Lau, K.-M.
- Abstract
The possible minimal range of reduction in snow surface albedo due to dry deposition of black carbon (BC) in the pre-monsoon period (March-May) was estimated as a lower bound together with the estimation of its accuracy, based on atmospheric observations at the Nepal Climate Observatory-Pyramid (NCO-P) sited at 5079 m a.s.l. in the Himalayan region. We estimated a total BC deposition rate of 2.89 μg m
-2 day-1 providing a total deposition of 266 μg m-2 for March-May at the site, based on a calculation with a minimal deposition velocity of 1.0×10-4 m s-1 with atmospheric data of equivalent BC concentration. Main BC size at NCO-P site was determined as 103.1-669.8 nm by correlation analysis between equivalent BC concentration and particulate size distribution in the atmosphere. We also estimated BC deposition from the size distribution data and found that 8.7% of the estimated dry deposition corresponds to the estimated BC deposition from equivalent BC concentration data. If all the BC is deposited uniformly on the top 2-cm pure snow, the corresponding BC concentration is 26.0-68.2 μg kg-1 assuming snow density variations of 195-512 kg m-3 of Yala Glacier close to NCO-P site. Such a concentration of BC in snow could result in 2.0-5.2% albedo reductions. From a simple numerical calculations and if assuming these albedo reductions continue throughout the year, this would lead to a runoff increases of 70-2 04 mm of water drainage equivalent of 11.6-33.9% of the annual discharge of a typical Tibetan glacier. Our estimates of BC concentration in snow surface for pre-monsoon season can be considered comparable to those at similar altitude in the Himalayan region, where glaciers and perpetual snow region starts in the vicinity of NCO-P. Our estimates from only BC are likely to represent a lower bound for snow albedo reductions, since a fixed slower deposition velocity was used and atmospheric wind and turbulence effects, snow aging, dust deposition, and snow albedo feedbacks were not considered. This study represents the first investigation about BC deposition on snow from atmospheric aerosol data in Himalayas and related albedo effect is especially the first track at the southern slope of Himalayas. [ABSTRACT FROM AUTHOR]- Published
- 2010
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45. Aerosol mass and black carbon concentrations, two year-round observations at NCO-P (5079 m, Southern Himalayas).
- Author
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Marinoni, A., Cristofanelli, P., Laj, P., Duchi, R., Calzolari, F., Decesari, S., Sellegri, K., Vuillermoz, E., Verza, G. P., Villani, P., and Bonasoni, P.
- Abstract
Aerosol mass and the absorbing fraction are important variables, needed to constrain the role of atmospheric particles in the Earth radiation budget, both directly and indirectly through CCN activation. In particular, their monitoring in remote areas and mountain sites is essential for determining source regions, elucidating the mechanisms of long range transport of anthropogenic pollutants, and validating regional and global models. Since March 2006, aerosol mass and black carbon concentration have been monitored at the Nepal Climate Observatory-Pyramid, a permanent high-altitude research station located in the Khumbu valley at 5079ma.s.l. below Mt. Everest. The first 10 two-year averages of PM
1 and PM1-10 mass were 1.94 μgm-3 and 1.88 μgm-3 , with standard deviations of 3.90 μgm-3 and 4.45 μgm-3 , respectively, while the black carbon concentration average is 160.5 ngm-3 , with a standard deviation of 296.1 ngm-3 . Both aerosol mass and black carbon show well defined annual cycles, with a maximum during the pre-monsoon season and a minimum during the monsoon. They also display a typical diurnal cycle during all the seasons, with the lowest particle concentration recorded during the night, and a considerable increase during the afternoon, revealing the major role played by thermal winds in influencing the behaviour of atmospheric compounds over the high Himalayas. The aerosol concentration is subject to high variability: in fact, as well as frequent "background conditions" (55% of the time) when BC concentrations are mainly below 100 ngm-3 , concentrations up to 5 μgm-3 are reached during some episodes (a few days every year) in the pre-monsoon seasons. The variability of PM and BC is the result of both short-term changes due to thermal wind development in the valley, and long-range transport/synoptic circulation. At NCO-P, higher concentrations of PM1 and BC are mostly associated with regional circulation and westerly air masses from the Middle East, while the strongest contributions of mineral dust arrive from the Middle East and regional circulation, with a special contribution from North Africa and South-West Arabian Peninsula in post-monsoon and winter season. [ABSTRACT FROM AUTHOR]- Published
- 2010
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46. Atmospheric Brown Clouds in the Himalayas: first two years of continuous observations at the Nepal-Climate Observatory at Pyramid (5079 m).
- Author
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Bonasoni, P., Laj, P., Marinoni, A., Sprenger, M., Angelini, F., Arduini, J., Bonafèe, U., Calzolari, F., Colombo, T., Decesari, S., Di Biagio, C., di Sarra, A. G., Evangelisti, F., Duchi, R., Facchini, M. C., Fuzzi, S., Gobbi, G. P., Maione, M., Panday, A., and Roccato, F.
- Abstract
South Asia is strongly influenced by the so-called Atmospheric Brown Cloud (ABC), a wide polluted layer extending from the Indian Ocean to the Himalayas during the winter and pre-monsoon seasons (November to April). This thick, grey-brown haze blanket substantially interacts with the incoming solar radiation, causing a cooling of the Earth's surface and a warming of the atmosphere, thus influencing the monsoon system and climate. In this area, the Himalayan region, particularly sensitive to climate change, offers a unique opportunity to detect global change processes and to analyse the influence of anthropogenic pollution on background atmospheric conditions through continuous monitoring activities. This paper provides a detailed description of the atmospheric conditions characterizing the high Himalayas, thanks to continuous observations begun in March 2006 at the Nepal Climate Observatory - Pyramid (NCO-P) located at 5079m a.s.l. on the southern foothills of Mt. Everest, in the framework of ABC-UNEP and SHARE-Ev-K2-CNR projects. Besides giving an overview of the measurement site and experimental activities, the work presents an in-depth characterization of meteorological conditions and air-mass circulation at NCO-P during the first two years of activity (March 2006-February 2008). The mean values of atmospheric pressure, temperature and wind speed recorded at the site were: 551 hPa,
-3 .0 °C, 4.7ms-1 , respectively. The highest seasonal values of temperature (1.7 °C) and relative humidity (94%) were registered during the monsoon season, which was also characterized by thick clouds present in about 80% of the afternoon hours and by a frequency of cloud-free sky less than 10%. The lowest temperature and relative humidity values were registered during winter, -6.3 °C and 22%, respectively, the season being characterised by mainly cloud-free sky conditions and rare thick clouds. The summer monsoon influenced the rain precipitation (seasonal mean 237 mm), while wind was dominated by flows from the bottom of the valley (S-SW) and upper mountain (N-NE). In relation to seasonal weather conditions, the time series variability of black carbon and dust particles (optical active aerosols) and ozone (regional greenhouse gas) were analysed, as they are significant constituents of the Atmospheric Brown Cloud and strongly influence the atmospheric radiative forcing. The highest seasonal values of black carbon (BC), ozone (O3 ) and dust particles were observed during the pre-monsoon season (316.9 ngm-3 , 60.9 ppbv, 0.37 cm-3 , respectively), while the lowest concentrations occurred during the monsoon for BC and O3 (49.6 ngm-3 and 33.6 ppbv, respectively) and postmonsoon for dust particles (0.07 cm-3 ). The seasonal cycles of these compounds are influenced both by the local mountain wind system and by the three principal largescale circulation regimes: Westerly, South-Westerly and Regional, as shown by the analysis of in-situ meteorological parameters and 5-day LAGRANTO back-trajectories.… [ABSTRACT FROM AUTHOR]- Published
- 2010
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47. Tropospheric ozone variations at the Nepal climate observatory -- pyramid (Himalayas, 5079ma.s.l.) and influence of stratospheric intrusion events.
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Cristofanelli, P., Bracci, A., Sprenger, M., Marinoni, A., Bonafè, U., Calzolari, F., Duchi, R., Laj, P., M.^Pichon, J., Roccato, F., Venzac, H., Vuillermoz, E., and Bonasoni, P.
- Abstract
The paper presents the first 2-years of continuous surface ozone (O
3 ) observations and systematic assessment of the influence of stratospheric intrusions (SI) at the Nepal Climate Observatory at Pyramid (NCO-P; 27°57′ N, 86°48′ E), located in the Southern 5 Himalayas at 5079ma.s.l. Continuous O3 monitoring has been carried out at this GAW-WMO station in the framework of the Ev-K2-CNR SHARE and UNEP ABC projects since March 2006. Over the period March 2006-February 2008, an average O3 value of 49±12 ppbv (±1δ) was recorded, with a large annual cycle characterized by a maximum during the pre-monsoon (61±9 ppbv) and a minimum during the mon10 soon (39±10 ppbv). In general, the average O3 diurnal cycles had different shapes in the different seasons, suggesting an important interaction between the synoptic-scale circulation and the local mountain wind regime. Short-term O3 behaviour in the middle/lower troposphere (e.g. at the altitude level of NCO-P) can be significantly affected by deep SI which, representing the most important 15 natural input for tropospheric O3 , can also influence the regional atmosphere radiative forcing. To identify days possibly influenced by SI at the NCO-P, analyses were performed on in-situ observations (O3 and meteorological parameters), total column O3 data from OMI satellite and air-mass potential vorticity provided by the LAGRANTO back-trajectory model. In particular, a specially designed statistical methodology was 20 applied to the time series of the observed and modelled stratospheric tracers. On this basis, during the 2-year investigation, 14.1% of analysed days were found to be affected by SI. The SI frequency showed a clear seasonal cycle, with minimum during the summer monsoon (1.2%) and higher values during the rest of the year (21.5%). As suggested by the LAGRANTO analysis, the position of the subtropical jet stream 25 could play an important role in determining the occurrence of deep SI transport on the Southern Himalayas. In order to estimate the fraction of O3 due to air-mass transport from the stratosphere at the NCO-P, the 30 min O3 concentrations recorded during the detected SI days were analysed. In particular, in-situ relative humidity and black carbon observations were used to exclude influence from wet and polluted air-masses transported by up-valley breezes. This analysis led to the conclusion that during SI O3 significantly increased by 27.1% (+13 ppbv) with respect to periods not affected by such events. Moreover, the integral contribution 5 of SI (O3 S) to O3 at the NCO-P was also calculated, showing that 13.7% of O3 recorded at the measurement site could be attributed to SI. On a seasonal basis, the lowest SI contributions were found during the summer monsoon (less than 0.1%), while the highest were found during the winter period (24.2%). These results indicated that, during non-monsoon periods, high O3 levels could affect NCO-P during 10 SI, thus influencing the variability of tropospheric O3 over the Southern Himalayas. Being a powerful regional greenhouse gas, these results indicate that the evaluation of the current and future regional climate cannot be assessed without properly taking into account the influence of SI to tropospheric O3 in this important area. [ABSTRACT FROM AUTHOR]- Published
- 2010
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48. Chemical composition of PM10 and PM1 at the high-altitude Himalayan station Nepal Climate Observatory-Pyramid (NCO-P) (5079 m a.s.l.).
- Author
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Decesari, S., Facchini, M. C., Carbone, C., Giulianelli, L., Rinaldi, M., Finessi, E., Fuzzi, S., Marinoni, A., Cristofanelli, P., Duchi, R., Bonasoni, P., Vuillermoz, E., Cozic, J., Jaffrezo, J. L., and Laj, P.
- Abstract
We report chemical composition data for PM
10 and PM1 from the Nepal Climate Observatory-Pyramid (NCO-P), the world's highest aerosol observatory, located at 5079 m a.s.l. at the foothills of Mt. Everest. Despite its high altitude, the average PM10 mass apportioned by the chemical analyses is of the order of 6 μgm-3 (i.e., 10 μg/scm), with almost a half of this mass accounted for by organic matter, elemental carbon (EC) and inorganic ions, the rest being mineral dust. Organic matter, in particular, accounted for by 2.0 μgm-3 (i.e., 3.6 μg/scm) on a yearly basis, and it is by far the major PM10 component beside mineral oxides. Non-negligible concentrations of EC were also observed (0.36 μg/scm), confirming that optically-active aerosol produced from combustion sources can be efficiently transported up the altitudes of Himalayan glaciers. The concentrations of carbonaceous and ionic aerosols follow a common time trend with a maximum in the premonsoon season, a minimum during the monsoon and a slow "ramp-up" period in the postmonsoon and dry seasons, which is the same phenomenology observed for other Nepalese Himalayan sites in previous studies. Such seasonal cycle can be explained by the seasonal variations of dry and moist convection and of wet scavenging processes characterizing the climate of north Indian subcontinent. We document the effect of orographic transport of carbonaceous and sulphate particles upslope the Himalayas, showing that the valley breeze circulation, which is almost permanently active during the out-of-monsoon season, greatly impacts the chemical composition of PM10 and PM1 in the high Himalayas and provides an efficient mechanism for bringing anthropogenic optically-active aerosols into the Asian upper troposphere (>5000 m a.s.l.). The concentrations of mineral dust are impacted to a smaller extent by valley breezes and follow a unique seasonal cycle which suggest multiple source areas in central and south-west Asia. Our findings, based on two years of observations of the aerosol chemical composition, provide clear evidence that the southern side of the high Himalayas are impacted by transport of anthropogenic aerosols which constitute the Asian brown cloud. [ABSTRACT FROM AUTHOR]- Published
- 2009
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49. Stratospheric intrusion index (SI2) from baseline measurement data.
- Author
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Cristofanelli, P., Calzolari, F., Bonafè, U., Duchi, R., Marinoni, A., Roccato, F., Tositti, L., and Bonasoni, P.
- Subjects
STRATOSPHERIC winds ,TROPOSPHERE ,STRATOSPHERE ,DATA analysis - Abstract
This work introduces an index to identify deep stratospheric intrusions (SI) from measurement data alone, without requiring additional model-based information. This stratospheric intrusion index (SI
2 ) provides a qualitative description of SI event behaviour by summarizing the information from different tracer variations. Moreover, being independent from any model constraint, the SI2 can also represent a valid tool to help in evaluating the capacity of chemistry-transport and chemistry-climate models in simulating deep stratosphere to troposphere transport. The in situ variations of ozone, beryllium-7 and relative humidity were used to calculate the index. The SI2 was applied on 8-year data recorded at the regional GAW station of Mt. Cimone (2165 m asl; 44.10N, 10.70E: Italy). The comparison of the SI2 behaviour with a pre-existing database obtained by also using model products, permitted us to tune a SI2 -threshold value capable of identifying SI events efficiently. In good agreement with previous climatological studies across Europe, at Mt. Cimone, the averaged monthly SI frequency obtained by the SI2 analysis showed a clear seasonal cycle with a winter maximum and a spring-summer minimum. These results suggest that the presented methodology is efficient for both identifying SI events and evaluating their annual frequency at the considered baseline measurement site. [ABSTRACT FROM AUTHOR]- Published
- 2009
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50. New atmospheric composition observations in the Karakorum region: Influence of local emissions and large-scale circulation during a summer field campaign
- Author
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P. Laj, Tony Christian Landi, U. Bonafè, Francescopiero Calzolari, Elisa Vuillermoz, Paolo Bonasoni, A. Broquet, Davide Putero, Paolo Cristofanelli, P. Villani, Marcello Alborghetti, G. P. Verza, Angela Marinoni, R. Duchi, Putero, D, Cristofanelli, P., Laj, P., Marinoni, A., Villani, P., Broquet, A., Alborghetti, M., Bonafè, U., Calzolari, F., Duchi, R., Landi, T.C., Verza, G.P., Vuillermoz, E., and Bonasoni, P.
- Subjects
Pollution ,Aerosol particle ,Atmospheric Science ,Particle number ,Anthropogenic pollution ,media_common.quotation_subject ,Atmospheric sciences ,Combustion ,Karakorum ,chemistry.chemical_compound ,Surface ozone ,Environmental Science(all) ,General Environmental Science ,media_common ,geography ,geography.geographical_feature_category ,Glacier ,Thermal wind ,Aerosol ,chemistry ,Carbon dioxide ,Climatology ,HYSPLIT ,Environmental science - Abstract
In this work we provide an overview of short lived climate forcers (SLCFs) and carbon dioxide variability in the Karakorum, by presenting results deriving from a field campaign carried out at Askole (3015 m a.s.l., Pakistan Northern Areas), by Baltoro glacier. By using an innovative embedded and transportable system, continuous measurements of aerosol particle number concentration (Np, 1571 ± 2670 cm−3), surface ozone (O3, 31.7 ± 10.4 nmol/mol), carbon dioxide (CO2, 394.3 ± 6.9 μmol/mol) and meteorological parameters have been performed from August 20th to November 10th 2012. The domestic combustion from the Askole village emerged as a possible systematic source of contamination in the valley, with short-lasting pollution events probably related to domestic cooking activities characterized by high values of Np (6066 ± 5903 cm−3). By excluding these local contamination events, mountain thermal wind regime dominated the diurnal variability of Np, O3 and CO2. In comparison to night-time, we observed higher Np (+354 cm−3) and O3 (+7 nmol/mol) but lower CO2 (−8 μmol/mol) in air-masses coming from the lower valley during the central part of the day. Part of the day-to-day atmospheric composition variability can be also ascribed to synoptic circulation variability, as observed by using HYSPLIT 5-day back-trajectories.
- Published
- 2014
- Full Text
- View/download PDF
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