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* old and bolonia study programme


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Alberto Prandi, Mirco Corazzin, Silvia Gazzin, Tanja Peric, Marta Montillo, Antonella Comin, published scientific conference contribution abstract

Abstract: Obesity and MetS (Metabolic Syndrome) are both linked to persistent long-term hormonal and metabolic changes. In most of the studies, cortisol (C) and dehydroepiandrosterone (DHEA) concentrations have been measured in obese and normal-weight subjects, obtaining heterogeneous results. Plasma, saliva and urine, matrices that represent timepoint or short-term steroids exposure, were used for these studies. The aim was to study C, DHEA and C/DHEA ratio of mice pups in the hair, matrix capable of providing cumulative hormonal exposure. Sixty C57Bl/6 mice pups (30 males and 30 females) were housed in a temperature-controlled environment (22±2°C) and on a 12h light/dark schedule, under ad-libitum access to food and water for 16 weeks (welfare: Italian Law Decree 116-92 and EC Directive 86-609-EEC). Control and experimental diets were offered immediately after weaning (3 weeks old pups), for 16 weeks. Four experimental checkpoints were established (T1: 4 weeks, T2: 8 weeks, T3: 12 weeks and T4: 16 weeks of diet). T3 and T4 showed the hormonal concentrations of pubertal animals. Twentyseven pups (13 females and 14 males) were randomly group-housed in cages (6 for T1, 5 for T2, 6 for T3, 10 for T4) and assigned to control diet (CTRL: D12328, Research Diets, New Brunswick, NJ). 33 pups (17 females and 16 males) were randomly group-housed in cages (5 for T1, 6 for T2 and T3, 16 for T4) and assigned to the HFHC diet (HFHC: D12331, Research Diets, New Brunswick, NJ - plus 42g/L fructose/sucrose in drinking water). At each experimental checkpoint, all the animals of one cage for each experimental group were suppressed. Hair strands were carefully cut with scissors as close as possible to the skin from the back of the mice, paying attention to not to wound the animals. Hair was stored in an envelope at RT in a dry room until use. C and DHEA hair concentrations was measured by a solid-phase microtiter RIA assay (Peric et al., 2016 adapted in the mouse). Only at 8 weeks the HFHC group showed significantly higher C concentrations than the CTRL group (1.56±0.06 vs 1.92±0.130 pg/mg; P<0.05). DHEA concentrations were significantly reduced in the HFHC group than the CTRL group at 4 (114.64±13.93 vs 69.08±5.33 pg/mg; P<0.05), 8 (71.67±7.08 vs 50.38±4.74 pg/mg; P<0.05), 12 (73.27±8.29 vs 41.59±1.60 pg/mg; P<0.01) and 16 weeks (65.26±3.35 vs 55.05±1.84 pg/mg; P<0.05). The C/DHEA ratio was significantly increased in the HFHC than the CTRL group, at 8 (0.023±0.002 vs 0.040±0.005; P<0.01), 12 (0.024±0.003 vs 0.036±0.004; P<0.05) and 16 weeks (0.019±0.001 vs 0.025±0.002; P<0.01). The gender effect was not significant. Taking into account the lag time required for the hair emersion from the skin (about 1 week), the significant stimulation of the C at 8 weeks in the HFHC group refers to its chronic elevation from 5 to 7 weeks of treatment when the animals were not pubertal. Conversely, DHEA shows a chronic reduction in obese mice leading to consider an independent adrenal regulation of C and DHEA, both stimulated by ACTH. Research was supported by the Project CBM (MIUR DM60643). Peric et al. 2016 J Appl Anim Welf Sci 18:1-8.
Found in: ključnih besedah
Published: 21.07.2016; Views: 2084; Downloads: 0
.pdf Fulltext (3,65 MB)

Katarzyna Rajkowska, 2018, doctoral dissertation

Abstract: The experimental work of this thesis was performed at the International Centre for Genetic Engineering and Biotechnology (ICGEB) in the Human Molecular Genetics Group, under the scientific direction of Prof. Franco Pagani. The project was developed during the academic years 2014-2017. Modified U1 RNAs, also named Exon Specific U1s (ExSpeU1s) represent a novel class of small RNA-based molecules that correct exons splicing defects. To evaluate their therapeutic potential focused on Familial Dysautonomia (FD), a rare autosomal recessive disorder characterized by progressive degeneration of the sensory and autonomic nervous system. More than 99% of patients are homozygous for the T to C transition in position 6 of the IKBKAP intron 20 (c.2204+6T>C). This substitution modifies the exon 20 5’ splice site (5’ss) inducing exon skipping in a tissue-specific manner and reducing the total amount of IKAP protein. The molecular mechanisms underlying the IKBKAP mis-splicing are not completely clear and there are no effective treatments. In this thesis, I investigated the therapeutic potential of ExSpeU1s and the role of cis- and trans-acting factors that regulates IKBKAP splicing. Using a splicing functional assay, I identified ExSpeU1s that bind to intron 20 sequences and rescue the exon 20 skipping defect. Interestingly, their rescue activity was modulated by several splicing factors and requires a critical exonic splicing enhancer element. Transfection experiment showed the involvement of both enhancing (TIA1, PTBP1 and PTB4) and inhibitory (SRSF3, hnRNPA1, FOX and FUS) splicing factors in IKBKAP splicing. To better evaluate the ExSpeU1s therapeutic efficacy, I transduced FD patient’s fibroblasts with a lentiviral vector expressing the most active ExSpeU1. This resulted in a complete rescue of the exon skipping defect and improvement in IKAP protein expression. Most importantly, intraperitoneal delivery of ExSpeU1s by AAV9 into a transgenic mouse model, that recapitulates the tissue-specific mis-splicing seen in FD patients, corrected the aberrant splicing patterns in several tissues increasing the amount of the corresponding IKAP protein. All together, these results identify novel regulatory splicing factors involved in the IKBKAP exon 20 regulation and provide the proof of principle that ExSpeU1s delivered in vivo by AAV vectors represent a novel therapeutic strategy for FD.
Found in: ključnih besedah
Summary of found: of ExSpeU1s by AAV9 into a transgenic mouse model, that recapitulates the tissue-specific mis-splicing seen in...
Keywords: Familial Dysautonomia, IKBKAP, IKAP, splicing, splicing defects, ExSpeU1, U1 snRNA, mouse model, AAV
Published: 26.03.2018; Views: 1733; Downloads: 49
.pdf Fulltext (13,96 MB)

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