DEVELOPING TOOLS FOR ACCELERATING THE DOMESTICATION OF TWO PROMISING BIOFUEL CROPS: ARUNDO DONAX (GIANT REED) AND PANICUM VIRGATUM (SWITCHGRASS)

The need for energy and the unsustainability in the long term of fossil fuels encouraged the quest for more environmental-friendly energy sources. One alternative is exploiting plants to produce biofuels. The screening of many perennial grasses suggested Miscanthus (Miscanthus spp), Arundo (Arundo donax), and reed canary grass (Phalaris arundinacea) in Europe and switchgrass (Panicum virgatum) in both Europe and US as good candidates for bioenergy production. This thesis focuses on two of these plants: Arundo and switchgrass. Both plants already show a good agronomic performance, but they underwent little domestication, so there is room for their genetic improvement. On the contrary to switchgrass, breeding, genetic transformation and in vitro cultivation of Arundo are lagging behind for the sterility and the recalcitrance of this plant. We developed a method for in vitro cultivation of Arundo that is faster and more efficient than the one previously reported in literature. Calli are induced from stem segments on an MS-based medium containing 9 \u3bcM 2,4-D and then a suspension culture can be obtained using the same medium supplemented with 4.4 \u3bcM BAP, leading to a 5-fold increase in cell mass over 14 days. This medium was the best in terms of growth rate and final cell density among the four tested media. Protocols for protoplast isolation from suspension cultures and electroporation were also established, allowing a transformation Tools for improving biofuel crops 4 efficiency up to 3.3\ub11.5 %. The use of a novel vector with two distinct fluorescent protein reporters (GFP and RFP) driven, respectively, by the Cauliflower Mosaic Virus 35S and the ubi2 promoter of switchgrass, allowed a direct comparison of the strength of the two promoters in Arundo. The switchgrass ubi2 promoter provided a much higher expression compared with the 35S promoter. Lastly, as a further methodological advancement, we developed a method to cryopreserve mesophyll protoplast of switchgrass. Both vitrification and slow-freezing methods were tested. Slow freezing is more efficient than vitrification with a percent recovery of alive protoplast of 45.8% vs. 5.2%

Methods for suspension culture, protoplast extraction, and transformation of high-biomass yielding perennial grass Arundo donax

Arundo donax L. is a promising biofuel feedstock in the Mediterranean region. Despite considerable interest in its genetic improvement, Arundo tissue culture and transformation remains arduous. The authors developed methodologies for cell- and tissue culture and genetic engineering in Arundo. A media screen was conducted, and a suspension culture was established using callus induced from stem axillary bud explants. DBAP medium, containing 9 \ub5M 2,4-D and 4.4 \ub5M BAP, was found to be the most effective medium among those tested for inducing cell suspension cultures, which resulted in a five-fold increase in tissue mass over 14 days. In contrast, CIM medium containing 13 \ub5M 2,4-D, resulted in just a 1.4-fold increase in mass over the same period. Optimized suspension cultures were superior to previously-described solidified medium-based callus culture methods for tissue mass increase. Suspension cultures proved to be very effective for subsequent protoplast isolation. Protoplast electroporation resulted in a 3.3 \ub1 1.5% transformation efficiency. A dual fluorescent reporter gene vector enabled the direct comparison of the CAMV 35S promoter with the switchgrass ubi2 promoter in single cells of Arundo. The switchgrass ubi2 promoter resulted in noticeably higher reporter gene expression compared with that conferred by the 35S promoter in Arundo

Greater impairment of postprandial triacylglycerol than glucose response in metabolic syndrome subjects with fasting hyperglycaemia

Abstract Objective: Studies have started to question whether a specific component or combinations of metabolic syndrome (MetS) components may be more important in relation to cardiovascular disease risk. Our aim was to examine the impact of the presence of raised fasting glucose as a MetS component on postprandial lipaemia. Methods: Men classified with the MetS underwent a sequential test meal investigation, in which blood samples were taken at regular intervals after a test breakfast (t=0 min) and lunch (t=330 min). Lipids, glucose and insulin were measured in the fasting and postprandial samples. Results: MetS subjects with 3 or 4 components were subdivided into those without (n=34) and with (n=23) fasting hyperglycaemia (≥ 5.6 mmol/l), irrespective of the combination of components. Fasting lipids and insulin were similar in the two groups, with glucose significantly higher in the men with glucose as a MetS component (P<0.001). Following the test meals, there was a higher maximum concentration (maxC), area under the curve (AUC) and incremental AUC (P≤0.016) for the postprandial triacylglycerol (TAG) response in men with fasting hyperglycaemia. Greater glucose AUC (P<0.001) and insulin maxC (P=0.010) was also observed in these individuals after the test meals. Multivariate regression analysis revealed fasting glucose to be an important predictor of the postprandial TAG and glucose response. Conclusion: Our data analysis has revealed a greater impairment of postprandial TAG than glucose response in MetS subjects with raised fasting glucose. The worsening of postprandial lipaemic control may contribute to the greater CVD risk reported in individuals with MetS component combinations which include hyperglycaemia

Is the pancreas affected in patients with septic shock? A prospective study

Hyperamylasemia can be observed anecdotally during the course of severe sepsis or septic shock. This study aimed to investigate the possibility of pancreatic involvement in patients with septic shock using serum pancreatic enzyme determinations and imaging techniques in 21 consecutive patients with septic shock and 21 healthy subjects as controls. METHODS: The serum activity of pancreatic amylase and lipase was assayed initially in all subjects and 24 and 48 hours after the initial observation in the 21 patients with septic shock. All patients also underwent radiological examination to detect pancreatic abnormalities. RESULTS: The serum activity of pancreatic amylase was significantly higher in the 21 patients with septic shock than in the 21 control subjects during the study period, while the serum activity of lipase was similar to that of the control subjects. Amylase and lipase serum activity did not significantly changed throughout the study period in the 21 patients with septic shock. None of the patients with pancreatic hyperenzymemia had clinical signs or morphological alterations compatible with acute pancreatitis. CONCLUSION: The presence of pancreatic hyperenzymemia in septic shock patients is not a biochemical manifestation of acute pancreatic damage, and the management of these patients should be dependent on the clinical situation and not merely the biochemical result

Cuerpo y disciplina, orden y poder: Del Instructor Popular a los Tribunales Infantiles

A fines del siglo XIX, en la República Argentina, el periódico mendocino El Instructor Popular publica el intercambio epistolar entre dos graduados de la Escuela Normal de Paraná: Carlos Norberto Vergara y Ernesto A.Bavio. Reprender, reformar y corregir el error, las faltas y la ignorancia, fueron las justificaciones para hacer uso de punteros y palmetas e incorporar la pena, el dolor y la culpa como correctivos, en las instituciones educativas de "la letra con la sangre entra" en manos de "maestros normales que quieren gobernar con el lático". El espistolario visibiliza y reprueba ciertas prácticas que tuvieron al cuerpo infantil como territorio de anclaje para la institucionalización educativa. Puntear esa conjetura nos permite trazar continuidades y discontinuidades entre los "principios de la disciplina" y "los castigos corporales" como antecedentes para los "tribunales infantiles", implementados en la Escuela Quintana de la Provincia de Mendoza, por Florencia Fossatti, en las primeras décadas del siglo XX.Fil: Alvarado, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Ciencias Humanas, Sociales y Ambientales; Argentin