The aim of this research was to identify the mechanisms that underpin changes in respiratory capacity during acclimation to temperature. Dark respiration, enzyme activities and leaf ultrastructure were measured from ryegrass (Lolium perenne) in controlled environmental chambers and two species of native grass (Chionochloa rubra & C. pallens) growing at different altitudinal ranges on Mount Hutt, Canterbury, New Zealand. The overall hypothesis was that the changes in both mitochondrial numbers and enzyme activity underpin the greater respiratory capacity observed in response to decreasing temperatures. Gas exchange measurements were carried out to measure rates of dark respiration (Rd) in leaves of both ryegrass and tussocks. Respiratory homeostasis (full acclimation) was achieved in ryegrass leaves but only partial acclimation in both species of tussock plants. Dark respiration rates for warm-grown ryegrass were greatly reduced compared to cool-grown grasses. Rd was lower for C. rubra growing at the base of the mountain (450m) compared to plants at a higher altitude (1060m). The dark respiration rates were also lower for C. pallens growing at 1070m than at 1600m. When comparing Rd between high and low altitude plants, it was significantly lower in low altitude plants at 450m than at 1600m. Oxygen consumption was measured in intact leaves and roots, crude mitochondria and isolated mitochondria from ryegrass to investigate whether a change in respiratory capacity was involved with changes in Rd. Mitochondrial respiratory capacity was slightly reduced in warm leaves and roots (not significantly). The respiratory capacity results from isolated mitochondria for C. rubra (at 450m and 1060m) and C. pallens (at 1070m and 1600m) were consistent with the hypothesis that plants from warm sites have lower respiratory capacity in comparison to plants from cool sites. Based on these results and those of previous studies, it was concluded that respiratory flux for any given temperature is not simply determined by maximal capacities of the respiratory apparatus but rather a combination of the availability of substrate supply, the demand for respiratory products (i.e. ATP) and/or the maximal capacity of respiratory enzymes. Utilizing transmission electron micrographs, it was found that mitochondria were significantly less abundant in warm-grown than cool-grown ryegrass mesophyll cells. Mitochondria dimensions increased slightly between the cool and warm treatment. At lower altitudes (C. rubra), there was a significant decrease in mitochondria numbers with decreasing elevation. At higher altitudes (C. pallens), there was no noticeable change in mitochondria numbers between 1070m and 1600m. It was concluded that mitochondrial abundance for the controlled and field experiments, and mitochondrial sizes in the field, were associated with changes in Rd. The maximal activities of fumarase and succinate dehydrogenase (SDH) in isolated mitochondria from leaves of ryegrass and tussock were measured spectrophotometrically. The results in the controlled experiment indicate that enzymes other than fumarase and SDH could be responsible for the increased respiratory capacity observed in cold acclimated leaves of ryegrass. However, fumarase maximal activity was significantly reduced in C. rubra at low altitude compared with C. pallens growing at high altitude - this suggests that it may be involved in the differences in respiratory capacity and Rd between the two sites. Succinate dehydrogenase did not differ significantly in response to altitude. The large difference between the two field sites for fumarase activity is comparable to the large difference in Rd and reduction in mitochondrial abundance and dimensions seen between the two sites. This supports the overall hypothesis that cool-grown plants keep up with energy demands at low temperatures by increasing enzyme concentrations/capacity. The results of this study are supportive of the hypothesis that growth in low altitudes and warm conditions will result in the reduction of Rd as a consequence of: (1) temperature sensitivity of the respiratory apparatus, resulting in the reduction of the respiratory capacities of mitochondria; (2) a reduction in mitochondria size and numbers; and as a consequence of this (3) a reduction in the activities of mitochondrial enzymes. However, these responses are species specific and vary according to the range of temperatures experienced by plants in the field and controlled environments.

This thesis deals with certain aspects of the solution chemistry of the simple dicarboxylate anions: oxalate, malonate and succinate, up to high concentrations. These ions are either significant impurities in the concentrated alkaline aluminate solutions used in the Bayer process for the purification of alumina, or are useful models for degraded organic matter in industrial Bayer liquors. Such impurities are known to have important effects on the operation of the Bayer process. To develop a better understanding of the speciation of oxalate (the major organic impurity in Bayer liquors) in concentrated electrolyte solutions, the formation constant (Log£]) of the extremely weak ion pair formed between sodium (Na+) and oxalate (Ox2ƒ{) ions was determined at 25 oC as a function if ionic strength in TMACl media by titration using a Na+ ion selective electrode. Attempts to measure this constant in CsCl media were unsuccessful probably because of competition for Ox2ƒ{ by Cs+. Aqueous solutions of sodium malonate (Na2Mal) and sodium succinate (Na2Suc) were studied up to high (saturation) concentrations at 25 oC by dielectric relaxation spectroscopy (DRS) over the approximate frequency range 0.1 „T £h/GHz „T 89. To complement a previous study of Na2Ox, formation constants of the Na+-dicarboxylate ion pairs were determined and they were shown to be of the solvent-shared type. Both the Mal2ƒ{ and Suc2ƒ{ ions, in contrast to Ox2ƒ{, were also shown to possess large secondary hydration shells Apparent molal volumes (Vf) and heat capacities at constant pressure (Cpf) of aqueous solutions of Na2Ox, Na2Suc, Na2Mal and K2Ox were determined at 25 oC up to their saturation limits using vibrating tube densitometry and flow calorimetry. These data were fitted using a Pitzer model. The adherence of Vf and Cpf of various Na+ and K+ salts to Young¡¦s rule was examined up to high concentrations using the present and literature data. Young¡¦s rule was then used to estimate hypothetical values of Cpf and Vf for the sparingly soluble Na2Ox at high ionic strengths, which are required for the thermodynamic modelling of Bayer liquors. The solubility of Na2Ox in various concentrated electrolytes was measured, at temperatures from 25 oC to 70 oC in media both with (NaCl, NaClO4, NaOH) and without a common ion (KCl, CsCl, TMACl). The common ion effect was found to dominate the solubility of Na2Ox. The solubility of calcium oxalate monohydrate (CaOx„ªH2O) was also determined. The solubilities of both Na2Ox and CaOx„ªH2O in media without a common ion increased with increasing electrolyte concentration, except in TMACl media, where they decreased. The solubility of Na2Ox was modelled using a Pitzer model assuming the Pitzer parameters for Na2SO4 and minimising the free energy of the system. The data were modelled successfully over the full concentration and temperature range of all the electrolytes, including ternary (mixed electrolyte) solutions.

Made available in DSpace on 2014-06-11T19:29:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-01-18Bitstream added on 2014-06-13T19:17:16Z : No. of bitstreams: 1 caires_fj_me_araiq.pdf: 493700 bytes, checksum: 3407a17ff8fb345606e061ffb146a15f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Esse trabalho descreve a síntese e caracterização dos succinatos de metais de transição bivalentes, MC2H4C2O4·nH2O (M = Mn(II), Fe(II), Co(II), Ni(II), Cu(II), Zn(II)), no estado sólido. Inicialmente prepararam-se os carbonatos dos respectivos íons metálicos adicionando-se lentamente e sob agitação solução saturada de hidrogeno carbonato de sódio à solução de cloreto (Mn, Co, Ni, Zn) ou sulfato (Fe, Cu), até precipitação completa dos íons metálicos. Os succinatos foram obtidos fazendo-se reagir os carbonatos dos íons metálicos com solução de ácido succínico. Os compostos sintetizados foram estudados empregando termogravimetria e análise térmica diferencial simultânea (TG-DTA), termogravimetria derivada (DTG), calorimetria exploratória diferencial (DSC), difratometria de raios X pelo método do pó, espectroscopia no infravermelho, termogravimetria acoplada à espectroscopia de absorção na região do infravermelho com transformada de Fourier (TG-FTIR) e complexometria. Os resultados forneceram informações sobre o comportamento térmico, cristalinidade, modos de coordenação etc. Também foi possível identificar os produtos liberados durante o aquecimento. Os resíduos finais foram Mn3O4, Fe2O3, Co3O4, NiO, CuO e ZnO / In this work, the synthesis and characterization of bivalent transition metal ion succinates in the solid state, MC2H4C2O4·nH2O (M = Mn(II), Fe(II), Co(II), Ni(II), Cu(II), Zn(II)), are described. Firstly, the respective metal ion carbonates were prepared by slow addition of sodium hydrogen carbonate saturate solution to the chloride (Mn, Co, Ni, Zn) or sulphate (Fe, Cu) solution under continuous magnetic stirring until complete precipitation of the metal ions. The succinates were obtained by reacting carbonate of the metal ions with succinic acid solution. The synthesized compounds were characterized by simultaneous thermogravimetry and differential thermal analysis (TGDTA), Thermogravimetry derivative (DTG), differential scanning calorimetry (DSC), Xray powder diffractometry, infrared spectroscopy, thermogravimetry coupled to absorption spectroscopy in the region of infrared with Fourier transform (TG-FTIR) and complexometry. The results provided information about the thermal behavior, crystallinity, modes of coordination etc. It was also possible to identify the products evolved during heating. The final residues were Mn3O4, Fe2O3, Co3O4, NiO, CuO and ZnO

Das Interesse an biotechnologisch hergestellter Bernsteinsäure als ein potenzielles intermediäres Ausgangsmaterial und als Alternative zu petrochemischen Produktionsprozessen in der Industrie steigt stetig. Für industrielle Zwecke wird Succinat derzeit noch hauptsächlich petrochemisch aus Maleinsäureanhydrid ausgehend von Butan gewonnen. Aufgrund ihrer Struktur als lineare, gesättigte Dicarbonsäure hat Bernsteinsäure allerdings das Potenzial als sogenannte Building-Block-Chemikalie zu fungieren und das als Ausgangssubstanz für viele Prozesse dienende Maleinsäureanhydrid zu verdrängen. Wichtige Vorstufen für die chemische Synthese, Polyesterproduktion und andere Prozesse können aus Succinat gewonnen werden, eine preiswerte und zu Maleinsäureanhydrid günstigere Bereitstellung von Succinat vorausgesetzt. Aufgrund der zahlreichen Anwendungsmöglichkeiten und der mit der petrochemischen Herstellung verbundenen Nachteile, wie z. B. hohe Kosten und der Einsatz umweltschädlicher Substanzen, ist die Forschung an succinatproduzierenden Organismen durch den voraussichtlichen ökologischen Nutzen und der besseren Kosteneffizienz einer biobasierten Succinatproduktion motiviert. Bernsteinsäure wird natürlicherweise durch viele Mikroorganismen, Pflanzen und Tiere als Intermediat im zentralen Stoffwechsel oder als Stoffwechselendprodukt gebildet. Die Mehrheit der natürlichen und optimierten Produzenten stellen Bakterienstämme dar, die Succinat unter anaeroben Bedingungen akkumulieren. In den letzten Jahren rückten auch Hefen immer stärker in den Fokus der Untersuchungen zur Entwicklung von biotechnologischen Verfahren zur Succinatproduktion. Neben der konventionellen Hefe Saccharomyces cerevisiae, mit der bisher trotz gentechnischer Veränderungen nur maximal 6,3 g Succinat je Liter produziert werden konnte, ist besonders die als Säureproduzent bekannte Hefe Yarrowia lipolytica von Bedeutung. Yarrowia lipolytica ist einzigartig in ihrer Fähigkeit zur Produktion und Sekretion von großen Mengen verschiedener organischer Säuren, wie Citrat, Isocitrat, α-Ketoglutarat und Pyruvat. Aufgrund dieser hohen Sekretionskapazität für Metabolite und Proteine und einer effizienten Verwertung eines breiten Substratspektrums, aber auch wegen ihrer Apathogenität und der guten molekularbiologischen und verfahrenstechnischen Handhabbarkeit ist diese Hefe schon seit einigen Jahren von hohem industriellen Interesse. Neben den bereits genannten organischen Säuren ist Yarrowia lipolytica unter geeigneten Bedingungen auch zur Produktion und Sekretion von Succinat fähig. In der vorliegenden Arbeit sollte das Potenzial dieser Hefe als Succinatproduzent untersucht werden. Der Fokus der Untersuchungen lag dabei vor allem auf dem succinat-metabolisierenden Enzym Succinatdehydrogenase, das im Tricarbonsäurezyklus die Oxidation von Succinat zu Fumarat katalysiert. Zu Beginn der Arbeiten wurden die Auswirkungen einer Reduktion der Aktivität der Succinatdehydrogenase durch das Antibiotikum Carboxin untersucht. Carboxin bewirkt in verschiedenen Organismen eine Hemmung der Succinatdehydrogenase. Dabei bindet Carboxin wahrscheinlich an die Ubichinon-Bindestelle der Succinatdehydrogenase und verhindert so eine Reduktion von Ubichinon. Die Wirkung von Carboxin auf die Succinatdehydrogenase und die Succinatproduktion der Hefe Yarrowia lipolytica wurde bisher allerdings nicht untersucht. Im Rahmen dieser Arbeit wurde jedoch nachgewiesen, dass Carboxin nicht nur auf das Wachstum des Wildtypstamms Yarrowia lipolytica H222, sondern auch auf die spezifische Aktivität der Succinatdehydrogenase hemmend wirkte. Es wurde außerdem gezeigt, dass eine Reduktion der Succinatdehydrogenaseaktivität zu einer deutlichen Steigerung der maximalen Succinatmengen und der Produktbildungsraten führte. So wurden mit 150 μg L-1 Carboxin maximale Succinatmengen von 31,6 ± 5,4 g L-1 mit durchschnittlichen Produktivitäten von 65,5 ± 7,6 mg L-1 h-1 und 4,3 ± 1,7 μg OD-1 h-1 im Vergleich zur Kultivierung ohne Carboxin mit 2,0 ± 0,7 g L-1, 4,3 ± 0,9 mg L-1 h-1 und 0,2 ± 0,1 μg OD-1 h-1 nachgewiesen. Um auf das für großtechnische Verfahren ungeeignete Antibiotikum Carboxin zu verzichten, sollte die Aktivität der Succinatdehydrogenase anschließend durch gezielte gentechnische Veränderungen reduziert werden. Um dies zu erreichen, wurde das Gen SDH2, das für die Eisen-Schwefel-Untereinheit der Succinatdehydrogenase kodiert, unter die Kontrolle ausgewählter Promotoren (des Isocitratlyasegens ICL1 oder des 3-Oxo-Acyl-Thiolasegens POT1) gesetzt, die unter bestimmten Bedingungen, z. B. bei Einsatz von Glycerol als Kohlenstoffquelle nur schwach expremiert werden. Die spezifische Aktivität der Succinatdehydrogenase konnte dadurch um 40 (pICL1-SDH2) bzw. 64 % (pPOT1-SDH2) herabgesetzt werden. Die so konstruierten Yarrowia lipolytica Stämme H222-AZ1 (pICL1-SDH2) und H222-AZ2 (pPOT1-SDH2) produzierten maximal 15,3 ± 0,5 g L-1 bzw. 30,0 ± 3,7 g L-1 Succinat. Die Untersuchungen mit Carboxin und gentechnisch bedingter verringerter Expression zeigten, dass es offenbar einen Zusammenhang zwischen Reduktion der Aktivität der Succinatdehydrogenase und Steigerung der Succinatproduktion gibt. Weiterhin waren die Produktbildungsraten bei beiden Stämmen im Vergleich zum Wildtyp signifikant erhöht. Eine Reduktion der Aktivität der Succinatdehydrogenase – sei es durch Carboxin oder Promotoraustausch – bewirkte außerdem eine Reduktion der Malat- und Fumaratbildung und eine Erhöhung der detektierbaren α-Ketoglutaratgehalte. Auf Basis dieser Ergebnisse wurde geschlussfolgert, dass die Succinatdehydrogenase das Schlüsselenzym zur Beeinflussung der Succinatproduktion zu sein scheint. Diese Schlussfolgerung wurde außerdem durch die Arbeiten von Yuzbashev et al. (2010) unterstützt. Parallel zu diesen Arbeiten wurden außerdem die Effekte der Überexpression der α-Ketoglutaratdehydrogenase, der Isocitratlyase und der Pyruvatcarboxylase untersucht. Trotz einer signifikanten Steigerung der spezifischen Aktivitäten hatten weder eine Überexpression der α-Ketoglutaratdehydrogenase noch der Isocitratlyase eine Steigerung der Succinatproduktion zur Folge. Nur die erhöhte Kopienzahl des Pyruvatcarboxylase kodierenden Gens und eine damit einhergehende Steigerung der Aktivität resultierte in einer geringen Erhöhung der gebildeten Succinatgehalte im Vergleich zum Wildtyp. Bei gleichzeitiger Reduktion der Succinatdehydrogenaseaktivität durch Carboxin wurde diese Tendenz eindeutig bestätigt. Aufgrund dieses Befunds wurde ausgehend von H222-AZ2 ein Stamm konstruiert, in dem zusätzlich zum Austausch des DH2-Promotors mehrere Kopien des Pyruvatcarboxylase-kodierenden Gens PYC1 eingeführt wurden. Die Überexpression von PYC1 resultierte nicht nur in einer gesteigerten Aktivität der Pyruvatcarboxylase, sondern auch in einer weiteren Steigerung der Succinatproduktion im Vergleich zu H222-AZ2. So wurden maximale Succinatmengen von 43,9 ± 7,9 g L-1 mit durchschnittlichen Produktivitäten von 129,4 ± 13,0 mg L-1 h-1 und 8,2 ± 1,5 μg OD-1 h-1 nachgewiesen. Im Vergleich zum Wildtyp entspricht dies einer Steigerung der detektierbaren Succinatmengen um das 34fache. Die in der vorliegenden Arbeit erzielten Ergebnisse verdeutlichen das große Potenzial der Hefe Yarrowia lipolytica für die biotechnologische Herstellung von Succinat. Es wurde gezeigt, dass eine Reduktion der Succinatdehydrogenaseaktivität allein oder kombiniert mit der gesteigerten Pyruvatcarboxylaseaktivität zu einer drastischen Steigerung der Succinatproduktion sowie zu einer Reduktion der Nebenprodukte Fumarat und Malat führte. Es wird davon ausgegangen, dass eine weitere Steigerung durch eine Optimierung der Kultivierungsbedingungen bzw. durch zusätzliche gentechnische Veränderungen möglich ist. So sollte durch nachfolgende gentechnische Veränderungen, z. B. eine erhöhte Expression der Gene der am Glyoxylatzyklus beteiligten Enzyme Malatsynthase und Isocitratlyase, vor allem die weitere Reduktion der Nebenprodukte, besonders eine Reduktion der α-Ketoglutaratgehalte angestrebt werden. Des Weiteren sollten auch die Transportprozesse der organischen Säuren, insbesondere durch die Membranen, untersucht und gegebenenfalls zugunsten der Succinatproduktion manipuliert werden.

Succinate is one of the intermediate in the Krebs cycle, which in recent years has been shown to interfere with other cellular events, some of which may affect cardiac ischemic tolerance. The aim of this project was to clarify its cardioprotective role in rat hearts subjected to acute ischemia-reperfusion. The myocardial resistance to acute ischemia (infarct size and incidence and severity of ischemic and reperfusion arrhythmias) was analyzed using the Langendorff method of isolated perfused heart at a constant flow with acute succinate administration. Local ischemia was induced by ligation of left anterior descending coronary artery. Acute administration of 1 mM succinate before 60 minutes of ischemia or before reperfusion only had a beneficial effect on reducing the infarct size by 25-30 % compared to the control group. At the same time, it had an adverse effect on the incidence and severity of ischemic and reperfusion arrhythmias. Key words: Succinate, heart, rat, heart-attack, ventricular arrhythmias

No description available.

The occurrence of plastic impaction in ruminants is a growing concern. As indiscriminate feeders, cattle may consume plastic foreign materials incorporated into their diets and it is currently estimated that 20% of cattle contain plastic foreign materials in their rumen. These materials are indigestible and accumulate for the lifetime of the animal. As these materials accumulate, they may reduce feed efficiency and production by erosion and ulceration of rumen epithelium, stunting of papillae, blockage of the reticulo-omasal orifice, and leaching of toxic heavy metals. It is necessary to reduce the incidences of plastic impaction in domestic ruminants. Using polyhydroxyalkanoate (PHA) and poly(butylene succinate-co-adipate) (PBSA) biodegradable materials for feed storage products such as bale netting could reduce the incidences and effects of polyethylene-based plastic impaction in ruminants. The objectives of these studies were to evaluate the degradability of PHA and PBSA materials in the reticulorumen via in vitro, in situ, and in vivo methods. Our hypothesis was that these materials would degrade in the rumen and that a melt-blend of PHA and PBSA may degrade faster than its individual components. An in vitro study incubated a proprietary PHA-based polymer, PBSA, and PBSA:PHA melt blend nurdles, and forage controls in rumen fluid for up to 240h in DaisyII Incubators. Mass loss was measured, and digestion kinetic parameters were estimated. Thermogravimetric and differential scanning calorimetry analyses were conducted on incubated samples. Results indicated that the first stage of degradation occurs within 24h and PHA degrades slowly. Degradation kinetics demonstrated that polymer treatments were still in the exponential degradation phase at 240h with a maximum disappearance rate of 0.0031%/h, and mass loss was less than 2% for all polymers. Melting temperature increased and onset thermal degradation temperature decreased with incubation time, indicating structural changes to the polymers starting at 24h. Further in situ degradation, however, indicated these biodegradable materials degrade at more accelerated rates in the rumen. Polyhydroxyalkanote, PBSA, PBSA:PHA blend, and low-density polyethylene (LDPE) films were incubated in the rumens of three cannulated, non-lactating Holsteins for 0, 1, 14, 30, 60, 90, 120, and 150d. In situ disappearance (ISD) and residue length were assessed after every incubation time. Polyhydroxyalkanoate achieved 100% degradation by 30d, with initiation occurring at 14d indicated by ISD and a reduction in residue length. The fractional rate of disappearance of PHA was 7.84%/d. Poly(butylene succinate-co¬-adipate) and Blend did not achieve any significant ISD, yet fragmentation of PBSA occurred at 60d and the blend at just 1d likely due to abiotic hydrolysis. Low-density polyethylene achieved no ISD and residue length did not change over incubation time. From these results, we proposed a PBSA:PHA blend is a valid alternative to polyethylene single-use agricultural plastic products based on its fragmentation within 1d of incubation. Administration of PBSA:PHA film boluses compared to LDPE films and a control further supported this dissemination. Holstein bull calves (n = 12, 62 ± 9d, 74.9 ± 8.0kg) were randomly allocated to one of three daily bolus treatments: 13.6g of PBSA:PHA in 4 gelatin capsules (Blend), 13.6g of LDPE in 4 gelatin capsules (LDPE), or 4 empty gelatin capsules (Control) for 30d. Hemograms were conducted on blood samples collected on d0 and d30. On d31, animals were sacrificed to evaluate gross rumen measurements and pathology, determine papillae length, and characterize polymer residues present in rumen contents. Feed intake, body weight, body temperature, and general health were determined throughout the study. No animals presented any symptoms related to plastic impaction and animal health was not particularly affected by treatment. Daily grain and hay intake, body weight, rectal temperature, hematological parameters, gross rumen measurements and pathology, and rumen pH and temperature were not affected by treatment. There was evidence that degradation of PBSA:PHA may release byproducts that support rumen functionality. Methylene blue reduction time of Blend calves tended to be decreased by 30% compared to LDPE calves, and caudal ventral papillae length of Blend calves were 50% longer than those of Control animals. Though studies are needed to specifically elucidate the production of byproducts due to degradation of PBSA:PHA and their correlations. Polymer accumulation and residue length differed among treatments. Calves dosed with LDPE retained 6.7% of the dosed polymer, undegraded, while Blend calves retained 0.4% of the dosed polymer. The polymer residues in Blend calves were 10% of their original size. Single-use agricultural plastics developed from PBSA:PHA may be a suitable alternative to LDPE-based products in the case of ingestion in ruminants due to no acute health inflictions, fragmentation of polymers with 1d, and improved clearance from the reticulorumen. As such, utilization of these materials may reduce the incidences of plastic impaction in ruminants in commercial operations. Further long-term feeding studies are needed to evaluate specific byproduct production of PBSA:PHA and their potential influences on rumen function and animal health and production in normal commercial conditions. / Master of Science in Life Sciences / Plastic feed-storage materials may unintentionally be incorporated into animal feeds. Net wraps and bale twines may be stuck or left on forages when they are ground and incorporated into mixed rations. As cattle are largely non-selective, they may inadvertently consume these plastic materials. Approximately 20% of cattle contain plastic foreign materials in their rumen. These materials are indigestible and accumulate for the animal's lifetime. As plastics build up in the rumen, they may reduce feed efficiency, body weight, and milk production by damaging the rumen lining, blocking the digestive tract, and leaching toxic heavy metals. Therefore, it is necessary to reduce the incidences of plastic impaction in domestic ruminants to improve their health and productivity. Using biodegradable materials that degrade by bacteria, such as polyhydroxyalkanoate (PHA) and poly(butylene succinate-co-adipate) (PBSA), for feed storage products could reduce the occurrence and effects of plastic impaction in ruminants due to the materials' potential degradation in and passage from the rumen. The objectives of these studies were to evaluate the breakdown of PHA and PBSA materials in the rumen. Our hypothesis was that these biodegradable materials would degrade in the rumen and that a blend of PHA and PBSA may degrade faster than its individual components. In our first study, PHA, PBSA, a PBSA:PHA blend, and forage controls were incubated in rumen fluid for up to 240h. Mass loss, degradation rate, and the structure of polymers were determined over incubation time. Results indicated that biodegradable polymers may begin to break down within 24h. Polymer treatments were still in the early stages of degradation at 240h with a maximum degradation rate of 0.0031%/h, and mass loss of polymers was less than 2%. However, within 24h, the structures of polymers may have altered to promote future degradation at longer incubation times. Accelerated degradation was observed when PHA, PBSA, PBSA:PHA (Blend), and polyethylene (LDPE) films were incubated in the rumens of three Holstein cows up to 150d. Mass loss and the length of the remaining polymers were assessed monthly. Polyhydroxyalkanoate began to degrade by 14d and completely degraded by 30d with a disappearance rate of 7.84%/d. The remaining polymer did not achieve any mass loss. However, PBSA and Blend residue size began to decrease by 60d and 1d, respectively. Based on Blend's structural degradation within 1d of incubation that may promote its clearance from the rumen if ingested, we proposed that the material may be an alternative to polyethylene single-use agricultural plastic products. When Blend films were fed to calves, breakdown of the material further supported our dissemination that PBSA:PHA may be a suitable alternative to LDPE in the case of animal ingestion. Holstein bull calves (n = 12, 62 ± 9d, 74.9 ± 8.0kg) were randomly allotted to one of three daily bolus treatments: 13.6g of PBSA:PHA (Blend), 13.6g of polyethylene (LDPE), or no polymer (Control) distributed over 4 gelatin capsules for 30d. Feed intake, body weight, body temperature, and general health were determined throughout the study. Blood analyses were conducted on blood samples collected before and after the experimental period. On d31, animals were sacrificed to evaluate rumen growth and health, measure rumen papillae length, and describe polymers that may reside in the rumen. No animals presented any signs related to plastic impaction and animal health was not particularly affected by treatment. Daily grain and hay intake, body weight, rectal temperature, blood parameters, and rumen growth and health were not affected by treatment. There was evidence that degradation of Blend may support rumen function. Methylene blue reduction time of Blend calves tended to be decreased by 30% compared to LDPE calves, which indicates the rumen microbiome of Blend calves may better ferment feeds. Papillae length of Blend calves were also 50% longer than those of Control animals, which would improve the absorption of nutrients. Byproduct formation from Blend degradation could explain this; however, studies are needed to specifically elucidate the production of byproducts and their relationship to rumen function. Polymer accumulation and residue length differed among treatments. Calves dosed with LDPE retained 6.7% of the dosed polymer, undegraded, while Blend calves retained 0.4% of the dosed polymer. The polymer residues in Blend calves were 10% of their original size. Single-use agricultural plastics developed from PBSA:PHA may be a suitable alternative to polyethylene-based products in the case of ingestion in ruminants due to no short-term health inflictions, the reduced polymer size within 1d, and improved clearance from the rumen. As such, utilization of these materials may reduce the incidences of plastic impaction in ruminants in commercial operations. Further long-term feeding studies are needed to evaluate specific byproduct production of PBSA:PHA and their potential influences on rumen function and animal health and production in normal commercial conditions.

No / A solid-phase Mitsunobu reaction between a resin-bound 1-thiosugar and an N-Fmoc protected amino alcohol was successfully employed for thio-linked glycopeptide synthesis. Facile cleavage and deprotection in one step afforded the target glycopeptide in good yield and purity.

Yes / We present hot melt extrusion (HME) for the design of floating multiparticulates. Metoprolol succinate was selected as the model drug. Our foremost objective was to optimize the components Eudragit® RS PO, polyethylene oxide (PEO) and hydroxypropyl methylcellulose (HPMC) to balance both buoyancy and controlled release. Gas generated by sodium bicarbonate in acidic medium was trapped in the polymer matrix to enable floating. Eudragit® RS PO and PEO with sodium bicarbonate resulted in multiparticulates which exhibited rapid flotation within 3 minutes but inadequate total floating time (TFT) of 3 hours. Addition of HPMC to the matrix did not affect floating lag time (FLT), moreover TFT increased to more than 12 hours with controlled release of metoprolol succinate. Floating multiparticulates exhibited t50% of 5.24 hours and t90% of 10.12 hours. XRD and DSC analysis revealed crystalline state of drug while FTIR suggested nonexistence of chemical interaction between the drug and the other excipients. The assay, FLT, TFT and the drug release of the multiparticulates were unchanged when stored at 40 °C/75%RH for 3 months confirming stability. We present floating multiparticulates by HME which could be extrapolated to a range of other drugs. Our approach hence presents platform technology for floating multiparticulates.

La succinate déshydrogénase (SDH) est à la fois une enzyme clé du cycle de Krebs oxydant le succinate en fumarate et le complexe II de la chaîne respiratoire mitochondriale impliqué dans le transfert des électrons et la réduction de l’ubiquinone. Des inhibiteurs de cette enzyme (SDHI) ont été développés ou sont en cours de développement comme antifongiques. Cette famille de fongicides est notamment utilisée pour lutter contre Botrytis cinerea, champignon phytopathogène responsable de la pourriture grise sur de nombreuses cultures dont la vigne. Des souches résistantes aux SDHI ont été isolées chez B. cinerea et d’autres champignons phytopathogènes. Chez ces isolats résistants, des mutations ont été identifiées dans les gènes codant la SDH. Au cours de cette thèse, nous avons étudié l’impact de mutations affectant la sous-unité B (SdhB) de la succinate déshydrogénase sur l’activité de l’enzyme, la biologie du champignon B. cinerea et la résistance aux inhibiteurs ciblant cette enzyme. Par mutagénèse dirigée du gène sdhB, nous avons obtenu des mutants dits « isogéniques » qui ont permis de confirmer l’implication de ces mutations dans la résistance aux différentes molécules SDHI. Par ailleurs, nos résultats montrent que les modifications de la sous-unité SdhB affectent l’affinité des SDHI pour la SDH et les niveaux d’inhibition de l’activité SDH par les molécules inhibitrices ; ce qui explique - in fine - les spectres de résistance des mutants aux SDHI. Actuellement, tous les mutants sont résistants au boscalid et les mutants les plus fréquemment retrouvés au vignoble, sdhBH272R/Y, sont sensibles au fluopyram. Les travaux réalisés sur les mutants sdhB montrent que les mutations étudiées ont également un impact sur l’activité de l’enzyme et sur le développement du champignon, conséquences dépendantes du résidu substitué et de la substitution. En particulier, les mutations sdhBH272L/R affectent fortement l’activité de l’enzyme et la fitness du champignon alors que le mutant sdhBH272Y est peu affecté. Enfin, l’analyse de populations de pourriture grise de différentes origines (région, plantes hôtes) par rapport à la résistance aux SDHI réalisée sur les années 2009/2010 montre que les mutants sdhBH272R/Y sont toujours les plus fréquents mais leurs fréquences varient en fonction des situations agronomiques. Notamment la fréquence du mutant sdhBH272R augmente avec la pression de sélection exercée par les fongicides. Ce mutant attire particulièrement notre attention du fait de sa relation non linéaire entre fitness et fréquence au champ. / Succinate dehydrogenase is both a key enzyme of the TCA cycle, oxidizing succinate into fumarate and complex II of the mitochondrial respiratory chain involved in electron transfer and ubiquinone reduction. Inhibitors of this enzyme (SDHIs) have been developed or are in the developmental process as fungicides. Actually, SDHIs are registered to deal with Botrytis cinerea, a phytopathogenic fungus responsible for grey mold on many crops including grapevine. Strains of B. cinerea and other pathogenic fungi have been isolated for their resistance to SDHI. They mainly harbor mutations in genes encoding SDH subunits. During this thesis, we studied the impact of mutations modifying subunit B of succinate dehydrogenase on enzyme activity, fungal biology and resistance to SDHIs. “Isogenic” mutants obtained through site-directed mutagenesis and homologous recombination allowed us to confirm the role of sdhB mutations in SDHIs resistance. Our results also show that the substitutions in the SdhB subunit impact respectively the affinity of SDHIs to SDH and the inhibition levels of SDH activity by inhibitors, which explain – in fine – the resistance spectra observed for the mutants. Up to now, all sdhB mutants are resistant to boscalid and the most frequent mutants observed in grapevines, sdhBH272R/Y, are susceptible to fluopyram. Studies on sdhB mutants reveal that the mutations also impact the enzymatic activity and the fungal development depending on the substitution. In particular, sdhBH272L/R mutations have the strongest impact on enzyme activity and the fitness of the fungus, whereas these parameters are almost not altered in the sdhBH272Y mutant. Finally, grey mold populations from different origins (country, plant host) were analyzed for their SDHI resistance pheno- and genotypes. Yet, the sdhBH272R/Y mutants were the most frequent, but these frequencies varied according to the agronomical situation. Interestingly, the frequencies of the sdhBH272R mutant seem to increase with the selective pressure exerted by fungicides. This mutant is of particular interest because of the absence of correlation between the fitness we measured and the frequencies we observed in natura.