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Es wird eine neue Methode zur Darstellung von Vinylthiocyanaten ueber [3,3]-sigmatrope Umlagerungen vorgestellt. Obwohl dabei aus einer energieaermeren Gruppe (Isothiocyanat) eine energiereichere (Thiocyanat) entsteht, isomerisiert das Senfoel zum Rhodanid. Das ist moeglich, weil der Energieunterschied zwischen den Isothiocyanaten und den entsprechenden Thiocyanaten vergleichsweise gering ist und strukturelle Eigenschaften die Gesamtenergie ebenfalls beeinflussen. Die Isothiocyanatvorlaeufer stellt man in vielen Faellen durch ein Eintopfverfahren her, welches ueber Azid- und Iminophosphoran- (bzw. Iminophosphit-) Verbindungen fuehrt. Die bereits erwaehnten Isomerisierungen der Isothiocyanate finden meistens in der Gasphase statt, um Reaktionen der Molekuele untereinander zu vermeiden. In einigen Faellen kommt es dabei zur Einstellung eines Gleichgewichtes, welches durch Analyse der Produkte unter Variation der Thermolysetemperatur bestimmt wird. Gleichzeitig kann dadurch die Ausbeute optimiert werden. In der Arbeit werden drei verschiedene Varianten [3,3]-sigmatroper Umlagerungen dargestellt: Typ A: die Propargylvorlaeufer reagieren zu Allenylthiocyanaten, Typ B: aus 2,3-Butadienyl-Verbindungen entstehen monosubstituierte 1,3-Butadiene, Typ C: aus 1,4-Diisothiocyanato-2-butinen werden disubstituierte 1,3-Butadiene. Im Anschluss daran beobachtet man elektrocyclische Reaktionen, ionische Isomerisierungen sowie eine Cope-Umlagerung. Als Folgereaktionen koennen auch Diels-Alder-Reaktionen durchgefuehrt werden. Zum Einsatz kommen dabei sowohl elektronenreiche als auch -arme Dienophile sowie ein ringgespannter Reaktionspartner.

In this project the factors influencing the stability and antimicrobial activity of allyl isothiocyanate (AITC) against Campylobacter jejuni, Salmonella or Listeria monocytogenes as well as factors that enhance sinigrin (glucosinolate in Oriental mustard) hydrolysis by these pathogens were investigated. The minimum inhibitory concentration (MIC) of AITC against 5 strains of each of Salmonella or L. monocytogenes, ranged from 60-100 ppm at 37 ºC. This was reduced to 10-40 ppm at 21 ºC and a further reduction to 5-10 ppm against strains of L. monocytogenes was observed at 4 ºC. This was attributed to greater stability of AITC as temperature was decreased. C. jejuni strains were more susceptible to AITC with MICs of 0.63-1.25 ppm and 2.5-5 ppm at 37 and 42 ºC, respectively. AITC was more inhibitory at ≤ 21 ºC against Salmonella with acidic pH or against L. monocytogenes with neutral pH. C. jejuni, Salmonella and L. monocytogenes strains and mixtures had the ability to degrade sinigrin to form inhibitory concentrations of AITC, and sinigrin hydrolysis was significantly enhanced by higher incubation temperature (21 ºC > 10 ºC > 4 ºC), the presence of 10 mM ferric or ferrous irons, and the presence of < 0.25% glucose. This project also investigated the antimicrobial activity of AITC or Oriental mustard extract alone or combined with ethylenediamine tetraacetic acid (EDTA), malic acid and acetic acid in edible antimicrobial coatings against C. jejuni and Salmonella on fresh, refrigerated, vacuum-packed chicken breasts or L. monocytogenes on refrigerated, cured roast chicken. Malic acid improved the antimicrobial activity of Oriental mustard extract against L. monocytogenes, while EDTA improved its activity against Salmonella. Incorporation of 25 to 50 µl/g AITC or 100 to 250 mg/g Oriental mustard extract in 0.5%κ-carrageenan/2%chitosan coatings, prepared using 1.5% malic or acetic acid, reduced L. monocytogenes on cooked, cured, vacuum-packed chicken slices 4.2 to > 7.0 log10 CFU/g, compared to uncoated chicken by 70 d at 4 ºC. In addition, 0.2%κ-carrageenan/2%chitosan coatings (prepared using a 1% acetic acid solution) containing 250 mg/g mustard extract or 50 µl/g AITC reduced Salmonella numbers on vacuum-packed chicken breasts 3.0 log10 CFU/g by 21 d at 4 ºC. Further, 0.2%κ-carrageenan/2%chitosan coatings containing 50 or 100 µl/g AITC reduced numbers of C. jejuni on fresh, vacuum-packed chicken breasts > 5.0 log10 CFU/g (C. jejuni cells were not detected) after 5 d storage at 4 ºC, while coatings containing 200 to 300 mg/g Oriental mustard extract or 25 µl/g AITC reduced C. jejuni numbers by 3.6 to 4.6 log10 CFU/g. Numbers of lactic acid and aerobic bacteria on poultry meat products were significantly reduced by the coatings. It is clear that κ-carrageenan/chitosan coatings containing either AITC, mustard extract alone or combined with EDTA, malic or acetic acid significantly reduced C. jejuni and Salmonella on fresh, refrigerated, vacuum-packed chicken breasts and L. monocytogenes on refrigerated, cured roast chicken, and consequently enhanced their safety.

Neste trabalho a síntese e caracterizaçcão de adutos entre tiocianatos de terras raras trivalentes (La, Ce Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm Yb, Lu e Y) e tripiperidinofosfinóxidos (tpppO) são reportados. Os adutos foram preparados pela reação entre tiocianatos de lantanídeos e tpppO em soluções etanólicas. Os complexos foram caracterizados por análises elementares, análises termogravimétricas, medidas de condutância em soluções milimolares, espectroscopias de absorção na região do infravermelho, espectroscopia de absorção na região do visível do complexos de neodímio, espectroscopia de emissão na região do visível do composto de európio, térbio e disprósio, difratograma de raios -X pelo método do pó e difratogramas de raios -X pelo método do mono cristal do composto de praseodímio. As análises elementares sugeriram fortemente a fórmula [Ln(SCN)3(tpppO)3] como representativa de toda a série de complexos. As medidas de condutividade de soluções milimolares em acetonitrila resultaram em valores entre 22 e 75 S cm2 mol-1. Esses valores sugeriram a natureza não eletrolítica dos adutos. Os difratogramas pelo método do pó sugeriram a existência de três séries isomorfas, formadas pelos seguintes elementos: 1) La; 2) Ce, Nd, Pr; 3) Sm, Gd, Eu, Ho, Er, To, Lu, Y. Todos os espectros de absorção na região do infravermelho dos adutos são semelhantes e exibem &#957;(CN)na região de 2050 cm-1.Isso é indicativo da coordenação do ânion a através do átomo de nitrogênio. Os complexos são portanto da classe dos isotiocianatos. Adicionalmente esses pico estão desdobrados em dois picos ( exceto para o Eu e Tb), evidenciando que esses ãnions não são todos equivalentes. Nos espectros de infravermelho observou-se ainda o pico &#948;(NCS) at ca. 470-480 nm e isso indica também a coordenação através do átomo de nitrogênio. O pico atribuído ao estiramento da ligação PO, &#957; (PO), está deslocado para regiões menos energética do espectro quando comparado ao espectro do ligante não coordenado. Isso indica a coordenação do tpppO pelo átomo de oxigênio. Nota-se também que os picos &#957; (PN) apresentando um desvio consistente para regiões mais energéticas para toda a série de complexos sintetizados. O espectro de absorção na região do visível do complexo de neodímio foi registrado e apresentou a banda atribuída á transição 4G5/2, 2G7/2 &#8592; 4I9/2. A banda atribuída à transição 2P1/2 &#8592; 4I9/2 não pode ser observada. A primeira transição foi observada a 17124 cm-1 Desse valor foram calculados: o parâmetro nefelauxético, &#946; = 0,989; o fator de covalência, b 1/2 = 0,072; e o fator de covalência de Sinha &#948; = 1,06. Esses valores mostram a natureza eletrostática das ligações entre o íon da terra rara e os ligantes. O espectro de emissão do composto de európio, a 77 K, apresenta uma pico atribuído à transição 5D0 &#8594;7D0 (578 nm), três picos à 5D0 &#8594; 7D1 (593 , 595, 597nm), e cinco à 5D0 &#8594; 7D2 (611, 614, 617, 618, 620 nm). Sabendo que a presença do pico 5D0 &#8594; 7D0 e os desdobramentos dos picos 5D0 &#8594; 7D1 e 5D0 &#8594; 7D2 a simetria C3v é a mais provável para o poliedro de coordenação. As análises térmicas foram conduzidas para os complexos de Ce, Nd, Gd. A curva de Tg-DTG mostra perdas de massa começando em 284 -320 ºC e terminando em 1000 ºC. Em 1200 ºC, outra perda de massa ocorre. As curvas, no entanto, mostram algumas diferenças entre si. Enquanto o complexo de cério apresenta cinco eventos não resolvidos entre 300 e 400 ºC, o complexo de Nd apresenta dois eventos não resolvidos e o complexo de Gd apresenta apenas um pico largo na mesma faixa de temperatura. As massas dos resíduos finais de aquecimento (temperaturas maiores que 1300 ºC) são compatíveis com as de fosofatos dos respectivos lantanídeos e o espectro de absorção na região do infravermelho desses resíduos confirmam esses resultados. A análise de difração de raios -X do composto de preseodímio mostrou como sitema critalino o monoclínico, o grupo espacial é o Cc, com quatro moléculas por célula unitária. O Poliedro de coordenação é um prisma trigonal levmente distrocido com simetria de C3V. O número de coordenação é seis com os ânions tiocianatos coordenados pelo nitrogênio, enquanto os os tpppO estão coodenados pelos átomo de hidrogênios. / In this work the synthesis and characterization of trivalent rare earth (La, Ce Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm Yb, Lu and Y) adducts between isothiocyanate with tripiperidinophosphinoxide (tpppO) are reported. The rare earth adducts were prepared by the reaction between lanthanides thiocyanates and tpppO in ethanolic solution. The complexes were characterized by elemental analysis, thermo gravimetric analysis, conductance measurements in millimolar acetonitrila solutions, IR absorption spectroscopy, uv-vis absorption spectrum of the neodymium compound, emission spectrum of europium, dysprosium and terbium compounds, X- ray powder patterns and X-ray diffraction through the monocrystal method of the praseodymium compound. Elemental analyses data strongly suggest the general formula [Ln(SCN)3(tpppO)3] for the complete series. Electrolytic conductivity measurements in milllimolar acetonitrile solutions yielded values in range 75 to 22 S cm2 mole-1. These values suggest the nonelectrolyte natures of the adducts. X-Ray powder diffraction pattern suggest the existence of three isomorphous series: 1) La; 2) Ce, Nd Pr; 3) Sm, Gd, Eu, Ho, Er, Tb, Lu and Y. All absortption spectra in IR region are similar and exhibit &#957;(CN) in the region of 2050 cm-1. This is indicative that the anion is coordinated through the nitrogen atom, or, in other words, the anion is in the isothiocyanate form. ln addition, these bands are split into two components in all complexes ( except for Eu and Tb ), evidencing that these anions are not all equivalent. In the IR spectra it is also possible to observe &#948;(NCS) at ca. 470-480 cm-1 and this also point out coordination through the nitrogen atom. In the IR spectra also &#957; (PO) is shifted to lower energies by ca. 10 cm1 with respect to free tpppO. This indicat coordination through the phosphoryl oxygen. It is also nortworthy that &#957; (PN) presents a consistent positive shift throghout the whole series of complexes. Absorption spectra of a neodymium compound were recorded. The hypersensitive band 4G5/2, 2G7/2 &#8592; 4I9/2 appears, while the 2P1/2&#8592; 4I9/2 could not be observed experimentally. The first transition was observed to be centered at 17124 cm-1. From this value, the nephelauxetic parameter, &#946; = 0,989, covalente factor, bl/2 = 0,072 and Sinha covalence parameter &#948; = 1,06 were calculated. These value point out the essentially electrostactic nature of metal-ligand bonds. The emission spectra of europium compound at 77 K presents one 5D0 &#8594; 7D0 peak (578 nm), three 5D0 &#8594; 7D1 peaks (593,595, 597nm) and five for 5D0 &#8594; 7D2 band (611,614, 617, 618, 620 nm). Taking into account the presence of 5D0 &#8594; 7D0 and the splits of 5D0 &#8594; 7D2 and 5D0 &#8594; 7D1 peaks the C3v symmetry can be proposed for this coordination polyhedron. Thermal analyses were carried out for Ce, Nd and Gd complexes. Tg-DTG curves show mass lasses starting at 284-320 ºC up to 1000 ºC. At 1200 ºC another mass loss occurs. The curves , however, are quite different whereas the Ce complex shows five non-resolved events between 300 and 400ºC, the Nd adducts shows two non-resolved events and the Gd only a broad peak. The mass of the final residues (above 1300ºC) are consistent with the corresponding lanthanide phosphate. Infrared spectra of these residues were recorded, confirming this assumption. Single- crystal X-ray diffraction analysis of the Pr complex was carried out. The crystal system is the monoclinic, space group Cc, with four molecules per unit cell. The coordination polyhedron is a slightly distorted trigonal antiprism with C3v symmetry. The coordination number is 6. All anion are in the thyocianate form and coordinated through their nitrogen atoms in one base of antiprism whereas the ligand (tpppO) are bonded to the other base through their oxygen atoms.

Neste trabalho a síntese e caracterizaçcão de adutos entre tiocianatos de terras raras trivalentes (La, Ce Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm Yb, Lu e Y) e tripiperidinofosfinóxidos (tpppO) são reportados. Os adutos foram preparados pela reação entre tiocianatos de lantanídeos e tpppO em soluções etanólicas. Os complexos foram caracterizados por análises elementares, análises termogravimétricas, medidas de condutância em soluções milimolares, espectroscopias de absorção na região do infravermelho, espectroscopia de absorção na região do visível do complexos de neodímio, espectroscopia de emissão na região do visível do composto de európio, térbio e disprósio, difratograma de raios -X pelo método do pó e difratogramas de raios -X pelo método do mono cristal do composto de praseodímio. As análises elementares sugeriram fortemente a fórmula [Ln(SCN)3(tpppO)3] como representativa de toda a série de complexos. As medidas de condutividade de soluções milimolares em acetonitrila resultaram em valores entre 22 e 75 S cm2 mol-1. Esses valores sugeriram a natureza não eletrolítica dos adutos. Os difratogramas pelo método do pó sugeriram a existência de três séries isomorfas, formadas pelos seguintes elementos: 1) La; 2) Ce, Nd, Pr; 3) Sm, Gd, Eu, Ho, Er, To, Lu, Y. Todos os espectros de absorção na região do infravermelho dos adutos são semelhantes e exibem &#957;(CN)na região de 2050 cm-1.Isso é indicativo da coordenação do ânion a através do átomo de nitrogênio. Os complexos são portanto da classe dos isotiocianatos. Adicionalmente esses pico estão desdobrados em dois picos ( exceto para o Eu e Tb), evidenciando que esses ãnions não são todos equivalentes. Nos espectros de infravermelho observou-se ainda o pico &#948;(NCS) at ca. 470-480 nm e isso indica também a coordenação através do átomo de nitrogênio. O pico atribuído ao estiramento da ligação PO, &#957; (PO), está deslocado para regiões menos energética do espectro quando comparado ao espectro do ligante não coordenado. Isso indica a coordenação do tpppO pelo átomo de oxigênio. Nota-se também que os picos &#957; (PN) apresentando um desvio consistente para regiões mais energéticas para toda a série de complexos sintetizados. O espectro de absorção na região do visível do complexo de neodímio foi registrado e apresentou a banda atribuída á transição 4G5/2, 2G7/2 &#8592; 4I9/2. A banda atribuída à transição 2P1/2 &#8592; 4I9/2 não pode ser observada. A primeira transição foi observada a 17124 cm-1 Desse valor foram calculados: o parâmetro nefelauxético, &#946; = 0,989; o fator de covalência, b 1/2 = 0,072; e o fator de covalência de Sinha &#948; = 1,06. Esses valores mostram a natureza eletrostática das ligações entre o íon da terra rara e os ligantes. O espectro de emissão do composto de európio, a 77 K, apresenta uma pico atribuído à transição 5D0 &#8594;7D0 (578 nm), três picos à 5D0 &#8594; 7D1 (593 , 595, 597nm), e cinco à 5D0 &#8594; 7D2 (611, 614, 617, 618, 620 nm). Sabendo que a presença do pico 5D0 &#8594; 7D0 e os desdobramentos dos picos 5D0 &#8594; 7D1 e 5D0 &#8594; 7D2 a simetria C3v é a mais provável para o poliedro de coordenação. As análises térmicas foram conduzidas para os complexos de Ce, Nd, Gd. A curva de Tg-DTG mostra perdas de massa começando em 284 -320 ºC e terminando em 1000 ºC. Em 1200 ºC, outra perda de massa ocorre. As curvas, no entanto, mostram algumas diferenças entre si. Enquanto o complexo de cério apresenta cinco eventos não resolvidos entre 300 e 400 ºC, o complexo de Nd apresenta dois eventos não resolvidos e o complexo de Gd apresenta apenas um pico largo na mesma faixa de temperatura. As massas dos resíduos finais de aquecimento (temperaturas maiores que 1300 ºC) são compatíveis com as de fosofatos dos respectivos lantanídeos e o espectro de absorção na região do infravermelho desses resíduos confirmam esses resultados. A análise de difração de raios -X do composto de preseodímio mostrou como sitema critalino o monoclínico, o grupo espacial é o Cc, com quatro moléculas por célula unitária. O Poliedro de coordenação é um prisma trigonal levmente distrocido com simetria de C3V. O número de coordenação é seis com os ânions tiocianatos coordenados pelo nitrogênio, enquanto os os tpppO estão coodenados pelos átomo de hidrogênios. / In this work the synthesis and characterization of trivalent rare earth (La, Ce Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm Yb, Lu and Y) adducts between isothiocyanate with tripiperidinophosphinoxide (tpppO) are reported. The rare earth adducts were prepared by the reaction between lanthanides thiocyanates and tpppO in ethanolic solution. The complexes were characterized by elemental analysis, thermo gravimetric analysis, conductance measurements in millimolar acetonitrila solutions, IR absorption spectroscopy, uv-vis absorption spectrum of the neodymium compound, emission spectrum of europium, dysprosium and terbium compounds, X- ray powder patterns and X-ray diffraction through the monocrystal method of the praseodymium compound. Elemental analyses data strongly suggest the general formula [Ln(SCN)3(tpppO)3] for the complete series. Electrolytic conductivity measurements in milllimolar acetonitrile solutions yielded values in range 75 to 22 S cm2 mole-1. These values suggest the nonelectrolyte natures of the adducts. X-Ray powder diffraction pattern suggest the existence of three isomorphous series: 1) La; 2) Ce, Nd Pr; 3) Sm, Gd, Eu, Ho, Er, Tb, Lu and Y. All absortption spectra in IR region are similar and exhibit &#957;(CN) in the region of 2050 cm-1. This is indicative that the anion is coordinated through the nitrogen atom, or, in other words, the anion is in the isothiocyanate form. ln addition, these bands are split into two components in all complexes ( except for Eu and Tb ), evidencing that these anions are not all equivalent. In the IR spectra it is also possible to observe &#948;(NCS) at ca. 470-480 cm-1 and this also point out coordination through the nitrogen atom. In the IR spectra also &#957; (PO) is shifted to lower energies by ca. 10 cm1 with respect to free tpppO. This indicat coordination through the phosphoryl oxygen. It is also nortworthy that &#957; (PN) presents a consistent positive shift throghout the whole series of complexes. Absorption spectra of a neodymium compound were recorded. The hypersensitive band 4G5/2, 2G7/2 &#8592; 4I9/2 appears, while the 2P1/2&#8592; 4I9/2 could not be observed experimentally. The first transition was observed to be centered at 17124 cm-1. From this value, the nephelauxetic parameter, &#946; = 0,989, covalente factor, bl/2 = 0,072 and Sinha covalence parameter &#948; = 1,06 were calculated. These value point out the essentially electrostactic nature of metal-ligand bonds. The emission spectra of europium compound at 77 K presents one 5D0 &#8594; 7D0 peak (578 nm), three 5D0 &#8594; 7D1 peaks (593,595, 597nm) and five for 5D0 &#8594; 7D2 band (611,614, 617, 618, 620 nm). Taking into account the presence of 5D0 &#8594; 7D0 and the splits of 5D0 &#8594; 7D2 and 5D0 &#8594; 7D1 peaks the C3v symmetry can be proposed for this coordination polyhedron. Thermal analyses were carried out for Ce, Nd and Gd complexes. Tg-DTG curves show mass lasses starting at 284-320 ºC up to 1000 ºC. At 1200 ºC another mass loss occurs. The curves , however, are quite different whereas the Ce complex shows five non-resolved events between 300 and 400ºC, the Nd adducts shows two non-resolved events and the Gd only a broad peak. The mass of the final residues (above 1300ºC) are consistent with the corresponding lanthanide phosphate. Infrared spectra of these residues were recorded, confirming this assumption. Single- crystal X-ray diffraction analysis of the Pr complex was carried out. The crystal system is the monoclinic, space group Cc, with four molecules per unit cell. The coordination polyhedron is a slightly distorted trigonal antiprism with C3v symmetry. The coordination number is 6. All anion are in the thyocianate form and coordinated through their nitrogen atoms in one base of antiprism whereas the ligand (tpppO) are bonded to the other base through their oxygen atoms.

Renal Cell Carcinoma (RCC) has low 5 year survival rate and is resistant to radiation and chemotherapy. Phenethyl Isothiocyanate (PEITC) is a naturally occurring phytochemical that has a variety of anti-cancer properties. Here we explore two anti-cancer properties of PEITC: induction of apoptosis and induction of cell cycle arrest in RCC cells and the underlying mechanisms. We used two human RCC cell lines Caki-1 and Caki-2. Survival and cell proliferation was assayed using Calcein AM. Annexin V staining was used to measure apoptosis. Caspase-3/7 induction was measured using a fluorescent substrate. Cell cycle was studied using Propidium Iodide staining. DNA damage was determined using phospho [gamma]-H2AX antibody. Protein expression and phosphorylation was determined using immunoblotting. PEITC significantly reduced survival of Caki-1 and Caki-2 cells and inhibited their proliferation as determined by Calcein AM. 15 and 20 [mu]M PEITC induced apoptosis in both cell lines and induced caspase-3/7 activity. Western blot analysis revealed caspase-8, caspase-9 and Bid cleavage as well as upregulation of the death receptors Fas and DR5. Lower doses (up to 10 [mu]M) arrested Caki-1 cells in G2/M phase, and this was associated with increased p38 and MK2 (Thr334) phosphorylation. The p38 inhibitor SB203850 inhibited this G2 arrest induced by PEITC. 15 and 20 [mu]M PEITC treatment resulted in increased [gamma]-H2AX phosphorylation suggesting DNA damage, but this was completely blocked by caspase inhibitor. In summary, our study shows that PEITC induces apoptosis in Caki-1 and Caki-2 cells by upregulating Fas and DR5 and activating the downstream apoptosis cascade. PEITC does not cause direct DNA damage to the cells; the observed DNA damage is a result of the apoptotic process and is blocked by caspase inhibitor. PEITC induces G2/M arrest in Caki-1 cells and the mechanism involves p38 phosphorylation which activates MK2. Inducing cell cycle arrest and apoptosis may play an important role in the anti-cancer properties of PEITC. Fully understanding the mechanism by which PEITC induces apoptosis and cell cycle arrest in RCC cells may lead to development of novel chemotherapeutic drugs against RCC. / text

Made available in DSpace on 2015-03-26T13:37:43Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2701343 bytes, checksum: b9c5cc5b07f66e4816776e04f12b0511 (MD5) Previous issue date: 2010-08-27 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Considering the importance of Meloidogyne enterolobii (= M. mayaguensis) to the culture of guava and the potential of Essential Oil of Mustard (EOM) on nematode control, was aimed to evaluate the efficiency of EOM on control of these nematodes in the field, on a orchard of plants uprooted and cut off at 0.3 m from soil level or in intact plants in production. An orchard of guava trees naturally infested with M. enterolobii, in Viçosa, MG, was selected. EOM was applied on soil at 100 &#956;g/mL, by strips of cardboard in the rhizosphere of intact plants, or by irrigating the cultivation area with EOM diluted in water after the plants have been cut off or uprooted. Samples were collected over 140 days after the product application and evaluated on the number of J2 in soil and number of galls and eggs in roots of tomato plants in the bioassay. The J2 population in soil underwent rapid reduction, regardless of treatment, and was configured as an unreliable variable. By the bioassay, was obtained a reduction of 71%, 81% and 99% for number of galls and 60%, 80% and 83% for number of eggs at 45, 75 and 140 days after application, respectively, for plots treated with EOM, in uprooted or cutted plants. The terbuphos only reduced the number of galls during 45 days. The biological evaluation at 140 days revealed a general trend of population reduction, considering the number of galls and eggs. In intact plants, there was no control of nematodes, as the average variations of J2, galls and eggs did not differ significantly between treatments. In conclusion, the EOM was effective in controlling M. enterolobii at the field in the absence of the host. / Considerando a importância de Meloidogyne enterolobii (= M. mayaguensis) para a cultura da goiabeira e o potencial do óleo essencial de mostarda (OEM) no controle de nematoides, objetivou-se avaliar a eficiência do OEM no controle do nematoide no campo, em pomares de plantas arrancadas, recepadas ou em produção. Selecionou-se um pomar de goiabeiras naturalmente infestado por M. enterolobii, em Viçosa, MG. O OEM foi aplicado no solo a 100 &#956;g/mL, por meio de tiras de papelão na rizosfera de plantas intactas ou irrigando a área de cultivo com OEM diluído em água, após as plantas serem recepadas ou arrancadas. Em amostras coletadas ao longo de 140 dias após aplicação do produto, avaliou-se o número de J2 no solo e número de galhas e ovos em raízes de tomateiros, no teste biológico. Como a população de J2 no solo sofreu rápida redução, independente do tratamento, ela foi considerada uma variável pouco confiável para a avaliação. Pelo teste biológico, obteve-se uma redução de 71%, 81% e 99% para número de galhas e 60%, 80% e 83% para número de ovos aos 45, 75 e 140 dias após a aplicação, respectivamente, para as parcelas tratadas com OEM, em plantas arrancadas ou recepadas. O terbufós reduziu apenas o número de galhas aos 45 dias, o qual voltou a aumentar posteriormente. Aos 140 dias, houve tendência generalizada de redução da população, considerando-se o número de galhas e ovos. Nas plantas intactas não houve controle do nematoide, pois as médias das variações de J2, galhas e ovos não diferiram significativamente entre os tratamentos. Em conclusão, o OEM foi eficiente em controlar M. enterolobii no campo na ausência do hospedeiro.

Es wird eine neue Methode zur Darstellung von Vinylthiocyanaten ueber [3,3]-sigmatrope Umlagerungen vorgestellt. Obwohl dabei aus einer energieaermeren Gruppe (Isothiocyanat) eine energiereichere (Thiocyanat) entsteht, isomerisiert das Senfoel zum Rhodanid. Das ist moeglich, weil der Energieunterschied zwischen den Isothiocyanaten und den entsprechenden Thiocyanaten vergleichsweise gering ist und strukturelle Eigenschaften die Gesamtenergie ebenfalls beeinflussen. Die Isothiocyanatvorlaeufer stellt man in vielen Faellen durch ein Eintopfverfahren her, welches ueber Azid- und Iminophosphoran- (bzw. Iminophosphit-) Verbindungen fuehrt. Die bereits erwaehnten Isomerisierungen der Isothiocyanate finden meistens in der Gasphase statt, um Reaktionen der Molekuele untereinander zu vermeiden. In einigen Faellen kommt es dabei zur Einstellung eines Gleichgewichtes, welches durch Analyse der Produkte unter Variation der Thermolysetemperatur bestimmt wird. Gleichzeitig kann dadurch die Ausbeute optimiert werden. In der Arbeit werden drei verschiedene Varianten [3,3]-sigmatroper Umlagerungen dargestellt: Typ A: die Propargylvorlaeufer reagieren zu Allenylthiocyanaten, Typ B: aus 2,3-Butadienyl-Verbindungen entstehen monosubstituierte 1,3-Butadiene, Typ C: aus 1,4-Diisothiocyanato-2-butinen werden disubstituierte 1,3-Butadiene. Im Anschluss daran beobachtet man elektrocyclische Reaktionen, ionische Isomerisierungen sowie eine Cope-Umlagerung. Als Folgereaktionen koennen auch Diels-Alder-Reaktionen durchgefuehrt werden. Zum Einsatz kommen dabei sowohl elektronenreiche als auch -arme Dienophile sowie ein ringgespannter Reaktionspartner.

Med hjälp av immunohistokemi kan antigen och antikroppar som är bundna till vävnaden detekteras. Autoimmuna hudsjukdomar är exempel på sjukdomar som diagnosticeras med immunohistokemi. På Falu lasarett användes immunofluorescens för diagnostik av autoimmuna hudsjukdomar. Syftet med denna studie var att optimera enzym-baserad immunohistokemi för epitoperna IgA, IgG, IgM och C3 och jämföra med immunofluorescens vad gäller specificitet, signalstyrka och upplösning. Vävnader som analyserades var tonsill, lever, tarmslemhinna och hudbiopsier. Fixering gjordes i 4% formaldehyd av vävnaderna som infärgades med ultraView DAB och ultraView DAB med FITC. Vävnad som infärgades med DIF med FITC sköljdes enbart i Reaction Buffer. Vävnaderna färgades enligt protokollen ultraView DAB, ultraView DAB med FITC och DIF med FITC. En manuell infärgning med aktiverat DAB utfördes. Resultatet visade bakgrundsinfärgning för samtliga infärgningar. DIF med FITC var tydligare infärgad och lättare att skilja mellan specifik och ospecifik infärgning. Det är svårt att optimera enzym-baserad IHC och epitopen IgA hade generellt starkare infärgning jämfört med epitopen IgM. För att erhålla tillförlitliga resultat krävs det att flera vävnadsprover analyseras. / Using immunohistochemistry, antigens and antibodies bound to the tissue can be detected. Autoimmune skin diseases are examples of diseases that are diagnosed with immunohistochemistry. At Falu Hospital, immunofluorescence was used to diagnose autoimmune skin diseases. The aim of this study was to optimize enzyme-based immunohistochemistry for the epitopes IgA, IgG, IgM and C3 and to compare with immunofluorescence in terms of specificity, signal strength and resolution. Tissues analyzed were tonsil, liver, intestinal mucosa and skin biopsies. Fixation was done in 4% formaldehyde of the tissues stained with ultraView DAB and ultraView DAB with FITC. Tissue stained with DIF with FITC was rinsed in Reaction Buffer only. A manual staining with activated DAB was performed. The result showed background staining for all stainings. DIF with FITC was more clearly stained and easier to distinguish between specific and nonspecific staining. It is difficult to optimize enzyme-based IHC and the epitope IgA generally had stronger staining compared to the epitope IgM. To obtain reliable results, several tissue samples must be analyzed.

La cartographie peptidique est une méthode qui permet entre autre d’identifier les modifications post-traductionnelles des protéines. Elle comprend trois étapes : 1) la protéolyse enzymatique, 2) la séparation par électrophorèse capillaire (CE) ou chromatographie en phase liquide à haute performance (HPLC) des fragments peptidiques et 3) l’identification de ces derniers. Cette dernière étape peut se faire par des méthodes photométriques ou par spectrométrie de masse (MS). Au cours de la dernière décennie, les enzymes protéolytiques immobilisées ont acquis une grande popularité parce qu’elles peuvent être réutilisées et permettent une digestion rapide des protéines due à un rapport élevé d’enzyme/substrat. Pour étudier les nouvelles techniques d’immobilisation qui ont été développées dans le laboratoire du Professeur Waldron, la cartographie peptidique par CE est souvent utilisée pour déterminer le nombre total de peptides détectés et leurs abondances. La CE nous permet d’avoir des séparations très efficaces et lorsque couplée à la fluorescence induite par laser (LIF), elle donne des limites de détection qui sont 1000 fois plus basses que celles obtenues avec l’absorbance UV-Vis. Dans la méthode typique, les peptides venant de l’étape 1) sont marqués avec un fluorophore avant l’analyse par CE-LIF. Bien que la sensibilité de détection LIF puisse approcher 10-12 M pour un fluorophore, la réaction de marquage nécessite un analyte dont la concentration est d’au moins 10-7 M, ce qui représente son principal désavantage. Donc, il n’est pas facile d’étudier les enzymes des peptides dérivés après la protéolyse en utilisant la technique CE-LIF si la concentration du substrat protéique initial est inférieure à 10-7 M. Ceci est attribué à la dilution supplémentaire lors de la protéolyse. Alors, afin d’utiliser le CE-LIF pour évaluer l’efficacité de la digestion par enzyme immobilisée à faible concentration de substrat,nous proposons d’utiliser des substrats protéiques marqués de fluorophores pouvant être purifiés et dilués. Trois méthodes de marquage fluorescent de protéine sont décrites dans ce mémoire pour étudier les enzymes solubles et immobilisées. Les fluorophores étudiés pour le marquage de protéine standard incluent le naphtalène-2,3-dicarboxaldéhyde (NDA), la fluorescéine-5-isothiocyanate (FITC) et l’ester de 6-carboxyfluorescéine N-succinimidyl (FAMSE). Le FAMSE est un excellent réactif puisqu’il se conjugue rapidement avec les amines primaires des peptides. Aussi, le substrat marqué est stable dans le temps. Les protéines étudiées étaient l’-lactalbumine (LACT), l’anhydrase carbonique (CA) et l’insuline chaîne B (INB). Les protéines sont digérées à l’aide de la trypsine (T), la chymotrypsine (CT) ou la pepsine (PEP) dans leurs formes solubles ou insolubles. La forme soluble est plus active que celle immobilisée. Cela nous a permis de vérifier que les protéines marquées sont encore reconnues par chaque enzyme. Nous avons comparé les digestions des protéines par différentes enzymes telles la chymotrypsine libre (i.e., soluble), la chymotrypsine immobilisée (i.e., insoluble) par réticulation avec le glutaraldéhyde (GACT) et la chymotrypsine immobilisée sur billes d’agarose en gel (GELCT). Cette dernière était disponible sur le marché. Selon la chymotrypsine utilisée, nos études ont démontré que les cartes peptidiques avaient des différences significatives selon le nombre de pics et leurs intensités correspondantes. De plus, ces études nous ont permis de constater que les digestions effectuées avec l’enzyme immobilisée avaient une bonne reproductibilité. Plusieurs paramètres quantitatifs ont été étudiés afin d’évaluer l’efficacité des méthodes développées. La limite de détection par CE-LIF obtenue était de 3,010-10 M (S/N = 2,7) pour la CA-FAM digérée par GACT et de 2,010-10 M (S/N = 4,3) pour la CA-FAM digérée par la chymotrypsine libre. Nos études ont aussi démontrées que la courbe d’étalonnage était linéaire dans la région de travail (1,0×10-9-1,0×10-6 M) avec un coefficient de corrélation (R2) de 0,9991. / Peptide mapping is a routine method for identifying post-translational modifications of proteins. It involves three steps: 1) enzymatic proteolysis, 2) separation of the peptide fragments by capillary electrophoresis (CE) or high performance liquid chromatography (HPLC), 3) identification of the peptide fragments by photometric methods or mass spectrometry (MS). During the past decade, immobilized enzymes for proteolysis have been gaining in popularity because they can be reused and they provide fast protein digestion due to the high ratio of enzyme-to-substrate. In order to study new immobilization techniques developed in the Waldron laboratory, peptide mapping by CE is frequently used, where the total number of peptides detected and their abundance are related to enzymatic activity. CE allows very high resolution separations and, when coupled to laser-induced fluorescence (LIF), provides excellent detection limits that are 1000 times lower than with UV-Vis absorbance. In the typical method, the peptides produced in step 1) above are derivatized with a fluorophore before separation by CE-LIF. Although the detection sensitivity of LIF can approach 10 12 M for a highly efficient fluorophore, a major disadvantage is that the derivatization reaction requires analyte concentrations to be approx. 10 7 M or higher. Therefore, it is not feasible to study enzymes using CE-LIF of the peptides derivatized after proteolysis if the initial protein substrate concentration is <10-7 M because additional dilution occurs during proteolysis. Instead, to take advantage of CE-LIF to evaluate the efficiency of immobilized enzyme digestion of low concentrations of substrate, we propose using fluorescently derivatized protein substrates that can be purified then diluted. Three methods for conjugating fluorophore to protein were investigated in this work as a means to study both soluble and immobilized enzymes. The fluorophores studied for derivatization of protein standards included naphthalene-2,3-dicarboxaldehyde (NDA), fluoresceine-5-isothiocyanate (FITC) and 6-carboxyfluorescein N-succinimide ester (FAMSE). The FAMSE was found to be an excellent reagent that conjugates quickly with primary amines and the derivatized substrate was stable over time. The studied substrates were -lactalbumin (LACT), carbonic anhydrase (CA) and insulin chain-B (INB). The CE-LIF peptide maps were generated from digestion of the fluorescently derivatized substrates by trypsin (T), chymotrypsin (CT) or pepsin (PEP), either in soluble or insoluble forms. The soluble form of an enzyme is more active than the immobilized form and this allowed us to verify that the conjugated proteins were still recognized as substrates by each enzyme. The digestion of the derivatized substrates with different types of chymotrypsin (CT) was compared: free (i.e., soluble) chymotrypsin, chymotrypsin cross-linked with glutaraldehyde (GACT) and chymotrypsin immobilized on agarose gel particles (GELCT), which was available commercially. The study showed that, according to the chymotrypsin used, the peptide map would vary in the number of peaks and their intensities. It also showed that the digestion by immobilized enzymes was quite reproducible. Several quantitative parameters were studied to evaluate the efficacy of the methods. The detection limit of the overall method (CE-LIF peptide mapping of FAM-derivatized protein digested by chymotrypsin) was 3.010-10 M (S/N = 2.7) carbonic anhydrase using insoluble GACT and 2.010-10 M (S/N = 4.3) CA using free chymotrypsin. Our studies also showed that the standard curve was linear in the working region (1.0×10-9-1.0×10-6 M) with a correlation coefficient (R2) of 0.9991.