Herbivorous fishes include a diverse assemblage of species that target primarily benthic autotrophs. This is perhaps one of the most well-studied groups of coral reef fishes, often reputed to be key components of coral reef communities, contributing to coral reef health in numerous ways. Through their feeding ecology and benthic interactions, they help mediate algae-coral interactions which can allow for improved coral survival and health. Despite the wealth of literature documenting the prominent roles of these fishes in coral reef ecosystems, studies from the Red Sea are surprisingly lacking. The Red Sea is a marginal reef environment, with a host of unique environmental and biological characteristics making it a unique environment where dynamics of herbivory may differ. This dissertation aims to fill key gaps in our knowledge of herbivorous fishes through the study of their distribution and trophic ecology. Herein, I describe habitat-specific partitioning of Red Sea herbivorous fish assemblages, discovering higher diversity and abundance found in reefs closer to shower, dissimilar to findings from other regions. Cross-shelf variation in assemblage structure seems to be quite robust through time, indicating short-term stability in herbivore assemblages. Through the use of stomach contents and stable isotope analyses, I then investigate the trophic ecology of browsing herbivores across the same shelf-gradient. I found higher trophic redundancy on nearshore reefs through time, with increased variation in diet and high levels of complementarity on offshore reefs where macroalgae are scarce. Stable isotope analyses of both liver and muscle revealed the stability of this resource partitioning through time, demonstrating for the first-time temporal stability of resource partitioning within this group. This dissertation broadens our knowledge of herbivorous fishes, filling important gaps. It offers new insight into the role of habitat in structuring trophic ecology and how flexible the diets of browsing species can be. Together, this information creates a foundation where improved knowledge of herbivorous fish ecology could be incorporated into future management plans of ongoing giga projects within the Kingdom. Incorporating herbivores into these plans could allow for increased resiliency for Red Sea coral reefs in the face of future development and shifting climatology.

<p> It has been suggested that combination treatment of high dose rate (HDR) intraluminal brachytherapy and PDT (Photodynamic therapy) in non-small cell lung cancer (NSCLC) may improve the efficacy of treatment, reduce the toxicity and improve quality of life for patients. To provide a cellular basis for this approach we have examined the in vitro sensitivity of normal lung fibroblasts (MRC5) and four NSCLC cell lines (SKMES-1, A549, NCIH460 and NCIH23) following, UVC treatment, HDR radiation, HDR radiation with Photofrin alone, PDT and combined HDR radiation and PDT. Cell sensitivity was measured using clonogenic survival. HDR radiation was cobalt-60 gamma rays (1.5-1.9 Gy/min). For PDT treatment, cells were exposed to 2.5 J.lg/ml Photofrin for 18-24 h followed by light exposure (20mW/cm2). D37 values calculated from the survival curves indicated a 2-fold difference in sensitivity to UVC, 6-fold difference in HDR radiation sensitivity and an 8-fold difference in PDT sensitivity. All cell lines showed a similar Photofrin uptake per cell when measured by flow cytometry using 488nm excitation and 620-675 nm emission wavelengths. Photofrin alone at concentrations up to 10 J.lg/ml had no significant effect on the survival of the NSCLC cell lines, whereas 10 J.lg/ml ofPhotofrin alone reduced survival significantly in MRC5 cells. A radiosensitizing effect of Photofrin was detected in MRC5 and NCIH460 cells, but not in A549, SKMES-1 and NCI-H23 cells. For combined treatment cells were exposed to Photofrin and then either exposed to light and 15-30 minutes later exposed to HDR radiation or exposed to HDR radiation and 15-30 minutes later exposed to light. Results showed that although light followed by gamma rays resulted in a somewhat greater tumor cell kill compared to gamma rays followed by light this difference was not significant for any of the cell lines tested. However, this difference was significant when data for all NSCLC cell lines were pooled. The combined treatment with high dose rate HDR radiation and PDT was not significantly different from an additive effect of the individual treatment modalities for in vitro survival of 4 NSCLC cells. In contrast the combined treatment was less than additive for the MRCS cells suggesting that the combined treatment would have the potential advantage of doing less damage to the normal lung cells and suggests that equivalent tumour cell kill in vivo may be possible at reduced systemic effects to patients. In preliminary experiments we have started to examine the effects of Photofrin-mediated PDT on the extra cellular signal-activated protein kinase (ERK) signaling pathway in NSCLC cells. The use of multiple NSCLC cell lines allows for the possible identification of cell line specific changes involved in resistance to PDT and HDR radiation and this will be explored in future work. </p> / Thesis / Master of Science (MSc)

Jusqu’à présent, la metformine a principalement été employée comme médicament contrôlant l’hyperglycémie des personnes atteintes de diabète de type II. Des études épidémiologiques ont démontré que les personnes, prenant de la metformine, développent moins de cancers. Par exemple, la prise de metformine réduit respectivement de 78% et de 46% les chances de développer un cancer hépatique ou pancréatique. Récemment, il a été montré que la metformine permet de réduire le développement de tumeur au niveau de la peau, suite à l’exposition à des rayons UVB. Dans cette étude, j’ai démontré que la présence de metformine permet une meilleure survie de la levure Saccharomyces cerevisiae suite à l’exposition à des rayons UVC ou UVA. De plus, j’ai démontré que la présence de metformine augmente le recrutement de l’histone Htz1 à la chromatine. Pour une souche htz1Δ, le niveau de survie suite à l’exposition aux rayons UVA est considérablement diminué. Htz1 permet le recrutement de Rad14 au site de dommages à l’ADN faits par les rayons UV. Htz1 est donc important pour la détection de ces sites. Enfin, le recrutement nucléaire de Rad14 en présence de metformine a considérablement augmenté. En absence de Rad14, le niveau de survie suite à l’exposition aux rayons UVA diminue significativement. Donc, Htz1 et Rad14 sont deux protéines clés dans la protection contre les rayons UV apportés par la metformine. En conclusion, avec les différents résultats de cette étude, il est possible de dire que la metformine permet une forme de protection contre les rayons UVC et UVA. / Recently, metformin has been widely used to treat hyperglycemia of humans that have type II diabetes mellitus. Recently, some epidemiological studies have showed that populations of individuals being treated with metformin showed lower incidence of cancer. For example, there is a 78 % and 46 % reduction in the incidences of liver and pancreatic cancers, respectively. It has also been demonstrated that metformin protects against skin cancers caused by ultraviolet radiation-B (UVB) DNA damage. In this study, I have demonstrated that metformin significantly protects the budding yeast Saccharomyces cerevisiae from ultraviolet radiation-C (UVC) and from ultraviolet radiation-A (UVA)-induced death. I also showed that metformin increases the recruitment of the histone Htz1 linked to chromatin. In an htz1Δ strain, the metformin protection from UVAinduced death is significantly reduced. Htz1 is important for the recruitment of Rad14 to sites of DNA damage, important for the detection of these sites. Furthermore, nuclear recruitment of Rad14 was significantly increased in cells previously treated with metformin. In the absence of Htz1, the survival rate to UVA exposure in presence of metformin drops significantly. So, Htz1 and Rad14 are two key proteins involved in the protection by metformin against UV DNA damage. With all the results of this study, it was shown that metformin can confer some protection against UVC and UVA for yeast.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Didaticamente, podemos dividir o espectro da radiação ultravioleta (UV) em três faixas: UVA (400 a 320 nm), UVB (320 a 290 nm) e UVC (290 a 100 nm). Apesar do UVC ou UV-curto ser eficientemente filtrado pela camada de ozônio da Terra e sua atmosfera, este é uma das faixas do espectro de UV mais usadas para explorar as consequências de danos causados ao DNA, já que a letalidade induzida por este agente está relacionada aos danos diretos no genoma celular, como as lesões dímero de pirimidina, que são letais se não reparadas. Contudo, demonstrou-se que a radiação UVC pode gerar espécies reativas de oxigênio (ERO), como o oxigênio singleto (1O2). Embora, o radical hidroxil (OH) cause modificações oxidativas nas bases de DNA, alguns trabalhos indicam que o 1O2 também está envolvido nos danos oxidativos no DNA. Esta ERO é produzida por vários sistemas biológicos e reações fotossensibilização, quando cromóforos são expostos à luz visível ou são excitados pela luz UV, permitindo que essa energia possa ser transferida para o oxigênio sendo convertido em 1O2, que é conhecido por modificar resíduos de guanina, gerando 8-oxoG, que caso não seja reparada pode gerar uma transversão GC-TA. O objetivo deste trabalho foi o de elucidar a participação de ERO nos efeitos genotóxicos e mutagênicos gerados pela radiação UVC, assim como as enzimas envolvidas no processo de reparação destas lesões em células de Escherichia coli. Nos ensaios as culturas foram irradiadas com o UVC (254 nm; 15W General Electric G15T8 germicidal lamp, USA). Nossos resultados mostram que o uso de quelantes de ferro não alterou a letalidade induzida pelo UVC. A azida sódica, um captador de 1O2, protegeu as cepas contra os danos genotóxicos gerados pelo UVC e também diminuiu a frequência de mutações induzidas no teste com rifampicina. A reversão específica GC-TA foi induzida mais de 2,5 vezes no ensaio de mutagênese. A cepa deficiente na proteína de reparo Fpg, enzima que corrige a lesão 8-oxoG, apresentou menos quebras no DNA do que a cepa selvagem no ensaio de eletroforese alcalina. A letalidade induzida pelo UVC foi aumentada nos mutantes transformados com o plasmídeo pFPG, ao mesmo tempo que representou uma redução na indução mutagênica. Houve dimuição na eficiência de transformação com plasmídeo pUC 9.1 na cepa fpg quando comparado a cepa selvagem. Assim como, um aumento da sensibilidade ao UVC na associação entre mutantes fpg e uvrA. Estes resultados mostram que o 1O2 participa dos danos induzidos pelo UVC, através da geração da lesão 8-oxoG, uma lesão mutagênica, que é reparada pela proteína Fpg / Didactically, we can divide the ultraviolet radiation (UV) spectrum into three bands: UVA (400 to 320 nm), UVB (320-290 nm) and UVC (290-100 nm). Despite the UVC or far-UV be efficiently filtered by Earths ozone layer and its atmosphere, this is one of bands of UV spectrum used to explore the consequences of DNA damages, since the UVC-induced lethality is related to direct damage in genome cells, such as pyrimidine dimers, which are lethal if not repaired. However, it was shown that UVC radiation can generate reactive oxygen species (ROS) such as singlet oxygen (1O2). Although hydroxyl radical (OH) cause oxidative modifications in DNA bases, some works suggests that 1O2 is also involved in oxidative DNA damage. This ROS is produced by several biological systems and photosensitivity reactions when chromophores are exposed to visible light or excited by UV light, allowing that energy can be transferred to the oxygen being converted to 1O2, which is known to modify guanine residues, generating 8-oxoG, if not repaired can lead to a GC-TA transversion. The objective of this work was to elucidate the ROS involvement in the genotoxic and mutagenic effects generated by UVC radiation, as well as the enzymes involved in the repair process of these lesions in Escherichia coli cells. In the assays, cultures were irradiated with UVC (254 nm, 15 W General Electric germicidal lamp G15T8, USA). Our results show that the use of iron chelators did not affect the UVC-induced lethality. The sodium azide, a 1O2 quencher, protected strains against the genotoxic damage produced by UVC and also decreased the frequency of mutations induced in rifampicin assay. Reversal specific GC-TA was induced more than 2.5 fold in the mutagenesis assay. The deficient strain in the repair protein Fpg, an enzyme that corrects 8-oxoG lesions, had less DNA breakage than the wild strain in electrophoresis alkaline assay. The UVC-induced lethality was increased in mutants transformed with the pFPG plasmid, while representing a decrease in mutagenic induction. There was a reduction in the transformation efficiency with plasmid pUC 9.1 on Fpg-strain compared to wild-type strain. Also there was a lethality increase in the association between fpg and uvrA mutants. These results shows that 1O2 participates in UVC-induced damages through the generation of 8-oxoG lesion, a mutagenic lesion that is repaired by the Fpg protein.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Didaticamente, podemos dividir o espectro da radiação ultravioleta (UV) em três faixas: UVA (400 a 320 nm), UVB (320 a 290 nm) e UVC (290 a 100 nm). Apesar do UVC ou UV-curto ser eficientemente filtrado pela camada de ozônio da Terra e sua atmosfera, este é uma das faixas do espectro de UV mais usadas para explorar as consequências de danos causados ao DNA, já que a letalidade induzida por este agente está relacionada aos danos diretos no genoma celular, como as lesões dímero de pirimidina, que são letais se não reparadas. Contudo, demonstrou-se que a radiação UVC pode gerar espécies reativas de oxigênio (ERO), como o oxigênio singleto (1O2). Embora, o radical hidroxil (OH) cause modificações oxidativas nas bases de DNA, alguns trabalhos indicam que o 1O2 também está envolvido nos danos oxidativos no DNA. Esta ERO é produzida por vários sistemas biológicos e reações fotossensibilização, quando cromóforos são expostos à luz visível ou são excitados pela luz UV, permitindo que essa energia possa ser transferida para o oxigênio sendo convertido em 1O2, que é conhecido por modificar resíduos de guanina, gerando 8-oxoG, que caso não seja reparada pode gerar uma transversão GC-TA. O objetivo deste trabalho foi o de elucidar a participação de ERO nos efeitos genotóxicos e mutagênicos gerados pela radiação UVC, assim como as enzimas envolvidas no processo de reparação destas lesões em células de Escherichia coli. Nos ensaios as culturas foram irradiadas com o UVC (254 nm; 15W General Electric G15T8 germicidal lamp, USA). Nossos resultados mostram que o uso de quelantes de ferro não alterou a letalidade induzida pelo UVC. A azida sódica, um captador de 1O2, protegeu as cepas contra os danos genotóxicos gerados pelo UVC e também diminuiu a frequência de mutações induzidas no teste com rifampicina. A reversão específica GC-TA foi induzida mais de 2,5 vezes no ensaio de mutagênese. A cepa deficiente na proteína de reparo Fpg, enzima que corrige a lesão 8-oxoG, apresentou menos quebras no DNA do que a cepa selvagem no ensaio de eletroforese alcalina. A letalidade induzida pelo UVC foi aumentada nos mutantes transformados com o plasmídeo pFPG, ao mesmo tempo que representou uma redução na indução mutagênica. Houve dimuição na eficiência de transformação com plasmídeo pUC 9.1 na cepa fpg quando comparado a cepa selvagem. Assim como, um aumento da sensibilidade ao UVC na associação entre mutantes fpg e uvrA. Estes resultados mostram que o 1O2 participa dos danos induzidos pelo UVC, através da geração da lesão 8-oxoG, uma lesão mutagênica, que é reparada pela proteína Fpg / Didactically, we can divide the ultraviolet radiation (UV) spectrum into three bands: UVA (400 to 320 nm), UVB (320-290 nm) and UVC (290-100 nm). Despite the UVC or far-UV be efficiently filtered by Earths ozone layer and its atmosphere, this is one of bands of UV spectrum used to explore the consequences of DNA damages, since the UVC-induced lethality is related to direct damage in genome cells, such as pyrimidine dimers, which are lethal if not repaired. However, it was shown that UVC radiation can generate reactive oxygen species (ROS) such as singlet oxygen (1O2). Although hydroxyl radical (OH) cause oxidative modifications in DNA bases, some works suggests that 1O2 is also involved in oxidative DNA damage. This ROS is produced by several biological systems and photosensitivity reactions when chromophores are exposed to visible light or excited by UV light, allowing that energy can be transferred to the oxygen being converted to 1O2, which is known to modify guanine residues, generating 8-oxoG, if not repaired can lead to a GC-TA transversion. The objective of this work was to elucidate the ROS involvement in the genotoxic and mutagenic effects generated by UVC radiation, as well as the enzymes involved in the repair process of these lesions in Escherichia coli cells. In the assays, cultures were irradiated with UVC (254 nm, 15 W General Electric germicidal lamp G15T8, USA). Our results show that the use of iron chelators did not affect the UVC-induced lethality. The sodium azide, a 1O2 quencher, protected strains against the genotoxic damage produced by UVC and also decreased the frequency of mutations induced in rifampicin assay. Reversal specific GC-TA was induced more than 2.5 fold in the mutagenesis assay. The deficient strain in the repair protein Fpg, an enzyme that corrects 8-oxoG lesions, had less DNA breakage than the wild strain in electrophoresis alkaline assay. The UVC-induced lethality was increased in mutants transformed with the pFPG plasmid, while representing a decrease in mutagenic induction. There was a reduction in the transformation efficiency with plasmid pUC 9.1 on Fpg-strain compared to wild-type strain. Also there was a lethality increase in the association between fpg and uvrA mutants. These results shows that 1O2 participates in UVC-induced damages through the generation of 8-oxoG lesion, a mutagenic lesion that is repaired by the Fpg protein.

Nach den Ergebnissen der zweiten Nationalen Verzehrsstudie sind in Deutschland bereits 66 % der Männer und 51 % der Frauen übergewichtig (BMI > 25) oder adipös (BMI > 30) [BMELV, 2008]. Bisher auf dem Markt befindliche „Light-Lebensmittel“ mit Fettaustausch- bzw. Fettersatzstoffen weisen jedoch häufig sensorische Mängel auf. Im Kooperationsprojekt „Pflanzliche Fettaustauschstoffe aus sphärischen Proteinmizellen“ (Universität Leipzig: Institut für Lebensmittelhygiene; Freising: Fraunhofer IVV) wurde ein Lupinenproteinisolat entwickelt, welches micellare Strukturen mit hydrophober Oberfläche ausbilden kann und sich aufgrund seiner fettähnlichen Eigenschaften als neuer proteinbasierter Fettaustauschstoff in Lebensmitteln eignet. Aufgrund der geringen mikrobiologischen Stabilität und einer hohen Belastung mit sporenbildenden Bakterien, z. T. Bacillus cereus, waren jedoch Maßnahmen zur Entkeimung der Rohstoffe sowie des Proteinisolats notwendig. Die Arbeit stellt diese Maßnahmen und deren Einfluss auf die mikrobiologische Beschaffenheit sowie sensorische, technofunktionelle und ausgewählte ernährungsphysiologische Eigenschaften dar. In der vorliegenden Arbeit wurde eine physikalische Methode der Saatgutentkeimung etabliert (130 °C/60 min), welche die mikrobielle Stabilisierung des lupinenproteinbasierten Fettaustauschstoffes sicherstellte, wobei die sensorische Qualität (Geschmack, Cremigkeit, Farbe) nur minimal, die ernährungsphysiologische (in-vitro-Verdaubarkeit, Maillard-Produkte, Polyphenolgehalt) jedoch nicht beeinflusst wurde. Starke Veränderungen der technofunktionellen Eigenschaften (z. B. Gelbildung, Wasserbindung, Emulgierbarkeit, Schaumbildung etc.) konnten sowohl im positiven als auch im negativen Sinne nicht beschrieben werden. Lichtmikroskopische Aufnahmen und Untersuchungen der Proteine mittels SDS-PAGE und DSC bestätigten eine nur geringfügige Beeinflussung der micellaren Struktur und Proteinzusammensetzung. Die Anwendung als Fettaustauschstoff in Lebensmitteln würde somit nicht beeinträchtigt. Der Einfluss der Saatgutbehandlung auf das Protein war wesentlich geringer als eine direkte thermische Behandlung des Proteinisolats. Im Hinblick auf den Gesamtprozess sollte eine Pasteurisierung der feuchten Proteinisolate im nichtproteinschädigenden Temperaturbereich (75 °C/5 min) dennoch durchgeführt werden, um während des Prozesses eingetragene Mikroorganismen zu inaktivieren.

Nach den Ergebnissen der zweiten Nationalen Verzehrsstudie sind in Deutschland bereits 66 % der Männer und 51 % der Frauen übergewichtig (BMI > 25) oder adipös (BMI > 30) [BMELV, 2008]. Bisher auf dem Markt befindliche „Light-Lebensmittel“ mit Fettaustausch- bzw. Fettersatzstoffen weisen jedoch häufig sensorische Mängel auf. Im Kooperationsprojekt „Pflanzliche Fettaustauschstoffe aus sphärischen Proteinmizellen“ (Universität Leipzig: Institut für Lebensmittelhygiene; Freising: Fraunhofer IVV) wurde ein Lupinenproteinisolat entwickelt, welches micellare Strukturen mit hydrophober Oberfläche ausbilden kann und sich aufgrund seiner fettähnlichen Eigenschaften als neuer proteinbasierter Fettaustauschstoff in Lebensmitteln eignet. Aufgrund der geringen mikrobiologischen Stabilität und einer hohen Belastung mit sporenbildenden Bakterien, z. T. Bacillus cereus, waren jedoch Maßnahmen zur Entkeimung der Rohstoffe sowie des Proteinisolats notwendig. Die Arbeit stellt diese Maßnahmen und deren Einfluss auf die mikrobiologische Beschaffenheit sowie sensorische, technofunktionelle und ausgewählte ernährungsphysiologische Eigenschaften dar. In der vorliegenden Arbeit wurde eine physikalische Methode der Saatgutentkeimung etabliert (130 °C/60 min), welche die mikrobielle Stabilisierung des lupinenproteinbasierten Fettaustauschstoffes sicherstellte, wobei die sensorische Qualität (Geschmack, Cremigkeit, Farbe) nur minimal, die ernährungsphysiologische (in-vitro-Verdaubarkeit, Maillard-Produkte, Polyphenolgehalt) jedoch nicht beeinflusst wurde. Starke Veränderungen der technofunktionellen Eigenschaften (z. B. Gelbildung, Wasserbindung, Emulgierbarkeit, Schaumbildung etc.) konnten sowohl im positiven als auch im negativen Sinne nicht beschrieben werden. Lichtmikroskopische Aufnahmen und Untersuchungen der Proteine mittels SDS-PAGE und DSC bestätigten eine nur geringfügige Beeinflussung der micellaren Struktur und Proteinzusammensetzung. Die Anwendung als Fettaustauschstoff in Lebensmitteln würde somit nicht beeinträchtigt. Der Einfluss der Saatgutbehandlung auf das Protein war wesentlich geringer als eine direkte thermische Behandlung des Proteinisolats. Im Hinblick auf den Gesamtprozess sollte eine Pasteurisierung der feuchten Proteinisolate im nichtproteinschädigenden Temperaturbereich (75 °C/5 min) dennoch durchgeführt werden, um während des Prozesses eingetragene Mikroorganismen zu inaktivieren.:1 Einleitung und Zielstellung 1 2 Stand des Wissens 4 2.1 Die Lupine 4 2.1.1 Anbau und Verbreitung 4 2.1.2 Einsatz von Lupinenprodukten und -proteinen in der Humanernährung 5 2.1.3 Inhaltsstoffe und deren Verteilung 5 2.1.4 Lupinenproteine 10 2.1.4.1 Einteilung und Struktur der Lupinenproteine 10 2.1.4.2 Lupinenproteine und Allergenität 12 2.1.5 Eigenschaften der verschiedenen Lupinenproteinfraktionen 13 2.1.5.1 Ernährungsphysiologische Eigenschaften 13 2.1.5.2 Funktionelle Eigenschaften 15 2.1.5.3 Modifikation der Proteinstruktur 15 2.1.5.4 Herstellung verschiedener Lupinenproteinpräparate 16 2.1.5.5 Micellare Proteine 17 2.2 Möglichkeiten der Fettreduktion in Lebensmitteln 18 2.2.1 Fettaustauschstoffe 18 2.2.1.1 Fettaustauschstoffe auf Proteinbasis (Mikropartikulierte Proteine) 18 2.2.1.2 Fettaustauschstoffe auf Kohlenhydratbasis 19 2.2.1.3 Quellstoffe 19 2.2.2 Fettersatzstoffe 19 2.2.2.1 Spezielle Triglyceride 20 2.2.2.2 Kohlenhydratpolyester 20 2.2.2.3 Retrofette 20 2.3 Herstellung des lupinenproteinbasierten Fettaustauschstoffes 20 2.4 Saatgutbehandlung 21 2.4.1 Methoden der Lebensmittelkonservierung 22 2.5 Proteinfunktionalität 25 2.5.1 Definition und Zusammenhang zu Proteinen 25 2.5.2 Ausgewählte funktionelle Eigenschaften 26 2.5.2.1 Wasserbindevermögen 26 2.5.2.2 Ölbindevermögen 26 2.5.2.3 Löslichkeit 27 2.5.2.4 Emulgiervermögen 27 2.5.2.5 Schaumbildungsvermögen 28 2.5.2.6 Gelbildungsvermögen 29 2.5.2.7 Oberflächenhydrophobität 30 2.5.2.8 Bedeutung für die Lebensmittelentwicklung 30 3 Material und Methoden 32 3.1 Material 32 3.1.1 Saatgut 32 3.1.2 Geräte, Chemikalien, Verbrauchsmaterial, Software 32 3.1.3 Pufferlösungen 39 3.1.4 Herstellung Bradford-Reagenz, 5-fach 39 3.1.5 Auswahl der Vergleichssubstanzen 39 3.2 Methoden 40 3.2.1 Herstellung der Proteinisolate 40 3.2.2 Mikrobiologische Analysen 41 3.2.3 Bestimmung der Trockenmasse 41 3.2.4 Bestimmung des Proteingehalts 42 3.2.5 Thermische Behandlungsmethoden im Prozess 42 3.2.5.1 UHT-Erhitzung des Extraktes 42 3.2.5.2 Pasteurisierung des Isolats 44 3.2.6 Saatgutentkeimung 44 3.2.6.1 UVC-Bestrahlung 44 3.2.6.2 Trockene Erhitzung 45 3.2.6.3 Autoklavieren 46 3.2.7 Sensorische Untersuchungen 46 3.2.8 Proteinfunktionalität 47 3.2.8.1 Ölbindevermögen 47 3.2.8.2 Wasserbindevermögen 47 3.2.8.3 Gelbildungsvermögen 47 3.2.8.4 Emulgiereigenschaften 47 3.2.8.5 Schaumbildungsvermögen 48 3.2.8.6 Proteinlöslichkeit 48 3.2.8.7 Oberflächenhydrophobität 49 3.2.9 Ernährungsphysiologische Eigenschaften 50 3.2.9.1 in-vitro-Verdaubarkeit 50 3.2.9.2 Maillard-Produkte 50 3.2.9.3 Nachweis reduzierender Zucker .50 3.2.9.4 Nachweis von Glykoproteinen 50 3.2.9.5 Polyphenolgehalt der Lupinenflocken und Proteinisolate 51 3.2.10 Proteincharakterisierung 51 3.2.10.1 Lichtmikroskopie 51 3.2.10.2 Dynamische Differenzkalorimetrie 51 3.2.10.3 Natriumdodecylsulfat-Polyacrylamidgelelektrophorese 52 4 Ergebnisse und Diskussion 54 4.1 Thermische Behandlungsmethoden im Prozess 54 4.1.1 UHT-Erhitzung des Extraktes: Einfluss auf Mikrobiologie und Proteinausbeute 54 4.1.2 Pasteurisierungsversuche: Einfluss auf Mikrobiologie und Proteinqualität 55 4.2 Saatgutentkeimung - Mikrobiologie und Proteinausbeute 56 4.2.1 Versuchsreihe I 56 4.2.2 Versuchsreihe II 61 4.3 Sensorische Untersuchungen 63 4.3.1 Verkostungen 64 4.3.2 Farbmessung der Proteinisolate und Flocken 65 4.4 Proteinfunktionalität 69 4.4.1 Wasser- und Ölbindevermögen 69 4.4.2 Gelbildungsvermögen 72 4.4.3 Emulgiereigenschaften 74 4.4.4 Schaumbildungsvermögen 78 4.4.5 Proteinlöslichkeit 81 4.4.6 Oberflächenhydrophobität 83 4.5 Ernährungsphysiologische Eigenschaften 86 4.5.1 Maillard-Produkte 86 4.5.2 Nachweis reduzierender Zucker 87 4.5.3 Nachweis von Glykoproteinen 87 4.5.4 Verdaubarkeit 88 4.5.5 Polyphenolgehalte 89 4.6 Proteincharakterisierung 91 4.6.1 Lichtmikroskopie 91 4.6.2 Dynamische Differenzkalorimetrie 95 4.6.3 Natriumdodecylsulfat-Polyacrylamidgelelektrophorese 98 5 Zusammenfassung 105 Anhang 109

Depuis quinze ans, de multiples instruments de mesure ont été envoyés vers Mars et explorent aujourd'hui sa surface ou y sont en orbite. Les données, renvoyées par ces sondes ont fourni une grande quantité d'informations quant aux conditions régnant aujourd'hui sur Mars, mais aussi sur la géologie des terrains et sur la minéralogie associée. Les minéraux découverts sur Mars se sont formés dans des conditions tout à fait différentes de celles qui y règnent aujourd'hui.Une approche associant modélisations numériques et expériences est proposée pour permettre d'identifier l'influence de divers processus comme le rayonnement UVC, la température et la pression partielle de CO2 ou de SO2. Des expériences en boîte à gants sont réalisées à des pressions partielles de CO2 de 10-6 bar, 10-3 bar et 10-2 bar, en présence ou non de rayonnement UVC. Conjointement à ces expériences, des expériences en capsules en or sont menées pour des températures de 20°C et 50°C avec des pressions partielles de CO2 d'environ 10-3 bar, 10-2 bar et 1 bar ainsi qu'avec une pression partielle de SO2 d'environ 1 bar. Les résultats ont montré qu'il était possible de reproduire expérimentalement et par les codes de calculs, la minéralogie supposée résultant de l'altération d'un verre basaltique de composition proche de roches martiennes. L'observation de carbonates de calcium dans les expériences réalisées à des pressions partielles de CO2 supérieures à 10-3 bar est en accord avec la prédiction de ces mêmes minéraux par des simulations numériques réalisées avec le logiciel DISSOL. Néanmoins, l'abondance de ces carbonates dans les deux types de modélisations, ainsi que leur composition à dominance calcique, contrastent avec les observations des rares minéraux carbonatés magnésiens à la surface de Mars. Ce contraste pose les questions concernant la composition de l'atmosphère, la composition initiale du verre et la préservation des carbonates après leur précipitation. Bien que des argiles n'aient pas été observées dans les expériences à cause d'un taux de dissolution du verre trop faible, des montmorillonites ferrifères et magnésiennes sont prédites dans les simulations numériques quelle que soit la pression partielle de CO2. De la beidellite sodique est également prédite dans le cas où les conditions atmosphériques martiennes seraient réductrices quelle que soit la pression partielle de CO2. Contrairement aux observations de la surface de Mars, aucune nontronite ne précipite. Les sulfates sont absents des paragenèses minérales obtenues lors des expériences avec une atmosphère à CO2. Dans les modélisations, aucun minéral sulfaté n'est néoformé pour les pressions partielles de CO2 imposées (> 10-6 bar) et la teneur en soufre du verre expérimental de synthèse. Toutefois, la précipitation de gypse peut être observée dans les simulations si la teneur en soufre est 100 fois supérieure à celle du verre utilisé pour ce travail. L'absence de sulfates magnésiens, de même que de carbonates magnésiens suggère que la composition chimique choisie pour le verre soumis à l'altération ne correspond pas totalement celle de la roche qui a pu donner les différents assemblages minéralogiques observés à la surface de Mars. En revanche, des sulfates de calcium et, des sulfates de fer et d'aluminium sont observés pour des expériences en capsules avec une atmosphère à SO2. Le rôle des atmosphères est essentiel quant à l'abondance des minéraux carbonatés et des minéraux sulfatés. Les expériences montrent que les radiations UVC accélèrent la dissolution du verre et favorise l'oxydation partielle du soufre du verre en pyrite / Process of deterioration on the primitive surface of Mars: experimental and digital simulations and mineralogical implications

Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-10-10T12:53:01Z No. of bitstreams: 1 DissIJSM.pdf: 4861351 bytes, checksum: f6e331316f2f40305ea71f2a5696632b (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T19:29:14Z (GMT) No. of bitstreams: 1 DissIJSM.pdf: 4861351 bytes, checksum: f6e331316f2f40305ea71f2a5696632b (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T19:29:19Z (GMT) No. of bitstreams: 1 DissIJSM.pdf: 4861351 bytes, checksum: f6e331316f2f40305ea71f2a5696632b (MD5) / Made available in DSpace on 2016-10-20T19:29:26Z (GMT). No. of bitstreams: 1 DissIJSM.pdf: 4861351 bytes, checksum: f6e331316f2f40305ea71f2a5696632b (MD5) Previous issue date: 2016-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / An electrochemical-photochemical system (EC-PC) using a dimensionally stable anode (DSA®) was used to oxidize and mineralize tebuthiuron (TBT) herbicide, which is a potential contaminant to the surface and ground water. The electrochemical process was conducted using a filter-press flow cell and the photochemical one by irradiation of the solution inside the reservoir using Hg vapor lamps (type UVC). The studied variables were: i) nominal power of Hg lamps (5, 9, 80, and 125 W), ii) pH (3, 7, 11, and no control), iii) NaCl concentration (0, 1, 2, and 4 g L–1), and iv) current density (10, 20, and 30 mA cm–2). Other parameters were kept constant, such as the flow rate (420 L h–1) and the solution temperature (25 °C). The electrolyzed solution was composed of 100 mg L–1 TBT and 0.1 mol L–1 Na2SO4, as supporting electrolyte. The oxidation and mineralization performances of the TBT and its intermediates using the hybrid process were assessed through high performance liquid chromatography coupled to mass spectrometry (HPLC-MS/MS), depending on the experiment, and total organic carbon (TOC) analyses. The experiment using a 9 W Hg lamp led to the complete oxidation and mineralization of TBT and its intermediate compounds in the presence of NaCl from acidic to neutral solutions, including the one with no control, and independently of the applied electrical current density. High CO2 conversions were attained after 6 h using the combined EC-PC process, due to the generation of hydroxyl radicals (HO•) from HOCl homolysis. The HO• also favored the formation of many hydroxylated organic compounds. Despite the formation of an organochlorine byproduct, this compound was completely eliminated after 3 h treatment. Moreover, no toxicity was observed when using the Artemia salina microcrustacean. Similar removal rates of TBT were attained when only using an electrochemical method with boron-doped diamond as anode. In addition, the EC-PC process showed a slightly higher electrical energy consumption for the removal of organic matter in comparison to the electrochemical one. Finally, only a chlorinated carboxylic acid was detected; however, this compound was completely eliminated in the final stages of the EC-PC treatment. All these features enables the classification of this method as an advanced oxidation process. / Um sistema eletroquímico-fotoquímico (EQ-FQ) utilizando um anodo dimensionalmente estável (ADE®) foi usado para oxidar e mineralizar o herbicida tebutiuron (TBT), que é um potencial contaminante de águas superficiais e subterrâneas. O processo eletroquímico foi realizado utilizando um reator do tipo filtro-prensa e o fotoquímico por meio da irradiação da solução no reservatório utilizando uma lâmpada de vapor de Hg (tipo UVC). As variáveis estudadas no processo foram: i) potência nominal da lâmpada de vapor de mercúrio (5, 9, 80 e 125 W), ii) pH (3, 7, 11 e sem controle), iii) concentração de NaCl (0, 1, 2 e 4 g L–1) e iv) densidade de corrente (10, 20 e 30 mA cm–2). Outros parâmetros constantes foram a vazão (420 L h–1) e a temperatura da solução (25 ºC). As soluções eletrolisadas continham 100 mg L–1 de TBT na presença de Na2SO4 0,1 mol L–1, como eletrólito suporte. A eficiência do processo híbrido quanto à oxidação e à mineralização do TBT e de seus intermediários foi acompanhada por análises de cromatografia líquida de alta eficiência acoplada a espectrometria de massas (CLAEEM/ EM), quando necessário, e de carbono orgânico total (COT). O experimento com a lâmpada de 9 W resultou na completa oxidação e mineralização do TBT e de seus intermediários na presença de NaCl e em soluções ácidas, neutras ou sem nenhum controle, independentemente da densidade de corrente elétrica aplicada. Altas conversões a CO2 foram atingidas ao final do tratamento utilizando-se o sistema EQFQ, devido à geração de radicais hidroxila (HO•), advindos da reação de homólise do HOCl, que também promoveram a formação de vários intermediários orgânicos hidroxilados. Apesar da formação de um composto organoclorado, este foi completamente eliminado após 3 h de tratamento; além disso, nenhuma toxicidade frente ao microcrustáceo Artemia salina foi constatada. As taxas de remoção do TBT também foram similares às obtidas em comparação com um sistema puramente eletroquímico e utilizando um anodo de diamante dopado com boro; contudo, o sistema EQ-FQ apresentou um consumo de energia elétrica ligeiramente mais alto para a remoção da carga orgânica. Finalmente, somente um ácido carboxílico clorado foi detectado, porém, este foi completamente eliminado ao final do tratamento EQ-FQ. Estes resultados permitem classificar o sistema EQ-FQ como sendo um processo oxidativo avançado.

Dans les cellules eucaryotes, la réponse aux lésions de l'ADN s'accompagne d'une réorganisation de la chromatine. Cette structure, associant l'ADN aux protéines histones, est porteuse de l'information épigénétique, qui définit l'identité cellulaire. Cependant, nos connaissances concernant les mécanismes impliqués dans la réorganisation de la chromatine dont l'intégrité structurale et fonctionnelle a été menacée par un stress génotoxique sont encore limitées, en particulier dans les cellules humaines. Au cours de ma thèse, je me suis donc intéressée à cette thématique en me concentrant sur l'étude de la dynamique des variants de l'histone H3 et de leurs chaperons associés après dommages UVC. En combinant une technologie innovante de suivi spécifique des histones parentales ou néo-synthétisées à des techniques de pointe d'induction de dommages locaux dans l'ADN, j'ai ainsi mis en évidence que le chaperon HIRA (Histone Regulator A) est recruté tôt aux sites de lésions où il stimule l'incorporation locale de nouveaux variants H3.3 et assure la reprise de la transcription après réparation des dommages UVC. Nous avons aussi démontré que les anciennes histones sont initialement redistribuées dans la chromatine autour des sites de lésions par un mécanisme faisant appel au facteur de détection des dommages DDB2 (DNA Damage Binding protein 2). A plus long terme, des histones parentales " reviennent " dans les régions de chromatine en cours de réparation où elles se mélangent aux nouvelles histones incorporées. Le " retour " d'histones préexistantes contribuerait ainsi au maintien de l'intégrité de l'information épigénétique véhiculée par la chromatine avant stress génotoxique. / In eukaryotic cells, the DNA damage response involves a reorganization of chromatin structure. This structure, in which DNA is associated with histone proteins, conveys the epigenetic information, which is critical for cell identity. However, we are still far from understanding the mechanisms underlying chromatin dynamics in response to DNA damage, which challenges both the structural and functional integrity of chromatin architecture. During my PhD, I thus decided to explore this issue in human cells, by deciphering the dynamics of histone H3 variants and their dedicated chaperones in response to UVC lesions. By combining local UVC irradiation with an innovative technology that allows specific tracking of parental and newly synthesized histones, I revealed that the histone chaperone HIRA (Histone Regulator A) is recruited early to UVC-damaged chromatin regions, where it promotes local deposition of new histone H3.3 variant and facilitates transcription recovery upon repair completion. We also demonstrated that old H3 histones are initially redistributed around the damaged chromatin zone, this conservative redistribution requiring the UVC damage sensor DDB2 (DNA Damage Binding protein 2). Later in the repair process, most parental histones recover and mix with newly deposited histones in repairing chromatin regions. The recovery of pre-existing histones may contribute to preserve the integrity of the epigenetic information conveyed by chromatin before genotoxic stress.