Triplet Excited Carbonyls and Singlet Oxygen Formation During Oxidative Radical Reaction in Skin

The skin is the largest organ in the body and is consistently exposed to aggressive environmental attacks (biological/physical/chemical, etc.). Reactive oxygen species (ROS) are formed during the normal oxidative metabolism which enhances to a lethal level under stress conditions referred to as oxidative stress. While, under normal conditions, cells are capable of dealing with ROS using non-enzymatic and enzymatic defense system, it can lead to a critical damage to cell system via the oxidation of cellular components under stress condition. Lipid peroxidation is a well-established mechanism of cellular injury in all kinds of organisms and it is often used as an indicator of oxidative stress in cells and tissues. In the presence of metal ions, ROS such as hydrogen peroxide (H2O2) produces highly reactive hydroxyl radical (HO•) via Fenton reaction. In the current study, we have used the porcine skin (intact pig ear/skin biopsies) as an ex vivo/in vitro model system to represent human skin. Experimental results have been presented on the participation of HO• in the initiation of lipid peroxidation and thereby leading to the formation of reactive intermediates and the formation of electronically excited species eventually leading to ultra-weak photon emission (UPE). To understand the participation of different electronically excited species in the overall UPE, the effect of a scavenger of singlet oxygen (1O2) on photon emission in the visible and near-infrared region of the spectrum was measured which showed its contribution. In addition, measurement with interference filter with a transmission in the range of 340–540 nm reflected a substantial contribution of triplet carbonyls (3L=O∗) in the photon emission. Thus, it is concluded that during the oxidative radical reactions, the UPE is contributed by the formation of both 3L=O∗ and 1O2. The method used in the current study is claimed to be a potential tool for non-invasive determination of the physiological and pathological state of human skin in dermatological research

Heterologous expression from Agrobacterium virulence promoters

The aim of this work was twofold: to construct plasmids with a gene encoding a pesticidal protein expressed from an Agrobacterium tumefaciens virulence promoter and to determine, in planta, the sites of Agrobacterium vir-induction. A number of methods were employed to detect in situ vir-induction and, to this end, genes encoding β-glucuronidase (GUS) and bioluminescence (lux) were linked in plasmid constructs to Agrobacterium vir-promoters. In each case, expression of the gene was shown to be induced by the v/r-inducing phenolic compound acetosyringone. An existing plasmid, in which the lacZ gene was under control of the virB promoter was utilised to demonstrate v/r-induction occurring at sites of injury on the roots of mung bean seedlings. Pesticidal genes expressed from Agrobacterium vimlence promoters would form the basis of a biological control system. A microbial inoculant harbouring such a construct would produce the pesticidal protein only when in the presence of vi-inducing compounds in plant wound exudates. A chitinase gene, chiB, from Serratia marcescens was characterised and sequenced and, following removal of its promoter region, was linked to an Agrobacterium virB promoter. Plasmids were also constructed in which the chiA gene of S. marcescens was brought under the control of a virB or virE promoter. All the constructs specified acetosyringone- inducible production of chitinase. Chitinase is effective in the biological control of chitin containing organisms such as fungi

The control of starch degradation in Arabidopsis thaliana leaves at night

The aim of this work was to understand how Arabidopsis thaliana plants control starch degradation at night. Starch is the major energy reserve in Arabidopsis. It is broken down at night to maintain growth and metabolism of the plant, when photosynthesis is not possible. The rate of starch degradation follows a linear pattern and is matched to the length of the night period such that almost all starch is exhausted by dawn. The mechanisms and the proteins involved in controlling starch degradation rates are largely unknown. With my work I wanted to identify components involved in the control of starch degradation rates. Using a forward genetic screen, I discovered several mutants with new starch degradation phenotypes. One of them was affected in the circadian clock component EARLY FLOWERING 3 (ELF3). It degraded its starch slower than wildtype plants and in a non-linear way. Two mutants degraded their starch at a much faster rate than wild-type plants and exhausted their reserves before dawn. One of them lacks a novel protein, which was named EXCESS STARCH TURNOVER 1 (EST1). This protein is required for normal starch degradation rates, but its function is still unknown. The second mutant is affected in BETA-AMYLASE 1 (BAM1) and produces aberrant BAM1 protein containing a serine to asparagine amino acid substitution in position 132. Faster starch degradation rates in this mutant depend on the presence of another protein of the pathway, LIKE SEX FOUR 1 (LSF1). The data indicate that modulation of BAM1 activity can strongly affect the rates of starch degradation, although starch is degraded normally in absence of BAM1. In a second approach, I analysed which known components of the starch degradation pathway are necessary for the adjustment of starch degradation rates. I found that PHOSPHOGLUCAN WATER DIKINASE (PWD) and BAM3 might play a role in adjusting starch degradation rates in response to an unexpectedly early night. In summary, in this thesis I introduce a novel protein necessary for normal starch degradation rates in Arabidopsis leaves and provide insights into proteins and mechanisms which might control starch degradation rates in response to an early night

Unraveling the regulatory mechanisms controlling the biosynthesis and emission of the volatile organic compound ’sodorifen’ produced by Serratia plymuthica 4Rx13

The aim of this work was to investigate the regulatory mechanisms underlying production of the unique volatile compound sodorifen in different Serratia species. For this purpose, expression analyses of the biosynthetic gene cluster were performed and global regulatory pathways in the sodorifen producing strain S. plymuthica 4Rx13 inactivated. As a result, biosynthesis of sodorifen was found to be predominantly regulated on a transcriptional level and the obtained results gave rise to the speculation that it might be involved in stress response of the producing bacteria.Ziel der Arbeit war es, die Regulation der ungewöhnlichen, volatilen Verbindung Sodorifen in verschiedenen Serratia Spezies zu untersuchen. Zu diesem Zweck wurden Expressionsanalysen des entsprechenden Biosynthese-Genclusters durchgeführt und zentrale Regulationsmechanismen im Sodorifen-Produzenten Serratia plymuthica 4Rx13 mittels Mutagenese inaktiviert. Dabei zeigte sich, dass die Biosynthese dieser ungewöhnlichen volatilen Verbindung hauptsächlich transkriptionell reguliert ist und dass Sodorifen eine mögliche Rolle in der Stress-Antwort der produzierenden Bakterien spielt