Browsing by Subject "ROS"

Tiivistelmä – Referat – Abstract ROS or Reactive Oxygen Species can be found throughout all living organisms on the planet. Without ROS, processes, which are essential for the sustainment of most living organisms, such as respiration would not be possible. On the other hand, uncontrolled ROS generation can cause severe damage to the cellular structure. The family of ROS includes multiple compounds, which share a common trait of high chemical activity. ROS can be produced on demand by specific enzymes which are localized within cellular structures, such as membranes. One group of enzymes is called NADPH (Nicotinamide adenine dinucleotide phosphate) oxidases. These enzymes possess common structure which is composed of transmembrane region with multiple loop helixes and usually two or more terminal motifs, which are devised into regulatory EF-hand motifs and catalytic motifs. NADPH oxidases are essential ROS producers and can be found throughout most clades of living organism and are widely represented in different cellular compartments and distributed across different tissues in multicellular organisms. As an example, Nox family of NADPH oxidases can be found in human tissues and immune cells. Another common group of NADPH oxidases is respiratory burst oxidase homologues (RBOH) can be found in plants. Members of this group play important role in plant immune defense against pathogens. One example is AtRBOHD, which is expressed in Arabidopsis genus of plants. Upon activation, these enzymes are known to produce hydrogen peroxide (H2O2) as mean of antibacterial defense. These host defense mechanisms are known to be driven by different signaling molecules. It has been determined that in some examples of NADPH Oxidases, including Nox5 and RBOHD, the state of activation can be induced through the effects of Ca2+ ions. Moreover, it has been determined, that ROS-producing state of these NADPH oxidases is achieved through change of conformation. This change in conformation is attributed to the different modes of interaction of motifs of oxidases, which are dependent on concentration of bivalent cation Ca2+. Previous research regarding intramolecular interactions within specific NADPH oxidase- Nox5β has been performed by multiple research teams and different sources appear to contradict each other on the exact mode of interaction of Nox5β EF-hand upon presence of Ca2+. Therefore the exact interaction model of terminals of Nox5β is unclear. In addition, the effect of presence of Ca2+ on the interaction terminals in another representative of NADPH oxidases- AtRBOHD, which possess highly analogous molecular structure of catalytic C-terminus to Nox5β, has never been thoroughly studied, as well as interactive cross-compatibility of the C and N terminals from these two distinct species of NADPH oxidases. The objectives of this research are to analyze intramolecular interactions of N- and C- terminals in Arabidopsis RBOHD and Human Nox5β upon presence of ionic calcium, compare Ca2+-induced terminals interactions in said oxidases and to establish possible cross-compatibility of terminals in these two distinct NADPH oxidase species. Practical aspects of this research included cloning the C- and N- cytoplasmic regions of Nox5β and AtRBOHD into bacterial expression vectors utilizing the PIPE cloning method, heterologous production of epitope-tagged tails of NOX5β and RBOHD in E. Coli BL21 and finally in-vitro pull-down assays to analyse the interactions of the tails upon the presence of Ca2+ as well as interactive cross-compatibility of these tails. By utilizing methods mentioned above, this research has demonstrated that interactions of terminals motifs both in Nox5β and AtRBOHD are possible even in calcium-deprived environment, which was achieved through use calcium-binding agent (EDTA) and the effect of calcium on interactions of terminals both in RBOHD and Nox5β is very limited if not insignificant. This research has also demonstrated that the cross-compatible interactions between terminals of Nox5β and AtRBOHD are possible. Results of this research indicate a strong structural conservation within NADPH oxidases, which indicates similar intramolecular interaction mechanisms within two highly diverged species. These findings may prove to be useful as a background for the future research regarding ROS producing enzymes and evolutional conservation in structures of oxidases.

Leaf senescence is a developmental and physiological phase in plants to end leaf development. Environment factors such as drought stress, extreme temperature, and pathogen threat and internal factors including age and reactive oxygen species induce leaf senescence. Some phytohormones such as jasmonic acid and salicylic acid play a key function in cell death in plants. WRKY transcription factors is known as one of the largest transcription factor family in plants which regulates a variety of plants processes. WRKY75 which belong to WRKY transcription factors has shown multiple functions in plant development like regulation of Pi starvation responses and root development and flowering. In my thesis, I focused on the role of WRKY75 in senescence and stress responses. WRKY75 was identified as a positive regulator of cell death in Arabidopsis. WRKY75 can promote salicylic acid biosynthesis by promote transcript levels of SID2 and also cause hydrogen peroxide accumulation by suppressing the transcription of CAT2. Hydrogen peroxide and salicylic acid can promote WRKY75 transcription at the same time. To evaluate the function of WRKY75 transcription factor in SA signalling and cell death, three lesion mimic mutants acd5, cat2, dnd1 and their corresponding wrky75 double mutant were used. Interestingly, no different phenotypes were found between acd5, cat2, dnd1 and their corresponding wrky75 double mutants in cell death and hydrogen peroxide accumulation detection in Arabidopsis leaves. Meanwhile, marker genes transcription levels were not different in both short day and long day growth condition. However, different phenotypes were observed in botrytis infection. Based on these results, we formed a hypothesis that gene redundancy could influence genetic characterization of WRKY75. To overcome this problem, SRDX-WRKY75 chimeric repressor transgenic lines were generated. The SRDX domain act as a dominant negative regulator to suppress WRKY75 target genes. In future research, these new lines can be used to test transcript levels for putative WRKY75 target genes.

An increased attention has been drawn towards porous silicon (PSi) based materials for biomedical applications, due to their promising features demonstrated through several scientific studies. Here, we further investigated the biological responses of PSi nanoparticles (NPs) with different surface chemistries, including immunomodulatory effects, inflammation mitigation and biocompatibility. In this collaborative study, the PSi NPs were investigated both in vitro and in vivo, using different molecular biology and biochemistry techniques, e.g., qPCR, ELISA, cell sorting and cell viability assays. Our results showed the capabilities of these PSi NPs to relieve the inflammatory conditions, whereas significant decrease was recorded of pro-inflammatory cytokines: TNF-α, IL-1β and IL-6. Likewise, these PSi NPs revealed a considerable consumption aptitude of pro-inflammatory reactive oxygen species molecules. Administrating PSi NPs in an acute liver inflammation (ALI) model, showed no conspicuous influence on cellular viability. Thus, the outcome of this study demonstrates the potential biocompatibility of PSi nanomaterials, in addition to their outstanding features as potential candidates for further incorporating in ALI applications.