Site-directed mutagenesis of the D2 protein in the green alga 'Chlamydomonas reinhardtii'

The D2 polypeptide together with the D1 polypeptide are key components of the photosystem II (PS II) complex which is involved in photosynthetic oxygen evolution. This thesis describes the use of site-directed mutagenesis to introduce specific amino acid changes in the D2 protein of Chlamydomonas reinhardtii. The mutations were constructed at positions D2-Leu205 and D2-Thr2. Plasmids carrying mutated copies of the psbD gene (which encodes D2) were introduced into the chloroplast genome of C. reinhardtii by means of a particle gun. In order to identify the transformants, a selectable marker encoding resistance to spectinomycin, was inserted either upstream or downstream of the psbD gene. Genetic characterisation of the mutants was carried out using Southern blotting, PCR and DNA sequencing. D2-Leu205 is thought to be analogous to residue Tyr210 of the M subunit of the reaction centre (RC) of the purple photosynthetic bacterium Rhodobacter sphaeroides. Previous mutagenesis experiments have shown that the replacement of M-Tyr210 by leucine reduces the rate of the primary charge separation in purple bacterial RCs. Urns, the mutation Leu205Tyr was constructed in order to study whether the rate of primary charge separation is increased in this mutant. The Leu205Tyr mutant could grow photoautotrophically at wild type rates. However, its light-saturated PS II activity was reduced to ~62% compared to the wild type. PS II RCs isolated from a "wild type” control transformant and from the Leu205Tyr mutant showed in ultrafast transient absorption measurements that the rate of formation of the radical pair was slowed down in the mutant (lifetime of 40±7 ps compared to 21±2 ps for the wild type PS II RCs). Also the quantum yield of charge separation was reduced by about 50%. Fluorescence and thermoluminescence measurements in whole cells were also consistent with a reduced quantum yield of charge separation in PS II. Mutations Thr2Ala and Thr2Ser were constructed in order to study the role of D2 phosphorylation in C. reinhardtii. Biochemical characterisation of the Thr2Ala and Thr2Ser mutants indicated that both strains could grow photoautotrophically at wild type rates. Moreover, oxygen evolution, fluorescence and thermoluminescence measurements suggested that these site-directed mutations have only minor effects on PS II function. In addition to these experiments, attempts were carried out to find out whether D2 can be phosphorylated in C. reinhardtii. These attempts included in vivo labelling of whole cells with [32P]-orthophosphate, in vitro labelling of thylakoid membranes with [y-32P]-ATP, pulse-chase labelling of cells with [uC]-acetate and the use of a monospecific antibody against phosphothreonine. The data obtained from these experiments did not provide any indication that D2 can be phosphorylated in C. reinhardtii.Open acces

PSTVd infection in Nicotiana benthamiana plants has a minor yet detectable effect on CG methylation

Viroids are small circular RNAs infecting a wide range of plants. They do not code for any protein or peptide and therefore rely on their structure for their biological cycle. Observed phenotypes of viroid infected plants are thought to occur through changes at the transcriptional/translational level of the host. A mechanism involved in such changes is RNA-directed DNA methylation (RdDM). Till today, there are contradictory works about viroids interference of RdDM. In this study, we investigated the epigenetic effect of viroid infection in Nicotiana benthamiana plants. Using potato spindle tuber viroid (PSTVd) as the triggering pathogen and via bioinformatic analyses, we identified endogenous gene promoters and transposable elements targeted by 24 nt host siRNAs that differentially accumulated in PSTVd-infected and healthy plants. The methylation status of these targets was evaluated following digestion with methylation-sensitive restriction enzymes coupled with PCR amplification, and bisulfite sequencing. In addition, we used Methylation Sensitive Amplification Polymorphism (MSAP) followed by sequencing (MSAP-seq) to study genomic DNA methylation of 5-methylcytosine (5mC) in CG sites upon viroid infection. In this study we identified a limited number of target loci differentially methylated upon PSTVd infection. These results enhance our understanding of the epigenetic host changes as a result of pospiviroid infection

Building an adverse outcome pathway network for COVID-19

The COVID-19 pandemic generated large amounts of data on the disease pathogenesis leading to a need for organizing the vast knowledge in a succinct manner. Between April 2020 and February 2023, the CIAO consortium exploited the Adverse Outcome Pathway (AOP) framework to comprehensively gather and systematically organize published scientific literature on COVID-19 pathology. The project considered 24 pathways relevant for COVID-19 by identifying essential key events (KEs) leading to 19 adverse outcomes observed in patients. While an individual AOP defines causally linked perturbed KEs towards an outcome, building an AOP network visually reflect the interrelatedness of the various pathways and outcomes. In this study, 17 of those COVID-19 AOPs were selected based on quality criteria to computationally derive an AOP network. This primary network highlighted the need to consider tissue specificity and helped to identify missing or redundant elements which were then manually implemented in the final network. Such a network enabled visualization of the complex interactions of the KEs leading to the various outcomes of the multifaceted COVID-19 and confirmed the central role of the inflammatory response in the disease. In addition, this study disclosed the importance of terminology harmonization and of tissue/organ specificity for network building. Furthermore the unequal completeness and quality of information contained in the AOPs highlighted the need for tighter implementation of the FAIR principles to improve AOP findability, accessibility, interoperability and re-usability. Finally, the study underlined that describing KEs specific to SARS-CoV-2 replication and discriminating physiological from pathological inflammation is necessary but requires adaptations to the framework. Hence, based on the challenges encountered, we proposed recommendations relevant for ongoing and future AOP-aligned consortia aiming to build computationally biologically meaningful AOP networks in the context of, but not limited to, viral diseases.info:eu-repo/semantics/publishedVersio

Association of microbiome organisms to Regulatory T Cell function in cancer and inflammation.

Regulatory T cells (Tregs) serve as negative regulators of T cell activation and play a crucial role in maintaining peripheral tolerance and regulating autoimmunity. In cancer, Tregs are implicated in the control of inflammation but mainly contribute to tumor escape. This creates a great challenge for immunotherapeutic strategies which aim to enhance anti-tumor immune responses by suppressing Tregs without compromising their ability to maintain peripheral immune homeostasis. Restoration of immune homeostasis is crucial for the management of chronic inflammatory disorders and inflammation-related cancers (e.g. colorectal), whereby Tregs seem to act by down-regulating inflammation. Gut microbiome organisms have recently shown prominence as key modulators of Treg function, conferring either a pro- or an anti-carcinogenic potential, thus influencing the fate of tumor immunotherapy approaches. Understanding the diverse role of Tregs and mapping relations with other cell types or microbiome organisms in various cancers, as well as inflammatory and autoimmune diseases is of paramount importance as there is need for novel combinatorial immunomodulatory strategies against cancer. We used Vizit, a free visual bibliographic search and science communication tool to identify related genes, cell types and microbiome organisms that could have an impact on the role of Tregs in colorectal cancer and inflammation. The graph is fully interactive. It can be reached at: http://www.biovista.com/vizit-research/#!bv_gid=f13b839e6c340aa0214fafa1451a5d80 and can be modified and shared, forming the basis of an open platform for science communication in the field of cancer immunotherapy

Relation mining over a corpus of scientific literature

The amount of new discoveries (as published in the scientific literature) in the area of Molecular Biology is currently growing at an exponential rate. This growth makes it very difficult to filter the most relevant results, and the extraction of the core information, for inclusion in one of the knowledge resources being maintained by the research community, becomes very expensive. Therefore, there is a growing interest in text processing approaches that can deliver selected information from scientific publications, which can limit the amount of human intervention normally needed to gather those results. This paper presents and evaluates an approach aimed at automating the process of extracting semantic relations (e.g. interactions between genes and proteins) from scientific literature in the domain of Molecular Biology. The approach, using a novel dependency-based parser, is based on a complete syntactic analysis of the corpus

Stress-related transcriptomic changes associated with GFP transgene expression and active transgene silencing in plants

Abstract Plants respond to biotic and abiotic stress by activating and interacting with multiple defense pathways, allowing for an efficient global defense response. RNA silencing is a conserved mechanism of regulation of gene expression directed by small RNAs important in acquired plant immunity and especially virus and transgene repression. Several RNA silencing pathways in plants are crucial to control developmental processes and provide protection against abiotic and biotic stresses as well as invasive nucleic acids such as viruses and transposable elements. Various notable studies have shed light on the genes, small RNAs, and mechanisms involved in plant RNA silencing. However, published research on the potential interactions between RNA silencing and other plant stress responses is limited. In the present study, we tested the hypothesis that spreading and maintenance of systemic post-transcriptional gene silencing (PTGS) of a GFP transgene are associated with transcriptional changes that pertain to non-RNA silencing-based stress responses. To this end, we analyzed the structure and function of the photosynthetic apparatus and conducted whole transcriptome analysis in a transgenic line of Nicotiana benthamiana that spontaneously initiates transgene silencing, at different stages of systemic GFP-PTGS. In vivo analysis of chlorophyll a fluorescence yield and expression levels of key photosynthetic genes indicates that photosynthetic activity remains unaffected by systemic GFP-PTGS. However, transcriptomic analysis reveals that spreading and maintenance of GFP-PTGS are associated with transcriptional reprogramming of genes that are involved in abiotic stress responses and pattern- or effector-triggered immunity-based stress responses. These findings suggest that systemic PTGS may affect non-RNA-silencing-based defense pathways in N. benthamiana, providing new insights into the complex interplay between different plant stress responses