# Review on Cyanobacterial Studies in Portugal: Current Impacts and Research Needs
## 2 Cyanobacterial Studies
### Proteomic Studies  
Proteomics research methods have been providing complementary information concerning the toxicology of cyanotoxins and mechanisms of action of these molecules (Figure 2). It has been an essential research strategy to elucidate ecotoxicological outcomes in aquatic invertebrates (bivalves) and plants, and also cytotoxic and genotoxic effects of cyanotoxins in cell models such as _Saccharomyces cerevisiae_. Methods of global protein analysis, based on two-dimensional gel electrophoresis (2 DE) and MALDI-TOF mass spectrometry (MS) were used, for example, to characterize the proteome of agricultural plant species, and describe the adverse effects related to the use of contaminated water in crop irrigation. This methodology enabled to reveal putative adverse alterations in the proteome of rice plants (12 and 20-day-old) exposed for 7 days to _M. aeruginosa_ bloom extract with 0.26-78 \(\mu\)g/L MCLR [34]. Moreover, the same methodology revealed alterations in ATP synthase, Cytochrome b6-f complex iron-sulfur and oxygen-evolving enhancer protein markers, and other proteins related to carbohydrate metabolism in tomato plants, pointing to possible damages in ATP synthesis, energy metabolism and photosynthesis caused by the toxin MCLR [35]. Yet, a leaf proteome profile in lettuce confirmed some of the molecular disturbances previously observed and additional alterations in stress/defense response, protein synthesis and signal transduction, related to MCLR and CYN exposure. A concomitant decrease in plant growth was also reported [36].  
A target proteomics approach, combining affinity chromatography and protein purification, 2 DE and MALDI-TOF mass spectrometry, was used to investigate the role of glutathione s-transferases (GSTs) in the response of bivalves [37; 38]. The studies carried out revealed protein profiles constituted by multiple GST isoforms in bivalve species. Alterations in individual isoforms were revealed by this methodology suggesting that the GST system may play a role in the detoxification processes of cyanotoxins in this group of aquatic invertebrates [37; 38]. A large-scale proteomic profiling, combining liquid chromatography and high-throughput MS analysis, was used to depict other molecular processes related to the response of bivalves to cyanotoxins like MCs and CYN. Sub-toxic effects of cyanotoxins were related for instance to changes in protein folding and stabilization, cytoskeleton structure, and gene transcription/translation [39].  
Protonic and gene expression studies shed more light concerning the response of eukaryotic cells to MCs. Using the cell model _Saccharomyces cerevisiae_, several metabolic responses were revealed related to exposure to low MCLR concentrations. These studies revealed dose responses in Base Excision Repair (BER) DNA-repair system, suggesting genotoxic effects [40; 41], and changes in a broad group of proteins with functions linked to gene translation and DNA replication, oxidative stress, cell cycle regulation and carbohydrate metabolism [41]. Alterations in protein markers such as translation initiation factor 1 (RLII), thioredoxin reductase 1 (TRR1) and UV excision repair protein (RAD23) confirmed the link between oxidative stress and DNA damage mechanisms [41].