Study of molecular mechanisms to increase carbon use efficiency in microalgae

In order to better understand alga\u2019s biology and allow to design biotechnological approaches to improve biomass yield, in this PhD thesis we investigated the molecular mechanism involved in the microalgae carbon use efficiency. In the Chapter 1 we studied the model algae C. reinhardtii. In section A Photosystem II assembly were investigated. Indeed, no detailed studies of the assembly factors of PSII have been performed. In this work we focus on a putative assembly factor of the CP43 subunit, called LPA2 (low PSII accumulation 2), previously identified in A. thaliana. A candidate lpa2 gene in C. reinhardtii was identified by homology and its role was studied in vivo thank to a CRISPR-cas9 mutant. The data collected demonstrated that LPA2 protein is involved in both de novo biogenesis and repair of PSII. In the section B the relationship between chloroplast and mitochondrion metabolism was explored studying a mutant of C. reinhardtii knockout for a mitochondrial transcription factor. Previous studies demonstrated that the mutation affect the mitochondrial respiration and resulted in a light-sensitive phenotype. In this work we investigated how a mutation affecting the mitochondrial respiration perturbed light acclimation of the strain. Chapter 2 regards two species of Chlorella genus. In the section A we elucidated the molecular basis of the improved growth and biomass yield in mixotrophic condition, where the cross-talk between chloroplast and mitochondria metabolism is essential for efficient biomass production. C. sorokiniana is able to combine an autotrophic metabolism with the utilization of reduced carbon source (mixotrophic condition). The de novo assembly transcriptome allowed to identify the regulation of several genes involved in control of carbon flux. In section B genetic basis of the highly productive phenotype of C. vulgaris in low light vs. high light condition was examined. Nuclear and organelle genomes were obtained combining short-reads Illumina, long PacBio reads and Bionano optical mapping, allowing to assembly a near-chromosome scale genome of 14 scaffolds and the two complete circular organelle genomes. All the genes encoding for photosynthetic subunit, as well as, genes involved in the key metabolic pathway were identified. In section C the two Chlorella species was compared for their adaptation to high CO2 level. In C. sorokiniana in 3% CO2 were observed several reorganizations of the photosynthetic machinery leading to an improved carbon fixation, while mitochondrial respiration was essentially unaffected. Instead, in C. vulgaris the 3% CO2 induced an improved uptake of reducing power by chloroplast leading to a reduced mitochondrial respiration. Chapter 3 is focused on the marine algae. In the section A was isolated a chemical mutant of N. gaditana with a reduction chlorophyll content per cell combined with increased lipids productivity. The mutant did not show an increased biomass accumulation but induced an increased lipid content, a class of macromolecules with a higher energy content per gram. This is in any case an indication of improved light energy conversion in line with an improved light penetration in the photobioreactor and more homogenous light availability due to the reduced chlorophyll content per cell in the mutant. Moreover, thank to Illumina sequencing, we found putative genes responsible of the observed phenotype. In the section B cells of T. weissflogii were grown together with an artificial cyanine molecular antenna (Cy5) that extends the absorbance range of the photosynthetic apparatus exploiting light energy in the orange spectral region. The dye was incorporate in the algae increasing light dependent growth, oxygen and biomass production. Time-resolved spectroscopy data indicates that a Cy5-chlorophyll a energy transfer mechanism happen, compatible with a FRET process

Impact of ABCG2 and ABCB1 Polymorphisms on Imatinib Plasmatic Exposure: An Original Work and Meta-Analysis

Adequate imatinib plasma levels are necessary to guarantee an efficacious and safe treatment in gastrointestinal stromal tumor (GIST) and chronic myeloid leukemia (CML) patients. Imatinib is a substrate of the drug transporters ATP-binding cassette subfamily B member 1 (ABCB1) and ATP-binding cassette subfamily G member 2 (ABCG2) that can affect its plasma concentration. In the present study, the association between three genetic polymorphisms in ABCB1 (rs1045642, rs2032582, rs1128503) and one in ABCG2 (rs2231142) and the imatinib plasma trough concentration (Ctrough) was investigated in 33 GIST patients enrolled in a prospective clinical trial. The results of the study were meta-analyzed with those of other seven studies (including a total of 649 patients) selected from the literature through a systematic review process. The ABCG2 c.421C>A genotype demonstrated, in our cohort of patients, a borderline association with imatinib plasma trough levels that became significant in the meta-analysis. Specifically, homozygous carriers of the ABCG2 c.421 A allele showed higher imatinib plasma Ctrough with respect to the CC/CA carriers (Ctrough, 1463.2 ng/mL AA, vs. 1196.6 ng/mL CC + AC, p = 0.04) in 293 patients eligible for the evaluation of this polymorphism in the meta-analysis. The results remained significant under the additive model. No significant association could be described between ABCB1 polymorphisms and imatinib Ctrough, neither in our cohort nor in the meta-analysis. In conclusion, our results and the available literature studies sustain an association between ABCG2 c.421C>A and imatinib plasma Ctrough in GIST and CML patients

BNC2 is a putative tumor suppressor gene in high-grade serous ovarian carcinoma and impacts cell survival after oxidative stress

Rs3814113 is the single-nucleotide polymorphism (SNP) showing the strongest association with high-grade serous ovarian carcinoma (HGSOC) incidence and is located in an intergenic region about 44\u2009kb downstream of basonuclin 2 (BNC2) gene. Lifetime number of ovulations is associated with increased risk to develop HGSOC, probably because of cell damage of extrauterine M\ufcllerian epithelium by ovulation-induced oxidative stress. However, the impact of low-penetrance HGSOC risk alleles (e.g. rs3814113) on the damage induced by oxidative stress remains unclear. Therefore, the purpose of this study was to investigate whether rs3814113 genetic interval regulates BNC2 expression and whether BNC2 expression levels impact on cell survival after oxidative stress. To do this, we analyzed gene expression levels of BNC2 first in HGSOC data sets and then in an isogenic cell line that we engineered to carry a 5\u2009kb deletion around rs3814113. Finally, we silenced BNC2 and measured surviving cells after hydrogen peroxide (H2O2) treatment to simulate oxidative stress after ovulation. In this paper, we describe that BNC2 expression levels are reduced in HGSOC samples compared with control samples, and that BNC2 expression levels decrease following oxidative stress and ovulation in vitro and in vivo, respectively. Moreover, deletion of 5\u2009kb surrounding rs3814113 decreases BNC2 expression levels in an isogenic cell line, and silencing of BNC2 expression levels increases cell survival after H2O2 treatment. Altogether, our findings suggest that the intergenic region located around rs3814113 regulates BNC2 expression, which in turn affects cell survival after oxidative stress response. Indeed, HGSOC samples present lower BNC2 expression levels that probably, in the initial phases of oncogenic transformation, conferred resistance to oxidative stress and ultimately reduced the clearance of cells with oxidative-induced damages

The evidence-based role of catecholaminergic PET tracers in Neuroblastoma. A systematic review and a head-to-head comparison with mIBG scintigraphy

Background: Molecular imaging is pivotal in staging and response assessment of children with neuroblastoma (NB). [123I]-metaiodobenzylguanidine (mIBG) is the standard imaging method; however, it is characterised by low spatial resolution, time-consuming acquisition procedures and difficult interpretation. Many PET catecholaminergic radiotracers have been proposed as a replacement for [123I]-mIBG, however they have not yet made it into clinical practice. We aimed to review the available literature comparing head-to-head [123I]-mIBG with the most common PET catecholaminergic radiopharmaceuticals. Methods: We searched the PubMed database for studies performing a head-to-head comparison between [123I]-mIBG and PET radiopharmaceuticals including meta-hydroxyephedrine ([11C]C-HED), 18F-18F-3,4-dihydroxyphenylalanine ([18F]DOPA) [124I]mIBG and Meta-[18F]fluorobenzylguanidine ([18F]mFBG). Review articles, preclinical studies, small case series (< 5 subjects), case reports, and articles not in English were excluded. From each study, the following characteristics were extracted: bibliographic information, technical parameters, and the sensitivity of the procedure according to a patient-based analysis (PBA) and a lesion-based analysis (LBA). Results: Ten studies were selected: two regarding [11C]C-HED, four [18F]DOPA, one [124I]mIBG, and three [18F]mFBG. These studies included 181 patients (range 5-46). For the PBA, the superiority of the PET method was reported in two out of ten studies (both using [18F]DOPA). For LBA, PET detected significantly more lesions than scintigraphy in seven out of ten studies. Conclusions: PET/CT using catecholaminergic tracers shows superior diagnostic performance than mIBG scintigraphy. However, it is still unknown if such superiority can influence clinical decision-making. Nonetheless, the PET examination appears promising for clinical practice as it offers faster image acquisition, less need for sedation, and a single-day examination

Impaired Mitochondrial Transcription Termination Disrupts the Stromal Redox Poise in Chlamydomonas

In photosynthetic eukaryotes, the metabolite exchange between chloroplast and mitochondria ensures efficient photosynthesis under saturating light conditions. The Chlamydomonas reinhardtii mutant stm6 is devoid of the mitochondrial transcription termination factor MOC1 and aberrantly expresses the mitochondrial genome, resulting in enhanced photosynthetic hydrogen production and diminished light tolerance. We analyzed the modulation of mitochondrial and chlororespiration during the acclimation of stm6 and the MOC1-complemented strain to excess light. Although light stress stimulated mitochondrial respiration via the energy-conserving cytochrome c pathway in both strains, the mutant was unable to fine-tune the expression and activity of oxidative phosphorylation complex I in excess light, which was accompanied by an increased mitochondrial respiration via the alternative oxidase pathway. Furthermore, stm6 failed to fully activate chlororespiration and cyclic electron flow due to a more oxidized state of the chloroplast stroma, which is caused by an increased mitochondrial electron sink capacity. Increased susceptibility to photoinhibition of PSII in stm6 demonstrates that the MOC1-dependent modulation of mitochondrial respiration helps control the stromal redox poise as a crucial part of high-light acclimation in C. reinhardtii

Effect of Rootstock expressing hairpin RNA in conferring fungal resistance in Vitis vinifera

Fungal and oomycetes pathogens are the major phytopathological problem in grapes cultivation and their control is mainly addressed by performing repeated fungicide treatments. Post-transcriptional gene silencing driven by the mechanism of RNA interference (RNAi) represents a powerful alternative, that can be employed to reduce the virulence of the pathogen. Considering modern viticulture, which is based on the cultivation of grapevine cultivars grafted onto hybrid Vitis rootstocks, the opportunity of having rootstocks capable of producing active long or small dsRNAs targeting fungal RNAs could have important implications as new defense strategies. The generation of grapevine plants expressing hairpin gene constructs aiming at the downregulation of Botrytis cinerea Dicer-like genes 1 and 2 has been optimized in the Thompson Seedless cultivar. The plants composed by a combination of different independent transformed lines used as rootstock down to Wild-type scions have been demonstrated to show a major tolerance to grey mold in detached leaves experiments. Moreover, we are monitoring the presence of specific dsRNA in non-transgenic leaves, sampled in different developmental stages. Although much remains to be elucidated about the trans-grafting technique, molecular and pathogen resistance analyses could indicate whether dsRNA is transferred from the producer HIGS rootstock to the combined non-transgenic scion

Circulating-Free DNA Analysis in Hepatocellular Carcinoma: A Promising Strategy to Improve Patients’ Management and Therapy Outcomes

Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide, representing the third leading cause of cancer-related deaths. HCC genetic characterization at the tumor level has been recently completed, highlighting how a number of genes are frequently mutated in this pathology. Actionable somatic mutations found in a HCC tumor may represent targets for innovative drugs as well as prognostic/predictive markers. Nonetheless, surgical or bioptic tissue is hardly accessible in HCC and a single tumor sample is poorly representative of the tumor genetic heterogeneity. In this context, analyzing the circulating cell-free DNA (ccfDNA) and its tumor-derived fraction (ctDNA) could represent a promising strategy of liquid biopsy. Recent data suggested that the fluctuation of the ccfDNA quantity in the plasma of HCC patients could anticipate the detection of tumor progression. The presence of somatic mutations in p53 signaling, Wnt/&beta;-catenin, chromatin remodeling, response to oxidative stress and telomerase maintenance pathways can also be studied in ccfDNA bypassing the need to perform a tumor biopsy. The profiling of ccfDNA fragmentation and the methylation pattern could further improve the clinical management of HCC patients. Performing a dynamic monitoring in the course of systemic treatment with sorafenib or regorafenib is a possible way to provide insights into the resistance mechanism, and to identify predictive and prognostic genetic alterations, helping the clinicians in terms of treatment decision making. This review will discuss the most recent literature data about the use of ccfDNA to monitor and improve the treatment of HCC

Astaxanthin and eicosapentaenoic acid production by S4, a new mutant strain of Nannochloropsis gaditana

Background Astaxanthin is a ketocarotenoid with high antioxidant power used in different fields as healthcare, food/feed supplementation and as pigmenting agent in aquaculture. Primary producers of astaxanthin are some species of microalgae, unicellular photosynthetic organisms, as Haematococcus lacustris. Astaxanthin production by cultivation of Haematococcus lacustris is costly due to low biomass productivity, high risk of contamination and the requirement of downstream extraction processes, causing an extremely high price on the market. Some microalgae species are also primary producers of omega-3 fatty acids, essential nutrients for humans, being related to cardiovascular wellness, and required for visual and cognitive development. One of the main well-known producers of omega-3 fatty eicosapentaenoic acid (EPA) is the marine microalga Nannochloropsis gaditana (named also Microchloropsis gaditana): this species has been already approved by the Food and Drug Administration (FDA) for human consumption and it is characterized by a fast grow phenotype. Results Here we obtained by chemical mutagenesis a Nannochloropsis gaditana mutant strain, called S4, characterized by increased carotenoid to chlorophyll ratio. S4 strain showed improved photosynthetic activity, increased lipid productivity and increased ketocarotenoids accumulation, producing not only canthaxanthin but also astaxanthin, usually found only in traces in the WT strain. Ketocarotenoids produced in S4 strain were extractible in different organic solvents, with the highest efficiency observed upon microwaves pre-treatment followed by methanol extraction. By cultivation of S4 strain at different irradiances it was possible to produce up to 1.3 and 5.2 mgL(-1) day(-1) of ketocarotenoids and EPA respectively, in a single cultivation phase, even in absence of stressing conditions. Genome sequencing of S4 strain allowed to identify 199 single nucleotide polymorphisms (SNP): among the mutated genes, mutations in a carotenoid oxygenase gene and in a glutamate synthase gene could explain the different carotenoids content and the lower chlorophylls content, respectively. Conclusions By chemical mutagenesis and selection of strain with increased carotenoids to chlorophyll ratio it was possible to isolate a new Nannochloropsis gaditana strain, called S4 strain, characterized by increased lipids and ketocarotenoids accumulation. S4 strain can thus be considered as novel platform for ketocarotenoids and EPA production for different industrial applications