A Loop-mediated Isothermal Amplification Method for Rapid Direct Detection and Differentiation of Non-pathogenic and Verocytotoxigenic Escherichia coli in Beef and Bovine Faeces

The aim of this study was to develop a multiplex loop-mediated isothermal amplification (LAMP) method capable of detecting Escherichia coli generally and verocytotoxigenic E. coli (VTEC) specifically in beef and bovine faeces. The LAMP assay developed was highly specific (100%) and able to distinguish between E. coli and VTEC based on the amplification of the phoA, and stx1 and/or stx2 genes, respectively. In the absence of an enrichment step, the limit of detection 50% (LOD50) of the LAMP assay was determined to be 2.83, 3.17 and 2.83-3.17 log CFU/g for E. coli with phoA, stx1 and stx2 genes, respectively, when artificially inoculated minced beef and bovine faeces were tested. The LAMP calibration curves generated with pure cultures, and spiked beef and faeces, suggested that the assay had good quantification capability. Validation of the assay, performed using retail beef and bovine faeces samples, demonstrated good correlation between counts obtained by the LAMP assay and by a conventional culture method, but suggested the possibility of false negative LAMP results for 12.5-14.7% of samples tested. The multiplex LAMP assay developed potentially represents a rapid alternative to culture for monitoring E.coli levels in beef or faeces and it would provide additional information on the presence of VTEC. However, some further optimisation is needed to improve detection sensitivity.

Microbial phylogenetic and functional responses within acidified wastewater communities exhibiting enhanced phosphate uptake

Acid stimulated accumulation of insoluble phosphorus within microbial cells is highly beneficial to wastewater treatment but remains largely unexplored. Using single cell analyses and next generation sequencing, the response of active polyphosphate accumulating microbial communities under conditions of enhanced phosphorus uptake under both acidic and aerobic conditions was characterised. Phosphorus accumulation activities were highest under acidic conditions (pH 5.5 > 8.5), where a significant positive effect on bioaccumulation was observed at pH 5.5 when compared to pH 8.5. In contrast to the Betaproteobacteria and Actinobacteria dominated enhanced biological phosphorus removal process, the functionally active polyP accumulators at pH 5.5 belonged to the Gammaproteobacteria, with key accumulators identified as members of the families Aeromonadaceae and Enterobacteriaceae. This study demonstrated a significant enrichment of key polyphosphate kinase and exopolyphosphatase genes within the community metagenome after acidification, concomitant with an increase in P accumulation kinetics.

Genomics and virulence factors of Fusobacterium necrophorum

Fusobacterium necrophorum, a Gram negative, anaerobic bacterium, is a common cause of acute pharyngitis and tonsillitis and a rare cause of more severe infections of the head and neck. At the beginning of the project, there was no available genome sequence for F. necrophorum. The aim of this project was to sequence the F. necrophorum genome and identify and study its putative virulence factors contained using in silico and in vitro analysis. Type strains JCM 3718 and JCM 3724,F. necrophorum subspecies necrophorum (Fnn) and funduliforme (Fnf), respectively, and strain ARU 01 (Fnf), isolated from a patient with LS, were commercially sequenced by Roche 454 GS-FLX+ next generation sequencing and assembled into contigs using Roche GS Assembler. Sequence data was annotated semi-automatically, using the xBASE pipeline, BLASTp and Pfam. The F. necrophorum genome was determined to be approximately 2.1 – 2.3 Mb in size, with an estimated 1,950 ORFs and includes genes for a leukotoxin, ecotin, haemolysin, haemagglutinin, haemin receptor, adhesin and type Vb and Vc secretion systems. The prevalence of the leukotoxin gene was investigated in strains JCM 3718, JCM 3724 and ARU 01, as well as a clinical collection of 25 Fnf strains, identified using biochemical and molecular tests. The leukotoxin operon was found to be universal within the strain collection by PCR. HL-60 cells subjected to aliquots of concentrated high molecular weight culture supernatant, predicted to contain the secreted leukotoxins of strains JCM 3718, JCM 3724 and ARU 01, were killed in a dose-dependent manner. The cytotoxic effect of the leukotoxin against human donor white blood cells was also tested to validate the HL-60 assay. The differences in the results between the two assays were not statistically significant. Ecotin, a serine protease inhibitor, was found to be present in 100 % of the strain collection and had a highly conserved sequence with primary and secondary binding sites exposed on opposing sides of the protein. During enzyme inhibition studies, a purified recombinant F. necrophorum ecotin protein inhibited human neutrophil elastase, a protease that degrades bacteria at inflammation sites, and human plasma kallikrein, a component of the host clotting cascade. The recombinant ecotin also prolonged human plasma clotting times by up to 7-fold for the extrinsic pathway, and up to 40-fold for the intrinsic pathway. The genome sequence data provides important information about F. necrophorum type strains and enables comparative study between strains and subspecies. Results from the leukotoxin and ecotin assays can be used to build up an understanding of how the organism behaves during infection.

Asthma and rhinitis have different genetic profiles for IL13, IL17A and GSTP1 polymorphisms

BACKGROUND: Asthma and rhinitis have a complex etiology, depending on multiple genetic and environmental risk factors. An increasing number of susceptibility genes are currently being identified, but the majority of reported associations have not been consistently replicated across populations of different genetic backgrounds. PURPOSE: To evaluate whether polymorphisms of IL4R (rs1805015), IL13 (rs20541), IL17A (rs2275913) and GSTP1 (rs1695) genes are associated with rhinitis and/or asthma in adults of Portuguese ancestry. METHODS: 192 unrelated healthy individuals and 232 patients, 83 with rhinitis and 149 with asthma, were studied. All polymorphisms were detected by real time polymerase chain reaction (PCR) using TaqMan assays. RESULTS: Comparing to controls, significant association with asthma was observed for GSTP1 rs1695 AA genotype (odds ratio (OR) - 1.96; 95% CI - 1.18 to 3.25; p=0.010). The association sustains for allergic asthma (OR - 2.17; 95% CI - 1.23 to 3.80; p=0.007). IL13 rs20541 GG genotype was associated with less susceptibility to asthma (OR - 0.55, 95% CI - 0.33 to 0.94, p=0.028). Among patients, IL17A rs2275913 AA genotype was less associated with asthma than with rhinitis (OR - 0.20; 95% CI of 0.07 to 0.56; p=0.002). A similar association was found for IL13 rs20541 GG genotype (OR - 0.48; 95% CI of 0.25 to 0.93; p=0.031). There were no significant differences in the distribution of allelic and genotypic frequencies between patients and controls for the IL4R polymorphism' analyzed. CONCLUSION: These results support the existence of a significant association between GSTP1 rs1695 and IL13 rs20541 SNPs, with susceptibility to asthma, in the population studied. Different genotype profiles of IL17A and IL13 genes seem to influence the clinical pattern of disease expression mainly confined to the upper airways, as rhinitis, or including the lower airways, as asthma.

Cell- and Tissue-Specific Transcriptome Analyses of Medicago truncatula Root Nodules

Legumes have the unique ability to host nitrogen-fixing Rhizobium bacteria as symbiosomes inside root nodule cells. To get insight into this key process, which forms the heart of the endosymbiosis, we isolated specific cells/tissues at different stages of symbiosome formation from nodules of the model legume Medicago truncatula using laser-capture microdissection. Next, we determined their associated expression profiles using Affymetrix Medicago GeneChips. Cells were collected from the nodule infection zone divided into a distal (where symbiosome formation and division occur) and proximal region (where symbiosomes are mainly differentiating), as well as infected cells from the fixation zone containing mature nitrogen fixing symbiosomes. As non-infected cells/tissue we included nodule meristem cells and uninfected cells from the fixation zone. Here, we present a comprehensive gene expression map of an indeterminate Medicago nodule and selected genes that show specific enriched expression in the different cells or tissues. Validation of the obtained expression profiles, by comparison to published gene expression profiles and experimental verification, indicates that the data can be used as digital "in situ''. This digital "in situ'' offers a genome-wide insight into genes specifically associated with subsequent stages of symbiosome and nodule cell development, and can serve to guide future functional studies.

CYR61 and TAZ Upregulation and Focal Epithelial to Mesenchymal Transition May Be Early Predictors of Barrett’s Esophagus Malignant Progression

Barrett's esophagus is the major risk factor for esophageal adenocarcinoma. It has a low but non-neglectable risk, high surveillance costs and no reliable risk stratification markers. We sought to identify early biomarkers, predictive of Barrett's malignant progression, using a meta-analysis approach on gene expression data. This in silico strategy was followed by experimental validation in a cohort of patients with extended follow up from the Instituto Português de Oncologia de Lisboa de Francisco Gentil EPE (Portugal). Bioinformatics and systems biology approaches singled out two candidate predictive markers for Barrett's progression, CYR61 and TAZ. Although previously implicated in other malignancies and in epithelial-to-mesenchymal transition phenotypes, our experimental validation shows for the first time that CYR61 and TAZ have the potential to be predictive biomarkers for cancer progression. Experimental validation by reverse transcriptase quantitative PCR and immunohistochemistry confirmed the up-regulation of both genes in Barrett's samples associated with high-grade dysplasia/adenocarcinoma. In our cohort CYR61 and TAZ up-regulation ranged from one to ten years prior to progression to adenocarcinoma in Barrett's esophagus index samples. Finally, we found that CYR61 and TAZ over-expression is correlated with early focal signs of epithelial to mesenchymal transition. Our results highlight both CYR61 and TAZ genes as potential predictive biomarkers for stratification of the risk for development of adenocarcinoma and suggest a potential mechanistic route for Barrett's esophagus neoplastic progression.

Asthma and rhinitis have different genetic profiles for IL13, IL17A and GSTP1 polymorphisms

BACKGROUND: Asthma and rhinitis have a complex etiology, depending on multiple genetic and environmental risk factors. An increasing number of susceptibility genes are currently being identified, but the majority of reported associations have not been consistently replicated across populations of different genetic backgrounds. PURPOSE: To evaluate whether polymorphisms of IL4R (rs1805015), IL13 (rs20541), IL17A (rs2275913) and GSTP1 (rs1695) genes are associated with rhinitis and/or asthma in adults of Portuguese ancestry. METHODS: 192 unrelated healthy individuals and 232 patients, 83 with rhinitis and 149 with asthma, were studied. All polymorphisms were detected by real time polymerase chain reaction (PCR) using TaqMan assays. RESULTS: Comparing to controls, significant association with asthma was observed for GSTP1 rs1695 AA genotype (odds ratio (OR) - 1.96; 95% CI - 1.18 to 3.25; p=0.010). The association sustains for allergic asthma (OR - 2.17; 95% CI - 1.23 to 3.80; p=0.007). IL13 rs20541 GG genotype was associated with less susceptibility to asthma (OR - 0.55, 95% CI - 0.33 to 0.94, p=0.028). Among patients, IL17A rs2275913 AA genotype was less associated with asthma than with rhinitis (OR - 0.20; 95% CI of 0.07 to 0.56; p=0.002). A similar association was found for IL13 rs20541 GG genotype (OR - 0.48; 95% CI of 0.25 to 0.93; p=0.031). There were no significant differences in the distribution of allelic and genotypic frequencies between patients and controls for the IL4R polymorphism' analyzed. CONCLUSION: These results support the existence of a significant association between GSTP1 rs1695 and IL13 rs20541 SNPs, with susceptibility to asthma, in the population studied. Different genotype profiles of IL17A and IL13 genes seem to influence the clinical pattern of disease expression mainly confined to the upper airways, as rhinitis, or including the lower airways, as asthma.

The metabolic and epigenetic effects of the isocitrate dehydrogenase-1 mutation in glioma

Cancer cells have been noted to have an altered metabolic phenotype for over ninety years. In the presence of oxygen, differentiated cells predominately utilise the tricarboxylic acid (TCA) cycle and oxidative phosphorylation to efficiently produce energy and the metabolites necessary for protein and lipid synthesis. However, in hypoxia, this process is altered and cells switch to a higher rate of glycolysis and lactate production to maintain their energy and metabolic needs. In cancer cells, glycolysis is maintained at a high rate, even in the presence of oxygen; a term described as “aerobic glycolysis”. Tumour cells are rapidly dividing and have a much greater need for anabolism compared to normal differentiated cells. Rapid glucose metabolism enables faster ATP production as well as a greater redistribution of carbons to nucleotide, protein, and fatty acid synthesis, thus maximising cell growth. Recently, other metabolic changes, driven by mutations in genes related to the TCA cycle, indicate an alternative role for metabolism in cancer, the “oncometabolite”. This is where a particular metabolite builds up within the cell and contributes to the tumorigenic process. One of these genes is isocitrate dehydrogenase (IDH) IDH is an enzyme that forms part of the tricarboxylic acid (TCA) cycle and converts isocitrate to α-ketoglutarate (α-KG). It exists in three isoforms; IDH1, IDH2 and IDH3 with the former present in the cytoplasm and the latter two in the mitochondria. Point mutations have been identified in the IDH1 and IDH2 genes in glioma which result in a gain of function by converting α-KG to 2-hydroxyglutarate (2HG), an oncometabolite. 2HG acts as a competitive inhibitor of the α-KG dependent dioxygenases, a superfamily of enzymes that are involved in numerous cellular processes such as DNA and histone demethylation. It was hypothesised that the IDH1 mutation would result in other metabolic changes in the cell other than 2HG production, and could potentially identify pathways which could be targeted for therapeutic treatment. In addition, 2HG can act as a potential competitive inhibitor of α-KG dependent dioxygenases, so it was hypothesised that there would be an effect on histone methylation. This may alter gene expression and provide a mechanism for tumourogenesis and potentially identify further therapeutic targets. Metabolic analysis of clinical tumour samples identified changes associated with the IDH1 mutation, which included a reduction in α-KG and an increase in GABA, in addition to the increase in 2HG. This was replicated in several cell models, where 13C labelled metabolomics was also used to identify a possible increase in metabolic flux from glutamate to GABA, as well as from α-KG to 2HG. This may provide a mechanism whereby the cell can bypass the IDH1 mutation as GABA can be metabolised to succinate in the mitochondria by GABA transaminase via the GABA shunt. JMJ histone demethylases are a subset of the α-KG dependent dioxygenases, and are involved in removing methyl groups from histone tails. Changes in histone methylation are associated with changes in gene expression depending on the site and extent of chemical modification. To identify whether the increase in 2HG and fall in α-KG was associated with inhibition of histone demethylases a histone methylation screen was used. The IDH1 mutation was associated with an increase in methylation of H3K4, which is associated with gene activation. ChiP and RNA sequencing identified an increase in H3K4me3 at the transcription start site of the GABRB3 subunit, resulting in an increase in gene expression. The GABRB3 subunit forms part of the GABA-A receptor, a chloride channel, which on activation can reduce cell proliferation. The IDH1 mutation was associated with an increase in GABA and GABRB3 subunit of the GABA-A receptor. This raises the possibility of GABA transaminase as a potential therapeutic target. Inhibition of this enzyme could reduce GABA metabolism, potentially reducing any beneficial effect of the GABA shunt in IDH1 mutant tumours, and increasing activation of the GABA-A receptor by increasing the concentration of GABA in the brain. This in turn may reduce cell proliferation, and could be achieved by using Vigabatrin, a GABA transaminase inhibitor licensed for use in epilepsy.

Soybean ferritin expression in saccharomyces cerevisiae modulates iron accumulation and resistance to elevated iron concentrations.

Fungi, including the yeast Saccharomyces cerevisiae, lack ferritin and use vacuoles as iron storage organelles. This work explored how plant ferritin expression influenced baker's yeast iron metabolism. Soybean seed ferritin H1 (SFerH1) and SFerH2 genes were cloned and expressed in yeast cells. Both soybean ferritins assembled as multimeric complexes, which bound yeast intracellular iron in vivo and, consequently, induced the activation of the genes expressed during iron scarcity. Soybean ferritin protected yeast cells that lacked the Ccc1 vacuolar iron detoxification transporter from toxic iron levels by reducing cellular oxidation, thus allowing growth at high iron concentrations. Interestingly, when simultaneously expressed in ccc1Δ cells, SFerH1 and SFerH2 assembled as heteropolymers, which further increased iron resistance and reduced the oxidative stress produced by excess iron compared to ferritin homopolymer complexes. Finally, soybean ferritin expression led to increased iron accumulation in both wild-type and ccc1Δ yeast cells at certain environmental iron concentrations.

Study of tRNA modifying enzymes and codon usage bias in cancer

Recent evidences indicate that tRNA modifications and tRNA modifying enzymes may play important roles in complex human diseases such as cancer, neurological disorders and mitochondrial-linked diseases. We postulate that expression deregulation of tRNA modifying enzymes affects the level of tRNA modifications and, consequently, their function and the translation efficiency of their tRNA corresponding codons. Due to the degeneracy of the genetic code, most amino acids are encoded by two to six synonymous codons. This degeneracy and the biased usage of synonymous codons cause alterations that can span from protein folding to enhanced translation efficiency of a select gene group. In this work, we focused on cancer and performed a meta-analysis study to compare microarray gene expression profiles, reported by previous studies and evaluate the codon usage of different types of cancer where tRNA modifying enzymes were found de-regulated. A total of 36 different tRNA modifying enzymes were found de-regulated in most cancer datasets analyzed. The codon usage analysis revealed a preference for codons ending in AU for the up-regulated genes, while the down-regulated genes show a preference for GC ending codons. Furthermore, a PCA biplot analysis showed this same tendency. We also analyzed the codon usage of the datasets where the CTU2 tRNA modifying enzyme was found deregulated as this enzyme affects the wobble position (position 34) of specific tRNAs. Our data points to a distinct codon usage pattern between up and downregulated genes in cancer, which might be caused by the deregulation of specific tRNA modifying enzymes. This codon usage bias may augment the transcription and translation efficiency of some genes that otherwise, in a normal situation, would be translated less efficiently.

Silencing the gip gene of Phytophthora cinnamomi by iRNA and studying the subcellular localization of GIP and NPP1 proteins

Ink Disease is considered one of the most important causes of the decline of chestnut orchards. The break in yield of Castanea sativa Mill is caused by two species: Phytophthora cinnamomi and Phytophthora cambivora, being the first one the foremost pathogen of ink disease in Portugal. P. cinnamomi is one of the most aggressive and widespread plant pathogen with nearly 1,000 host species. This oomycete causes enormous economic losses and it is responsible for the decline of many plant species in Europe and worldwide. Up to now no efficient treatments are available to fight these pathogens. Because of the importance of chestnut at economical and ecological levels, especially in Portugal, it becomes essential to explore the molecular mechanisms that determine the interaction between Phytophthora species and host plants through the study of proteins GIP (glucanase inhibitor protein) and NPP1 (necrosis-inducing Phytophthora protein 1) produced by P. cinnamomi during the infection. The technique of RNA interference was used to knockdown the gip gene of P. cinnamomi. Transformants obtained with the silenced gene have been used to infect C. sativa, in order to determine the effect of gene silencing on the plant phenotype. To know more about the function of GIP and NPP1 involved in the mechanism of infection, the ORF’s of gip and npp1 genes have been cloned to the pTOR-eGFP vector for a future observation of P. cinnamomi transformants with fluorescent microscopy and determination of the subcellular localization. Moreover the prediction by bioinformatics tools indicates that both GIP and NPP1 proteins are secreted. The results allow to predict the secretory destination of both GIP and NPP1 proteins and confirm RNAi as a potential alternative biological tool in the control and management of P. cinnamomi. Keywords:

Role of Aeromonas hydrophila in bacterial septicemia of cultured carps in Khouzestan Province and investigation on protectivity of bacterin (s) prepared from acute isolate(s) of it

Motile Aeromonas are the most common bacteria of freshwater in the world that cause disease in fish and other cold-blooded and warm-blooded hosts. Among this group of bacteria, Aeromonas hydrophila is important in causing complications such as fin rot, skin ulcers and lethal hemorrhagic septicemia in fish. Several virulence factors involved in the pathogenesis of Aeromonas hydrophila, including extracellular enzymes (protease, lipase, elastase, gelatinase and nuclease) and toxins. From the exotoxins, hemolysin, aerolysin and cytolytic enterotoxin play an important role in pathogenesis. Detection of virulence markers by PCR as a key component of determining the pathogenesis of the bacteria and using indigenous vaccines for better immunization against this disease is important. In this study, a total of 200 fanned carps (126 common carp. 39 silver carp and 35 of grass carp) with symptoms suspected aeromonas septicemia were isolated from Khouzestan province farms. 125 bacteria belong to Aeromonas genus detected by biochemical and PCR methods. 31 of all isolates recognized as Aeromonas hydrophila with biochemical methods, I6srRNA detection and Lipase genes. Results showed that the role of Aeromonas sp. and Aeromonas hydrophila in fish with disease symptoms were 62.5% and 15.5% respectively. By using specific primers, three virulence genes including hemolysin, aerolysine and cytolytic enterotoxin were detected in these confirmed isolates, that 18 isolates (58/06%) hemolysin positive (hlyA +), 16 isolates (51/61%) aerolysine positive (aerA+) and 23 isolates (74/19%) for cytolytic enterotoxin gene (act+) were positive. The result of present study showed that most of the confirmed isolates genotype was hlyA+ act- with frequency equal to 51/61%. For investigating the protection effect of acut strain of bacteria, UV inactivated bacterin was used.

Identificação e expressão de genes relacionados à via esteroidogênica e resposta antioxidante após a exposição à Atrazina em Poecilia vivipara (Poeciliidae, Cyprinodontiformes)

O herbicida Atrazina (ATR) é um agrotóxico utilizado há cerca de 50 anos, responsável pelo controle seletivo de plantas daninhas em cultivo de arroz, milho e cana-de-açúcar, principalmente. Estudos recentes apontam diversos efeitos desse herbicida em invertebrados e vertebrados, através da contaminação do solo, bem como da lixiviação para os ecossistemas aquáticos. Foi demonstrado que a ATR é um desregulador endócrino, além de causar efeitos como estresse oxidativo, imunotoxicidade e distúrbios no metabolismo energético. No presente estudo, a espécie nativa Poecilia vivipara foi utilizada como modelo experimental para identificar e analisar a expressão de genes atuantes na via esteroidogênica (StAR e Cyp19a1) e genes atuantes no sistema de defesa antioxidante enzimático (SOD-1 e CAT), frente a exposição à diferentes concentrações de ATR. Sequências parciais dos genes-alvo foram obtidas e comparadas com sequências disponíveis de espécies próximas. Foram analisadas a expressão órgãoespecífica para cada um dos genes isolados, bem como a expressão dos genes frente à exposição ao herbicida atrazina. Os animais foram expostos a ATR em concentrações de 2, 10 e 100 µg/L e a expressão dos genes em gônadas e fígado desses animais foram analisadas em 24 e 96 horas de exposição. As sequências obtidas dos genes StAR, Cyp19a1, SOD-1 e CAT apresentaram 821, 80, 954, 350 pares de bases respectivamente, com identidades que variam de 86 a 100% com espécies filogeneticamente próximas a P. vivipara. Os animais apresentaram uma maior expressão dos genes StAR e Cyp19a1 nas gônadas e no fígado, enquanto a menor expressão se mostrou em órgãos como intestino e baço. Já os genes SOD e CAT apresentaram uma maior expressão no fígado, e menor expressão no intestino. Em relação à expressão gênica frente à exposição à ATR, os resultados apontaram para uma indução dos genes StAR, SOD e CAT em 24 horas, nas gônadas e no fígado, enquanto 8 que a expressão do gene Cyp19a1 foi aumentada apenas após 96 horas de exposição. Foi demonstrado que o herbicida ATR, mesmo em baixas concentrações, é capaz de desregular a expressão de genes que codificam tanto para proteínas componentes da via de síntese de hormônios esteróides, quanto para enzimas atuantes na resposta antioxidante celular de P. vivipara. 

Transcription of alternative oxidase (AOX) and plastid terminal oxidase (PTOX) during stress-regulated root tissue growth in Daucus carota L. - An approach to identify functional marker candidates for breeding on carrot yield stability

A presente tese explora a hipótese de utilização dos genes da oxidase alternativa (AOX) e da oxidase terminal da plastoquinona (PTOX) como genes-alvo para o desenvolvimento de marcadores funcionais (MF) para avaliar a performance do crescimento em cenoura, fator determinante da produtividade. Para avaliar se os referidos genes estão associados com o crescimento da cenoura procedeu—se ao seu isolamento e posterior análise dos seus perfis de transcrição em diversos sistemas biológicos. O sistema in vitro selecionado, denominado sistema de culturas primárias, permitiu avaliar alterações na quantidade de transcritos desses genes durante os processos de reprogramação celular e crescimento. Ao nível da planta foi também estudado o efeito do frio na expressão precoce dos genes AOX. Ambos os genes DcAOX1 e DcAOX2a revelaram uma resposta rápida e um padrão semelhante apos stresse (inoculação in vitro e resposta ao frio). Foi igualmente verificado um incremento na expressão do gene DcPTOX durante a fase inicial do processo de reprogramação celular. Estudos de expressão dos genes AOX durante o desenvolvimento da raiz da cenoura revelaram que o gene DcAOX2a será potencialmente o gene mais envolvido neste processo. De modo a avaliar a hipótese de envolvimento do gene DcPTOX no crescimento da raíz procederam—se a estudos de expressão ao nível do tecido meristemático. Todavia, para um mais completo entendimento da ligação entre DcPTOX e o crescimento secundário e/ou acumulação de carotenos, a expressão do gene DcPTOX foi também avaliada em raízes de cenoura durante o desenvolvimento, utilizando cultivares caracterizadas por distintos conteúdos de carotenos. Os resultados obtidos demonstraram a associação do gene DcPTOX a ambos os processos. O envolvimento da PTOX no crescimento adaptativo da raiz foi analisado com um ensaio que permitiu identificar, no tecido meristemático, uma resposta precoce do gene DcPTOX face a uma diminuição da temperatura. Adicionalmente, foi efetuada a seleção de genes de referência para uma analise precisa da expressão génica por RT-qPCR em diversos sistemas biológicos de cenoura, e a importância do seu estudo ao nível do sistema biológico foi realçada. Os resultados desta tese são encorajadores para prosseguir os estudos de utilização dos genes AOX e PTOX como MF no melhoramento da performance do crescimento adaptativo em cenoura, fator determinante para a produtividade; ABSTRACT: This thesis explores the hypothesis of using the alternative oxidase (AOX) and theplastid terminal oxidase (PTOX) as target genes for functional marker (FM) development for yield-determining growth performance in carrot. To understand if these genes are associated to growth, different AOX gene family members and the single PTOX gene were isolated, and their expression patterns evaluated in diverse carrot plant systems. An in-vitro primary culture system was selected to study AOX and PTOX transcript changes during cell reprogramming and growth performance. At plant level, a putative early response of AOX to chilling was also evaluated. In fact, both DcAOXl and DcAOXZa were early responsive and showed similar patterns under stress conditions (in vitro inoculation and chilling). A role for DcPTOX during earliest events of cell reprogramming was also suggested. Next, the expression profiles of AOX gene family members during carrot tap root development were investigated. DcAOXZa was identified as the most responsive gene to root development. In order to evaluate if DcPTOX is associated with carrot tap root growth performance, DcPTOX transcript levels were measured in the central root meristem. To further understand whether DcPTOX is associated with secondary growth and/or carotenoids accumulation, DcPTOX expression was also studied in deveIOping carrot tap roots in cultivars with different carotenoids contents. The results indicated that DcPTOX associates to both carotenoid biosynthesis and secondary growth during storage root development. To obtain further insights into the involvement of PTOX on adaptive growth, the early effects of temperature decrease were explored in the root meristem, where a short—term early response in DcPTOX was found, probably associated with adaptive growth. Furthermore, a selection of the most suitable reference genes for accurate RT—qPCR analysis in several carrot experimental systems was performed and discussed. The present research provides the necessary toolbox for continuing studies in carrot AOX and PTOX genes as promising resources for FM candidates in order to assist breeding on yield—determining adaptive growth performance.

A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in the rpoB, katG, inhA, ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 for rpoB, katG, inhA, ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.