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.

Biogeographical distribution of Rimicaris exoculata resident gut epibiont communities along the Mid-Atlantic Ridge hydrothermal vent sites

Rimicaris exoculata is a deep-sea hydrothermal vent shrimp which enlarged gill chamber houses a complex trophic epibiotic community. Its gut harbours an autochthonous and distinct microbial community. This species dominates hydrothermal ecosystems megafauna along the Mid-Atlantic Ridge, regardless of contrasted geochemical conditions prevailing in them. Here, the resident gut epibiont community at four contrasted hydrothermal vent sites (Rainbow/TAG/Logatchev/Ashadze) was analysed and compiled with previous data to evaluate the possible influence of site location, using 16S rRNA surveys and microscopic observations (TEM, SEM and FISH analyses). Filamentous epibionts inserted between the epithelial cells microvilli were observed on all examined samples. Results confirmed resident gut community affiliation to Deferribacteres, Mollicutes, Epsilonproteobacteria and to a lesser extent Gammaproteobacteria lineages. Still a single Deferribacteres phylotype was retrieved at all sites. Four Mollicutes-related OTUs were distinguished, one being only identified on Rainbow specimens. The topology of ribotypes median-joining networks illustrated a community diversification possibly following demographic expansions, suggesting a more ancient evolutionary history and/or a larger effective population size at Rainbow. Finally, the gill chamber community distribution was also analysed through ribotypes networks based on sequences from R. exoculata collected at Rainbow/Snake Pit/TAG/Logatchev/Ashadze sites. Results allow refining hypotheses on the epibiont role and transmission pathways.

Understanding the role of auxins and oxidative enzymes on adventitious root formation in olive (olea europaea L.) cultivars

Olive (Olea europaea L.), one of the main crops in the Mediterranean basin, is mainly propagated by cuttings, a classical propagation method that relies on the ability of the cuttings to form adventitious roots. While some cultivars are easily propagated by this technique, some of the most interesting olive cultivars are considered difficult-to-root which poses a challenge for their preservation and commercialization. Therefore, increasing the current knowledge on adventitious root formation is extremely important for species like olive. This research focuses on evaluating the role of free auxins and oxidative enzymes on adventitious root formation of two olive cultivars with different rooting ability - ‘Galega vulgar’ (difficult-to-root) and ‘Cobrançosa’ (easy-to-root). In this context, free auxin levels and enzyme activities were determined in in vitro-cultured ‘Galega vulgar’ microshoots and in semi-hardwood cuttings of cvs. ‘Galega vulgar’ and ‘Cobrançosa’. To attain this goal, an analytical method for the quantification of free indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) was developed, which is based on dispersive liquid-liquid microextraction followed by microwave derivatization (DLLME-MAD) and gas chromatography-mass spectrometry (GC/MS) analysis. The developed method was validated in terms of linearity, recovery, limit of detection (LOD) and limit of quantification (LOQ) and proved to be useful in the analysis of two very different types of plant tissues. The results from auxin quantification in olive samples point at a relationship between free auxin levels and rooting ability of both microshoots and semihardwood cuttings. A defective IBA-IAA conversion, resulting in a peak of free IAA during initiation phase, seems to be associated with low rooting ability. Likewise, differences in the activity of oxidative enzymes also appear to be related with rooting ability. Higher polyphenol oxidases (PPO) activity is likely related with an easyto- root behavior, while the opposite is true for peroxidases (POX) (including IAA oxidase (IAAox)) activity. A possible hypothesis for adventitious root formation in olive microcuttings is presented herein for the first time. Free auxins, oxidative enzymes, alternative oxidase (AOX) and reactive oxygen species (ROS) are some of the factors that may be involved in this highly complex physiological process. Interestingly, while temporal changes in auxin levels were similar between microshoots and semihardwood cuttings, the conclusions obtained from enzyme activity results in microshoots didn’t translate to semi-hardwood tissues, showing the emerging need for adaptation of classical agronomical research studies to modern techniques; Resumo: Procurando compreender o papel das auxinas e enzimas oxidativas na formação de raízes adventícias em cultivares de oliveira (Olea europaea L.) A oliveira (Olea europaea L.) é uma das principais culturas da bacia Mediterrânica e é propagada maioritariamente por estacaria, um processo altamente dependente da capacidade das estacas para formar raízes adventícias. Enquanto algumas cultivares são fáceis de propagar desta forma, algumas das cultivares de oliveira mais interessantes são consideradas difíceis de enraizar, o que dificulta a sua preservação e comercialização e torna extremamente importante aprofundar o conhecimento sobre o enraizamento adventício desta espécie. Este trabalho foca-se na avaliação do papel das auxinas livres e das enzimas oxidativas na formação de raízes adventícias em duas cultivares de oliveira com diferente capacidade de enraizamento - ‘Galega vulgar’ (difícil de enraizar) e ‘Cobrançosa’ (fácil de enraizar). Neste contexto, determinaram-se os níveis de auxinas livres e as actividades de enzimas oxidativas em microestacas de ‘Galega vulgar’ cultivadas in vitro bem como em estacas semi-lenhosas das cvs. ‘Galega vulgar’ e ‘Cobrançosa’. Para tal foi necessário desenvolver uma metodologia analítica para a quantificação de ácido indol-3-acético (IAA) e ácido indol-3-butírico (IBA), baseada em microextracção dispersiva líquido-líquido (DLLME) seguida de derivatização em microondas (MAD) e análise por cromatografia gasosa acoplada a espectrometria de massa (GC/MS). O método desenvolvido foi validado em termos de linearidade, recuperação, limite de detecção (LOD) e limite de quantificação (LOQ), e mostrou-se eficaz na análise de dois tipos de tecidos vegetais bastante diferentes. Os resultados da análise de auxinas em amostras de oliveira apontam para uma possível relação entre os níveis de auxinas livres e a capacidade de enraizamento, tanto em microestacas como em estacas semi-lenhosas. Uma conversão IBA-IAA deficiente, que resulta num pico de IAA durante a fase de iniciação, parece estar associada à baixa capacidade de enraizamento. Por outro lado, a capacidade de enraizamento também parece estar relacionada com diferenças na actividade de enzimas oxidativas. Comportamentos fáceis de enraizar estão associados a actividade mais elevada das polifenoloxidases (PPO), enquanto o oposto é verdade para a actividade das peroxidases (POX) (incluindo a IAA oxidase (IAAox)). Neste trabalho propõe-se pela primeira vez uma possível explicação para o enraizamento adventício em microestacas de oliveira. Auxinas livres, enzimas oxidativas, oxidase alternativa (AOX) e espécies reactivas de oxigénio (ROS) são alguns dos factores envolvidos neste processo fisiológico altamente complexo. Curiosamente, enquanto as alterações temporais nos níveis de auxinas foram semelhantes entre microestacas e estacas semi-lenhosas, o mesmo não se observou relativamente à actividade enzimática, o que mostra a necessidade de adaptação dos estudos agronómicos tradicionais às técnicas correntes.

Ich hoffe dass alles da gut lauft. Eine Analyse von Aufsätzen finnischer Gymnasiasten

Diese Pro-Gradu-Arbeit basiert auf Deutschaufsätzen von finnischen Gymnasiasten. Es soll der Versuch unternommen werden, die Schreibkompetenz der Lernenden zu analysieren. Es wird betrachtet, welche Verbstrukturen sie in ihren Aufsätzen benutzen und wie sie sie anwenden. Auch die Stellung des Verbs wird untersucht. Diese werden mit den Inhalten, die im Unterricht gelehrt worden sind, verglichen. Das Beherrschen der Verbstrukturen hat einen starken Einfluss auf die Bestimmung der sprachlichen Kompetenz. Das Primärmaterial besteht aus zwölf Aufsätzen und neun Lehrbüchern. Die Aufsätze wurden während des dritten und sechsten B2-Deutschkurses geschrieben. Die SchülerInnen haben unterschiedliche Hintergründe: Einige lernen Deutsch erst seit ein Paar Jahren, während andere sogar deutsche Verwandte haben. Der deutsche Hintergrund hat die Sprachkompetenz positiv beeinflusst, aber auch z. B. die Motivation, sprachliche Begabung und die Lehrwerke tragen hierzu bei. Auch Transfer wurde untersucht. Die Aufsätze wurden Satz per Satz analysiert. Es wurde betrachtet, welche Strukturen vorkamen und wie vielseitig sie benutzt wurden. Die Lernenden hatten in den ersten Texten sehr einfache Sätze geschrieben, aber sie haben Variation z. B. durch die Inversion gebracht. In den näcshten Aufsätzen war der Satzbau komplexer, aber dadurch waren die Sätze manchmal nicht sofort zu verstehen. Der sprachliche Fortschritt wurde mit Hilfe des Analysemodells von Rösch (2011) analysiert und die Sprachkompetenz wurde in die Niveaus des gemeinsamen europäischen Referenzrahmens eingeordnet. Der finnische Lehrplan stellt eigene Ziele zum Fremdsprachenunterricht und die Lernenden dieser Untersuchungsgruppe haben die Ziele schon nach dem dritten Kurs erreicht. Zwischen dem dritten und sechsten Kurs haben sie das folgende Niveau nicht erreicht, aber die Schreibkompetenz hat sich vertieft. Mit ein bisschen Übung würden sie bestimmt das folgende Niveau erreichen. Die Kompetenzunterschiede zwischen den Lernenden waren deutlich zu sehen.

Diet, microbes and gut immune responses in the pathogenesis of experimental type 1 diabetes

Type 1diabetes (T1D) is an autoimmune disease, which is influenced by a variety of environmental factors including diet and microbes. These factors affect the homeostasis and the immune system of the gut. This thesis explored the altered regulation of the immune system and the development of diabetes in non-obese diabetic (NOD) mice. Inflammation in the entire intestine of diabetes-prone NOD mice was studied using a novel ex-vivo imaging system of reactive oxygen and nitrogen species (RONS), in relation to two feeding regimens. In parallel, gut barrier integrity and intestinal T-cell activation were assessed. Extra-intestinal manifestations of inflammation and decreased barrier integrity were sought for by studying peritoneal leukocytes. In addition, the role of pectin and xylan as dietary factors involved in diabetes development in NOD mice was explored. NOD mice showed expression of RONS especially in the distal small intestine, which coincided with T-cell activation and increased permeability to macromolecules. The introduction of a casein hydrolysate (hydrolysed milk protein) diet reduced these phenomena, altered the gut microbiota and reduced the incidence of T1D. Extra-intestinally, macrophages appeared in large numbers in the peritoneum of NOD mice after weaning. Peritoneal macrophages (PM) expressed high levels of interleukin-1 receptor associated kinase M (IRAK-M), which was indicative of exposure to ligands of toll-like receptor 4 (TLR-4) such as bacterial lipopolysaccharide (LPS). Intraperitoneal LPS injections activated T cells in the pancreatic lymph nodes (PaLN) and thus, therefore potentially could activate islet-specific T cells. Addition of pectin and xylan to an otherwise diabetes-retarding semisynthetic diet affected microbial colonization of newly-weaned NOD mice, disturbed gut homeostasis and promoted diabetes development. These results help us to understand how diet and microbiota impact the regulation of the gut immune system in a way that might promote T1D in NOD mice.

Implication of gut microbiota in nonalcoholic fatty liver disease

International audience

Avaliação da ação bioinseticida de SBTI e vicilina de Erythrina velutina em enzimas digestivas e membrana peritrófica de larvas de Plodia interpunctella (Lepidoptera: Pyralidae)

Plodia interpunctella (Indian meal moth) is a cosmopolitan pest that attacks not only a wide range of stored grain as well other food products. Due to its economic importance several researches have focused in a method with ability to control this pest with few or no damage to the environment. The study of digestive enzymes inhibitors, lectins and chitin-binding proteins, has often been proposed as an alternative to reduce insect damage. In this study we report the major classes of digestive enzymes during larval growth in P. Interpunctella, being those proteinases actives at pH 9.5 and optimum temperature of 50 oC to both larvae of the 3rd instar and pre-pupal stage of development. In vitro and zymogram assays presented the effects of several inhibitors, such as SBTI, TLCK and PMSF to intestinal homogenate of 3rd instar larvae of 62%, 92% and 87% of inhibition and In pre-pupal stage of 87%, 62 % and 55% of inhibition, respectively. Zymograms showed inhibition of two low molecular masses protein bands by TLCK and that in presence of SBTI were retarded. These results are indicative of predominance of digestive serine proteinases in gut homogenate from Plodia interpunctella larvae. This serine proteinase was then used as a target to evaluate the effect of SBTI on larvae in in vivo assay. Effect of SBTI on mortality and larval mass was not observed at until 4% of concentration (w/w) in diets. Chitin, another target to insecticidal proteins, was observed by chemical method. Moreover, optic microscopy confirmed the presence of a peritrophic membrane. Established this target, in vivo effect of EvV, a chitin binding vicilin, evaluated during the larval development of P. interpunctella and was obtained a LD50 of 0,23% and WD50 of 0,27% to this protein. Mechanism of action was proposed through of the in vivo digestibility of EvV methodology. During the passage through the larval digestive tract was observed that EvV was susceptible to digestive enzymes and a reactive fragment, visualized by Western blotting, produced by digestion was recovered after dissociation of the peritrophic membrane. The bound of EvV to peritrophic membrane was confirmed by immunohystochemical assays that showed strong immunofluorescent signal of EvV-FITC binding and peritrophic membrane. These results are a indicative that vicilins could be utilized as potential insecticide to Plodia interpunctella and a control methods using EvV as bioinsecticide should be studied to reduce lost caused by storage insect pests

Optimization of Enzymes Inactivation in High Pressure Processes

The demand of highest quality foods in terms of taste and their properties preservation without the use of additives is constantly increasing. Consequently, new approaches to food processing have been developed, as for example high-pressure technology which has proven to be very valuable because it allows to maintain good properties of food like some vitamins and, at the same time, to reduce some undesirable bacteria. This technology avoids the use of high temperatures during the process (not like Pasteurization), which may have adverse effect on some nutritional properties of the food, its flavour, etc. The models for some enzymatic inactivations, which depend on the pressure and temperature profiles are presented. This work deals with the optimization of the inactivation of certain enzymes when high pressure treatment on food processing is applied. The optimization algorithms will minimize the inactivation not only of a certain isolated enzyme but also to several enzymes that can be involved simultaneously in the high-pressure process.

Influence of enzymes and technology on the composition of Cobrançosa and Galega vulgar virgin olive oils

Doutoramento em Engenharia Alimentar - Instituto Superior de Agronomia - UL

Influence of Pseudomonas putida AF7 inoculation on soil enzymes.

There has been some concern about the environmental impact of microbial agents. Pseudomonas may be used as bioremediator and as biopesticide. In this study, we report the use of soil enzyme assays as biological indicator of possible negative effects in soil functioning after the P. putida AF7 inoculation. For that, P. putida AF7 was originally isolated from the rizosphere of rice and was inoculated on three soil types: Rhodic Hapludox (RH), Typic Hapludox (TH); and Arenic Hapludult (AH). The acid phosphatase, b-glucosidase and protease enzymes activities were measured for three period of evaluation (7, 14 and 21 days). In general, the enzymatic activities pre- sented variation among the tested soils. The highest activities of b-glucosidase and acid phosphatase were observed in the RH and AH soils, while the protease activity was higher in the TH soil. Also, the soil charac- teristics were measured for each plot. The activity of enzymes from the carbon cycle was positively correlated with the N and the P and the enzyme from the nitrogen cycle was negatively correlated with N and C.org. The presented data indicate that soil biochemical properties can be an useful tool for use as an indicator of soil perturba- tions by microbial inoculation in a risk assessment.

Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

In this review, we detail the efforts performed to couple the purification and the immobilization of industrial enzymes in a single step. The use of antibodies, the development of specific domains with affinity for some specific supports will be revised. Moreover, we will discuss the use of domains that increase the affinity for standard matrices (ionic exchangers, silicates). We will show how the control of the immobilization conditions may convert some unspecific supports in largely specific ones. The development of tailor-made heterofunctional supports as a tool to immobilize–stabilize–purify some proteins will be discussed in deep, using low concentration of adsorbent groups and a dense layer of groups able to give an intense multipoint covalent attachment. The final coupling of mutagenesis and tailor made supports will be the last part of the review.

Importance of the Support Properties for Immobilization or Purification of Enzymes

Immobilization and purification of enzymes are usual requirements for their industrial use. Both purification and immobilization have a common factor: they use a solid activated support. Using a support for enzyme purification means having mild conditions for enzyme release and a selective enzyme–support interaction is interesting. When using a support for immobilization, however, enzyme desorption is a problem. The improvement of enzyme features through immobilization is a usual objective (e.g., stability, selectivity). Thus, a support designed for enzyme purification and a support designed for enzyme immobilization may differ significantly. In this review, we will focus our attention on the requirements of a support surface to produce the desired objectives. The ideal physical properties of the matrix, the properties of the introduced reactive groups, the best surface activation degree to reach the desired objective, and the properties of the reactive groups will be discussed.

A Mutational Hotspot and Strong Selection Contribute to the Order of Mutations Selected for during Escherichia coli Adaptation to the Gut

The relative role of drift versus selection underlying the evolution of bacterial species within the gut microbiota remains poorly understood. The large sizes of bacterial populations in this environment suggest that even adaptive mutations with weak effects, thought to be the most frequently occurring, could substantially contribute to a rapid pace of evolutionary change in the gut. We followed the emergence of intra-species diversity in a commensal Escherichia coli strain that previously acquired an adaptive mutation with strong effect during one week of colonization of the mouse gut. Following this first step, which consisted of inactivating a metabolic operon, one third of the subsequent adaptive mutations were found to have a selective effect as high as the first. Nevertheless, the order of the adaptive steps was strongly affected by a mutational hotspot with an exceptionally high mutation rate of 10-5. The pattern of polymorphism emerging in the populations evolving within different hosts was characterized by periodic selection, which reduced diversity, but also frequency-dependent selection, actively maintaining genetic diversity. Furthermore, the continuous emergence of similar phenotypes due to distinct mutations, known as clonal interference, was pervasive. Evolutionary change within the gut is therefore highly repeatable within and across hosts, with adaptive mutations of selection coefficients as strong as 12% accumulating without strong constraints on genetic background. In vivo competitive assays showed that one of the second steps (focA) exhibited positive epistasis with the first, while another (dcuB) exhibited negative epistasis. The data shows that strong effect adaptive mutations continuously recur in gut commensal bacterial species.

Antibiotic resistance in the gut microbiota

Antibiotic resistance is an increasing threat to our ability to treat infectious diseases. Thus, understanding the effects of antibiotics on the gut microbiota, as well as the potential for such populations to act as a reservoir for resistance genes, is imperative. This thesis set out to investigate the gut microbiota of antibiotic treated infants compared to untreated controls using high-throughput DNA sequencing. The results demonstrated the significant effects of antibiotic treatment, resulting in increased proportions of Proteobacteria and decreased proportions of Bifidobacterium. The species diversity of bifidobacteria was also reduced. This thesis also highlights the ability of the human gut microbiota to act as an antibiotic resistance reservoir. Using metagenomic DNA extracted from faecal samples from adult males, PCR was employed to demonstrate the prevalence and diversity of aminoglycoside and β-lactam resistance genes in the adult gut microbiota and highlighted the merits of the approach adopted. Using infant faecal samples, we constructed and screened a second fosmid metagenomic bank for the same families of resistance genes and demonstrated that the infant gut microbiota is also a reservoir for resistance genes. Using in silico analysis we highlighted the existence of putative aminoglycoside and β-lactam resistance determinants within the genomes of Bifidobacterium species. In the case of the β- lactamases, these appear to be mis-annotated. However, through homologous recombination-mediated insertional inactivation, we have demonstrated that the putative aminoglycoside resistance proteins do contribute to resistance. In additional studies, we investigated the effects of short bowel syndrome on infant gut microbiota, the immune system and bile acid metabolism. We also sequenced the microbiota of the human vermiform appendix, highlighting its complexity. Finally, this thesis demonstrated the strain specific nature of 2 different probiotic CLA-producing Bifidobacterium breve on the murine gut microbiota.

The impact of whey protein consumption and exercise on the composition and diversity of the gut microbiota: a high through-put DNA sequencing approach

Advances in culture independent technologies over the last decade have highlighted the pivotal role which the gut microbiota plays in maintaining human health. Conversely, perturbations to the composition or actions of the ‘normal/functioning’ microbiota have been frequently associated with the pathogenesis of several disease states. Therefore the selective modulation of enteric microbial communities represents a viable target for the development of novel treatments for such diseases. Notably, while bovine whey proteins and exercise have been shown to positively influence several physiological processes, such as energy balance, their effect on the composition or functionality of the gut microbiota remains largely unknown. In this thesis, a variety of ex vivo, murine and human models are used in conjunction with high-throughput DNA sequencing-based analysis to provide valuable and novel insights into the impact of both whey proteins and exercise on enteric microbial communities. Overall the results presented in this thesis highlight that the consumption both whey protein isolate (WPI), and individual component proteins of whey such as bovine serum albumin (BSA) and lactoferrin, reduce high fat diet associated body weight gain and are associated with beneficial alterations within the murine gut microbiota. Although the impact of exercise on enteric microbial communities remains less clear, it may be that longer term investigations are required for the true effect of exercise on the gut microbiota to be fully elucidated.