Studies on the potential of alternative oxidase (AOX) as a functional marker candidate for efficient aventitious rooting in olive (Olea europaea L.)

O envolvimento e a importância da oxidase alternativa (AOX) na resposta das plantas a diversas condições de stress têm vindo a ser demonstrado. Tendo em conta esse conhecimento, colocou-se a seguinte hipótese: “o enraizamento adventício (EA) é a uma reação da planta ao stress abiótico no qual a AOX tem um papel determinante”. Com a finalidade de testar a hipótese e avaliar o potencial da AOX para o desenvolvimento de um marcador funcional (MF), relacionado com a eficiência no enraizamento adventício (EA) em Olea europaea L., quatro genes AOX foram isolados (OeAOX1a, OeAOX1b, OeAOX1c e OeAOX2). O envolvimento da AOX na resposta fisiológica ao EA foi estudado em dois sistemas distintos, num utilizando estacas semi-lenhosas e noutro microestacas. As cultivares de oliveira, ‘Galega vulgar’, recalcitrante ao enraizamento por estacaria semilenhosa e ‘Cobrançosa’, de fácil enraizamento por este processo, foram utilizadas nos ensaios. Os resultados obtidos em ambos os sistemas mostraram que o EA é significativamente reduzido pelo tratamento com um inibidor da AOX, o ácido salicilhidroxâmico (SHAM), enquanto que tratamentos com peróxido de hidrogénio ou piruvato, ambos indutores da AOX, resultaram num aumento das taxas de EA. Resultados similares foram obtidos de microestcas de ‘Galega vulgar’. O SHAM, reduziu significativamente o EA, resultado que foi confirmado por análises histológicas, porém não inibiu a formação de calli. As análises de metabólitos mostraram uma redução significativa na quantidade de fenilpropanóides e de lignina após o tratamento com SHAM, indicando que a atividade da AOX durante o EA estará associada ao metabolismo de adaptação desses metabólitos. O estudo da variabilidade nos genes AOX, realizado ao nível do ADN e ARN, centrou-se no gene OeAOX2. A maior frequência de polimorfismos foi identificada na extremidade 3'UTR. Diferenças no nível de acumulação de transcrito durante o EA foram observadas apenas quando se utilizou um primer reverse localizado na 3'UTR, o que indica a ocorrência de uma modificação pos-transcripcional. Em suma, os resultados obtidos permitem considerar a AOX como enzima chave no processo de reprogramação celular associado ao EA. Em suma, os resultados obtidos permitem considerar a AOX como enzima chave no processo de reprogramação celular associado ao EA. Os resultados desta tese são encorajadores e abrem uma linha de investigação promissora com um grande potencial de aplicação que deverá ser considerado a nível regional/nacional/internacional; ‘Studies on the potential of alternative oxidase (AOX) as a functional marker candidate for efficient adventitious rooting in olive (Olea europaea L.)’ Abstract :It has been suggested that alternative oxidase (AOX) plays an important role during plant stress responses. In this thesis the hypothesis is that root induction is a plant cell reaction linked to abiotic stress and that the activity of stress-induced AOX is important for adventitious rooting (AR). In order to investigate AOX as a functional marker (FM) to assist AR efficiency in Olea europaea L., four AOX gene members (OeAOX1a, OeAOX1b, OeAOX1c e OeAOX2) were isolated from ‘Galega vulgar’, a cultivar with low AR efficiency. The involvement of AOX in AR physiological responses was studied in two different system, using a semi-hardwood cuttings and another microcuttings. The olive cultivars 'Galega Vulgar', recalcitrant rooting by semi-hardwood and 'Cobrançosa', easy to root for this process were used in the tests. Rooting was significantly reduced by treatment with salicylhydroxamic acid (SHAM), whereas treatment with hydrogen peroxide or pyruvate, increased the degree of rooting. A similar approach was adopted using microshoots of the cultivar ‘Galega vulgar’. SHAM significantly reduced AR, which was confirmed by histological analysis, but failed to exhibit any effect on the preceding calli stage. Metabolite analyses showed that the amounts of phenylpropanoids and lignin were significantly reduced following SHAM treatment, indicating that the influence of AOX activity on root formation was associated with adaptive phenylpropanoid- and lignin- metabolism. A detailed study of DNA and RNA level was carried out on OeAOX2. 3’-UTR was the most important source for polymorphisms. Differential transcript accumulation of OeAOX2 during AR induction was observed when using primers in the 3’UTR indicating posttranscriptional modification. In summary, the results obtained allow to consider AOX as a key enzyme in cellular reprogramming associated with the EA process. The results of this thesis are encouraging and open a promising line of research with a great potential for application to be considered at regional / national / international level

Unlocking nature’s glycosylation potential : characterization and engineering of novel sucrose/trehalose synthases

Glycosylation - the addition of a sugar molecule onto an acceptor substrate - is a promising strategy to improve the activity, solubility, stability, flavor and/or pharmacokinetic behavior of chemicals such as pharmaceuticals, neutraceuticals or cosmetics. Glycosylation can efficiently be performed in an aqueous environment under mild reaction conditions by enzymes called GlycosylTransferases (GTs). However, the industrial application of these enzymes in vitro is mainly hampered by their need for nucleotide-activated sugars (e.g. UDP-glucose) as donor substrates, which are highly expensive and rarely available in large quantities. In this doctoral thesis, two strategies to make in vitro reactions with GTs more cost-efficient were evaluated: the use of Sucrose Synthase (SuSy) as intermediate enzyme to produce UDP-glucose from the cheap substrate sucrose and the engineering of GTs to alter the sugar donor specificity towards cheaper glycosyl-phosphates. To this end, several new bacterial SuSy enzymes were cloned, expressed, purified and characterized. In addition, one of them was subjected to extensive mutagenesis to improve or change properties such as substrate affinity, substrate specificity and stability. The enzyme Trehalose glycosylTransferring synthase, on the other hand, was used as a test-case to scrutinize the possibility of changing the donor specificity of GTs from nucleotide sugars towards glycosyl-phosphates through mutagenesis

Survey of Clustering and Motif Finding for Microarray Technologies

Department of Computer Scienc

Plant Responses and Tolerance to Salt Stress: Physiological and Molecular Interventions

Overall, the 19 contributions in this Special Issue “Plant Responses and Tolerance to Salt Stress: Physiological and Molecular Interventions” discuss the various aspects of salt stress responses in plants. It also discusses various mechanisms and approaches to conferring salt tolerance on plants. These types of research studies provide further directions in the development of crop plants for the saline environment in the era of climate change

The development and optimization of microbial molecular biomarkers for the in situ assessment of trace metal toxicity

Merged with duplicate record 10026.1/2607 on 06.20.2017 by CS (TIS)Microorganisms are fundamental components of many geochemical transformations occurring in the aquatic environment. Microbial redox and methylation of metals within the environment can alter metal speciation, mobility and ultimately, toxicity to eukaryotes. It is therefore practical that any environmental monitoring framework advocating the application of `early-warning biomarker system' should incorporate a holistic view of the environment beginning with microbial activity. This thesis describes the development of protocols for assessing the in situ condition of microbial ecosystems within a gradient of metal contaminated sites radiating downstream of the Anaconda Smelter, a USEPA-designated superfund site and within two control sites. Experiments focus on evaluating the incidence (i. e. prevalence and absence) of genes related to general stress and specific metal detoxification reactions. Moreover, a number of selected genes were quantified directly from the environment and statistically correlated with metal concentrations. Furthermore, the influence of metals on structuring microbial communities was also investigated by evaluating temporal communities shifts in response to changing metal concentrations using denaturant gradient gel electrophoresis (DGGE). The data recorded the highest prevalence of all genes was found at the most polluted site directly downstream of the Anaconda Smelter. Furthermore, significant correlations were observed between gene prevalence and metals (arsenic, copper and zinc) (P < 0.05) and organic carbon concentration (P < 0.05). A number of genes were successfully amplified from sediment with significantly higher gene copy number (/ ng DNA) at the more polluted sites when compared to corresponding control sites. Examination of community diversity found that long-term metal-contaminated sediments, adjacent to the Smelter, had microbial communities twice as diverse as corresponding reference sites. In addition, multivariate statistical techniques identified factors important to community structuring, concluding that geographic position and localized geochemistry fundamentally influence the structuring of communities. This thesis represents a significant advance in the use of microorganisms as `early warning systems' of deterioration in ecosystem health, while the application of advanced molecular methods facilitates their intergration within a traditional ecotoxicological framework.Montana State Universit

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

Relevance of IgA in allergy: regulation by mucosal factors

Allergy is mediated by allergen-specific antibodies of various classes of which immunoglobulin E (IgE) produced by B cells upon stimulation by T helper type 2 cells is of crucial importance. Immunoglobulins have varying antigen and allergen specificity (introduced in chapter 1). IgE has to be specific for an allergen (sensitization) and bind simultaneously to several epitopes on the allergen to cause cross-linking of IgE immobilized on high affinity IgE receptors expressed on mast cells and basophils to induce degranulation (Chapter 2). IgE molecules can be specific for a protein of one species or bind to similar regions on allergens from different species, thereby causing cross-reactivity as discussed for shrimp and mussel tropomyosin in chapter 2 and fig and ficus in chapter 3. In most cases of cross-reactivity the primary sensitizing allergen can be identifying by inhibition assays (Chapter 3). The presence of allergen-specific IgE in serum of allergic patients does not necessarily cause clinically relevant symptoms of allergy when blocking immunoglobulins of non-IgE isotypes are present, like IgG4, which is induced by specific immunotherapy. Until now, serum-derived IgA has not been shown to be able to block IgE-allergen interactions and the potential protective role of allergen-specific IgA is still debated. The aim of this thesis was to study the potential protective role of the mucosal immune system and especially IgA production by B cells against allergy. The mechanisms underlying differential regulation of the two IgA subclasses IgA1 and IgA2 production characteristic for humans are largely unknown. Most likely factors in the local mucosal tissue are involved in differentiating between IgA1 and IgA2 production. Evidence for the role of dendritic cells (DC) in IgA production is increasing. We generated and characterized two DC types not previously described in literature by using the mucosal factors retinoic acid (RA) and transforming growth factor (TGF)-ß (Chapter 5). Those RA- and TGF-ß-derived dendritic cells have tolerogenic characteristics, as shown by reduced inflammatory cytokine production (IL-12 and TNF-α), but non-significant reduction in IL-10 production, and reduced expression of activation marker CD86 and maturation marker CD83 after stimulation with bacterial ligands. To study the role of epithelial cell- and DC-derived molecules in induction of production of IgA by B cells and plasma cells, the mucosa-related cytokines APRIL, BAFF, IL-10, TGF-ß, and vitamin A-derived RA were added to B cells. Addition of RA resulted in differentiation of B cells into plasma cells (CD38+) and enhanced secretion of IgA1 and IgA2 when also IL-10 and APRIL or BAFF was present. Our data indicate that production of IgA1 is increased in the presence of BAFF (probably by plasma cells), whereas IgA2 production is increased in the presence of APRIL which is produced by either DCs or epithelial cells in vivo (probably by de-novo induction). When RA and IL-10 were added, B cells upregulated homing integrin ß7 on the membrane, which is believed to preferably direct homing to the small intestine. In literature, increased levels of APRIL have been associated with decreased prevalence of eczema, whereas increased levels of TSLP have been associated with presence of eczema. In chapter 6, it is shown that APRIL is involved in induction of IgA2, and that TSLP potently inhibits IgA1, and to a lesser extent also IgA2 production, which is consistent with reports showing that increased levels of IgA correlate with less eczema. Also in chapter 4, an association was found between reduced levels of allergen-specific IgA2 in serum and presence of eczema and asthma. Also the tissue (systemic, e.g. serum versus mucosal, e.g. saliva) where IgA is determined is differentially linked to the clinical status in house dust mite-allergic patients. In addition, allergen-specific levels of IgA2 rather than IgA1 appeared to be associated with absence of allergy. Within house dust mite-allergic patients, the ratio between serum-IgE and saliva-IgA2 was associated with severity of local (mucosal) clinical symptoms. IgA production is diminished in the absence of intestinal bacteria and colonization of the gut induces production of IgA. The ontogeny of the bacteria-specific repertoire of intestinal IgA in relation to composition of the intestinal microbiota has not been studied before. In chickens we analysed the CDR3-repertoire development in the first ten weeks post hatch. No association between bacterial composition and IgA CDR3 repertoire was found, indicating that bacteria may induce IgA production but not cause extensive modification in the specificity of IgA (Chapter 7). Transitional changes in the composition of the microbiota were restored once IgA production was initiated, suggesting that IgA is directly involved in regulation of the intestinal microbiota composition. Diet-derived RA is a key regulator of tolerance and IgA production in the mucosa. In addition, dietary ingredients can directly interact with cells of the intestinal immune system. In chapter 8 we show that bovine IL-10 binds to the human IL-10 receptor and thereby modulates bacterial ligand induced activation of monocytes and DCs. Bovine milk also contains immunoglobulins that are specific for bacterial ligands and potential allergens that are also encountered by the human mucosal immune system. Bovine IgG efficiently binds to human IgG receptors and can modulate myeloid cell activation by LPS (Chapter 9). Immunoglobulin from bovine milk may therefore provide potent opportunities to either induce tolerance or antigen-specific immune responses resulting in the production of protective IgA, thereby assisting in providing protection against pathogenic infections. This protection may be mediated by bovine IgG alone, or in cooperation with IL-10, TGF-ß and vitamin A-derived RA. The findings from chapters 2-9 are discussed and applied for the field of allergy, both for the clinic and experimental research. The finding that human IgA1 and IgA2 are differentially regulated by innate factors and differentially correlated with the presence or absence of allergy and severity of clinical symptoms is a promising finding. This finding may provide new opportunities for allergen-specific immunotherapy. In addition, efficacy of other mucosal vaccination strategies could be affected by the innate mucosal mechanisms of regulation of IgA production. </p

The microbiology of diabetic foot infections: a Ghanaian perspective

Diabetic foot ulcer (DFU), a major complication of both types 1 and 2 diabetes, develops in about 15–25% of people living with the disease. In Ghana, DFUs contribute to most hospital admissions (53%) among diabetics with high rates of amputation (33.3%) and death (8.8%). Diabetic foot ulcers are predisposed to infections from bacteria in the environment which normally colonise these wounds as multicellular communities called biofilms. Biofilms have been found to have increased resistance to antimicrobial agents probably due to the presence of an extracellular matrix that retards or prevents the entry of antimicrobial agents into the bacterial community, antibiotic resistance genes and/or the presence of persister cells that are unresponsive to antimicrobial agents. The work presented here studied the role of 2 multidrug resistant DFU isolates, Klebsiella pneumoniae and Proteus mirabilis in maintaining the chronicity of diabetic foot ulcers. Using 3 in vitro biofilm models; the conventional microtitre plate and Minimum Biofilm Eradication Concentration (MBEC™) High-Throughput assays and the Quasi–Vivo® continuous flow system, K. pneumoniae and P. mirabilis were found to be positive for acyl–homoserine lactone production, biofilm and persister cell producers and could resist and/or tolerate antibiotics such as ceftazidime and levofloxacin up to 1280 times their minimum inhibitory concentration. K. pneumoniae and P. mirabilis were also found to express the interspecies AI–2 quorum sensing molecules which significantly increased biofilm formation and fold induction of bioluminescence in a luxS mutant V. harveyi reference strain. Quorum sensing (QS) inhibition assays using baicalin hydrate, cinnamaldehyde and 2(5H)–furanone showed considerable inhibition of K. pneumoniae and P. mirabilis biofilm formation but failed to completely inhibit their growth. The combinatorial effects of antibiotics and QS inhibitors/antimicrobial peptides such as polymyxin B and polymyxin B nonapeptide determined as fractional inhibitory concentration (FIC) index suggests that, additive and synergistic effects produced by the combination of two antimicrobial agents have the potential to eradicate biofilms. Data from the FIC indices determined from the combination assays can provide the basis for the formulation of topical treatment for DFUs