Browsing by Subject "Altern"

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    Membrane transport and long-distance translocation of urea in Arabidopsis thaliana
    (2011) Bohner, Anne; von Wirén, Nicolaus
    Urea is a soil nitrogen (N) form available to plant roots and a secondary N metabolite liberated in plant cells by protein degradation, especially during senescence. Despite the fact that urea also represents the most widespread form in N fertilizers used in agricultural plant production, membrane transporters that might contribute to urea uptake in plant roots or urea retranslocation in senescent leaves have so far been characterized only in heterologous systems. The first part of the thesis investigated a role of the H+/urea cotransporter AtDUR3 in N nutrition of Arabidopsis thaliana plants. T-DNA insertion lines with a defective expression in AtDUR3 showed impaired growth on urea as a sole nitrogen source. In transgenic lines expressing an AtDUR3-promoter-GFP construct, promoter activity was upregulated under N deficiency and localized to the rhizodermis, including root hairs, as well as to the cortex in more basal root zones. The AtDUR3 protein accumulated in plasma membrane-enriched protein fractions, and AtDUR3 gene expression in N-deficient roots was repressed by ammonium and nitrate but induced after supply of urea. Higher urea accumulation in roots of wild-type plants relative to the T-DNA insertion lines confirmed that urea was the transported substrate of AtDUR3. Influx of 15N-labeled urea allowed the calculation of an affinity constant of 4 µM. These results indicated that AtDUR3 is the major transporter for high-affinity urea uptake in Arabidopsis roots and suggested that the high substrate affinity of AtDUR3 reflects an adaptation to the low urea levels usually found in unfertilized soils. A physiological function of urea and its transporters in leaves was investigated in the second part of the thesis. Currently it is unclear whether transport and metabolism of urea might limit the overall retranslocation of N during senescence. AtDUR3 transcript levels were only slightly de-repressed under N starvation, but strongly increased in senescent leaves. Urea concentrations in leaf samples of different plant and leaf age showed a strong increase after plants turned into generative growth. In parallel, mRNA as much as the protein abundance of AtDUR3 increased with leaf age. The analysis of leaf petiole exudates revealed that urea was indeed a translocated N form and urea-N represented approx. 13% of the total amino acid-N irrespective of the N status of the plant. Urea concentrations determined in apoplastic wash fluids supported a role of AtDUR3 in urea retrieval from the leaf apoplast, and transgenic AtDUR3-promoter-GUS lines indicated a localization of AtDUR3 promoter activity in the vasculature of old leaves. Thus, AtDUR3 might keep internal urea in the cytosol by urea retrieval from the apoplast, allowing urea to be transported to the vascular bundle, where it is either passively loaded to the phloem or converted into amino acids for long-distance N translocation. A strong daytime-dependent phenotype with shorter leaf petioles of an Arabidopsis line overexpressing AtDUR3 led to an in silico analysis of the AtDUR3 promoter sequence revealing that salicylic acid (SA) appears to induce AtDUR3 gene expression in senescent leaves. SA is well known for its involvement in the initiation of senescence. A strongly enhanced uptake capacity for 15N-labeled urea in N-sufficient Arabidopsis roots after SA pretreatment indicated that SA might be able to mimic N-deficiency conditions, paving the way to the possibility that SA builds a regulatory link between developmental and N deficiency-induced senescence.
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    The effect of aging in the murine gut microbiome
    (2020) Hernández Arriaga, Angélica; Camarinha-Silva, Amélia
    Aging is characterized by several physiological changes. During the lifespan, the biological systems from the body of humans and other animals remain dynamic. Throughout the early stages of life, the microbiome develops into a complex ecosystem with thousands of species. Variations related to diet, environmental changes, medications affect the diversity and composition of the microbiota through the lifespan. Some old individuals with higher incidence of chronic diseases have a loss of the stability of the microbiome and an imbalance occurs between the different colonizers of the gut, also named dysbiosis. One of the most distinctive changes occurring with age is the prevalent low grade inflammation, which is named inflamm-aging. This not only changes the microbial composition of the GIT but also affects the permeability. Murine models are well established and help us to understand the complex dynamics between the host and the microbial communities inhabiting the gastrointestinal tract. These models allow us to analyze microbial communities from tissue and mucosa, from all sections of the gut, which is limited in humans. Methods standardization is an important topic in microbiome research. In chapter 2 it was compared the efficiency of two sample methods, cotton swab and tissue biopsy, in characterizing the mouth microbiota. In recent years, the mouth microbiome is being seen as a diagnostic tool for not only oral diseases but also systemic diseases. As physiological changes occur with aging, the microbiome from the mouth is affected and there is an increase of pathogens present in the oral surfaces. In murine models, cotton swab is a common tool used for sampling the microbiome of the oral cavity. In our study, we observed similar microbial community structure using both methodologies. However, the species Streptococcus danieliae, Moraxella osloensis, and some unclassified members of Streptococcus were affected by the different sampling procedures. In this trial, we included mice at two different ages, 2 months old being considered young and 15 months old considered middle aged mice. We observed changes in the genera Actinobacillus, Neisseria, Staphylococcus, and Streptococcus related to the age of the animal and the sampling type. These results showed the importance of sampling standardization in microbiome research and that age has a strong effect on the microbial ecology of the oral cavity. In chapter 3, it was studied the bacterial communities from duodenum and colon of mice at 2, 15, 24 and 30 months of age in combination with the results of the expression levels of antimicrobial peptides in small intestine and markers of intestinal barrier function. Besides, in this chapter were also assessed the indices of liver damage, inflammation and expression levels of lipopolysaccharide binding protein (Lbp) as well as of toll-like receptors (Tlr) 1-9 in liver tissues. At 24 and 30 months of age there was an increase in inflammation, they developed fibrosis and the levels of endotoxin in plasma were higher. Regarding changes in the microbiome, the duodenum had more changes than the colon related to age. Allobacullum, Bifidobacterium, Olsenella, Corynebacterium were the genera that differed statistically in the duodenum through the murine lifespan. Fewer changes were observed in the colon, as Allobaculum was the only genus that showed differences between young and old mice. Additionaly, it was analyzed the impact of aging in the active microbial communities of mouth, duodenum and colon at 2, 9, 15, 24 and 30 months of age (chapter 4). Changes were observed at every age and different taxonomical levels, with a greater shift at 15 months of age. This is related to the age of the mice, as at middle age systemic changes related to the aging process start to occur. At old ages, there was an increment of the pathobiontic species Helicobacter hepaticus and Helicobacter ganmani in the duodenum and colon. The oral, duodenal and colonic microbial communities are important pieces of information that can be related to the health status of the host. Research that focuses on assessing the changes in the different niches and not only in the feces, gives a broader overview of the microbial community of the host.