Non-digestible polysaccharides to support the intestinal immune barrier: in vitro models to unravel molecular mechanisms
Tang, Yongfu - \ 2017
Wageningen University. Promotor(en): H.J. Wichers, co-promotor(en): J.J. Mes; C.C.F.M. Govers. - Wageningen : Wageningen University - ISBN 9789463437134 - 166
polysaccharides - health - immunomodulatory properties - homeostasis - intestinal diseases - human nutrition research - polysacchariden - gezondheid - immunomodulerende eigenschappen - homeostase - darmziekten - voedingsonderzoek bij de mens
Non-digestible polysaccharides (NDPs) are considered as important ingredients to support health. Among these health effects, immunomodulatory effects raised interests in the past decade. The intestine is the primary organ that interact with NDPs. The intestinal epithelial cells (IECs) form a dynamic physical barrier and together with associated immune cells determine for a large part our immune homeostasis. Studying the direct interaction between NDPs and intestinal and immune cells could help us to uncover the mechanism by which NDPs exert immunomodulatory effects and how NDPs can differ in this activity. In this thesis, we investigated the immunomodulatory effects of NDPs through interaction with intestinal immune cells using in vitro methods in order to characterise the NDPs and preselect NDPs with differential activity for further in vivo evaluations.
The intestinal immune barrier is formed by various IECs and immune cells, which are introduced and their specific functions discussed in Chapter 1. NDPs could interact directly with both IECs and immune cells that sample in or from the lumen. The majority of IECs are enterocytes and most relevant immune cells responsible for sampling in the lumen have been characterised as macrophages, which leads us to focus on these cell types by in vitro approaches. In addition, basic information on NDPs and current status on health effects of NDPs both in vitro and in vivo are discussed.
In Chapter 2, the direct response of IEC to NDPs stimulation was investigated. IECs form the largest surface of the body that, with a crucial role as barrier also, perform a role in signalling towards immune cells. We used 21-day transwell cultured Caco-2 to resemble the small intestinal enterocytes that form largest part of this intestinal layer. We first characterized the chemical composition of five NDPs which revealed different mono sugar composition, linkages of backbone and side chains and a wide range of MW (from 17 KDa to 2100 KDa). The NDPs could reduce translocation of FITC-Dextran of 4 kDa across the epithelial layer, potentially through physical interference. Gene expression analysis indicated the induction of unique gene expression characteristics in Caco-2 cells upon exposure to different NDPs. An arabinoxylan preparation from wheat and a lentinan-containing extract from shiitake mushrooms showed upregulation of gene expression of the NF-κB family and chemokines CCL20 and CXCL10. Besides these immune related changes by some NDPs, we also observed changes in receptor expression (like TLR2, CD14 and GPCRs) and other pathways, amongst which the cholesterol biosynthesis pathway.
Macrophages, as the resident population of immune cells penetrating between or associating with close contact with the IECs, are generally classified as inflammatory (M1) or as tolerant (M2) macrophages. In Chapter 3, we set up a macrophage differentiation method based on primary blood cells and selected and validated M1 and M2 specific gene expression markers. Next, we analysed the effect when macrophages are exposed to NDPs and compared the resulting macrophages with M1 and M2 macrophages. Based on M1 and M2 markers we identified an alternative subset that we named MNDP. This MNDP was further studied by microarray analysis and revealed a commonly modulated set of genes, involved in migration, metabolic processes, cell cycle, and inflammatory immune function.
In Chapter 4, we further functionally characterize these MNDP in comparison to M1 and M2 macrophages based on a set of functional assays. NDP-treated macrophages showed no IDO activity and showed an inhibited antigen uptake and processing capacity compared to M1 and M2 macrophages. Also their phagocytic capacity was reduced compared to both M1 and M2 macrophages. Furthermore, the alternative expression pattern for NDP-treated macrophages, as demonstrated by gene expression, was confirmed by protein measurements. The signature mix of the chemokines CCL1, CCL5, CCL20, CCL24, CXCL8, and IL1β secreted by MNDP, and in particular when macrophages were treated with Naxus, was shown to induce a recruitment of monocytes.
As macrophage plasticity could be essential for intestinal immune homeostasis, resolving activity of inflammatory responses upon a challenge is important. Besides, redirecting differentiation and function of tolerant macrophages can also be beneficial to the intestinal immune status. In Chapter 5, we analysed plasticity of M1 and M2 macrophages to NDPs exposure. Macrophage plasticity was demonstrated as M1 and M2 could be skewed to an alternative subset indicated by a dedicated set of gene expression markers, selected to characterize M1, M2 and MNDP macrophages. In addition, phagocytosis and antigen processing capacity of both M1 and M2 were decreased by the NDP Naxus. Besides, Naxus could change the secretion of cytokines by macrophages that previously were differentiated towards M1 and M2. For M2, this resulted in an increase of recruitment of monocytes by M2 macrophages.
In Chapter 6, we discussed the important findings in each chapter of this thesis together with current literature, and gave a general perspective on this research line focussing on the immunomodulating activity of NDPs and the direction for future research. We suggested NDPs in terms of Naxus as candidate for guiding investigations in ex vivo and in vivo studies for immunomodulation of intestinal disease.
Mucus and gut barrier in health and disease
Sovran, B. - \ 2015
Wageningen University. Promotor(en): Jerry Wells; P. de Vos, co-promotor(en): J. Dekker. - Wageningen : Wageningen University - ISBN 9789462574892 - 233
slijm - spijsverteringskanaal - darmen - muizen - probiotica - eilandjes van peyer - colitis - transcriptomen - immunohistologie - veroudering - geslacht (sex) - homeostase - gezondheid - ziekten - mucus - digestive tract - intestines - mice - probiotics - peyer patches - colitis - transcriptomes - immunohistology - senescence - sex - homeostasis - health - diseases
This publication describes his work as a PhD student in the Host-Microbe Interactomics Chair group at Wageningen University within the Gastrointestinal Health theme. It has been completed under the supervision of Prof. Dr Jerry M Wells, Dr Jan Dekker and the TIFN project leader, Prof. Dr Paul de Vos.
Mucus serves as a protective layer between the intestinal content and the intestinal wall. It facilitates the passage of the luminal content through the intestine, reducing the risk of mechanical damage to the intestinal epithelium. The overarching goal of this thesis was to investigate the role of mucus in the maintenance of the intestinal immune barrier and the effects of ageing and gender differences on mucus production and the gut barrier.
We found by using a mouse model that decreased mucus production leads to changes in microbiota and mucosal stress responses, without the appearance of pathology, demonstrating the importance of mucus in intestinal homeostasis. The mucus barrier was shown to deteriorate during aging but this could be prevented with specific probiotics. Furthermore gender-specific differences in the effects of ageing on the mucosal barrier were found. Increased knowledge on these mechanisms might contribute significantly to disease prevention and treatment, for instance by optimizing gender-specific dietary and pharmacological requirements.
The study presented in this thesis was performed within the framework of Top Institute Food and Nutrition, within the GH002 project.
Vitamin D-tour : cognition and depression: the role of vitamin D and its interplay with glucose homeostasis
Brouwer-Brolsma, E.M. - \ 2014
Wageningen University. Promotor(en): Lisette de Groot; Edith Feskens, co-promotor(en): Teun Schuurman; Wilma Steegenga. - Wageningen : Wageningen University - ISBN 9789462571082 - 215
vitamine d - depressie - glucose - homeostase - gezondheid - hersenen - vitaminetekorten - vitamin d - depression - glucose - homeostasis - health - brain - vitamin deficiencies
According to recent estimations approximately 35.6 million people have dementia worldwide. Globally, 350 million people experience one or more depressive episodes during their life. As the therapeutic options for dementia and depression are limited, these conditions form a major challenge for public health and society. More and more researchers have initiated research on potential preventive factors for dementia and depression, including the potential effects of nutritional factors. The aim of this PhD-thesis was to study the role of vitamin D and its potential interplay with glucose homeostasis, in the development of cognitive decline and depression, using epidemiological data as well experimental animal data.
Chapter 2 recapitulates a debate between vitamin D experts that was organized to make a step towards the harmonization on the formulation of optimal vitamin D intake levels and serum 25(OH)D concentrations across Europe. It was concluded that based on the current evidence-base 25(OH)D concentrations ≥50 nmol/L are sufficient with respect to optimal bone health. For health outcomes beyond bone health evidence was considered insufficient to formulate optimal levels. In order to achieve and maintain a 25(OH)D concentration ≥50 nmol/L, older adults aged ≥65 years were recommended to adhere to a vitamin D intake of 20 μg/day.
Chapter 3 shows that there is a high prevalence of 25(OH)D inadequacy in a population of Dutch older adults that participated in the B-PROOF study (n=2857), namely 45% had 25(OH)D concentrations <50 nmol/L. Mean vitamin D intake was 4.9±2.9 µg/day and only 20% of the participants reported to use vitamin D containing supplements. Exploration of the determinants of 25(OH)D status showed significant associations between vitamin D ‘raising’ SNPs (n=2530), higher sun exposure (n=1012), vitamin D intake (n=596) and higher 25(OH)D concentrations. Including all the potential relevant predictors in one model explained 35% of the variance in 25(OH)D status (R2=0.35).
In chapter 4 the associations between 25(OH)D status and global cognitive performance (n=116), depressive symptoms (n=118), and surrogate markers of glucose intolerance (n=593) were evaluated using data of European adults aged 70-75 years. None of the associations reached significance.
Studying the potential role of vitamin D in domain-specific cognitive performance and depression in 127 Dutch pre-frail and frail older adults aged ≥65 years (chapter 5), showed an association between 25(OH)D concentration and executive functioning, and a tendency towards an association with information processing speed. Stratification for ‘low’ and ‘high’ fasting glucose concentrations did not suggest an interaction between vitamin D and glucose homeostasis in the association with domain-specific cognitive performance. Moreover, adding fasting glucose or insulin did not substantially influence the associations between 25(OH)D status and domain-specific cognitive performance, and hence a mediation effect of glucose homeostasis was considered unlikely.
We furthermore observed associations of 25(OH)D status with attention and working memory (n=787) (chapter 6), depression (n=2839) (chapter 7) and grey matter volume of the brain (n=217) (chapter 8) in a population community-dwelling Dutch older adults aged ≥65 years. Again, these studies did not provide evidence that the associations were modified or mediated by glucose intolerance. However, it should be emphasized that glucose intolerance in these three chapters was defined sub-optimally, specifically using blood samples that may have been collected in a non-fasting state, or by using self-reported diabetes data. Hence, the mediation and interaction effects should be interpreted cautiously.
Finally, chapter 9 shows the results of a proof of principle study on the effect of a long-term vitamin D deficiency on cognitive decline and emotional reactivity in old C57BL/6j mice. Modest tendencies were shown for a relation between vitamin D and spatial learning, but these tendencies did not reach significance. Vitamin D deficiency did not affect recognition memory, spatial memory or emotional reactivity. Mice that received a higher dietary fat load, which was given to induce an impaired glucose tolerance, did not respond differently to a vitamin D deficiency than mice that received a low fat diet did.
Overall, it is concluded that the evidence for an effect of vitamin D on cognitive performance/decline, depression or brain volume is insufficient to formulate disease specific cut-off values for vitamin D intake or 25(OH)D status. However, given the high prevalence of 25(OH)D concentrations <50 nmol/L we do call for a more active promotion of the current vitamin D intake recommendations.
An evolutionary perspective on differential regulation of zinc and cadmium homeostatis genes in Arabidopsis thaliana and Noccaea caerulescens
Lin, Y.F. - \ 2014
Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts. - Wageningen : Wageningen University - ISBN 9789461738950 - 220
arabidopsis thaliana - zink - cadmium - homeostase - genen - evolutie - nucleotidenvolgordes - tolerantie - arabidopsis thaliana - zinc - cadmium - homeostasis - genes - evolution - nucleotide sequences - tolerance
Some plants can tolerate and accumulate unusually high levels of toxic metals, and the analysis of such plants can provide insights into the ecology of environments that are polluted with heavy metals due to human industrial activities. The study of heavy metal hyperaccumulators such as Noccaea caerulescens can show how plants cope with excess metals and increase their fitness when growing in metalliferous environments. In this thesis, I compared the molecular mechanisms of zinc (Zn) homeostasis and cadmium (Cd) response in the hyperaccumulator species N. caerulescens and its non-accumulator relative Arabidopsis thalianaby investigating the regulation of the ZNT1/ZIP4gene that promotes Zn uptake and Zn/Cd tolerance.I also studied the ecological advantages of metal hyperaccumulators in nature and determined the DNA sequences of the N. caerulescens transcriptometo find candidate genes that control metal hyperaccumulation and provide an evolutionary perspective on the emergence of this trait.
The functional characterization of the N. caerulescens and A. thaliana Zn-transporter genes NcZNT1 and AtZIP4 (and their promoters) showed how their differential expression pattern contributed to their role in metal tolerance and accumulation. The NcZNT1 gene is induced by Zn deficiency and the NcZNT1 protein is localized in the plasma membrane. Transgenic N. caerulescens roots containing a transgene for the green fluorescent protein (GFP) driven by the NcZNT1promoter revealed GFP fluorescence localized to pericycle and vascular tissues. This suggests that NcZNT1 contributes to metal loading into the xylem and long-distance metal transport. The overexpression of NcZNT1 in A. thaliana increased Zn and Cd tolerance and the capacity to accumulate these metals compared to wild-type plants. These results suggest that NcZNT1 plays an important role in Zn and Cd hypertolerance and hyperaccumulation in N. caerulescens, where it is expressed in both Zn-sufficient and Zn-excess conditions. The differential activity of the NcZNT1 and AtZIP4 promoters in N. caerulescens and A. thaliana implies that different cis-regulatory elements and trans-regulatory factors are present in both species.
The ecological advantage of hyperaccumulators in metal-contaminated soils was investigated by studying a natural nas1mutant, in which the Nicotianamine Synthase1 gene is disrupted by a transposon insertion. This mutant allele was found in three natural N. caerulescens populations, which were compared to wild-type plants in terms of phenotype and adaptive advantage. Although the transposon disrupted the gene, the loss of NAS1 gene activity was compensated by enhanced expression of NAS3 and NAS4, resulting in increased nicotianamine (NA) production, which enhanced the Zn and Cd accumulation in the nas1 mutants. This increased their metal sensitivity compared to wild-type plants, but also made them more toxic towards Pieris rapae caterpillars, which developed more slowly and gained less weight when fed on mutant plants exposed to excess Zn or Cd. Therefore, the possible selective advantage of the nas1 mutant in nature is high metal accumulation and the protection of plants from herbivores. Differences in nas1 allele frequency among the three natural populations suggests that nas1 alleles experience different degrees of natural selection or may be at different stages on the route to fixation.
Molecular evolutionary studies involve the identification of candidate genes that play a role in adaptation. Therefore, a comprehensive set of transcript sequences was obtained from N. caerulescens accession Ganges (GA) by 454 pyrosequencing. In total, the collected 23,836 isotigs (putative transcripts) were grouped into 20,378 isogroups and 93.2% of them could be matched to Brassicaceae protein sequences, which allowedtheir functional annotation. A total of 87 isogroups was annotated as metal homeostasis related genes, including metal transporter families, metal chelator biosynthesis families, and metal tolerance gene families, which are candidate genes for the molecular analysis of heavy metal homeostasis mechanisms. A group of genes required for the synthesis of glucosinolates, which are important secondary metabolites that protect plants against herbivores, were also shown to be expressed in N. caerulescens. The METALLOTHIONEIN3 (MT3) gene was found to have been duplicated in the N. caerulescens genome, when compared to related Brassicaceae. These transcript sequences will provide an important tool to annotate the genome sequence of the N. caerulescensGanges accession, which is in progress. This genome sequence will also be the basis of genome comparisons between the different N. caerulescens accessions that have different levels of metal accumulation and metal tolerance and which may therefore differ in terms of gene expression levels or modes of actions that affect metal homeostasis.
Identification of traits and QTLs contributing to salt tolerance in barley (Hordeum vulgare L.)
Nguyen Viet Long, L. - \ 2012
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Gerard van der Linden; Oene Dolstra. - S.l. : s.n. - ISBN 9789461734099 - 137
hordeum vulgare - gerst - zouttolerantie - loci voor kwantitatief kenmerk - groei - plantensamenstelling - genotypen - chromosomen - homeostase - plantenveredeling - hordeum vulgare - barley - salt tolerance - quantitative trait loci - growth - plant composition - genotypes - chromosomes - homeostasis - plant breeding
Salinity is the most severe abiotic stress perceived by plants and affects about 800 million hectares of land worldwide, including 20% of the world’s highly productive irrigated land. Significant crop yield losses are observed due to salinity. Salinization is increasing because of poor irrigation management and climate change. Improving salt tolerance in crops is for these reasons an important target for plant breeding in the near future. However, salinity tolerance in plants is not easy to breed for due to its interaction with many physiological processes controlled by many genes and their interaction with the environment. Barley is a good model crop to study different mechanisms conferring salt tolerance in cereals. A traditional QTL mapping approach in combination with a new association mapping method allowed us to efficiently explore the genetics and genetic diversity of salt tolerance in barley. Improvements of the association mapping technology highly increased detection power and mapping accuracy. The traits and QTLs identified in this thesis point out both osmotic and ionic stress tolerant genes as important targets for salt tolerance breeding. This thesis provides tools to plant breeders for the application of marker-assisted introgression breeding of salt tolerance genes in their breeding programs. Some QTLs were found to be syntenic with the important QTLs/genes for salt tolerance found in wheat and rice such as Na+ and K+ transporter gene families. Other QTLs were new and suggest the presence of novel genes that play an important role in plant ion homeostasis, transportation of Cl- and Ca2+ and osmotic tolerance. We demonstrated that association mapping can be a powerful approach to dissect the complexity of salt tolerance in barley. The newly available high-density SNP map of barley and the barley genome sequence in the near future further increases the accuracy of mapping studieswith the association panel and will greatly facilitate the cloning of the genes underlying salt tolerance in barley. This thesis thus contributes to better a understanding of the physiological and genetic basis of salt tolerance and improved breeding strategies for the development of salt tolerant varieties.
The dynamic interplay of microbiota and mucosa drives establishment of homeostasis in conventionalized mice
Aidy, S.F. El - \ 2012
Wageningen University. Promotor(en): Michiel Kleerebezem, co-promotor(en): Peter van Baarlen; Erwin Zoetendal. - S.l. : s.n. - ISBN 9789461731951 - 168
kiemvrije dieren - muizen - darmmicro-organismen - homeostase - slijmvlies van het spijsverteringskanaal - transcriptomica - metabolomica - germfree animals - mice - intestinal microorganisms - homeostasis - digestive tract mucosa - transcriptomics - metabolomics
The intimate interplay between gut microbiota, host, and nutrient flow is crucial in defining the health status of the host. During microbial conventionalization of germfree mice, tightly regulated molecular responses assure the establishment of homeostasis and immune tolerance towards the microbiota. To decipher the temporal and regional dynamics of host-microbiota communication during the process of conventionalization, a combination of transcriptomics, (immune-)histology, metabonomics (tissue, urine, and plasma), as well as MITchip (Mouse Intestinal Tract chip) based microbiota profiling was employed. To this end, C57/B6 J germfree mice were conventionalized with mouse fecal microbiota and responses were followed in a time-resolved manner for thirty days. The colonizing microbiota was characterized by a shift from low towards higher diversity of its composition, over the period of conventionalization. Microbial colonization was rapidly (after one day) reflected by increased concentrations of specific urine and jejunal metabolites as well as by biologically relevant changes in jejunal tissue transcriptome profiles. Conversely, ileal and colonic transcriptome responses could be measured later, after four days post-conventionalization, and led towards stable molecular profiles at sixteen and thirty days of conventionalization, albeit with region-specific differences. The major molecular responses included strong induction of innate immune response followed by stimulation of adaptive and regulatory immune functions, as well as modulation of metabolic pathways involved in lipid, carbohydrate, and anabolic metabolism. Conventionalization was characterized by two stages separated by one stage of a single day which, particularly in the colon, resembled a transient stage of inflammation, based on transcriptomes, histology and transiently elevated levels of specific plasma markers. This state coincided with temporal domination of specific microbial groups that have previously been identified as “pathobionts”, suggestive of a transient state of dysbiosis. Extensive transcriptome profile analyses throughout the GI tract enabled the identification of central gene regulatory networks that govern the molecular responses during conventionalization and are proposed to serve as genetic signatures for the control of intestinal homeostasis in mice. Nearly all genes in these regulatory networks have human orthologues, suggesting that the biological findings of this study is also relevant for human intestinal biology. In support of this hypothesis, in the jejunum, the identified gene regulatory network appeared to be strongly associated with human metabolic disorders. This notion also suggests that at least in mice, possibly also in human, there is a prominent role of the proximal small intestine in systemic metabolic control.
This thesis exemplifies the pivotal role of the dynamic molecular interactions between the microbiota and the intestinal mucosa, in the establishment and maintenance of mucosal homeostasis in healthy mice. The molecular signatures obtained from these studies in mice may provide novel diagnostic tools and/or therapeutic targets in humans for specific disorders associated with intestinal dysbiosis and loss of mucosal homeostasis.
Keywords: C57/BL6 J mice, conventionalization, transcriptomics, (immune-)histology, metabonomics, microbiota
Lichaam in balans
Keijer, J. - \ 2009
Wageningen : Wageningen Universiteit, Wageningen UR - ISBN 9789085852803
mens - dieren - energiebalans - homeostase - vetweefsel - quetelet index - obesitas - fysiologische functies - voedingsfysiologie - gezondheid - man - animals - energy balance - homeostasis - adipose tissue - body mass index - obesity - physiological functions - nutrition physiology - health
Functional characterisation of three zinc transporters in Thlaspi caerulescens
Talukdar, S. - \ 2007
Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts. - [S.l.] : S.n. - ISBN 9789085046691 - 154
thlaspi - zink - zware metalen - actief transport - genen - genexpressie - homeostase - bioaccumulatie - thlaspi - zinc - heavy metals - active transport - genes - gene expression - homeostasis - bioaccumulation
Heavy metal hyperaccumulation in plants is a poorly understood phenomenon. Transmembrane metal transporters are assumed to play a key role in this process. In the research described in this thesis, genes encoding Zn transporters of Thlaspicaerulescens, a heavy metal hyperaccumulator plant,are studied and compared to their orthologues in Arabidopsis thaliana, a non-hyperaccumulator plant.The TcZNT1 and TcZNT2 genes of T. caerulescens are members of the Zrt, Irt ( ZIP) -like gene family and show 89% and 87% similarity with the AtZIP4 and AtIRT3 genes of A. thaliana,respectively. AtZIP4 and AtIRT3 genes are induced exclusively by Zn-deficiency in Arabidopsis. In limiting Zn conditions both Arabidopsis and Thlaspi show characteristic symptoms of Zn deficiency manifested both in vegetative and reproductive tissues. However, in T. caerulescens , TcZNT1 and TcZNT2 are constitutively and highly expressed in roots, irrespective of the Zn concentration in the medium. This observation suggested a role forthesegenes in Zn uptake and possibly in explaining the Zn hyperaccumulation trait in Thlaspi . The proteins are localized in the plasma membrane, conferring zinc uptake into the cytoplasm. TcZTP1, another Zn transporter gene shows 85% sequence similarity in its coding region with the AtZAT/ MTP1 ofA. thaliana and is assumed to be localized in the vacuolar membrane. This gene belongs to the Cation Diffusion Facilitator (CDF) family.The responses of the over-expression lines of TcZNT1,TcZNT2 and TcZTP1 in A. thalianawereinvestigated with respect to deficiency and toxicity of Zn, Fe and Cd.Over-expression of TcZNT1showed higher Zn accumulation in A. thaliana , whereas this effect was small and not significant for TcZNT2.TcZNT1 over-expression lines also showed increased Cd accumulation.In addition, the TcZNT1, TcZNT2 and TcZNT1/TcZNT2 silenced T. caerulescens showedreduced Zn content in roots. These results, together with the plasma membrane localisation of TcZNT1 and TcZNT2 in cowpea protoplast, suggest a role in metal uptake of these two proteins in the plant. Over-expression of the TcZTP1 confers Zn and Cd tolerance and accumulation in A. thaliana. The regulation of expression of these genes was studied in comparison to the orthologous genes in A. thaliana by the study of the TcZNT1 and AtZIP4 promoter. The TcZNT1 promoter was isolated by PCR using forward primers designed on the A. thaliana gene upstream of AtZIP4 and a reverse primer on the T.caerulescensTcZNT1 cDNA). The promoters of ZIP4 orthologues were isolated by a similar method from Arabidopsis halleri, Arabidopsis lyrata and Cochleria pyrenaica. The sequence analysis performed on the ZIP4 promoters shows the presence of two conserved palindromic sequences in all five species, suggesting that these are essential promoter elements for both the hyperaccumulators and non-accumulators. Using (deleted) promoter: GUS fusions transformed to A. thaliana , the cis elements of AtZIP4 were identified between-263 bp and -115 bp from the start codon andfor TcZNT1between -223 bp and -98bp by 5' deletion study of the promoters. These regions also contain the two palindromic sequences in both promoters, indicating the importance of these conserved palindromes in the hyperaccumulators and non-accumulators. The AtZIP4pro-GUS transformed A. thaliana plants showed high expression in Zn-deficient roots, shoots, flowers and seeds. Similar expression pattern was observed in TcZNT1pro-GUS transformed A. thaliana plants. This suggests both the cis elements in the AtZIP4 and TcZNT1 promoters are conserved. To explain the high expression of TcZNT1 in T. caerulescens,the mode of cis/trans interaction and/or the expression profiles of thetransgenes in both species need to be studied. Most likely, theexpression profile of the transcription factors and consequently many other genes functioning in metal uptake, transport and sequestration have been changed, in the hyperaccumulator plants as an adaptation strategy to survive in the highly contaminated soil. It is expected that the knowledge obtained can be used to design strategies to generate genetically engineered plants that can be used for phytoremediation and/or that have an improved nutritional quality.
Genetic Variation in Bile Acid Metabolism: Implications for Lipoprotein Homeostasis
Hofman, M.K. - \ 2005
Wageningen University. Promotor(en): Evert Schouten; Frans Kok. - - 168
cholesterolmetabolisme - galzuren - metabolisme - lipoproteïnen - homeostase - genetische variatie - genetica - atherosclerose - cholesterol metabolism - bile acids - metabolism - lipoproteins - homeostasis - genetic variation - genetics - atherosclerosis
Genetic factors play an important role in the homeostasis of cholesterol in the human body. An important pathway for eliminating cholesterol from the body is to convert it into bile acids in the liver. The rate-limiting enzyme in this catabolism of cholesterol is CYP7A1. In the gene of CYP7A1, a sequence variation was found: the CYP7A1 A-278C polymorphism. We found that this polymorphism affects triglyceride concentrations in healthy individuals and cholesterol concentrations in patients with Hypertriglyceridemia. However, we found that this polymorphism probably plays no role in the response of serum lipid levels to diet. Interestingly, subjects with the genotype CC of the CYP7A1 A-278C polymorphism have an almost twice as high risk of a new clinical event, as compared to subjects with the genotype AA. In addition, subjects with the genotype CC display more progression of atherosclerosis. The results of this thesis contribute to the understanding of the role of variations in genes in cholesterol homeostasis in human.