Browsing by Subject "Microarray"

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Biometrical tools for heterosis research
(2010) Schützenmeister, André; Piepho, Hans-Peter
Molecular biological technologies are frequently applied for heterosis research. Large datasets are generated, which are usually analyzed with linear models or linear mixed models. Both types of model make a number of assumptions, and it is important to ensure that the underlying theory applies for datasets at hand. Simultaneous violation of the normality and homoscedasticity assumptions in the linear model setup can produce highly misleading results of associated t- and F-tests. Linear mixed models assume multivariate normality of random effects and errors. These distributional assumptions enable (restricted) maximum likelihood based procedures for estimating variance components. Violations of these assumptions lead to results, which are unreliable and, thus, are potentially misleading. A simulation-based approach for the residual analysis of linear models is introduced, which is extended to linear mixed models. Based on simulation results, the concept of simultaneous tolerance bounds is developed, which facilitates assessing various diagnostic plots. This is exemplified by applying the approach to the residual analysis of different datasets, comparing results to those of other authors. It is shown that the approach is also beneficial, when applied to formal significance tests, which may be used for assessing model assumptions as well. This is supported by the results of a simulation study, where various alternative, non-normal distributions were used for generating data of various experimental designs of varying complexity. For linear mixed models, where studentized residuals are not pivotal quantities, as is the case for linear models, a simulation study is employed for assessing whether the nominal error rate under the null hypothesis complies with the expected nominal error rate. Furthermore, a novel step within the preprocessing pipeline of two-color cDNA microarray data is introduced. The additional step comprises spatial smoothing of microarray background intensities. It is investigated whether anisotropic correlation models need to be employed or isotropic models are sufficient. A self-versus-self dataset with superimposed sets of simulated, differentially expressed genes is used to demonstrate several beneficial features of background smoothing. In combination with background correction algorithms, which avoid negative intensities and which have already been shown to be superior, this additional step increases the power in finding differentially expressed genes, lowers the number of false positive results, and increases the accuracy of estimated fold changes.
Safety assessment of coagulase-negative staphylococci used in food production
(2015) Seitter, Marion; Hertel, Christian
Coagulase-negative staphylococci (CNS) are used in starter cultures for the production of fermented meat products due to their involvement in the development of desired red color, characteristic flavor as well as ensuring stability. But also other CNS species like S. condimenti, S. piscifermentans, S. equorum and S. succinus have a potential for future use in starter cultures. The safety of fermented food products is principally proven by long-term experience as traditional methods are considered safe based on their long “history of safe use”. However, for the last mentioned species long-term experience concerning sanitary harmlessness exists only with limitations. To get an insight in safety relevant properties of food associated CNS in Chapter III-V strains of the species S. carnosus, S. condimenti and S. piscifermentans (S. carnosus-group) as well as S. equorum, S. succinus and S. xylosus (S. xylosus-group) were phenotypically and partly genotypically investigated. Based on these insights in Chapter VI a DNA microarray was developed for rapid and simultaneous detection of various safety relevant properties in CNS with future use in the food production. To increase the application potential of this microarray, additionally technological relevant properties were considered in the array design. Subsequently, the designed microarray was used for the genotypic investigation of phenotypically characterized CNS concerning the presence of safety relevant properties. In Chapter III, antibiotic resistances of 330 CNS belonging to S. carnosus- and S. xylosus-group isolated from food and starter cultures were examined. Resistances to 21 antibiotics were phenotypically determined and resistance genes blaZ, lnuA and tetK were detected in strains showing phenotypic resistances to ß-lactam antibiotics, lincomycin and tetracycline. Antibiotic resistance profiles in strains of the species S. equorum, S. succinus and S. piscifermentans are described and due to the high number of investigated strains an insight regarding the occurrence of antibiotic resistances in food associated CNS is given. In Chapter IV toxin production of food associated CNS belonging to S. carnosus- and S. xylosus-group was investigated. First, 330 strains isolated from food, starter cultures and clinical isolates have been analyzed to hemolytic activity on human and sheep blood agar plates. Secondly, the ability of 35 selected strains to produce staphylococcal enterotoxins, toxic shock syndrome toxin 1 and exfoliative toxin A has been examined by immunoblot analysis. The chapter demonstrates that CNS strains present in high numbers in fermented food cannot necessarily be regarded as safe. Thus, strains used in the production of fermented food should be analyzed with respect of their toxigenic potential to avoid negative effects on human health. Chapter V is dealing with the formation of binding proteins to extracellular matrix proteins (ECM) and the production of biogenic amines (BA) by 32 CNS of S. carnosus- and S. xylosus-group. Binding capacity of CNS to the ECM fibronectin and fibrinogen was investigated by detection of fluorescent labeled cells which were added to microtiter plates coated with ECM. The formation of six important BA was examined by HPLC using growing and resting cells. By the results of this chapter the ability of food associated CNS to develop undesired properties like the formation of binding proteins to ECM and BA was demonstrated. Thus, further research is needed concerning potential risks and the importance on human health if strains with these properties are used in the production of fermented food. In Chapter VI, the design of a polynucleotide based DNA microarray as screening tool to detect genes of potential health concern and technological relevance in food associated CNS is described. The array considered 220 genes encoding for antibiotic resistances, hemolysins, toxins, amino acid decarboxylases (involved in the formation of BA), binding proteins to ECM, lipases, proteases, stress response factors, and nitrate dissimilation. Hybridization experiments were performed using genomic DNA isolated of 32 in Chapter III-V phenotypically characterized CNS allowing the detection of e.g. antibiotic resistance genes blaZ, lnuA, and tetK. Genes coding for decarboxylases as well as fibronectin and fibrinogen binding proteins were rarely correlated with the phenotype. Toxin genes could not be detected, whereas technological relevant genes like genes coding for proteases, lipases, catalase, superoxide dismutase or genes involved in dissimilatory nitrate reduction resulted in hybridization signals. The present thesis provides data concerning safety relevant properties in food associated CNS which are important for accurate safety assessment. Comparison of the results of Chapter III-V with them of Chapter VI showed that antibiotic resistances, formation of toxins and binding proteins to ECM are more present in strains of S. xylosus- than in S. carnosus-group. In context with safety assessment of food associated CNS, the designed microarray can be used as screening tool for the detection of safety relevant combined with technologically important properties (nitrate dissimilation, control of oxidative damage by catalase, flavor formation by proteases and lipases). Summarizing, the array is able to make a contribution in enhancing the selection criteria of CNS used as starter organisms in respect to food safety as well as technologically relevant properties.
Transcriptional and proteomic responses towards early nitrogen depletion in Arabidopsis thaliana
(2016) Menz, Jochen; Ludewig, Uwe
Plant roots acquire nitrogen predominantly as ammonium and nitrate, which besides serving as nutrients, also have signaling roles. Re-addition of nitrate to starved plants rapidly and di-rectly transcriptionally re-programs the metabolism and induces root architectural changes, but the earliest responses to nitrogen deprivation are unknown. In this thesis, the early transcriptional response of developed roots to nitrate or ammonium deprivation were analyzed in two Arabidopsis ecotypes contrasting in their nitrogen use efficiency: the inefficient genotype Col-0 and the efficient Tsu-0. The rapid transcriptional repression of known nitrate-induced genes proceeded the tissue NO3- concentration drop, with the transcription factor genes LBD37/38 and HRS1/HHO1 among those with earliest significant change. Some transcripts were stabilized by nitrate, but similar rapid transcriptional repression occurred in loss-of-function mutants of the nitrate response factor NLP7. In contrast, an early transcriptional response to ammonium deprivation was almost completely absent. In Col-0, the analysis was extended with the proteome and phospho-proteome resulting in a rapid and transient perturbation of the proteome induced by ammonium deprivation and a differential phosphorylation pattern in proteins involved in adjusting the pH and cation homeostasis, plasma membrane H+, NH4+, K+ and water fluxes. Fewer differential phosphorylation patterns in transporters, kinases and other proteins occurred with nitrate deprivation. The deprivation responses are not just opposite to the resupply responses, identify NO3--deprivation induced mRNA decay and signaling candidates potentially reporting the external nitrate status to the cell. Transcrip-tome comparison revealed only few N-nutrition related genes between both ecotypes contributing the increased NUE of Tsu-0, which probably relies on higher biomass accumulation. Besides, Tsu-0 confirmed the transcriptional depletion response of Col-0.
Transcriptomic analyses during infectious anemia in pigs
(2019) Mack, Sarah-Lena; Hölzle, Ludwig
Mycoplasma suis (M. suis) is a uncultivable hemotrophic bactreia parasiting red blood cells in pigs and a small range of other animals. It becomes more and more important because of leading to big economic losses in swine industry. M. suis causes anemia in pigs and is accompanied with other immunosuppressive diseases. A once infected animals is a life-long carrier and could infect other animals as well. To date, there is less information about the pathogenesis and reproduction of the bacteria and it is not possible to cultivate M. suis in vitro. One objective of the present study was to get more information about the transcriptomic changes in a pig during an infection course. Therefore, 3 splenectomized piglets were infected with the highly virulent strain KI_3806. After 2, 4 and 8 days post infection (p.i.) blood samples were taken and total RNA of blood was extracted. Microarray analyses were performed with a commercial Affymetrix array. Using microarrays more than 7000 DE genes from infected pigs could be detected. With M. suis in its host cells – the erythrocyte – we had a perfect model showing molecular interactions or signaling pathways in the M. suis infection process. With the help of the Ingenuity pathway analyses service many genes involved in immune and inflammatory response were found. Moreover typical genes involved in anemia, psoriasis and endothelial cell damage could be observed. The detection of these genes verified the depression and alteration of the immune system by M. suis resulting in evading the immune system and therefore in persisting among the organism. Another aim was to go deeper on the transcriptional level of M. suis and to get insights of the behavior of the bacteria at the time point of and after infection. RNA Sequencing was performed on a HiSeq 2000 Genome Analyzer from Illumina an resulting reads were mapped to reference sequences M. suis KI_3806 and Sus scrofa. Moreover, differential expression analysis was performed using the edgeR package. After mapping, it could be observed that on day 4 p.i. M. suis transcripts seem to be overlapped by porcine transcripts, whereas on day 8 p.i. most of the reads could be allocated to the M. suis genome resulting in almost all M. suis genes were found to be transcribed at the end of infection. When looking at the COG categories the group of proteins with “unknown function” (hypothetical proteins) represented the largest group on both days. Also a high number within the differentially expressed genes were hypothetical genes showing that these genes play an important role during infection. Further investigations are needed to confirm that the hypothetical genes also are involved in M. suis replication and recombination. In conclusion, our analysis revealed several thousand genes differentially expressed during acute IAP and numerous altered pathways and cellular processes throughout the course of host response to acute M. suis infections, thus contributing to a better understanding of the IAP pathogenesis. Moreover, this could lead to new approaches towards cultivation of M. suis as well as therapeutic treatments.