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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Quantification and signaling of alternatively spliced GFRα2 isoforms

Too, Heng-Phon, Fung, Winnie Kar Yee 2003 (has links)
Neurturin (NTN) belongs to the glial cell-line derived neurotrophic factor (GDNF) family of growth factors. Both NTN and GDNF have been shown to potently prevent the degeneration of dopaminergic neuron in vitro and in vivo. The GDNF family receptor alpha 2 (GFRα-2) is the preferred receptor for NTN. In addition to the known full-length isoform (GFRα-2a), we have previously reported the isolation of two novel alternatively spliced isoforms (GFRα-2b and GFRα-2c). The expression levels of these isoforms have yet to be quantified and the functional properties determined. In this report, we have developed a real time polymerase chain reaction (PCR) using SYBR Green I to detect the expression levels of the three splice variants (GFRα-2a, GFRα-2b and GFRα-2c) in murine tissues. Both GFRα-2a and GFRα-2c were expressed at similar levels in all tissues examined. GFRα-2b was found to be 10 fold lower in expression. All three isoforms activated MAPK (ERK1/2) and Akt. Transcriptional profiling with DNA microarrays demonstrated that the spliced isoforms do not share similar profiles. In conclusion, we have now shown the expression levels of the spliced variants. All three isoforms are functional. However, each isoform appeared to have unique transcriptional profiles when activated. Singapore-MIT Alliance (SMA)
2

A quantitative real-time PCR assay for Ehrlichia ruminantium using pCS20

Steyn, HC, Pretorius, A, McCrindle, CME 10 April 2008 (has links)
Heartwater is a tick borne disease that affects ruminants and wild animals in Africa south of the Sahara. It is caused by Ehrlichia ruminantium and transmitted by the tick Amblyomma hebraeum. The protocols currently used to detect heartwater take several days to complete. Here, we describe the development of a pCS20 quantitative real-time PCRTaqMan probe assay to detect E. ruminantium in livestock blood and ticks from the field. The assay is based on the conserved pCS20 gene region of E. ruminantium that contains two overlapping genes, rnc and ctaG [Collins, N.E., Liebenberg, J., De Villiers, E.P., Brayton, K.A., Louw, E., Pretorius, A., Faber, F.E., Van Heerden, H., Josemans, A., Van Kleef, M., Steyn, H.C., Van Strijp, M.F., Zweygarth, E., Jongejan, F., Maillard, J.C., Berthier, D., Botha, M., Joubert, F., Corton, C.H., Thomson, N.R., Allsopp, M.T., Allsopp, B.A., 2005. The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number. PNAS 102, 838–843]. The pCS20 quantitative real-time PCRTaqMan probe was compared to the currently used pCS20 PCR and PCR/32P-probe test with regards to sensitivity, specificity and the ability to detect DNA in field samples and in blood from experimentally infected sheep. This investigation showed that the pCS20 quantitative real-time PCRTaqMan probe was the most sensitive assay detecting seven copies of DNA/ml of cell culture. All three assays, however, cross react with Ehrlichia canis and Ehrlichia chaffeensis. The pCS20 real-time PCR detected significantly more positive field samples. Both the PCR and pCS20 real-time PCR could only detect E. ruminantium parasites in the blood of experimentally infected sheep during the febrile reaction. The PCR/32P-probe assay, however, detected the parasite DNA 1 day before and during the febrile reaction. Thus, because this new quantitative pCS20 real-time PCRTaqMan probe assay was the most sensitive and can be performed within 2 h it is an effective assay for epidemiological surveillance and monitoring of infected animals.
3

Correlating Gene Transfection Efficiency and the Physical Properties of Various Cationic Poly(methacrylate) Systems

Tan, J. F., Too, Heng-Phon, Hatton, T. Alan, Tam, K. C. 16 December 2005 (has links)
Transfection efficiencies of several polymeric gene carriers were compared and correlated quantitatively to the amounts of cellular accumulation of plasmid DNA and to the expression of mRNA by quantitative real time PCR. Three cationic methacrylate polymer systems with similar chemical structure were used in this study, namely: poly(dimethylamino)ethyl methacrylate (PDMA) homopolymer, PEO-b-PDMA copolymer and PEO-b-poly(diethylamino)ethyl methacrylate (PEO-b-PDEA) copolymer. Despite their similar chemical structures, their transfection efficiencies were significantly different. PEO-b-PDEA copolymer was significantly less efficient as gene carrier compared to both PDMA and PEO-b-PDMA systems. Results from quantitative real-time polymerase chain reaction (real-time PCR), cytotoxicity and Zeta potential measurements showed correlations between the physical properties of the polymers and the efficiencies of cellular uptake of the transgene and transfections. In the case of PEO-b-PDEA system, cytotoxicity was due primarily to the excess polymers that did not participate in the DNA binding. In addition, the inability of the polymer/DNA complexes to interact with cell effectively was identified as the main barrier for high efficiency of transfection. This study demonstrated that the use of quantitative real-time PCR in combination with other physical characterization techniques can provide greater insights into the transfection barrier at different cellular levels. Singapore-MIT Alliance (SMA)
4

Investigating the role and activity of CC-Type glutaredoxins in the redox regulation of TGA1/TGA4 in Arabidopsis thaliana

Hahn, Kristen Rae 7 July 2009
Plants respond to and defend themselves against a wide range of disease-causing microbes. In order to do so, massive reprogramming of cellular protein expression patterns, which underpin various defense pathways, must occur. A family of basic leucine zipper transcription factors, called TGA factors, has been implicated in mediating this response. The TGA factors themselves are subject to complex regulation; of note, TGA1 and TGA4 are regulated via a reduction of conserved cysteines after treatment with the phenolic signaling molecular salicylic acid, which accumulates following pathogen challenge. Previous studies indicate that TGA factors physically interact in the yeast two-hybrid system with the plant-specific CC-type of glutaredoxin (Grx)-like proteins. Grx are a family of oxidoreductases that are important for maintaining the cellular redox status and often are required to modulate protein activity. The goal of this study was to ascertain the role of these Grx-like proteins in regulating TGA1 redox state. To this end, the expression patterns of several Grx genes were analyzed.

Quantitative-reverse-transcriptase PCR (q-RT-PCR) experiments indicated that TGA1 and TGA4 may be involved in down-regulating levels Grx-like gene transcripts after exposure to pathogens or salicylic acid (SA). Furthermore, qRT-PCR experiments also indicated that expression of some Grx-like genes is induced by SA, jasmonic acid (JA), and Pseudomonas syringae. Overexpression of the Grx-like protein, CXXC9, in Arabidopsis thaliana revealed that it is a regulatory factor in the cross-talk between vi theSA/JA pathways as it is able to suppress expression of PDF1.2, a marker for the JA defense pathway, as determined by qRT-PCR. The â-hydroxy ethyl disulfide (HED) assay was utilized to determine if the CC-type of Grx-like proteins have oxidoreductase activity in vitro. These studies revealed that that the Grx-like proteins do not exhibit oxidoreductase activity in this assay.

5

Investigating the role and activity of CC-Type glutaredoxins in the redox regulation of TGA1/TGA4 in Arabidopsis thaliana

Hahn, Kristen Rae 7 July 2009 (has links)
Plants respond to and defend themselves against a wide range of disease-causing microbes. In order to do so, massive reprogramming of cellular protein expression patterns, which underpin various defense pathways, must occur. A family of basic leucine zipper transcription factors, called TGA factors, has been implicated in mediating this response. The TGA factors themselves are subject to complex regulation; of note, TGA1 and TGA4 are regulated via a reduction of conserved cysteines after treatment with the phenolic signaling molecular salicylic acid, which accumulates following pathogen challenge. Previous studies indicate that TGA factors physically interact in the yeast two-hybrid system with the plant-specific CC-type of glutaredoxin (Grx)-like proteins. Grx are a family of oxidoreductases that are important for maintaining the cellular redox status and often are required to modulate protein activity. The goal of this study was to ascertain the role of these Grx-like proteins in regulating TGA1 redox state. To this end, the expression patterns of several Grx genes were analyzed.

Quantitative-reverse-transcriptase PCR (q-RT-PCR) experiments indicated that TGA1 and TGA4 may be involved in down-regulating levels Grx-like gene transcripts after exposure to pathogens or salicylic acid (SA). Furthermore, qRT-PCR experiments also indicated that expression of some Grx-like genes is induced by SA, jasmonic acid (JA), and Pseudomonas syringae. Overexpression of the Grx-like protein, CXXC9, in Arabidopsis thaliana revealed that it is a regulatory factor in the cross-talk between vi theSA/JA pathways as it is able to suppress expression of PDF1.2, a marker for the JA defense pathway, as determined by qRT-PCR. The â-hydroxy ethyl disulfide (HED) assay was utilized to determine if the CC-type of Grx-like proteins have oxidoreductase activity in vitro. These studies revealed that that the Grx-like proteins do not exhibit oxidoreductase activity in this assay.

6

Molecular Characterization of Toxic Cyanobacteria in North American and East African Lakes

Chhun, Aline 2007 (has links)
Toxic cyanobacterial blooms constitute a threat to the safety and ecological quality of aquatic environments worldwide. Cyclic hepatotoxin, especially microcystin, is the most widely occurring of the cyanotoxins. The aim of this study was to identify the cyanobacterial genotypes present including how many toxic genotypes were present in two North American lakes and one African Lake. All three lakes are prone to cyanobacterial blooms and were sampled in 2005 and 2006: Lake Ontario (Bay of Quinte, Canada), Lake Erie (Maumee Bay, Canada) and Lake Victoria (Nyanza Gulf, Kenya). The cyanobacterial genotypic community was assessed using DNA based analyses of the hypervariable V3 region of the 16S rRNA gene. In addition, the aminotransferase (AMT) domain in modules mcyE and ndaF of the microcystin and nodularin gene cluster respectively was used to detect the presence of hepatotoxic genotypes. Denaturing gradient gel electrophoresis (DGGE) results from this study suggested that hepatotoxin producers were present in all study sites sampled and were most likely members of the genus Microcystis. This study was the first to report the potential for microcystin production in the in-shore and off-shore open lake of Nyanza Gulf in Kenya. A seasonal study of the Bay of Quinte and Maumee Bay showed differences in the cyanobacterial genotypic community from early to late summer. In addition, the cyanobacterial genotypic community from the Bay of Quinte differed from 2005 to 2006 and quantification of the North American samples revealed an increase in cyanobacterial cells from early to late summer. The Bay of Quinte saw relatively no change in hepatotoxic cells from early to late summer but in Maumee Bay hepatotoxic cells increased from undetectable in early summer to dominating the cyanobacterial community by late summer. This study demonstrated the use of DGGE and qPCR of the 16S rRNA-V3 and AMT gene region in monitoring the cyanobacterial community of waterbodies susceptible to toxic cyanobacterial blooms.
7

Endometrial gene expression related to recurrent miscarriage

Nordqvist, Anna 2014 (has links)
A woman’s uterus is a safe place for the baby to grow and get nourished which is not always the case since the endometrium can, due to some underlying causes repel the fertilised egg which in other words means that the woman undergoes miscarriage. Recurrent miscarriage is defined as three or more pregnancy losses before 22 weeks of gestation. Multiple etiologies is thought to cause recurrent miscarriage although still 50 % of these cases remains unknown. The aim of this study was to measure the gene expression of DKK1, STC1, TK1, IL8 and OLFM1 in the endometrium of women with recurrent miscarriage compared to fertile women by using quantitative real-time PCR (qPCR) with TaqMan probes and primers. Immunohistochemical staining of paraffin embedded endometrium tissue was performed with a primary antibody anti-IL8 for detection of the IL8-protein. No significant difference was seen in mRNA expression between the two groups, only the IL8 mRNA showed a tendency to significant difference between the groups, p=0,063. On protein level, the immunohistochemical staining of IL8 showed few stained cells in both groups. Interestingly, the number of cells was clearly more abundant in women with recurrent miscarriage than in fertile women, p=0,036. The main conclusion from this study is that the high number of IL8 produced cells in the endometrium may be a contributing factor to recurrent miscarriage and need to be investigated further.
8

Metabolismo respiratório de bradirrizóbios em processos "in vitro" e simbióticos analisado por PCR quantitativo em tempo real

Moreira, Wellington Marcelo Queixas. 2009 (has links)
Resumo: O Brasil é o segundo maior produtor de soja no mundo, cuja cultura requer o elemento nitrogênio em quantidades elevadas para manutenção do alto teor protéico dos grãos. A entrada de nitrogênio nos sistemas agrícolas pode ocorrer pela adição de fertilizantes nitrogenados ou por processos naturais como a Fixação Biológica do Nitrogênio, que se constitui como supridor de nitrogênio mais viável para a cultura da soja, tanto economicamente como ecologicamente. Este processo ocorre graças à simbiose que ocorre entre esta leguminosa e as bactérias do gênero Bradyrhizobium, resultando na formação de nódulos radiculares onde se dá a obtenção de todo o nitrogênio que a cultura necessita para alta produtividade. A introdução destas bactérias no solo se dá através da utilização de inoculantes comerciais, que incluem as bactérias Bradyrhizobium elkanii e Bradyrhizobium japonicum em sua composição. Aspectos relacionados à formulação e fabricação dos inoculantes comerciais reúnem os fatores mais importantes para a obtenção de um produto de qualidade, obedecendo à legislação vigente. Tendo em vista a análise de qualidade de inoculantes comerciais para soja em diferentes períodos de armazenamento, um estudo do metabolismo respiratório de bradirrizóbios foi realizado in vitro e em simbiose. Inoculantes comerciais com 12, 27 e 48 meses de idade foram analisados quanto suas características bioquímicas e fisiológicas, assim como testados em casa de vegetação. Adicionalmente, os mesmos testes foram realizados com Bradyrhizobium elkanii sob diferentes condições de oxigênio. Análise da expressão gênica indicou que um processo de expressão de genes relacionados à fixação biológica de nitrogênio (genes sensores a baixas tensões de oxigênio) foram expressos, entretanto não ocorrendo expressão do gene nifH, este só expresso em condições... (Resumo completo, clicar acesso eletrônico abaixo) Abstract: Brazil is the second major soybean producer in the world, whose culture requires the element nitrogen in large amounts for the maintenance of the high protein content in grains. The input of nitrogen in agricultural systems can occur by adding nitrogen fertilizer or by natural processes such as biological nitrogen fixation (BNF). BNF is the most feasible way to delivery nitrogen for the soybean crop, both economically and ecologically. This process occurs through the symbiosis between the legume and the bacteria of the genus Bradyrhizobium, resulting in the formation of root nodules where all nitrogen that the crop needs for high productivity is acquired. The introduction of these bacteria in soil is given by use of commercial inoculants, which include the bacteria Bradyrhizobium japonicum and Bradyrhizobium elkanii in its composition. Aspects related to formulation and manufacture of inoculants include the most important factors affecting the product quality, according to law. In order to analysis of the quality of commercial inoculants for soybean in different storage periods, a study of respiratory metabolism of bradyrhizobia was performed in vitro and in symbiosis. Inoculants with 12, 27 and 48 months old were analyzed accessing their biochemical and physiological characteristics, and tested at greenhouse. In addition, the same tests were conducted on cultures of Bradyrhizobium elkanii under different oxygen conditions. Analysis of gene expression have indicated that genes related to biological nitrogen fixation (genes sensors at low oxygen tension) were intensively expressed, but not occurring nifH gene expression which is only expressed under symbiosis. Similar data were found for the expression of these genes when B. elkanii grown at different oxygen tensions. These data indicate that as the oxygen level is reduced some genes related to nitrogen fixation are expressed... (Complete abstract click electronic access below) Orientadora: Eliana Gertrudes de Macedo Lemos Coorientador: Jackson Antônio Marcondes de Souza Banca: Jesus Aparecido Ferro Banca: Emanuel Maltempi de Souza Mestre
9

In-vitro-Untersuchungen zu transkriptionellen und translationalen Zusammenhängen von COX2 und MUC4 im Pankreaskarzinom Transcriptional and translational in-vitro analyses of COX2 and MUC4 in pancreatic cancer

Jo, Yong-Jun Peter 23 June 2011 (has links)
No description available.
10

Improvement of Winter Oilseed Rape Resistance to Verticillium longisporum - Assessment of Field Resistance and Characterization of Ultrastructural Plant Responses

Knüfer, Jessica 21 July 2011 (has links)
Die Intensivierung des Rapsanbaus in den letzten Jahren hat zu einem verstärkten Aufkommen des bodenbürtigen Gefäßpathogens V. longisporum geführt. Die für den Pilz charakteristischen Mikrosklerotien können langjährig im Boden überdauern, akkumulieren und somit zur fortdauernden Bodenkontamination führen. Eine Infektion mit V. longisporum kann bereits im Herbst erfolgen, wenn durch Wurzelexsudate stimulierte Mikrosklerotien auskeimen und direkt die Wurzelepidermis der Rapspflanze penetrieren. Einer sowohl intra-als auch interzellulär gerichteten Ausbreitung bis zu den Gefäßelementen schließt sich eine langanhaltende Phase des Pilzes im Gefäßsystem an. In dieser latenten Phase zeigen sich keine auffälligen Symptome an der Pflanze, erst zum Ende der Pflanzenentwicklung zeigt sich halbseitige Stängelverbräunung und vorzeitige Abreife kann zu Ertragseinbußen führen. Der Pilz bleibt so lange auf die Gefäße beschränkt bis die Pflanze in die Seneszenzphase eintritt. Dann erfolgt eine Besiedelung der angrenzenden parenchymatischen Zellen und die Bildung von Mikrosklerotien. Mit Pflanzenresten können diese wieder in den Boden gelangen. Da derzeit keine adequaten Pflanzenschutzmittel zur Verfügung stehen, ist der Anbau resistenter Sorten eine wirkungsvolle Maßnahme die Verbreitung des Pilzes einzudämmen und der Anreicherung von Mikrosklerotien im Boden entgegenzuwirken. Im Rahmen dieser Arbeit wurde ein entscheidender Beitrag zur Züchtung neuer resistenter Genotypen geleistet. Phänotypisierungen zur Identifizierung resistenter B. napus-Linien (darunter auch DH-Linien) erfolgten unter kontrollierten Bedingungen im Gewächshaus in Göttingen. Darüber hinaus wurde die Resistenz ausgewählter B. napus-Linien in zwei aufeinander folgenden Jahren anhand von Feldversuchen in Göttingen, an verschiedenen Standorten in Norddeutschland und an einem Standort in Südschweden evaluiert. Eine Untersuchung der von 2004 bis 2009 im Gewächshaus getesteten B. napus Akzessionen wurde hinsichtlich der Häufigkeitsverteilungen der berechneten normierten AUDPC-Werte betrachtet. So konnte deutlich gezeigt werden, dass sich das Resistenzlevel in den aktuellsten Screenings deutlich verbessert hat im Vergleich zum Beginn der Screenings. Die Reproduzierbarkeit der Screenings wurde deutlich durch die Betrachtung der normierten AUDPC-Werte der Referenzsorten ‘Falcon’ und ‘Express’. So waren die normierten AUDPC-Werte der mittelgradig resistenten Referenzsorte ‘Express’ durchgängig niedriger im Vergleich zu der anfälligen Sorte ‘Falcon’, was für die Robustheit der Methodik spricht. Der Vergleich zwischen Gewächshaus- und Feldversuchen zeigte, dass eine geringe Korrelation zwischen den im Feld und Gewächshaus getesteten Akzessionen besteht und macht die Komplexität der Untersuchungen deutlich. Ein Screening von Genotypen kann jedoch nur schnell und in großem Umfang unter Gewächshaus-Bedingungen erfolgen. Die erweiterte Testung im Feld ist dann jedoch nötig, um die Resistenz unter zusätzlichem abiotischem Stress zu evaluieren. Neben der Bewertung des Befallsgrades (Befallshäufigkeit, Befallsstärke) mittels Stoppelbonitur wurde eine alternative Bewertungsmethode zur Evaluierung der Resistenz im Feld kultivierter Rapspflanzen gegenüber V. longisporum entwickelt. Die Entwicklung einer sensitiven real-time PCR (qPCR)-Methode zur Detektion von V. longisporum in Rapsstängeln beinhaltete die Bewertung zweier unterschiedlicher Primer, abzielend auf die internal transcribed spacer (ITS) Region bzw. auf die β-Tubulin-Region, die hinsichtlich ihrer Sensitivität und Spezifität analysiert wurden. Die hier getesteten ITS-Primer wiesen eine hohe Sensitivität gegenüber genomischer Pilz-DNA auf, jedoch wurde keine Spezifität gegenüber V. longisporum Isolaten festgestellt; vielmehr wurden V. dahliae Isolate und zwei weitere Verticillium Arten mit ITS-Primern detektiert. Das zweite getestete Primerpaar zeigte hingegen eine hohe Spezifität gegenüber V. longsiporum Isolaten, lediglich 3 von 15 getesteten V. longisporum Isolaten wurden nicht erfasst. Die Sensitivität dieser Primer war jedoch im Vergleich zu den ITS-Primern stark verringert. Die ITS-basierte qPCR Analyse führte zur Detektion des Pathogens noch vor der Symptomausbildung im Feld. So konnte in der Saison 2008/09 am Standort Göttingen gezeigt werden, dass frühe Infektionen bereits zu BBCH 65 auftraten und innerhalb weniger Wochen eine massive Besiedelung anfälliger Sorten erfolgte. Zudem konnte die pilzliche DNA-Konzentration in infizierten Rapsstängeln verschieden anfälliger Sorten quantifiziert und eine Korrelation zwischen der herkömmlichen Stoppelbonitur und dem Screening im Gewächshaus hinsichtlich der Einordnung der Resistenzniveaus hergestellt werden. Dies unterstützt die Verwendung der molekularen Methode als Alternative zur Stoppelbonitur. Neben der Verbesserung der Detektion von V. longisporum im Feld wurde die Pathogen-Wirt-Interaktion hinsichtlich der Ausbildung von Resistenzmechanismen charakterisiert. Dazu wurden zwei verschieden anfällige B. napus-Linien nach Inokulation mit V. longisporum sowohl auf histologischer als auch auf molekularbiologischer Ebene im Hypokotylbereich untersucht. Dieser Abschnitt, der den Bereich vom Wurzelhals bis zum Keimblattansatz markiert, konnte in vorangegangenen Untersuchungen als Schlüsselgewebe für die Ausbildung von Resistenzstrukturen identifiziert werden (Eynck et al., 2009). Anknüpfend an diese Untersuchungen wurden mittels Transmissionselektronenmikroskopie (TEM) genotypabhängige Resistenzstrukturen wie Gefäßverschlüsse und morphologische Veränderungen des Gefäßbereiches untersucht und begleitende qPCR-Messungen dokumentierten die Pathogenausbreitung. Diese ließen erkennen, dass der anfällige Genotyp im Vergleich zum resistenten Genotyp schneller besiedelt wird. Jedoch zeigten beide mit V. longisporum inokulierten Genotypen ähnliche ultrastrukturelle Veränderungen im vaskulären Bereich. So konnten Veränderungen an vaskulären Zellwänden wie elektronendichte Ablagerungen und Degradation primärer Zellwände im Bereich der Tüpfel beobachtet werden. Zudem konnte das Verschließen von Gefäßelementen mittels gelartiger Strukturen nachgewiesen werden. Unsere Untersuchungen lassen vermuten, dass der resistente Genotyp fähig ist Infektionen schneller zu erkennen und Resistenzmechanismen zielgerichteter und intensiver zu aktivieren. Da eine V. longisporum-Infektion in dem untersuchten resistenten Genotyp SEM 05-500256 u. a. zu einer verstärkten Bildung von Gefäßbarrieren im Hypokotylbereich führt (Eynck et al., 2009), wurde eine Beeinträchtigung des pflanzlichen Wassertransportes vermutet. Zur Klärung dieser Frage wurde der resistente Genotyp zusätzlich zu einer Infektion mit V. longisporum Trockenstressbedingungen (30% Feldkapazität) ausgesetzt und physiologische Parameter (Gaswechselmessungen), Befallswerte (AUDPC, Stauchung) und agronomische Parameter (Phänologisches Entwicklungsstadium, Anzahl Seitentriebe, Ertragsparameter) erfasst und im Vergleich zu der anfälligen Sorte ‘Falcon’ evaluiert. Weder die Befallsparameter noch die agronomischen Parameter zeigten eine Beeinträchtigung der Resistenz von SEM bei V. longisporum-Infektion in Kombination mit Trockenstress an.

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