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)

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.

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)

Nutrient transporters in the small intestine are responsible for dietary nutrient assimilation and therefore the expression of these transporters can influence the overall nutrient status as well as the growth and development of the animal. This thesis examined correlated responses to selection in the developmental gene expression of the peptide transporter PepT1, the glutamate/aspartate transporter EAAT3, the sodium-dependent glucose transporter SGLT1, and the fructose transporter GLUT5 in the small intestine of chickens from lines divergently selected for high (HH) or low (LL) eight-week body weight and their reciprocal crosses, (HL and LH). Chicks were weighed and killed on embryonic day 20 (E20), day of hatch (DOH with no access to feed), and days 3 (D3), 7(D7), and 14 (D14) post hatch. Duodenum, jejunum, ileum and liver were collected. DNA extracted from liver was used to sex birds by PCR. RNA was extracted from the intestinal segments of four males and four females from each mating combination (MC) and time point except E20 HL males (n = 3) and D7 LL females (n = 2). Expression of nutrient transporters was assayed by real-time PCR using the relative quantification method. In comparing HH and LL males and females there was a line by segment interaction in PepT1 gene expression, with no segment difference in HH and greatest expression in the ileum of the LL (P < 0.05). There was also a MC by age by sex interaction for PepT1 gene expression (P < 0.0001) with peak gene expression occurring on DOH for LL females, on D7 for HH females, on D7 for LL males and D14 for HH males. Overall, females had greater EAAT3 expression (P < 0.03). Gene expression of EAAT3 was greatest in the ileum, intermediate in the jejunum, and least in the duodenum (P < 0.0007). There was an age by segment interaction for EAAT3 expression (P = 0.0002) and a MC by segment interaction (P < 0.02), with LL having greater expression than HH in the ileum. Females had greater SGLT1 expression than males (P < 0.0001). There was a sex by age interaction for the expression of SGLT1 (P < 0.0001). Females induced SGLT1 expression on DOH and maintained this level through D14, while males gradually increased expression through D7 and decreased expression by D14. These results indicate that expression of PepT1, EAAT3, SGLT1 are differentially expressed in male and female chickens regardless of selection for high or low juvenile body weight. These results also show a sexual dimorphism in the capacity to absorb peptides, anionic amino acids, and glucose from the intestine, which has implications for the poultry industry with regard to diet formulations for straight-run and sex-separate grow-out operations. In comparing male HH, HL, LH, and LL chicks, overall LL had the greatest level of expression (P <0.06), HH had the least level of expression (P < 0.006) and HL and LH had intermediate levels of expression (P < 0.06). Greatest PepT1 gene was expression in the ileum (P < 0.0003) and there was a MC by segment interaction with expression increasing from duodenum to ileum in LL, but there was no segment difference in any other MC (P < 0.08). Within each intestinal segment there was a MC difference (P < 0.02). There was an effect of sire for PepT1 expression, with progeny from low weight selected sires (LWS) having greater expression than progeny from high weight selected (HWS) sires (P = 0.0008). There was no difference between intestinal segments in progeny from HWS sires, however, greatest PepT1 gene expression was seen in the ileum of progeny from LWS sires (P < 0.0001). Overall, expression of EAAT3 was greatest in the ileum, intermediate in the jejunum and least in the ileum (P < 0.0001) and there was a segment by age interaction for EAAT3 expression (P < 0.0001). In all MCs except HH, EAAT3 gene expression increased from duodenum to ileum (P < 0.08). Within the ileum, the LL had greatest EAAT3 gene expression, LH and HL had intermediate gene expression, and HH had least expression (P < 0.08). Expression of SGLT1 gradually increased through D7 and decreased by D14 (P < 0.0001) and overall, was greatest in the distal small intestine (P < 0.0001). There was a MC by segment interaction, with SGLT1 gene expression being greatest in the distal small intestine in LL, LH, and HL, but greatest in the jejunum of HH (P < 0.04). Within the ileum, LL had greater SGLT1 gene expression than HH (P < 0.06). Overall, greatest GLUT5 expression was in the distal small intestine (P < 0.0001) and there was a MC by segment interaction, with expression being greatest in the distal small intestine in LL and HL (P < 0.02), greatest in the ileum of LH (P < 0.08), and greatest in the jejunum of HH (P < 0.09). Within the ileum there was a MC difference (P < 0.07). These results indicate that selection for high or low juvenile body weight may have influenced the gene expression pattern of these nutrient transporters in the small intestine, which may contribute to the overall differences in the growth and development of these lines of chickens. / Master of Science

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.<p> 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 <i>Pseudomonas syringae</i>. Overexpression of the Grx-like protein, CXXC9, in <i>Arabidopsis thaliana</i> 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 <i>in vitro</i>. These studies revealed that that the Grx-like proteins do not exhibit oxidoreductase activity in this assay.

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.<p> 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 <i>Pseudomonas syringae</i>. Overexpression of the Grx-like protein, CXXC9, in <i>Arabidopsis thaliana</i> 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 <i>in vitro</i>. These studies revealed that that the Grx-like proteins do not exhibit oxidoreductase activity in this assay.

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.

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

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.

In der vorliegenden Dissertation sollte der Krankheitsverlauf einer KHV-Infektion untersucht werden sowie der Einfluss den Haltungsbedingungen von Nutzkarpfen auf dessen Krankheitsverlauf (KHV-Disease; KHVD) haben. Zusätzlich sollte untersucht werden, ob Plötzen (Rutilus rutilus), Schleien (Tinca tinca) und Karauschen (Carassius carassius) als potentielle Carrierfische, ebenso als empfängliche Spezies eine Rolle spielen können.