Infection with challenge virus standard-11 (CVS) strain, a laboratory fixed rabies virus strain, induces neuronal process degeneration in both in vivo and in vitro models. CVS-induced axonal swellings of primary rodent dorsal root ganglion neurons are associated with 4-hydroxy-2-nonenal staining indicating a critical role of oxidative stress. Mitochondrial dysfunction is one of the most important causes of oxidative stress. We hypothesized that CVS infection induces mitochondrial dysfunction leading to oxidative stress. We investigated the effects of CVS infection on several mitochondrial parameters in different cell types. CVS infection increased electron transport chain capacity, Complex I and IV activities, but did not affect Complex II-III, citrate synthase, and malate dehydrogenase activities. CVS maintained normal oxidative phosphorylation capacity and proton leak, indicating a tight mitochondrial coupling. Possibly as a result of enhanced Complex activity and efficient coupling, a high mitochondrial membrane potential was generated. CVS infection reduced the intracellular ATP level and altered the cellular redox state as indicated by high NADH/NAD+ ratio. CVS infection was associated with a higher rate of hydrogen peroxide production. We conclude that CVS infection induces mitochondrial dysfunction leading to ROS overgeneration, oxidative stress and neuronal process degeneration.

Spinobulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of a polyglutamine (polyGln) tract in the human androgen receptor (hAR). One mechanism by which polyGln-expanded proteins are believed to cause neuronotoxicity is through aberrant interaction(s) with, and possible sequestration of, critical cellular protein(s). / Our goal was to confirm and further characterize the interaction between hAR and cytochrome c oxidase subunit Vb (COXVb), a nuclear-encoded mitochondrial protein. We had previously isolated COXVb as an AR-interacting protein in a yeast two-hybrid search to identify candidates that interact with normal and polyGln-expanded AR. Using the mammalian two-hybrid system, we confirm that COXVb interacts with normal and mutant AR and demonstrate that the COXVb-normal AR interaction is stimulated by heat shock protein 70 (Hsp70). Also, BFP-tagged AR specifically co-localizes with cytoplasmic aggregates formed by GFP-labelled polyGln-expanded AR in androgen-treated cells. / Mitochondrial dysfunction may precede neuropathological findings in polyGln-expanded disorders and may thus represent an early event in neuronotoxicity. Interaction of COXVb and hAR, with subsequent sequestration of COXVb, may provide a mechanism for putative mitochondrial dysfunction in SBMA.

Infection with challenge virus standard-11 (CVS) strain, a laboratory fixed rabies virus strain, induces neuronal process degeneration in both in vivo and in vitro models. CVS-induced axonal swellings of primary rodent dorsal root ganglion neurons are associated with 4-hydroxy-2-nonenal staining indicating a critical role of oxidative stress. Mitochondrial dysfunction is one of the most important causes of oxidative stress. We hypothesized that CVS infection induces mitochondrial dysfunction leading to oxidative stress. We investigated the effects of CVS infection on several mitochondrial parameters in different cell types. CVS infection increased electron transport chain capacity, Complex I and IV activities, but did not affect Complex II-III, citrate synthase, and malate dehydrogenase activities. CVS maintained normal oxidative phosphorylation capacity and proton leak, indicating a tight mitochondrial coupling. Possibly as a result of enhanced Complex activity and efficient coupling, a high mitochondrial membrane potential was generated. CVS infection reduced the intracellular ATP level and altered the cellular redox state as indicated by high NADH/NAD+ ratio. CVS infection was associated with a higher rate of hydrogen peroxide production. We conclude that CVS infection induces mitochondrial dysfunction leading to ROS overgeneration, oxidative stress and neuronal process degeneration.

The study of leptospiral pathogenesis is hampered by the lack of efficient mutagenesis methodologies. Thus research has focused on alternative approaches including genome sequencing, comparative genomics, transcriptomics and proteomics. In this thesis a comparative proteomic approach was used to identify leptospiral proteins with a potential role in the leptospiral infection process. Identification of proteins was followed by characterization of target proteins with potential roles in the infection process and ultimately led to the identification of a novel leptospiral virulence factor. Specifically, comparative proteomics using isobaric tags for relative and absolute quantitation complemented with two-dimensional gel electrophoresis were used for mass spectrometry-based protein identification and quantitation. These methodologies were utilised to identify and quantitate leptospiral proteins altered in expression in response to growth media limited in iron supply and/or supplemented with serum. These conditions were designed to mimic a subset of variables encountered by the bacteria within the host. These experiments led to the identification of five proteins with potentially novel roles in the leptospiral infection process. One of these proteins was further characterized as a periplasmic catalase, KatE. Using insertion mutagenesis it was demonstrated that KatE enhances extracellular H2O2 resistance and is required for virulence in guinea pigs and hamsters. Proteomic analyses also led to the identification of glutamic acid methylation of a protein that was further characterised to be surface exposed and expressed during leptospiral colonization of hamster liver and kidneys. This was the first description of glutamic acid methylation of a surface exposed protein in Leptospira. / Graduate

Abiotic stress is one of the major factors that affect food production worldwide and, therefore understanding stress responsive proteins and engineering plants for abiotic stress tolerance is very important. In the present study, the biological role of pea pathogenesis-related 10.4 (PR-10.4; also known as abscisic acid responsive 17; ABR17) in abiotic stress tolerance has been investigated. Our investigation on ribonuclease (RNase) activity of ABR17 suggested that highly conserved histidine-69 and glutamic acid-148 are important for RNase activity. In order to further investigate the biological role(s) of ABR17, transcriptional profiling of pea ABR17-mediated gene expression changes in ABR17-transgenic Arabidopsis thaliana plants was carried out using microarrays. Our results indicated that pea ABR17 modulates many plant growth/development genes most of which are cytokinin (CK) responsive. These results agree very well with previously reported enhanced endogenous CKs in these transgenic plants. However, no significant changes in transcript abundance of CK biosynthetic genes were observed between transgenic and wild-type plants, suggesting an alternate source of CK in ABR17-transgenic plants. It is speculated that ABR17 may act as either a CK reservoir (through its reported CK binding property) or may be responsible for isopentenylated-tRNA degradation (through its demonstrated RNase activity) thereby increasing endogenous CK pools. Furthermore, microarray analysis of salinity stressed ABR17-Arabidopsis indicated that ABR17 modulates many stress responsive genes that included four putative AP2 family genes (RAP2.6-At1g43160, RAP2.6L-At5g13330, DREB26-At1g21910 and DREB19-At2g38340). Functional characterization of these genes suggested that they are transcription factors and they play very important roles in abiotic stress response in addition to growth and development. Moreover, overexpression of RAP2.6L and DREB19 genes enhanced salinity and drought tolerance in Arabidopsis. Taken together, our results suggest that pea ABR17 proteins are important in abiotic stress responses as they may act as source of enhanced CKs and they may also modulate expression of stress responsive genes to enhance stress tolerance in plants. However, additional research aimed at deciphering the links between ABR17 and CK biosynthesis as well as the mechanism of ABR17-mediated gene expression changes should be conducted in order to get more insights into the biological roles of PR10 proteins in planta. / Plant Science

No description available.

Improved methods for the in situ hybridisation detection of messenger RNA ( mRNA ) in sections of liver tissue, were derived by use of an experimental system. This involved the use of tritiated-poly ( dT ) probes to detect poly ( A ) sequences attached to the 3 ' end of mRNA in sections of mouse liver that had been processed in various ways. The improved - methods were applied to the detection of hepatitis B virus ( HBV ) - and hepatitis delta virus ( HDV ) - RNA. In situ hybridisation and immunostaining techniques were then applied to studies of the pathogenesis of HBV and duck hepatitis B virus ( DHBV ) infection. In situ hybridisation studies of liver biopsy tissue from HBV - infected immunosuppressed renal transplant patients demonstrated an anatomical association between piecemeal necrosis and HBV replication at the cellular level in some patients. However, widespread replicative infection of hepatocytes also occurred in some patients in the presence of normal hepatocyte morphology and mild inflammatory changes indicating that at the cellular level virus replication was not necessarily a direct cytopathic process. These findings supported the view that hepatocyte Injury may : ( i ) result from immune - mediated damage directed against cells undergoing replicative, but not restricted infection ; ( ii ) eliminate cells undergoing replicative infection and favour clonal regeneration of cells undergoing restricted infection. Localisation of interferon - alpha ( IFN - alpha ) expression in liver tissue chronically infected with HBV and HDV, identified mononuclear cells and fibroblasts ( but not hepatocytes ) as the main producers of IFN - alpha. IFN - alpha - positive cells were associated with areas of liver tissue containing cells supporting virus replication and exhibiting the greatest degree of liver damage, suggesting that locally produced IFN - alpha may be a natural regulator of virus replication in chronic liver disease. Experimental DHBV infection of Pekin - Aylesbury ducks showed that virus inoculated either intravenously or intraperitoneally, gained access to randomly distributed hepatocytes without first replicating in other cell types in the liver. Virus was seen to disseminate to contiguous cells following anatomical boundaries by the third day post - inoculation. Markers of DHBV infection in liver and serum showed reproducible kinetics, and duck hepatocytes in this system appeared to be highly permissive as large amounts of DHBV DNA and DHBsAg were produced intracellularly without the development of ongoing cytopathology. Hepatocytes were the major cell type responsible for early significant DHBV replication, in contrast to pancreas, kidney, spleen and circulating mononuclear cells where significant levels of infection were detected only after the first week of infection and the onset of viraemia. / Thesis (Ph.D.)--Department of Microbiology and Immunology, 1989.

Studies presented in this thesis looked at developing new methods for the diagnosis of virulent footrot (VFR) in sheep and identification of serogroups of Dichelobacter nodosus, the principal causative agent of footrot. Earlier studies had shown that immunological memory response in sheep recovered from VFR can be aroused by natural or recurrent infection or by injection of outer membrane protein (OMP) antigens to be used as a retrospective diagnostic test for VFR. But OMP antigen was found to be non-specific in older animals. To overcome this non-specificity of OMP antigen in anamnestic response, pilus antigen was evaluated in a trial at Camden. The results of this trial indicated that antibodies to pilus antigen can be detected over time in a manner similar to OMP antibodies so a retrospective assessment of VFR status can be made by anamnestic test with pilus antigens. The anamnestic response to pilus was similar in character to OMP antigen but unlike OMP was highly specific. The response to anamnestic challenge with pilus was determined by severity of the lesions they had expressed, with severe lesions triggering the greater responses. However, there was variation between individuals, with some (6 of 46 with severe lesions) failing to respond. This individual variation is probably mediated genetically as is response to vaccination. This anamnestic test was tested in flocks of sheep in Nepal that had a history of VFR which had apparently been eradicated. That assessment, based on clinical findings, was confirmed by the uniformly negative results in the pilus anamnestic test applied to a representative sample of the population. This allowed a conclusion that the virulent strains of D. nodosus involved had been eliminated from these flocks. As mentioned in the preceding study pilus antigen was found to be very specific and ideal for retrospective diagnosis of virulent footrot with an anamnestic challenge ELISA test. However, serogroup specificity was seen as a disadvantage of using pilus antigen for the anamnestic test. The possibility of using multivalent pilus antigens was tested in another trial. These animals had been involved in a clinical expression experiment conducted by another research group and had a clinical and bacteriological history extending over more than 12 months. After these initial trials all these animals were treated for footrot and managed for 5 months as a single flock at Camden. These were then challenged with multivalent pilus antigen (serogroup A - I) as a single injection. The results obtained indicate that multivalent pilus anamnestic ELISA is equally effective as monovalent pilus. This has the added advantage that prior knowledge of the serogroups present in the flock is not required. It has the possibility of being used as an indirect test to check the presence of serogroups in a flock without doing the bacterial cultures. This test can identify most animals with pre-existing underrunning lesions (Scores of 3 or higher). However, the sensitivity and specificity of this test need to be tested extensively in flocks of known clinical history before it could be adopted as a routine test. As a key component of a larger study to determine the role of fimbrial genes (fimA and fimB) of D. nodosus in the pathogenesis of footrot using allelic exchange to disrupt these genes of a strain (serogroup G), the study presented in this thesis contributed a detailed characterisation of the resultant mutant and the wild strains and tested these strains for virulence in sheep. The results presented provided the first definitive evidence that the fimA gene is essential for virulence of D.nodosus in sheep. In vivo virulence testing of two fimA mutants showed that they were not able to establish any footrot whereas the wild type of the same strain produced virulent footrot in the same trial conducted under similar conditions. These mutant bacteria were not re-isolated from interdigital skin after in vivo challenge. This indicated that fimA mutant strains could not colonise the ovine foot, and the simplest and most likely explanation for these results was that colonisation of the interdigital skin and subsequent penetration of the stratum corneum requires the adhesive activity of type IV fimbriae. However, since these mutants also had altered ability to secrete extracellular proteases, and perhaps other as yet unknown extracellular proteins, the possibility of the involvement of these factors as major determinants of host colonisation or invasion cannot be excluded. Current methods for the identification of the serogroup of D. nodosus present in the bacterial population requires isolation of the organism and after purification by subculture, antigenic analysis with agglutination tests. This usually takes at least 3 to 4 weeks. With the objective of developing a rapid serogroup specific PCR assay, the basis of serogroup variation in D. nodosus localised in the fimbrial gene region was exploited. A common forward primer and 9 serogroup specific reverse primers were selected from the fimbrial gene sequences of the prototype strains. Analytical sensitivity of the serogroup specific primers on chromosomal DNA was similar to PCR tests in other bacterial species reported before. Immuno-magnetic bead capture PCR method was able to detect 5 to 10 cells in cell lysates. Specificity within and between the serogroups of D. nodosus was tested with all the prototype strains. They were found to be very specific to each serogroup and specific only to D. nodosus when tested with 84 commonly found bacterial strains or strains related to D. nodosus. To overcome the time delay in conducting 9 different amplifications to find out the prevalence of all possible serogroups in a flock multiplex PCR reactions with common forward primer and groups of 3, 4 and 5 reverse primers were successful in reducing the number of reactions to 2 (with groups of 4 and 5) or 3 (with groups of 3) primers. A drawback of the multiplex reaction was that if a template was 1000 times less concentrated that the others in the mixture it was not amplified but the margin for difference is very high. The main aim of developing rapid serogroup specific PCR was to apply these tests directly on footrot lesion samples to make it a rapid diagnostic test for field samples. The sensitivity of the test on lesion samples was found to be very low. To try and improve the sensitivity an overnight or four days old pre-enrichment culture in broth was tested but was found to be no better than direct PCR. The immuno-magnetic capture method which improved the sensitivity of pure culture samples by 10 -100 fold also had very low sensitivity with lesion samples. However, this drawback can be overcome by picking up colonies from 4 days old lesion cultures on hoof agar (HA) plates for serogroup specific multiplex PCR. If the colonies are too small/ too few on the lesion cultures these can be sub cultured onto a quarter of 4 percent HA plates and then used for the PCR test which also reduces the time taken for serogrouping at least by 2 weeks. The other advantage is that individual colonies do not need to be isolated. A PCR test can be done on pooled colonies just as well and can be used to identify all serogroups present in the sample. Serogroup specific PCR is much faster and is more sensitive and accurate than slide agglutination tests which take 3 to 4 weeks to complete. Multiplex PCR makes it easier to detect different serogroups in a sample with a maximum of 3 PCR tests. Serogroup specific multiplex PCR will be a useful tool for footrot control based on specific vaccination. The difficulty in using the test on direct lesion swabs needs to be further looked into. There may be future advances in the application of PCR tests to clinical samples.

Diss. (sammanfattning) Alnarp : Sveriges lantbruksuniversitet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2004. / Härtill 3 uppsatser.