The binding of a novel GABAs receptor radioligand eH]-CGP62349 to human hippocampal control and epileptic sections was investigated using quantitative receptor autoradiography. Kinetic analyses performed on rat brain sections, to conserve the use of human tissues, demonstrate that eH]-CGP62349 associated rapidly. The same radioligand dissociated rapidly initially, then very slowly. Utilising human hippocampus it was shown that CH]-CGP62349 bound with high affinity (O.SnM) to human control hippocampal sections. The kinetics of GABAs receptor in human hippocampus using the novel compound confirmed previous studies performed in rat membrane. The localisation of GABAB receptors in human hippocampal control partially supported former studies using agonist ligands such as CH]-GABA and eH]-baclofen, despite differences have been noticed. Hippocampal slices from surgical resected specimens obtained from hippocampal sclerotic (HS)/temporal lobe epilepsy (TLE) patients were compared with neurologically normal post-mortem control subjects for neuropathology and GABAs receptor density and affinity. Neuronal loss was observed in most of the hippocampal subregions, whereas in subiculum any significant difference was detected. For GABAa receptor density (Bmax) a significant reduction was reported in CA3, CA4, and DG; the affinity was increased exclusively in DG. After the correction of Bmax value for the neuronal loss a significant increase was seen in CA3. Oligonucleotides were designed to investigate the two GABAB1 isoforms (la, and lb), and the GABAa2 subunit in human hippocampal control and HSITLE tissues, obtained as well as for the autoradiography. GABAsla, GABAB1b, and GABAB2 transcript distribution was in agreement with the receptor protein localisation, even though in the human hippocampus GABAa2 has to be yet localised and to verify if it is associated with GABAsla or GABABlbto form the dimeric active receptor. The present study suggests an involvement of GABABreceptors in HS/TLE in some not all the hippocampal subregions in terms of receptor density and affinity. Further investigations in regard to the quantitative in situ hybridisation data and the immunocytochemical results are fundamental to gain insight into the pathological role of GABAs receptors.

No description available.

No description available.

Dysfunctional or over and under expressed enzymes play a crucial role in a variety of diseases. A tool that can identify dis-regulated enzymes in individual patients would be beneficial and would allow personalised treatment. For this purpose, a 10,000 membered ‘spit-and-mix’ PNA-encoded peptide library with a cell penetrating peptide was synthesised and interrogated with K562 cell lysate and intact K562 cells. This allowed the specific enzyme activity pattern for ABL tyrosine kinase from both inside a cell and a lysate to be obtained. Hybridisation of this library with a DNA-microarray resulted in bio-fouling by the cell lysate, thereby preventing analysis of the phosphorylation pattern. To allow extraction and purification of the peptide library from the cell lysates, a His-tag was incorporated into the library, and enabled successful library analysis. In addition to this 10,000 member library, a focused 100 PNA-encoded peptide library was synthesised. The library included peptide sequences known to be phosphorylated by specific tyrosine kinases deregulated in acute lymphoblastic leukaemia (ALL) with a PNA-tag complementary to a DNA microarray. Different ligation methods to conjugate the peptides to PNA-tags were screened – this included amide coupling, copper catalysed azide–alkyne cycloaddition, strain promoted azide–alkyne cycloaddition and Diels–Alder cycloaddition. The inverse electron demand Diels–Alder cycloaddition between a tetrazine and norbornene was chosen as the preferred ligation method, and the reaction conditions optimised. To purify the library from cell lysate, a His-tag was again coupled to each member using the strain promoted azide–alkyne cycloaddition. To test the tetrazine ligation, fluorescence in situ hybridisation (FISH) was used in cells, whereby a fluorophore was ligated onto a tetrazine–conjugated PNA probe. This was hybridised onto an mRNA in fixed cells. Results indicated that the ligation needed further optimisation.

A cryptic haplosporidian parasite was detected infecting rock oysters from the Montebello Islands in north-western Australia using a PCR targeting the parasite’s small ribosomal subunit gene. The PCR products were cloned and sequenced along with the remaining sections of the parasite’s SSU rRNA gene. Phylogenetic analysis of the sequence generated indicated a Minchinia species (Haplosporidia). The SSU sequence generated was used to develop two in situ hybridisation assays to visualise the parasite in H/E sections as well as a PCR assay to detect the parasite. The molecular assays were assessed for specificity and sensitivity and were then used to compare the parasite to previous haplosporidian parasite infections of pearl oysters. Both assays produced positive results from the infected pearl oysters but not from other closely related haplosporidian species. An SEM and TEM electron microscopy analysis was performed on spores from both parasite species. The spores of the pearl oyster parasite had two spore wall filaments wound around the spore originating for a posterior thickening while the spores of the rock oyster parasite were covered in microtubule-like structures. These data suggests pearl oysters where co-infected with both the Haplosporidium sp. and the Minchinia sp. detected in rock oysters. No evidence of a posterior thickening could be found on the spores of the rock oyster parasite. Attempts to detect the parasite at the previous geographic sites of its detection in pearl oysters resulted in detection of the Minchinia species in tropical oysters in the Kimberley region of Western Australia by in-situ hybridisation.

Microbial communities associated with gastrointestinal tract of animals play a critical role in gut development, digestion and resistance to disease, thus the prospect of altering these communities beneficially by using probiotics is attractive. In terrestrial animals, the gut provides a stable, moist habitat in an otherwise moisture-limited environment, thus microbial communities tend to be very stable. In contrast, farmed aquatic animals reside within an environment that can support microbes in high densities, and as many marine animals drink continuously for osmoregulation, they are subjected to potential re-inoculation. Consequently, little is known of the stability of gut microbial communities in marine shrimp or whether it is possible to establish beneficial bacteria in the gut. The aims of this thesis were therefore to examine the midgut microbial community associated with farmed black tiger shrimp, Penaeus monodon, and to investigate whether the introduction of potentially probiotic Bacillus could alter the species diversity or abundance of the present microbes. Using culture methods it was found that B. pumilus was able to transfer between animals via the water column and persisted in the midgut for at least 7 days, while B. subtilis was only recovered from animals directly fed the bacteria and persisted for less than 24 h in the midgut. V. parahaemolyticus, a known shrimp pathogen,remained in the tanks it was originally found in, and did not transfer via the water column to other tanks and is therefore tightly associated with its host. A bacterium with apparent probiotic qualities was isolated from control animals in the above study and identified as a strain of B. pumilus. Its safety for food animal use was confirmed due to the absence of B. cereus toxin genes, and the isolate’s pH and salt tolerances were investigated. Moreover, the isolate was highly inhibitory to crustacean pathogens in the family Vibrionaceae. Methods to investigate the gut microbiota using the full cycle 16S rRNA methodology were optimized. Fluorescence in situ hybridization (FISH) probes designed specifically targeting B. pumilus, B. subtilis and B. licheniformis, commercially available probiotics, were validated for specificity and optimal hybridization conditions. For FISH analysis of bacteria in situ in histological sections of shrimp midgut trunks, fixation times in 4 % paraformaldehyde wereoptimizedfor bacterial RNA retention whilst maintaining tissue integrity. Due to the broad range of autofluorescence in the shrimp tissue, spectral imaging is required to adequately differentiate between host tissue and multiple bacterial probes. The richness and diversity of the midgut microbiota of animals treated with the novel strain of B. pumiluswere analyzed using 16S rRNA gene clone libraries and FISH analysis of histological sections. It was confirmed that B. pumilus can enter the midgut via top-coated feed and through water inoculation. In the tanks that were treated with B. pumilus the proportion of Vibrio sp. in the microbial community decreased, however, only in the systems in which B. pumilus was recovered from the shrimp midgut did the proportion of pathogenic Vibrio species decrease. The application of the B. pumilus caused a shift in the shrimp midgut microbiota, but the community returned to its initial diversity over time. The midgut microbiota of P. monodon is relatively stable but can be adjusted using probiotics. The transience or residence of the probiotics is strain-specific and should be tested for any new strains before determining optimum application protocols. The methods designed in this study are applicable to future research in this field.

All animals have evolved mechanisms to recognise and eliminate nonself in order to defend against invading pathogens and to prevent chimerism, the fusion between genetically distinct conspecifics. Like other metazoans, sponges are known to rely on sophisticated systems to maintain their self-integrity. As poriferans are also considered one of the most ancient extant metazoan phyla, they represent a critical comparative model for understanding the early evolution of immunity and self/nonself recognition in animals. The Toll-like receptor (TLR) signalling cascade plays a crucial role in immunity, and recent findings in the sponge Suberites domuncula suggest that its origin could predate eumetazoan cladogenesis. My genome and expressed sequence tag (EST) screens of the demosponge Amphimedon queenslandica detected homologues to most components of this pathway, supporting the notion that a primordial TLR signalling cascade emerged at the dawn of the Metazoa. The sponge also encodes a couple of putative TLR-related proteins (AmqIgTIRs) that consist of at least one extracellular immunoglobulin (Ig) and an intracellular Toll/Interleukin-1 receptor/resistance (TIR) domain. The presence of other unconventional TLRs in S. domuncula and in cnidarian representatives, implies that an ancestral TLR probably existed in the last common ancestor of all living metazoans, and independent duplication and divergence events led to the variety of forms observed in animals. Among the putative transcription factors present in Amphimedon, which are known to be activated by the TLR signalling cascade in other eumetazoans, I detected a single member of the Rel/nuclear factor-kappaB (NF-κB) family, AmqNF-κB, which is also the only Rel homology domain (RHD)-containing gene present in the sponge. This gene encodes a protein that is equipped with both a RHD and ankyrin (ANK) repeats, suggesting that the ancestral metazoan NF-κB was configured identically to contemporary vertebrate and sponge forms, and that the truncated NF-κB found in Nematostella vectensis resulted from the secondary loss of ANK. Aside from immunity, the Toll and TLR pathways contribute to a variety of biological processes in bilaterians, however their functions have only been investigated in detail in a limited number of metazoan model organisms. While studies have tested the immune role of various sponge genes, including components of the TLR cascade, no research has yet established whether they are also involved in development. Therefore, I investigated the expression of some of the immunity-related genes I isolated in Amphimedon in a developmental and immune context to shed light on the potential ancestral function(s) of the proteins they encode. Using in situ hybridisation, I demonstrate that AmqIgTIR2, AmqMyD88, AmqTollip, AmqPellino and AmqNF-ĸB are expressed during A. queenslandica early development. In contrast, the spatial and temporal expression of AmqIgTIR1 suggests it might encode a receptor that is specifically involved in the detection of metamorphic cues in larvae. A real-time quantitative PCR (qPCR) study performed on a pool of adult sponge cDNAs indicates that the expression levels of AmqIgTIR1, AmqIgTIR2, AmqMyD88 and AmqTollip are significantly affected by a nine-hour incubation in 50 µg/ml of lipopolysaccharide (LPS), and to a lesser extent by 105 colony forming units (cfu)/ml of live Vibrio harveyi. The gene expression of AmqIgTIR1 and AmqIgTIR2 suggests that they may encode proteins with antagonistic immunological functions. While AmqPellino and AmqNF-ĸB do not appear to be affected by LPS and Vibrio exposure, it is possible that these genes do not participate in the early immune response of poriferans. Together, my data indicate that the sponge genes surveyed might encode proteins that perform developmental, sensory and immunological functions, suggesting their roles could have also been multifaceted in the last common ancestor to all living metazoans. As is observed in other invertebrates, poriferans display an ontogenic shift in allorecognition; genetically different individuals can fuse during early development but, in most instances, not as adults. However, there is a limited understanding of the cellular organisation of sponge chimeras and the onset of poriferan allorecognition response. By following the fates of fluorescently tagged cells derived from genetically distinct Amphimedon larvae that are fused together at metamorphosis, I establish that there is a rapid ontogenic shift in the sponge allogeneic response about two weeks after the initiation of metamorphosis. Moreover, the molecular basis of the poriferan allorecognition system is possibly involved in creating differential cell affinities, which underlie the construction of the sponge body plan. Compatible with this scenario is the observation that cells from postlarvae that are allowed to develop for two weeks before contact do not fuse, and form a distinct boundary between genotypes. The molecules responsible for sponge cell reaggregation, the aggregation factors (AFs), have been proposed to drive the allorecognition response in poriferans. Notably, the Amphimedon genome encodes six putative AFs, of which five occur in a cluster. These findings indicate that the polymorphic variation observed in other poriferan AFs is probably the result of allelic variations of multiple genes belonging to the same family.

Trematode peptidases are important molecules responsible for biocatalysis in many basal biological processes and are crucial in host-parasite interactions. Therefore, these enzymes are intensively studied in order to characterize their biological functions and to use them as potential diagnostic or therapeutic targets. Lately, investigation of transcriptome and secretome revealed, that adult Fascioloides magna (giant liver fluke) expresses and secretes a variety of peptidases. Thus, this thesis focuses on three newly identified enzymes: cathepsin L (FmCL), cathepsin B (FmCB) and cathepsin D (FmCD). In other trematode species, these cathepsins are being linked mainly with the digestion of host blood. We applied quantitative PCR (qPCR) to investigate relative expression levels of the three enzymes among three developmental stages - egg, miracidium and adult. It was revealed that all cathepsins have the highest expression in adult flukes in comparison to eggs and miracidia. We also localized the place of transcription of FmCL, FmCB and FmCD in adult fluke using RNA in situ hybridization. All of the peptidases were detected in gastrodermis, and in addition, they were localized in the reproductive system. The latter surprising finding is suggesting that these enzymes might have multiple functions in adult F....

Non-small cell lung carcinoma is a leading cause of cancer mortality worldwide. There are gender and smoking associated differences both in tumour types and clinical outcome. Squamous cell carcinomas (SCC) are more frequent among smoking men while females develop adenocarcinomas (ADCA). NSCLC among never smokers are mainly ADCA, and occurs mostly in females. The present thesis elucidates the role of estrogen receptor (ER) and epidermal growth factor receptor family (EGFR/HER2-4) in NSCLC in the perspective of gender and histology as well as the influence of smoking on those biomarkers. A recently developed technique, tissue micro array (TMA), was employed.The question of how much of a tumour tissue that needed to be included in a TMA for biomarker analysis was analyzed by a statistical approach. Data indicates a sample size of three cylinders of tumour tissue with a diameter of 0.6 mm each as being appropriate and cost-effective. In order to optimally use the up to thousands of different tumour samples within a TMA, it would be optimal to serially cut and store slides before performing in situ detection of proteins and nucleic acids. Applying up to date methodology, and by evaluation with image analysis, data are presented that shows that such handling of TMA slides would be possible without any loss of biomarker information. ERα is more frequently observed in ADCA and in females and a local estradiol synthesis is supported by the presence of aromatase. ERβ is identified as a positive prognostic marker in ADCA. Smoking is associated to increased levels of ERβ mRNA. EGFR over expression is associated with a ligand. Independent phosporylation of ERα. HER-4 intracellular domain may also act as a co-activator to ERα in ADCA, especially among neversmokers. The question of ER and EGFR family signalling crosstalk as a potential target for combined targeted therapy is raised.

<p>Human endogenous retroviruses (HERVs) represent approximately 7% of the human genome. This investigation was focused on one particular HERV, ERV3, with the main purpose of characterising its gene expression patterns and genomic distribution of ERV3-like sequences. Furthermore, this careful expression study should provide insights into the biological role of HERVs. The impact of HERVs in health and disease is not yet clarified. ERV3 is expressed as three envelope (<i>env</i>) transcripts, of which two also contain a cellular gene, <i>H-plk</i> (human proviral linked <i>Krüppel</i>). ERV3 <i>env</i> expression was mainly investigated at the RNA level. The gene expression of two other HERVs, HERV-K and HERV-E was analysed and compared with ERV3 activity.</p><p>Real-time PCRs were developed and in combination with in situ hybridisation, it was found that ERV3 is expressed in a tissue- and cell-specific way. High levels of ERV3 mRNA (up to six times over Histone3.3) were demonstrated in placenta, sebaceous glands, foetal and adult adrenal glands, brown adipose tissue, corpus luteum, pituitary gland, thymus and testis. In monocytic cells including both normal monocytes and malignant U-937 cells, elevated mRNA levels were observed after retinoic acid (RA)-induced differentiation. ERV3-encoded Env protein was detected in selected cases, one following RA-treatment. In addition, several new ERV3-like sequences were discovered in the human genome. </p><p>ERV3 was found to have conserved open reading frames in contrast to other ERV3-like sequences in the human genome. This suggests that ERV3 may be involved in important cellular processes such as differentiation, cell fusion, immunomodulation and protection against infectious retroviruses. The developed techniques and obtained results will allow further studies of HERV expression to better correlate HERV activity to both normal development and disease. </p>