Comparative gene expression in brain regions of human alcoholics

The mesocorticolimbic system is the reward centre of the brain and the major target for drugs of abuse including alcohol. Neuroadaptive changes in this region are thought to underlie the process of tolerance and dependence. Recently, several research groups have searched for alcohol-responsive genes using high-throughput microarrays and well-characterized human post-mortem material. Comparison of data from these studies of cortical regions highlights the differences in experimental approach and selection of cases. However, alcohol-responsive gene sets associated with transcription, oxidative stress and energy production were common to these studies. In marked contrast, alcohol-responsive genes in the nucleus accumbens and the ventral tegmental area are primarily associated with changes in neurotransmission and signal transduction. These data support the concept that, within cortical regions, changes in gene expression are associated with alcoholism-related pathology. In the dopaminergic tract of the mesocorticolimbic system, alcohol-responsive gene sets suggest long-term neuroplastic changes in synaptic transmission.

PerR controls Mn-dependent resistance to oxidative stress in Neisseria gonorrhoeae

In previous studies it has been established that resistance to superoxide by Neisseria gonorrhoeae is dependent on the accumulation of Mn(II) ions involving the ABC transporter, MntABC. A mutant strain lacking the periplasmic binding protein component (MntC) of this transport system is hypersensitive to killing by superoxide anion. In this study the mntC mutant was found to be more sensitive to H2O2 killing than the wild-type. Analysis of regulation of MntC expression revealed that it was de-repressed under low Mn(II) conditions. The N. gonorrhoeae mntABC locus lacks the mntR repressor typically found associated with this locus in other organisms. A search for a candidate regulator of mntABC expression revealed a homologue of PerR, a Mn-dependent peroxide-responsive regulator found in Gram-positive organisms. A perR mutant expressed more MntC protein than wild-type, and expression was independent of Mn(II), consistent with a role for PerR as a repressor of mntABC expression. The PerR regulon of N. gonorrhoeae was defined by microarray analysis and includes ribosomal proteins, TonB-dependent receptors and an alcohol dehydrogenase. Both the mntC and perR mutants had reduced intracellular survival in a human cervical epithelial cell model.

Expression and methylation pattern of TSLC1 cascade genes in lung carcinomas

TSLC1 (tumor suppressor in lung cancer-1, IGSF4) encodes a member of the immunoglobulin superfamily molecules, which is involved in cell-cell adhesion. TSLC1 is connected to the actin cytoskeleton by DAL-1 (differentially expressed in adenocarcinoma of the lung-1, EPB41L3) and it directly associates with MPP3, one of the human homologues of a Drosophila tumor suppressor gene, Discs large. Recent data suggest that aberrant promoter methylation is important for TSLC1 inactivation in lung carcinomas. However, little is known about the other two genes in this cascade, DAL-1 and MPP3. Thus, we investigated the expression and methylation patterns of these genes in lung cancer cell lines, primary lung carcinomas and nonmalignant lung tissue samples. By reverse transcription-polymerase chain reaction, loss of TSLC1 expression was observed in seven of 16 (44%) non-small-cell lung cancer (NSCLC) cell lines and in one of 11 (9%) small-cell lung cancer (SCLC) cell lines, while loss of DAL- 1 expression was seen in 14 of 16 (87%) NSCLC cell lines and in four of 11 (36%) SCLC cell lines. By contrast, MPP3 expression was found in all tumor cell lines analysed. Similar results were obtained by microarray analysis. TSLC1 methylation was seen in 13 of 39 (33%) NSC LC cell lines, in one of 11 (9%) SCLC cell lines and in 100 of 268 (37%) primary NSCLCs. DAL-1 methylation was observed in 17 of 39 (44%) NSCLC cell lines, in three of 11 (27%) SCLC cell lines and in 147 of 268 (55%) primary NSCLCs. In tumors of NSCLC patients with stage II-III disease, DAL-1 methylation was seen at a statistically significant higher frequency compared to tumors of patients with stage I disease. A significant correlation between loss of expression and methylation of the genes in lung cancer cell lines was found. Overall, 65% of primary NSCLCs had either TSLC1 or DAL-1 methylated. Methylation of one of these genes was detected in 59% of NSCLC cell lines; however, in SCLC cell lines, methylation was much less frequently observed. The majority of nonmalignant lung tissue samples was not TSLC1 and DAL-1 methylated. Re-expression of TSLC1 and DAL-1 was seen after treatment of lung cancer cell lines with 5-aza-2$-deoxy-cytidine. Our results suggest that methylation of TSLC1 and/or DAL-1, leading to loss of their expression, is an important event in the pathogenesis of NSCLC.

Ascorbic acid induces an oxidative stress in CCRF cells activating the transcription factors AP-1 and NFRB

We have previously tested the effects of high dose AA supplements on human volunteers in terms of reducing DNA damage, as a possible mechanism of the vitamin’s proposed protective effect against cancer and detected a transient, pro-oxidant effect at high doses (500 mg/day). Herein, we present evidence of a pro-oxidant effect of the vitamin when added to CCRF cells at extracellular concentrations which mimic those present in human serum in vivo (50–150AM). The activation of the transcription factor AP-1 was optimal at 100 AM AA following 3h exposure at 37jC. A minimum dose of 50 AM of AA activated NFnB but there appeared to be no dose-dependent effect. Increases of 2–3 fold were observed for both transcription factors when cells were exposed to 100 AM AA for 3h, comparing well with the pro-oxidant effect of H2O2 at similar concentrations. In parallel experiments the activation of AP-1 (binding to DNA) was potentiated when cells were pre-incubated with AA prior to exposure with H2O2. Cycloheximide pretreatment (10 Ag/ml for 15min) caused a 50% inhibition of AP-1 binding to DNA suggesting that it was due to a combination of increasing the binding of pre-existing Fos and Jun and an increase in their de novo synthesis. Cellular localisation was confirmed by immunocytochemistry using antibodies specific for c-Fos and c-Jun proteins. These results suggest that extracellular AA can elicit an intracellular stress response resulting in the activation of the oxidative stress-responsive transcription factors AP-1 and NFnB. These transcription factors are involved in the induction of genes associated with an oxidative stress response, cell cycle arrest and DNA repair confirmed by our cDNA microarray analysis (Affymetrix). This may explain the abilty for AA to appear to inhibit 8-oxodG, yet simultaneously generate another oxidative stress biomarker, 8-oxo-dA. These results suggest a completely novel DNA repair action for AA. Whether this action is relevant to our in vivo findings will be the subject of our future research.

Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level

Background: The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale. Results: We performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol- or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol. Conclusions: P. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very different. The main response to different growth conditions in P. pastoris is transcriptional; translational regulation was not transcript-specific. The high proportion of mRNAs associated with polysomes in methanol-grown cells is a major finding of this study; it reveals that high productivity during methanol induction is directly linked to the growth condition and not only to promoter strength.

Intraclonal heterogeneity and distinct molecular mechanisms characterize the development of t(4;14) and t(11;14) myeloma.

We have used whole exome sequencing to compare a group of presentation t(4;14) with t(11;14) cases of myeloma to define the mutational landscape. Each case was characterized by a median of 24.5 exonic nonsynonymous single-nucleotide variations, and there was a consistently higher number of mutations in the t(4;14) group, but this number did not reach statistical significance. We show that the transition and transversion rates in the 2 subgroups are similar, suggesting that there was no specific mechanism leading to mutation differentiating the 2 groups. Only 3% of mutations were seen in both groups, and recurrently mutated genes include NRAS, KRAS, BRAF, and DIS3 as well as DNAH5, a member of the axonemal dynein family. The pattern of mutation in each group was distinct, with the t(4;14) group being characterized by deregulation of chromatin organization, actin filament, and microfilament movement. Recurrent RAS pathway mutations identified subclonal heterogeneity at a mutational level in both groups, with mutations being present as either dominant or minor subclones. The presence of subclonal diversity was confirmed at a single-cell level using other tumor-acquired mutations. These results are consistent with a distinct molecular pathogenesis underlying each subgroup and have important impacts on targeted treatment strategies. The Medical Research Council Myeloma IX trial is registered under ISRCTN68454111.

Gelsolin-mediated activation of PI3K/Akt pathway is crucial for hepatocyte growth factor-induced cell scattering in gastric carcinoma

In gastric cancer (GC), the main subtypes (diffuse and intestinal types) differ in pathological characteristics, with diffuse GC exhibiting early disseminative and invasive behaviour. A distinctive feature of diffuse GC is loss of intercellular adhesion. Although widely attributed to mutations in the CDH1 gene encoding E-cadherin, a significant percentage of diffuse GC do not harbor CDH1 mutations. We found that the expression of the actin-modulating cytoskeletal protein, gelsolin, is significantly higher in diffuse-type compared to intestinal-type GCs, using immunohistochemical and microarray analysis. Furthermore, in GCs with wild-type CDH1, gelsolin expression correlated inversely with CDH1 gene expression. Downregulating gelsolin using siRNA in GC cells enhanced intercellular adhesion and E-cadherin expression, and reduced invasive capacity. Interestingly, hepatocyte growth factor (HGF) induced increased gelsolin expression, and gelsolin was essential for HGF-medicated cell scattering and E-cadherin transcriptional repression through Snail, Twist and Zeb2. The HGF-dependent effect on E-cadherin was found to be mediated by interactions between gelsolin and PI3K-Akt signaling. This study reveals for the first time a function of gelsolin in the HGF/cMet oncogenic pathway, which leads to E-cadherin repression and cell scattering in gastric cancer. Our study highlights gelsolin as an important pro-disseminative factor contributing to the aggressive phenotype of diffuse GC.

Transcriptome-Wide Mapping of Pea Seed Ageing Reveals a Pivotal Role for Genes Related to Oxidative Stress and Programmed Cell Death

Understanding of seed ageing, which leads to viability loss during storage, is vital for ex situ plant conservation and agriculture alike. Yet the potential for regulation at the transcriptional level has not been fully investigated. Here, we studied the relationship between seed viability, gene expression and glutathione redox status during artificial ageing of pea (Pisum sativum) seeds. Transcriptome-wide analysis using microarrays was complemented with qRT-PCR analysis of selected genes and a multilevel analysis of the antioxidant glutathione. Partial degradation of DNA and RNA occurred from the onset of artificial ageing at 60% RH and 50 degrees C, and transcriptome profiling showed that the expression of genes associated with programmed cell death, oxidative stress and protein ubiquitination were altered prior to any sign of viability loss. After 25 days of ageing viability started to decline in conjunction with progressively oxidising cellular conditions, as indicated by a shift of the glutathione redox state towards more positive values (>-190 mV). The unravelling of the molecular basis of seed ageing revealed that transcriptome reprogramming is a key component of the ageing process, which influences the progression of programmed cell death and decline in antioxidant capacity that ultimately lead to seed viability loss.

A 1.77 Mb deletion in 3p26.3 encompassing CNTN6 and CNTN4 genes: case report

Chromosome microarray analysis is a powerful diagnostic tool and is being used as a first-line approach to detect chromosome imbalances associated with intellectual disability, dysmorphic features and congenital abnormalities. This test enables the identification of new copy number variants (CNVs) and their association with new microdeletion/microduplication syndromes in patients previously without diagnosis. We report the case of a 7 year-old female with moderate intellectual disability, severe speech delay and auto and hetero aggressivity with a previous 45,XX,der(13;14)mat karyotype performed at a younger age. Affymetrix CytoScan 750K chromosome microarray analysis was performed detecting a 1.77 Mb deletion at 3p26.3, encompassing 2 OMIM genes, CNTN6 and CNTN4. These genes play an important role in the formation, maintenance, and plasticity of functional neuronal networks. Deletions or mutations in CNTN4 gene have been implicated in intellectual disability and learning disabilities. Disruptions or deletions in the CNTN6 gene have been associated with development delay and other neurodevelopmental disorders. The haploinsufficiency of these genes has been suggested to participate to the typical clinical features of 3p deletion syndrome. Nevertheless inheritance from a healthy parent has been reported, suggesting incomplete penetrance and variable phenotype for this CNV. We compare our patient with other similar reported cases, adding additional value to the phenotype-genotype correlation of deletions in this region.

Development of dendritic cells in the intestine

The intestinal tract is exposed to a large variety of antigens such as food proteins, commensal bacteria and pathogens and contains one of the largest arms of the immune system. The intestinal immune system has to discriminate between harmless and harmful antigens, inducing tolerance to harmless antigens and active immunity towards pathogens and other harmful materials. Dendritic cells (DC) in the mucosal lamina propria (LP) are central to this process, as they sample bacteria from the local environment and constitutively migrate to the draining mesenteric lymph nodes (MLN), where they present antigen to naïve T cells in order to direct an appropriate immune response. Despite their crucial role, understanding the function and phenotype of LP DC has been hampered by the fact that they share phenotypic markers with macrophages (mφ), which are the dominant population of mononuclear phagocyte (MP) in the LP. Recent work in our own and other laboratories has established gating strategies and phenotyping panels that allow precise discrimination between intestinal DC and mφ using the mφ specific markers CD64 and F4/80. In this way four bona fide DC subsets with distinct functions have been identified in adult LP based on their expression of CD11b and CD103 and a major aim of my project was to understand how these subsets might develop in the neonatal intestine. At the beginning of my PhD, the laboratory had used these new methods to show that signal regulatory protein α (SIRPα), an inhibitory receptor expressed by myeloid cells, was expressed by mφ and most DC in the intestine, except for those expressing CD103 alone. In addition, mice carrying a non-signalling mutation in SIRPα (SIRPα mt) had a selective reduction in CD103+CD11b+ DC, a subset which is unique to the intestinal LP. This was the basis for the initial experiments of my project, described in Chapter 3, where I investigated if the phenotype in SIRPα mt mice was intrinsic to haematopoietic cells or not. To explore this, I generated bone marrow (BM) chimeric mice by reconstituting irradiated WT mice with SIRPα mt BM, or SIRPα mt animals with WT BM. These experiments suggested that the defect in CD103+CD11b+ DC was not replicated in DC derived from BM of SIRPα origin. However as this seemed inconsistent with other data, I considered the possibility that 18 the phenotype may have been lost with age, as the BM chimeric mice were considerably older than those used in the original studies of SIRPα function. However a comparison of DC subsets in the intestine of WT and SIRPα mt mice as they aged provided no conclusive evidence to support this idea. As these experiments did show age-dependent effects on DC subsets, in Chapter 4, I went on to investigate how the DC populations appeared in the intestine and other tissues in the neonatal period. These experiments showed there were few CD103+CD11b+ DC present in the LP and migratory DC compartment of the MLN in the neonate and that as this population gradually increased in proportion with age, there was a reciprocal decrease in the relative proportion of CD103-CD11b+ DC. Interestingly, most of the changes in DC numbers in the intestine were found during the second or third week of life when the weaning process began. To validate my findings that there were few CD103+CD11b+ DC in the neonate and that this was not merely an absence of CD103 upregulation, I examined the expression of CD101 and Trem-1, markers that other work in the laboratory had suggested were specific to the CD103+CD11b+ DC lineage. My work showed that CD101 and Trem-1 were co- expressed by most CD103+CD11b+ DC in small intestine (SI) LP, as well as a small subset of CD103-CD11b+ DC in this tissue. Interestingly, Trem-1 was highly specific to the SI LP and migratory DC in the MLN, but absent from the colon and other tissues. CD101 expression was also only found on CD11b+ DC, but showed a less restricted pattern of distribution, being found in several tissues as well as the SI LP. The relative timing of their development suggested there might be a relationship between CD103+CD11b+ and CD103-CD11b+ DC and this was supported by microarray analysis. I hypothesised that the CD103-CD11b+ DC that co-expressed CD101 and Trem-1 may be the cells that developed into CD103+CD11b+ DC. To investigate this I analysed how CD101 and Trem-1 expression changed with age amongst the DC subsets in SI LP, colonic LP (CLP) and MLN. The proportion of CD101+Trem-1+ cells increased amongst CD103+CD11b+ DC in the SI LP and MLN with age, while amongst CD103+CD11b+ DC in the CLP this decreased. This was not the same in CD103-CD11b+ DC, where CD101 and Trem-1 expression was more varied with age in all tissues. CD101 and Trem-1 were not expressed to any great extent on CD103+CD11b- or CD103-CD11b- DC. The phenotypic development of the 19 intestinal DC subsets was paralleled by the gradual upregulation of CD103 expression, while the production of retinoic acid (RA), as assessed by the AldefluorTM assay, was low early in life and did not attain adult levels until after weaning. Thus DC in the neonatal intestine take some time to acquire the adult pattern of phenotypic subsets and are functionally immature compared with their adult counterparts. In Chapter 5, I used CD101 and Trem-1 to explore the ontogeny of intestinal DC subsets in CCR2-/- and SIRPα mt mice, both of which have selective defects in one particular group of DC. The selective defect seen amongst CD103+CD11b+ DC in adult SIRPα mt mice was more profound in mice at D7 and D14 of age, indicating that it may be intrinsic to this population and not highly dependent on environmental factors that change after birth. The expression of CD101 and Trem-1 by both CD103+CD11b+ and CD103-CD11b+ DC was reduced in SIRPα mt mice, again indicating that this entire lineage was affected by the lack of SIRPα signalling. However there was also a generalised defect in the numbers of all DC subsets in many tissues from early in life, suggesting there was compromised development, recruitment or survival of DC in the absence of SIRPα signalling. In contrast to the findings in SIRPα mt mice, more CD103+CD11b+ DC co-expressed CD101 and Trem-1 in CCR2-/- mice, while there were no differences in the expression of these molecules amongst CD103-CD11b+ DC. This may suggest that CCR2+ CD103-CD11b+ DC are not the cells that express CD101 and Trem-1 that are predicted to be the direct precursors of CD103+CD11b+ DC. I also examined the expression of DC growth factor receptors on DC subsets from mice of different ages, but no clear age or subset- related patterns of the expression of mRNA for Csf2ra, Irf4, Tgfbr1 and Rara could be observed. Next, I investigated whether Trem-1 played any role in DC development. Preliminary experiments in Trem-1-/- mice show no differences between any of the DC subsets, nor were there any selective effects on individual subsets when DC development from Trem-1-/- KO and WT BM was compared in competitive chimeras. However these experiments were difficult to interpret due to viability problems and because I found an unexpected defect in the ability of Trem-1-/- BM to generate all DC, irrespective of whether they expressed Trem-1 or not. 20 The final experiments I carried out were to examine the role of the microbiota in driving the differentiation of intestinal DC subsets, based on the hypothesis that this could be one of the environmental factors that might influence events in the developing intestine. To this end I performed experiments in both antibiotic treated and germ free adult mice, both of which showed no significant phenotypic differences amongst any of the DC subsets. However the study of germ free mice was compromised by recent contamination of the colony and may not be the conclusive answer. Together the data in this thesis have shown that the population of CD103+CD11b+ DC, which is unique to the intestine, is not present at birth. These cells gradually increase in frequency over time and as this occurs there is a reciprocal decrease in the frequency of CD103-CD11b+ DC. Along with other results, this leads to the idea that there may be a linear developmental pathway from CD103-CD11b+ DC to CD103+CD11b+ DC that is driven by non-microbial factors that are located preferentially in the small intestine. My project indicates that markers such as CD101 and Trem-1 may assist the dissection of this process and highlights the importance of the neonatal period for these events.

A Livestock-Associated, Multidrug-Resistant, Methicillin-Resistant Staphylococcus aureus Clonal Complex 97 Lineage Spreading in Dairy Cattle and Pigs in Italy

Pandemic methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 97 (CC97) lineages originated from livestock-to-human host jumps. In recent years, CC97 has become one of the major MRSA lineages detected in Italian farmed animals. The aim of this study was to characterize and analyze differences in MRSA and methicillin-susceptible S. aureus (MSSA) mainly of swine and bovine origins. Forty-seven CC97 isolates, 35 MRSA isolates, and 6 MSSA isolates from different Italian pig and cattle holdings; 5 pig MRSA isolates from Germany; and 1 human MSSA isolate from Spain were characterized by macrorestriction pulsed-field gel electrophoresis (PFGE) analysis, multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial resistance pattern analysis. Virulence and resistance genes were investigated by PCR and microarray analysis. Most of the isolates were of SCCmec type V (SCCmec V), except for two German MRSA isolates (SCCmec III). Five main clusters were identified by PFGE, with the German isolates (clusters I and II) showing 60.5% similarity with the Italian isolates, most of which (68.1%) grouped into cluster V. All CC97 isolates were Panton-Valentine leukocidin (PVL) negative, and a few (n = 7) tested positive for sak or scn. All MRSA isolates were multidrug resistant (MDR), and the main features were erm(B)- or erm(C)-mediated (n = 18) macrolide-lincosamide-streptogramin B resistance, vga(A)-mediated (n = 37) pleuromutilin resistance, fluoroquinolone resistance (n = 33), tet(K) in 32/37 tet(M)-positive isolates, and blaZ in almost all MRSA isolates. Few host-associated differences were detected among CC97 MRSA isolates: their extensive MDR nature in both pigs and dairy cattle may be a consequence of a spillback from pigs of a MRSA lineage that originated in cattle as MSSA and needs further investigation. Measures should be implemented at the farm level to prevent spillover to humans in intensive farming areas.

The role of chondrocyte senescence in the pathogenesis of canine osteoarthritis

The aims of this study were to (1) evaluate cellular senescence in chondrocytes from osteoarthritic articular cartilage, (2) investigate the hypothesis that oxidative stress is a feature of canine OA chondrocytes and that oxidative stress contributes to cellular senescence in canine chondrocytes, (3) investigate the hypothesis that osteoarthritic chondrocytes alter the gene expression of adjacent normal chondrocytes in OA joints leading to modulation of genes known to play a role in the pathogenesis of OA and (4) evaluate the presentation of dogs undergoing femoral head excision in veterinary referral practice in the UK as a treatment for osteoarthritis of the coxofemoral joint, and to categorise the distribution and severity of associated pathological lesions. Chondrocytes from osteoarthritic and normal cartilage were examined for levels of senescence. Initially chondrocytes were cultured using an alginate bead culture system, thought to mimic the extracellular matrix of articular cartilage. However, these chondrocytes showed almost no growth as compared to monolayer culture where they grew rapidly. OA chondrocytes entered the senescent state after 1.5 to 4.9 population doublings in monolayer culture, while normal chondrocytes underwent 4.8 to 14.6 population doublings before entering the senescent state. Osteoarthritic chondrocytes had increased levels of markers of cellular senescence (senescence associated beta-galactosidase accumulation and p16 protein accumulation) as compared to normal chondrocytes, suggesting that chondrocyte senescence is a feature of canine osteoarthritis. An experimental model for the induction of oxidative stress in chondrocyte cell culture was developed using tert-Butyl hydroperoxide and total cellular glutathione was measured as an indicator of cellular oxidative stress levels. Experimental induction of oxidative stress in both normal and osteoarthritic chondrocytes in cell culture resulted in increased amounts of cellular senescence, shown by an increase in levels of senescence associated beta-galactosidase accumulation and decreased replicative capacity. Experimental induction of oxidative stress also resulted in altered gene expression of three genes important to the degradation of the extracellular matrix; MMP-13, MMP-3 and Col-3A1, measured by RT-PCR, in normal canine chondrocytes in monolayer cell culture. MMP-3 showed the greatest relative expression change, with a fold-change of between 1.43 and 4.78. MMP-13 had a fold change of 1.16 to 1.38. Col-3A1 was down regulated, with a fold-change of between 0.21 and 0.31. These data demonstrate that experimentally induced oxidative stress in chondrocytes in monolayer culture increases levels of cellular senescence and alters the expression of genes relevant to the pathogenesis of canine OA. Coculture of osteoarthritic chondrocytes with normal canine chondrocytes resulted in gene modulation in the normal chondrocytes. Altered gene expression of ten genes known to play a role in the pathogenesis of osteoarthritis was detected in the normal chondrocytes (fold change shown in brackets); TNF-alpha (11.95), MMP-13 (5.93), MMP-3 (5.48), IL-4 (7.03), IL-6 (5.3), IL-8 (4.92), IL-F3 (4.22), COL-3A1 (4.12), ADAMTS-4 (3.78) and ADAMTS-5 (4.27). In total, 594 genes were significantly modulated suggesting that osteoarthritic chondrocytes contribute to the disease propagation by altering the gene expression of adjacent normal chondrocytes, thus recruiting them into the disease process. Gene expression changes were measured by microarray analysis and validated by RT-PCR and Western blot analysis. An epidemiological study of femoral heads collected from dogs undergoing total hip replacement surgery as a treatment for osteoarthritis of the coxofemoral joint secondary to canine hip dysplasia revealed that there was no characteristic pattern of cartilage lesion for canine hip dysplasia. Severe pathology of the femoral head with cartilage erosion occurred in 63.9% of cases and exposure of subchondral bone in 31.3% of cases. The work presented in this thesis has demonstrated that cellular senescence is a feature of chondrocytes from canine osteoarthritic cartilage and suggests that cellular senescence and oxidative stress play an important role in the pathogenesis of osteoarthritis in dogs.

Tissue Microarray Is A Reliable Method For Immunohistochemical Analysis Of Pleomorphic Adenoma.

To determine the most adequate number and size of tissue microarray (TMA) cores for pleomorphic adenoma immunohistochemical studies. Eighty-two pleomorphic adenoma cases were distributed in 3 TMA blocks assembled in triplicate containing 1.0-, 2.0-, and 3.0-mm cores. Immunohistochemical analysis against cytokeratin 7, Ki67, p63, and CD34 were performed and subsequently evaluated with PixelCount, nuclear, and microvessel software applications. The 1.0-mm TMA presented lower results than 2.0- and 3.0-mm TMAs versus conventional whole section slides. Possibly because of an increased amount of stromal tissue, 3.0-mm cores presented a higher microvessel density. Comparing the results obtained with one, two, and three 2.0-mm cores, there was no difference between triplicate or duplicate TMAs and a single-core TMA. Considering the possible loss of cylinders during immunohistochemical reactions, 2.0-mm TMAs in duplicate are a more reliable approach for pleomorphic adenoma immunohistochemical study.

Production of DNA microarray and expression analysis of genes from Xylella fastidiosa in different culture media

DNA Microarray was developed to monitor the expression of many genes from Xylella fastidiosa, allowing the side by-side comparison of two situations in a single experiment. The experiments were performed using X. fastidiosa cells grown in two culture media: BCYE and XDM2. The primers were synthesized, spotted onto glass slides and the array was hybridized against fluorescently labeled cDNAs. The emitted signals were quantified, normalized and the data were statistically analyzed to verify the differentially expressed genes. According to the data, 104 genes were differentially expressed in XDM2 and 30 genes in BCYE media. The present study showed that DNA microarray technique efficiently differentiate the expressed genes under different conditions.

Gene expression profile analysis of primary glioblastomas and non-neoplastic brain tissue: identification of potential target genes by oligonucleotide microarray and real-time quantitative PCR

The prognosis of glioblastomas is still extremely poor and the discovery of novel molecular therapeutic targets can be important to optimize treatment strategies. Gene expression analyses comparing normal and neoplastic tissues have been used to identify genes associated with tumorigenesis and potential therapeutic targets. We have used this approach to identify differentially expressed genes between primary glioblastomas and non-neoplastic brain tissues. We selected 20 overexpressed genes related to cell cycle, cellular movement and growth, proliferation and cell-to-cell signaling and analyzed their expression levels by real time quantitative PCR in cDNA obtained from microdissected fresh tumor tissue from 20 patients with primary glioblastomas and from 10 samples of non-neoplastic white matter tissue. The gene expression levels were significantly higher in glioblastomas than in non-neoplastic white matter in 18 out of 20 genes analyzed: P < 0.00001 for CDKN2C, CKS2, EEF1A1, EMP3, PDPN, BNIP2, CA12, CD34, CDC42EP4, PPIE, SNAI2, GDF15 and MMP23b; and NFIA (P: 0.0001), GPS1 (P: 0.0003), LAMA1 (P: 0.002), STIM1 (P: 0.006), and TASP1 (P: 0.01). Five of these genes are located in contiguous loci at 1p31-36 and 2 at 17q24-25 and 8 of them encode surface membrane proteins. PDPN and CD34 protein expression were evaluated by immunohistochemistry and they showed concordance with the PCR results. The present results indicate the presence of 18 overexpressed genes in human primary glioblastomas that may play a significant role in the pathogenesis of these tumors and that deserve further functional investigation as attractive candidates for new therapeutic targets.