Despite significant advances in diagnosis and treatment, breast cancer remains the leading cause of cancer-related deaths in Australian women. Colorectal cancer is the second most common cancer in both males and females; after prostate and breast cancer, respectively, and excluding non-melanocytic skin cancer. Both breast cancer and colorectal cancer follow a common progressive course of illness; presenting (at least initially) with benign symptoms that can be treated by ablation (or removal) of the affected area. Cancer progression is associated with breakdown of tissue barriers (such as basement membranes), leading to the spread of cancer cells (via the vasculature or lymphatic system), and the establishment of secondary metastatic disease at green-field sites. Secondary tumours presenting in the lungs, ovaries, liver, bone, or brain are associated with chronic-debilitating symptoms that are difficult to treat, and will result in death. In the case of breast and colon cancer, effective early therapeutic intervention does have a significant impact upon patient survival. Tumour progression in breast and colon carcinomas is characterised by invasion of the surrounding stroma, and the acquisition of stromal characteristics, by previously epithelial cells. This progression is associated with the expression of extracellular proteases (ECPs) and increased motility. The process of mesenchymal transformation that tumour cells undergo is also referred to as the epithelial to mesenchymal transition (EMT). In general terms the aim of the study, presented in this thesis, was to investigate gene expression in cancer biology; and to characterise changes in breast cancer and colon cancer, with a focus on those genes, and gene products that may play a role in metastasis, including a family of ECPs, the matrix metalloproteinases (MMPs). In our laboratory, we have applied methods in microdissection, differential display polymerase chain reaction amplification (DD-PCR), and array hybridisation analysis to identify gene expression patterns in late stage archival formalin fixed paraffin embedded (FFPE) breast tumour biopsies that may be indicative of the EMT; or the response to the surrounding stroma/interstitium to the presence of the tumour.' The quality of nucleic acid obtainable from FFPE material presents a considerable challenge for gene expression studies. In order to identify tissue specific gene expression patterns, DD-PCR products, amplified from message obtained following segregation of tumour tissue from surrounding stroma, was hybridised to arrayed cDNA libraries created from stromal tumours, or sarcomas. In this way, 21 known genes, or expressed sequence tags (ESTs), were identified. These included the cytoskeletal element and EMT marker, vimentin, the mammary developmental factor and, signal transducer and activator of transcription (STAT)-3, and the cargo selection protein (TIP47). Seventeen genes showed differential expression in either the tumour, or stromal fractions. When applied to transformed breast cancer cell lines (MDA-MB-435 & T47D) DD-array analysis revealed a further 17 genes that were differentially regulated in invasive cells, compared with those displaying a less invasive phenotype. Six of the ESTs identified by DD-PCR array analysis, had no known (or predicted) function. For example, bcaf-2 was identified as the 3'-end of a putative open reading frame (ORF) localised to chromosome 6, while bcaf-10 showed homology with a known ORF. In order to analyse the expression of these bcafs further, a stromal cell culture model, representative of the original osteosarcoma cDNA libraries from which they were obtained, was used. In this model, CD14' (or adherent) peripheral blood mononuclear cells (PBMCs) treated with macrophage colony stimulating factor (M-CSF), can be allowed to differentiate into macrophage-like (ML) cells; while cells treated with M-CSF, and the receptor activator of NF-KB ligand (RANKL) will differentiate into multinucleate osteoclast-like (OCL) multinucleate giant cells. Uniquely, the stromal EST, bcaf-2 was expressed only by RANKL-treated (or OCL) cells. bcaf-2 and other ESTs, identified by DD-PCR analysis (and recently published) are the subject of on going research in our laboratory. The role of RANKL in mammary gland development and bone metastasis suggested that the identification of a RANKL-regulated stromal factor in breast tissue (bcaf-2) was not an artefact. RANKL is a membrane-bound, member of the tumour necrosis factor (TNF)-a cytokine super family. In order to test the hypothesis that RANKL might act as an inflammatory cytokine to regulate clinically significant stromal gene expression in the breast, we employed quantitative real time PCR analysis to examine the relative levels of selected members of a group of metal dependent ECPs, the matrix metalloproteinases (MMPs). RNA was extracted from ML cells and OCL cells, as well as RANK positive breast cancer cell lines (T47D, MDA-MB-435 & MCF-7). When the relative levels of protease mRNA were compared we demonstrated a significant (>20- fold) specific increase in collagenase (collagenase 2lMMP-8 and collagenase 3lMMP-13), and the tissue inhibitor of MMP (TIMP)-2 expression in M-CSF and RANKL treated PBMCs cells. When the assay was applied to RANKL treated breast cancer cell lines (MCF-7, T47D & MDAMB- 231), minor (40-fold) but potentially significant alterations in stromal protease gene expression were observed. The changes observed did not however, support the hypothesis that RANKL might act as an inflammatory cytokine to induce significant alterations in ECP expression in breast cancer cells. To investigate the role of RANKL as a driver of EMT in aberrant breast epithelium, total message (mRNNcDNA) from T47D, MCF-7, MDA-MB-231 cells, and message from the same cell lines treated with RANKL were compared by comparative fluorescent cDNA microarray analysis. Of the 1,700 targets available on the arrays, this study identified 160 that were differentially expressed in RANKL treated cells. The results suggest that RANKL may promote rather than suppress a mammary epithelial phenotype in breast cancer. In fact a putative mesenchymal to epithelial transition (MET) was observed following microscopic analysis, and this finding is the subject of on going research in our laboratory. Sporadic structural alterations in certain mitogenic factors represent important early events in cancer progression, while inherited mutations govern familial susceptibility to disease. In colon cancer, a close link exists between Winglessllnt (WNT) signalling, disease pathology, and the expression of MMPs. To examine the relationship between protease expression and structural genetic alterations in this EMT-linked signalling pathway, and others, we applied combined QPCR analysis of MMP expression and PCR-Single Strand Conformation Analysis (SSCA) to 26 colonic tumours, and patient-matched normal colonic mucosa. In this study, significant correlations between the expression of ECPs, and a key mediator of WNT signalling (p-catenin) were identified. While tumours possessing specific functional mutations in K-Ras, were found to group with phenotypic clustering based on protease gene expression. This result may be due to an interruption of normal interactions between RasIRaf signalling and transforming growth factor (TGF) P signalling, via Sma- and Mad- related protein (SMAD) signalling. These results demonstrate that the already identified link between mutations in kinase signalling, and aspects of gross colon tumour morphology (such as dysplasia) may be due to aberrant MMP expression patterning. The final aim of this research was to utilise methods developed in microdissection and specific Q-PCR analysis, to identify whether tumour-stroma differences in MMP gene expression might be used as markers of disease pathology. Total RNA from tumour, and biopsy-matched adjacent stromal tissue were segregated from 35 FFPE archival breast tumour biopsies. Comparison with stroma identified specific associations between TIMP-2 expression in the stroma and lymph node involvement, as well as stromelysin-3 (MMP-I I ) and TIMP-I expression and calcification of the tumour. Furthermore, a significant correlation was identified in the pattern of gelatinase (gelatinase AIMMP-2 & gelatinaseB1MMP-9) expression; while no significant correlation was identified in tumour-stroma MMP gene expression differences, and tumour grade, or hormone receptor status. These results suggest that coordinated changes within the tumour, and proximal stromal tissues (rather than tissue specific changes per se), regulate pathologically significant changes in breast carcinogenesis. In conclusion, this thesis describes the use of novel techniques in specific and global gene expression analysis that permitted examination of stromal gene expression changes in epithelial tumour progression. Microdissection facilitated localisation of expression to particular tissues, while cell culture models provided material with which to optimise and demonstrate the efficacy of techniques used (where tumour material itself was not abundant). Furthermore, we have identified significant and specific correlations between general stromal protease gene expression changes, a putative mammary epithelial differentiation factor (RANKL), alterations in growth factor signalling, and epithelial tumour pathology in the breast and colon. The combination of techniques developed in this study may assist in improvement of categorisation of tumours in clinical pathology. Specifically, the development of novel grading systems that link underlying molecular genetic changes with changes in tumour pathology. These processes may assist to improve diagnosis and provide more effective patient/tumour-specific drug therapies.

Übergewicht und Adipositas führen zu Insulinresistenz und erhöhen deutlich das Risiko für die Entwicklung von Typ-2-Diabetes und kardiovaskulären Erkrankungen. Sowohl Adipositas als auch die Suszeptibilität gegenüber Diabetes sind zu einem erheblichen Teil genetisch determiniert. Die relevanten Risikogene, deren Interaktion mit der Umwelt, insbesondere mit Bestandteilen der Nahrung, und die Pathomechanismen, die zur Insulinresistenz und Diabetes führen, sind nicht vollständig aufgeklärt. In der vorliegenden Arbeit sollte durch Genexpressionsanalysen des weißen Fettgewebes (WAT) und der Langerhansschen Inseln die Entstehung und Progression von Adipositas und Typ-2-Diabetes untersucht werden, um relevante Pathomechanismen und neue Kandidatengene zu identifizieren. Zu diesem Zweck wurden Diät-Interventionsstudien mit NZO- und verwandten NZL-Mäusen, zwei polygenen Mausmodellen für das humane metabolische Syndrom, durchgeführt. Eine kohlenhydrathaltige Hochfett-Diät (HF: 14,6 % Fettanteil) führte in beiden Mausmodellen zu früher Adipositas, Insulinresistenz und Typ 2 Diabetes. Eine fettreduzierte Standarddiät (SD: 3,3 % Fettanteil), welche die Entstehung von Adipositas und Diabetes stark verzögert, sowie eine diabetesprotektive kohlenhydratfreie Hochfett-Diät (CHF: 30,2 % Fettanteil) dienten als Kontrolldiäten. Mit Hilfe der Microarray-Technologie wurden genomweite Expressionsprofile des WAT erstellt. Pankreatische Inseln wurden durch laserbasierte Mikropräparation (Laser Capture Microdissection; LCM) isoliert und ebenfalls hinsichtlich ihres Expressionsprofils analysiert. Differenziell exprimierte Gene wurden durch Real-Time-PCR validiert. Im WAT der NZO-Maus bewirkte die HF-Diät eine reduzierte Expression nukleärer Gene der oxidativen Phosphorylierung und von lipogenen Enzymen. Dies deutet auf eine inadäquate Fettspeicherung und -verwertung in diesen Tieren hin. Die Reduktion in der Fettspeicherung und -oxidation ist spezifisch für das adipöse NZO-Modell und konnte bei der schlanken SJL Maus nicht beobachtet werden, was auf eine mögliche Beteiligung an der Entstehung der Insulinresistenz hinweist. Zusätzlich wurde bestätigt, dass die Expansion des Fettgewebes bei der adipösen NZO-Maus eine zeitlich verzögerte Infiltration von Makrophagen in das WAT und dort eine lokale Immunantwort auslöst. Darüber hinaus wurde die Methode der LCM etabliert und zur Gewinnung hochangereicherter RNA aus den Langerhansschen Inseln eingesetzt. In erstmalig durchgeführten genomweiten Expressionsanalysen wurde zu einem frühen Zeitpunkt in der Diabetesentwicklung der Einfluss einer diabetogenen HF-Diät und einer diabetesprotektiven CHF-Diät auf das Expressionsprofil von pankreatischen Inselzellen verglichen. Im Gegensatz zum WAT bewirkt die diabetogene HF-Diät in Inselzellen einerseits, eine erhöhte Expression von nukleären Genen für die oxidative Phosphorylierung und andererseits von Genen, die mit Zellproliferation assoziiert sind. Zudem wurden 37 bereits annotierte Gene identifiziert, deren differenzielle Expression mit der Diabetesentwicklung korreliert. Das Peptidhormon Cholecystokinin (Cck, 11,8-fach erhöht durch die HF) stellt eines der am stärksten herauf regulierten Gene dar. Die hohe Anreicherung der Cck-mRNA in Inselzellen deutet auf eine bisher unbekannte Funktion des Hormons in der Regulation der Inselzellproliferation hin. Der Transkriptionsfaktor Mlxipl (ChREBP; 3,8-fach erniedrigt durch die HF) stellt in Langerhansschen Inseln eines der am stärksten herunter regulierten Gene dar. Ferner wurde ChREBP, dessen Funktion als glucoseregulierter Transkriptionsfaktor für lipogene Enzyme bislang in der Leber, aber nicht in Inselzellen nachgewiesen werden konnte, erstmals immunhistochemisch in Inselzellen detektiert. Dies deutet auf eine neue, bisher unbekannte regulatorische Funktion von ChREBP im Glucosesensor-Mechanismus der Inselzellen hin. Eine durchgeführte Korrelation der mit der Diabetesentwicklung assoziierten, differenziell exprimierten Inselzellgene mit Genvarianten aus humanen genomweiten Assoziationsstudien für Typ-2-Diabetes (WTCCC, Broad-DGI-T2D-Studie) ermöglichte die Identifizierung von 24 neuartigen Diabetes-Kandidatengenen. Die Ergebnisse der erstmals am polygenen NZO-Mausmodell durchgeführten genomweiten Expressionsuntersuchungen bestätigen bisherige Befunde aus Mausmodellen für Adipositas und Diabetes (z.B. ob/ob- und db/db-Mäuse), zeigen in einigen Fällen aber auch Unterschiede auf. Insbesondere in der oxidativen Phosphorylierung könnten die Ergebnisse relevant sein für das Verständnis der Pathogenese des polygen-bedingten humanen metabolischen Syndroms. / Overweight and obesity cause insulin resistance and increase the risk of developing type 2 diabetes and cardiovascular diseases. Both, obesity and susceptibility to diabetes, are to a major part genetically predisposed. The relevant genes, their interaction with the environment – especially with food components – and the pathomechanisms causing insulin resistance and diabetes are not fully known yet. In the present study the development and progression of obesity and type 2 diabetes should be investigated by the means of gene expression analyses of the white adipose tissue (WAT) and the islets of Langerhans to identify underlying pathomechanisms and new causative candidate genes. For this purpose diet intervention studies on NZO- and related NZL-mice – two polygenic mouse models for the human metabolic syndrome – were performed. A carbohydrate containing high fat-diet (HF: 14.6 % fat) caused early obesity, insulin resistance and type 2 diabetes in both mouse models. A fat reduced standard chow (SD: 3.3 % fat) which strongly delayed the onset of obesity and diabetes, and a diabetes protective carbohydrate free high fat-diet (CHF: 30.2 % fat) served as control diets. Using microarray technology genome wide expression profiles of the WAT were generated. Pancreatic islets were isolated by the means of laser capture microdissection (LCM) and expression profiles of them were created, too. Differentially expressed genes were validated by quantitative real time PCR. The HF-diet reduced the expression of nuclear genes of the oxidative phosphorylation and lipogenic enzymes in the WAT of the NZO-mouse. This suggests an inadequate storage and utilization of fat in these animals. This is specific for the obese NZO-model and wasn’t observed for the lean SJL-mouse, indicating a role in the development of insulin resistance. Additionally, there was proof that the enlargement of the WAT triggers a retarded infiltration of macrophages into the WAT and there a local immune response. Moreover, the LCM technique was established and used for the isolation of highly enriched RNA from islets of Langerhans. For the first time the influence of carbohydrates in a high fat-diet on the expression profile of pancreatic islets was investigated by the use of genome wide expression analyses at an early time point at the onset of diabetes. Contrary to the WAT the diabetogenic HF-diet in islets cells increased the expression of both nuclear genes coding for the oxidative phosphorylation and genes associated with cell proliferation. Furthermore 37 already annotated genes correlated with diabetes progression were identified. The peptide hormone cholecystokinin (Cck: 11.8-fold enriched by the HF-diet) is one of the most up-regulated genes. The strong enrichment of Cck-mRNA in islets suggests a previously unknown function of the hormone in the regulation of the islet cell proliferation. The transcription factor ChREBP (Mlxipl: 3.8-fold reduced by the HF-diet) is one of the most down-regulated genes in the islets of Langerhans. Moreover, ChREBP, which has been already identified as a glucose regulated transcription factor for lipogenic enzymes in the liver but not in islets of Langerhans, was detected for the first time in islet cells, using immunohistochemistry. This points to an until now unknown regulatory function of ChREBP in the glucosesensor mechanism of the islet cells. Correlation of the differentially expressed genes associated with diabetes progression with gene variants from human genome wide association studies for type 2 diabetes (WTCCC, Broad-DGI-T2D-study) made the identification of 24 new diabetes candidate genes possible. The results of the genome wide expression analyses, which were done for the first time on a polygenic mouse-model, corroborated previous results for monogenic mouse-models for obesity and diabetes (e.g. ob/ob- and db/db-mice), however also demonstrated differences in some instances. Especially the results concerning the oxidative phosphorylation could be relevant for the comprehension of the pathogenesis of the polygenic human metabolic syndrome.

With over a billion people infected world wide, hookworms are considered as important human pathogens, particularly in developing countries which have the highest rates of infections. Hookworms reside in the gastrointestinal tract of the host where they continuously feed on blood, leading to conditions such as chronic irondeficiency anaemia. The majority of blood-feeding parasites rely on proteins found in blood to provide many of their nutritional requirements for growth, reproduction and survival. Of the numerous proteins found in blood, haemoglobin (Hb) is one of the most abundant. In order to acquire amino acids for protein synthesis, it is thought that haematophagous parasites degrade Hb using various classes of endo- and exoproteases, in a manner similar to that which occurs in catabolism of proteins in mammalian cellular lysosomes. This study identified and characterised proteases involved in the Hb degradation process in the human hookworm, Necator americanus, in order to identify potential candidate antigens for a vaccine that interrupts blood-feeding. Red blood cells ingested by hookworms are lysed to release Hb, which is cleaved by various proteases into dipeptides or free amino acids and these are taken up through the gut membrane by amino acid transporters. Proteases expressed in the intestinal tract of hookworms are thought to play a major role in this process and would therefore make good targets for vaccine candidates aimed at interrupting blood-feeding. To identify these proteases, adult hookworms (both N. americanus and Ancylostoma caninum) were sectioned and intestinal tissue was dissected via laser microdissection microscopy. RNA extracted from the dissected tissue was used to generate gut-specific cDNA, which then was used to create plasmid libraries. Each library was subjected to shotgun sequencing, and of the 480 expressed sequence tags (ESTs) sequenced from each species, 268 and 276 contigs were assembled from the N. americanus and A. caninum libraries, respectively. Nine percent of N. americanus and 6.5% of A. caninum contigs were considered novel as no homologues were identified in any published/accessible database. The gene ontology (GO) classification system was used to categorise the contigs to predicted biological functions. Only 17% and 38% of N. americanus and A. caninum contigs, respectively, were assigned GO categories, while the rest were classified as being of unknown function. The most highly represented GO categories were molecular functions such as protein binding and catalytic activity. The most abundant transcripts encoded fatty acid binding proteins, C-type lectins and activation associated secreted proteins, indicative of the diversity of functions that occur in this complex organ. Of particular interest to this study were the contigs that encoded for cysteine and metalloproteases, expanding the list of potential N. americanus haemoglobinases. In the N. americanus cDNA library, four contigs encoding for cathepsin B cysteine proteases were identified. Three contigs from the A. caninum and one contig from the N. americanus cDNA libraries encoded for metalloproteases, including astacin-like and O-sialoglycoprotein endopeptidases, neither of which had previously been reported from adult hookworms. Apart from haemoglobinases, other mRNAs encoding potential vaccine candidate molecules were identified, including anti-clotting factors, defensins and membrane proteins. This study confirmed that the gut of hookworms encodes a diverse range of proteases, some of which are likely to be involved in Hb digestion and have the potential to be hidden (cryptic) vaccine antigens. Four cysteine proteases (Na-CP-2, -3, -4 and -5) were identified from the gut cDNA library of N. americanus. All four proteases belong to the clan CA, family C1, share homology with human cathepsin B and possess a modified occluding loop. Real-time PCR indicated that all transcripts were up-regulated in the adult stage of the hookworm parasite with high levels of mRNA expression detected in gut cDNA. All four proteases were expressed in recombinant form, but only Na-CP-3 was successfully expressed in soluble form in the yeast Pichia pastoris. Proteolytic activity for Na-CP-3 was detected on a gelatin zymogen gel, however no catalytic activity was detected against the class-specific fluorogenic peptides Z-Phe-Arg-AMC and Z-Arg-Arg-AMC. Mass spectrometry analysis of the purified protein suggested that the pro-region had not been processed in trans when the protein was secreted by yeast. Incubation of Na-CP-3 in salt buffers containing dextran sulfate resulted in autoprocessing of the pro-region as detected by Western blot and catalytic activity was detected against Z-Phe-Arg-AMC. Activated Na-CP-3 did not digest intact tetrameric human Hb. The other three cysteine proteases (Na-CP-2, -4, and -5) were expressed in insoluble form in Escherichia coli. Antibodies to all four proteins (Na- CP-2 to 5) immunolocalised to the gut region of the adult worm, supporting mRNA amplification results and strongly indicated that they might play a role in nutrient acquisition. Hb digestion in blood feeding parasites such as schistosomes and Plasmodium spp. occurs via a semi-ordered cascade of proteolysis involving numerous enzymes. In Plasmodium falciparum, at least three distinct mechanistic classes of endopeptidases have been implicated in this process, and at least two classes have been implicated in schistosomes. A similar process is thought to occur in hookworms. An aspartic protease, Na-APR-1, was expressed in P. pastoris and purified protein was shown to cleave the class-specific fluorogenic peptide 7- Methoxycoumarin-4-Acetyl-GKPILFFRLK(DNP)-D-Arg-Amide. Recombinant Na- APR-1 was able to cleave intact human Hb and completely degrade the 16 kDa monomer and 32 kDa dimer within one hour. Recombinant Na-CP-3 was not able to cleave intact Hb, but was able to further digest globin fragments that had previously been digested with Na-APR-1. A clan MA metalloprotease, Na-MEP-1, was identified in gut tissue of N. americanus and was expressed in recombinant form in Hi5 insect cells using the baculovirus expression system. Recombinant Na-MEP-1 displayed proteolytic activity when assessed by gelatin zymography, but was incapable of cleaving intact Hb. However, Na-MEP-1 did cleave globin fragments which had previously been incubated with Na-APR-1 and Na-CP-3. Hb digested with all three proteases was subjected to reverse phase HPLC and peptides were analysed using Liquid Chromatography-Mass Spectrometry (LC-MS). A total of 74 cleavage sites were identified within Hb ƒ¿ and ƒÀ chains. Na-APR-1 was responsible for cleavage of Hb at the hinge region, probably unravelling the molecule so that Na- CP-3 and Na-MEP-1 could gain access to globin peptides. All three proteases were promiscuous in their subsite specificities, but the most common P1-P1Œ residues were hydrophobic and/or bulky in nature, such as Phe, Leu and Ala. Antibodies to all three proteins (Na-APR-1, -CP-3, -MEP-1) immunolocalised to the gut region of the worm, further supporting their roles in Hb degradation. These results suggest that Hb degradation in N. americanus follows a similar pattern to that which has been described in Plasomdium falciparum. Studies conducted in this project have identified a number of potential haemoglobinases and have demonstrated that the gut region of the hookworm contains a multitude of proteases which could be targeted for production of new chemotherapies or as vaccine candidates. Results presented here also suggest that the Hb degradation process occurs in an ordered cascade, similar to those which have been reported in other haematophagous parasites. More importantly, it has been confirmed that Na-APR-1 plays a crucial role in the initiation of the Hb degradation process and therefore targeting this molecule as a vaccine candidate could provide high levels of protection against hookworm infection.

Primary aldosteronism is the most common secondary cause of hypertension with a prevalence of about 10%. About half of PA cases are caused by aldosterone-producing adenomas (APA). Two APA subtypes, ZG-like and ZF-like APAs, have been described, according to the histological resemblance to normal zona glomerulosa (ZG) and zona fasciculata (ZF), underlying somatic mutations (KCNJ5 commonly found in ZF-like, CACN1AD, ATP1A1, ATP2B3, CTNNB1 in ZG-like APAs), and transcriptome profile. It is unknown if the process of tumorigenesis differs between ZG- and ZF-like APAs. In order to define ZG specific genes, we have compared the transcriptome of APAs and their adjacent adrenal glands by microarray assay. RNA was isolated by laser capture microdissection (LCM) from adjacent ZG, ZF and APAs from 14 patients with Conn’s and 7 patients with phaeocromocytoma. Two top hit genes from the comparison of ZG vs ZF were functionally studied, ANO4 and NEFM. NEFM, encoding neurofilament medium, was the fourth most up-regulated gene in ZG vs ZF, showing 14.8-fold-fold higher expression levels (p=9.16-12) in ZG than ZF. NEFM was also one of the most down-regulated genes in ZF-like vs ZG-like APAs. Immunohistochemistry (IHC) confirmed selective high expression of NEFM in ZG and ZG-like APAs. Silencing NEFM in H295R cells increased aldosterone secretion and cell proliferation. In addition, it increased stimulation and inhibition, respectively, of aldosterone secretion from H295R cells by the dopamine receptor D1R agonist fenoldopam and antagonist SCH23390. IHC showed predominantly intracellular staining for D1R in NEFM-rich ZG-like APAs, but membranous staining in NEFM-poor ZF-like APAs. Aldosterone secretion in response to fenoldopam in primary cells from ZG-like APAs was lower than in cells from ZF-like APAs. NEFM expression levels directly correlate with KCNJ5 phenotype: KCNJ5 mutations down-regulate NEFM mRNA and protein levels in H295R cells and in primary cells from ZG-like APAs. ANO4,encoding a Ca2+-activated chloride channel family member, was the third most upregulated gene, showing 19.9-fold higher expression levels (p=6.6x10-24) in ZG than ZF. IHC confirmed ZG selectivity of ANO4 protein in the adrenal cortex. The staining was mainly cytoplasmic. Unlike NEFM, there was no difference in expression of ANO4 between ZG- and ZF-like APAs, the levels being mid-way between those of ZF and ZG. Overexpression of ANO4 in H295R cells caused an increase in CYP11B2 and NR4A2 gene expression levels but basal aldosterone secretion was unchanged. In the presence of calcium agonists, ANO4 reduced aldosterone secretion. ANO4 subcellular localisation was confirmed as cytoplasmic by immunofluorescence microscopy of transfected cells. When exposed to calcium ionophores, ANO4 generated small chloride currents as detected by YFP assay. In summary, the comparison of transcriptome of ZG with paired ZF found unexpected up-regulated genes. Most of the highly up regulated genes in human ZG, including NEFM and ANO4, inhibit either basal or stimulated aldosterone secretion, and this may reflect an adaptive response to high salt intake. No clear-cut correspondence was found between transcriptome of APAs and their resembling zone of adrenal cortex. The down-regulation of NEFM following transfection of mutant KCNJ5 suggests that ZF-like properties may be a consequence of mutation, rather than tissue of origin.

Cromossomos sexuais evoluíram repetidas vezes independentemente nos grandes grupos de vertebrados. Sistemas sexuais altamente diferenciados e antigos são caracterizados por grandes diferenças morfológicas e de conteúdo gênico entre os dois cromossomos homólogos onde a recombinação é restrita a uma pequena região homóloga. Os sistemas recentes característicos de peixes caracterizam-se pela similaridade entre os cromossomos X e Y (ou Z e W), nos quais as diferenças observadas freqüentemente envolvem a presença de heterocromatina, translocações e inversões. A recombinação ocorre entre o par sexual na maior parte de sua extensão, sendo inibida apenas na região diretamente relacionada com a determinação sexual. Notavelmente, sistemas diferentes de determinação podem ser encontrados em espécies, ou mesmo populações. O gênero Eigenmannia compreende grupos de espécies crípticas do ponto de vista morfológico que exibem variação no número cromossômico e podem apresentar sistemas sexuais XY ou ZW, incluindo sistemas múltiplos (com translocação Y-autossomo). Estes sistemas estão entre os mais recentes descritos (<16ma) e estão dispostos de forma desordenada em árvores de relações filogenéticas, sugerindo origens múltiplas. No presente estudo, a técnicas de pintura cromossômica usando sondas obtidas por microdissecção de cromossomos sexuais foram empregadas para testar a homologia de dois sistemas XY encontrados nos citótipos (ou espécies) E. virescens e E. sp.2. Os resultados mostram que, de fato, ambos são não homólogos. A fusão Y-autossomo provavelmente ocorreu após a separação de E. sp.2 com sua espécie irmã, E. sp.1 uma vez que um evento de fusão independente, envolvendo um dos cromossomos homólogos ao Y, foi detectado em E. sp.1. A hibridação in sit&#956; do cromossomo X de E. virescens em sua população mais próxima (também com 38 cromossomos, mas sem cromossomos sexuais heteromórficos) mostrou que o cromossomo X é homólogo a um par de acrocêntricos, condizente com o modelo proposto de diferenciação por acúmulo de heterocromatina. Essa heterocromatina foi caracterizada e mostrou um padrão complexo de seqüências CG-ricas. Dois fragmentos de DNA repetitivo GC-ricos presentes no cromossomo X foram isolados e seqüenciados. Não foram detectadas similaridades em comparações com bases de dados e entre os fragmentos obtidos. Estes mostraram-se concentrados nas regiões cromomicina-positivas de E. virescens, incluindo regiões periteloméricas de sete pares e os dois maiores blocos heterocromáticos (nos cromossomos X e par n. 8), além de um cromossomo acrocêntrico, possivelmente o Y. Curiosamente, essas seqüências foram detectadas em apenas três pares cromossômicos na população mais próxima, incluindo um par acrocêntrico de morfologia semelhante à condição ancestral do X, sugerindo que processos dinâmicos de expansão e homogenização genômica ocorreram após a separação dessas populações / Sex chromosomes have evolved independently several times in all major groups of vertebrates. Highly differentiated sex chromosomes are characterized by extensive differences in morphology and gene content, whereas recombination is restricted to a small homologous region. Recent sex chromosomes are characteristic of fish, and display a high level of homology between X and Y (or Z and W) chromosomes, recombination is restricted only in a small sex determining region. Notably, different sex chromosome systems can be found in closely related groups, such as species or even populations. The genus Eigenmannia comprises a group of morphologically cryptic species that display a variety of diploid numbers and different sex chromosome systems, including XY, ZW and a multiple XY system (with a Y-autosome fusion). These systems are among the most recent known (<16ma) and occur with a lack of phylogenetic pattern, whereas frequently populations bearing heteromorphic sex chromosomes are closest related to populations displaying no sex chromosomes. In the present study, chromosome painting using probes derived from the microdissection of two different sex chromosomes where used to investigate the homology of both systems. Results show that, in fact, they are non-homologous and evolved independently. The Y-autosome hypothesis gained further support from the observation that a chromosome homologous to the Y in a close population is involved in yet a different fusion event. The X chromosome present in the E. virescens karyotype was found to be homologous to acrocentric chromosomes in all populations analyzed, thus supporting the notion that its differentiations is mainly due to the accumulation of heterochromatin. The X heterochromatic block was shown to form a complex pattern of GC-rich sequences, different from what was previously described. Two GC-rich fragments were isolated and sequenced; both showed no similarities to known sequences and to one another. These sequences were shown to be concentrated viii on the two largest heterochromatic blocks, those of the X and n.8 chromosomes besides peri-telomeric regions of seven additional pairs and the putative Y. Curiously, these sequences were detected in only three pairs in the closest population, including an acrocentric pair morphologically similar to undifferentiated sex pair. This suggests that dynamic evolutionary processes of expansion and genomic homogenization have occurred after the separation of these populations.

INTRODUÇÃO: A exposição prolongada às partículas ambientais está associada à mortalidade prematura devido às causas cárdio-respiratórias e ao câncer do pulmão. O tamanho e composição destas partículas estão relacionados à maior toxicidade, agravada pela retenção prolongada destas partículas no pulmão. Pode-se postular que partículas com diferentes composições elementares podem ter distribuição diferente ao longo da árvore traqueobrônquica. A captura por micro-dissecção a laser (CML) aparece neste cenário como uma alternativa rápida e eficaz para se obter amostras para estudo da distribuição destas partículas no pulmão. OBJETIVOS: Testar a eficácia da CML no estudo de partículas retidas no pulmão comparando com outra técnica de análise de cortes de parafina (P), já consagrada na literatura. Comparar também o perfil elementar de partículas retidas ao longo da árvore traqueobrônquica e linfonodos em duas cidades com perfis distintos de poluição, através da CML, associada à espectometria de raios-X por dispersão de energia (RX-DE) aliada à microscopia eletrônica de varredura (MEV). MÉTODOS: Vinte e quatro casos autopsiados foram obtidos em duas cidades com perfis distintos de poluição (São Paulo e São José do Rio Preto). As amostras de parênquima pulmonar obtidas do lobo médio do pulmão direito foram coletadas em três regiões: tecido peribrônquico, parênquima pulmonar periférico e linfonodos hilares. Para P, a análise foi feita em áreas visualizadas no MEV correspondentes às áreas de antracose. Estas áreas de antracose foram também micro-dissecadas, usando CML e analisadas através do RX-DE aliado ao MEV. RESULTADOS: Quando os dois métodos foram comparados, a maioria dos elementos encontrados, mostrando diferença significante, foi vista na CML, exceto pelo Ca e Mg (mais comumente encontrados em P). A análise elementar das partículas depositadas ao longo da árvore traqueobrônquica mostra dois grupos de distribuição dos elementos, com deposição predominante peribronquiolar ou com deposição predominante linfonodal. A análise elementar das áreas peribrônquicas descrimina melhor (95.8 %), o tipo de exposição à poluição, sem diferença significante para os outros locais estudados. CONCLUSÃO: Os perfis elementares das partículas retidas ao longo da árvore traqueobrônquica mostram dois grupos com padrão de distribuição distinta. A região proximal é o principal local para discriminação do tipo de exposição do indivíduo, sendo a antracose o carimbo da exposição à poluição. A CML se mostrou também uma ferramenta útil para discriminar áreas de interesse no estudo da análise elementar de partículas retidas no pulmão quando comparada com a análise nos cortes de parafina. / RATIONALE: Prolonged exposure to ambient particles is associated with premature mortality due to cardio respiratory diseases and lung cancer. Size and composition of these particles determine toxicity, aggravated by their long term retention in the lungs. In this context, it is plausible to postulate that different particles with different elemental composition must have distinct distribution patterns along the bronchial tree. Laser capture microdissection (LCM) appears as a rapid and efficient alternative for obtaining representative lung tissue for particle analysis. OBJECTIVES: To test laser capture microdissection (LCM) as a new tool for studying particle retention, comparing to a classical method using paraffin sections (PS). To compare the elemental profile of particles retained along the bronchial tree and lymph nodes in two cities with distinct pollution backgrounds by using LCM, through energy dispersive x-ray (EDX) and scanning electron microscopy (SEM). METHODS: Twenty-four right lung middle lobes from autopsied cases were obtained in two cities with different pollution backgrounds. Lung samples were collected from three distinct regions in the lungs at the time of autopsy: peribronchial tissue, peripheral parenchyma and hylar lymph nodes. For PS, analysis was performed in areas visualized in the SEM correspondent to anthracotic areas. These areas of anthracosis were also microdissected using LCM and analyzed for elemental composition through EDX allied to SEM. RESULTS: When the two methods were compared, the majority of the elements showing significant difference was predominantly found when LCM was used, except for Ca and Mg (more related to PS) Elemental analysis of particles deposited along the bronchial tree shows two groups of distribution: elements with preferable peribronchiolar or preferable lymph node deposition. Elemental profile of peribronchial areas discriminate accurately (95.8 %) the type of pollution exposure, with no statistical difference noted for the other sites. CONCLUSIONS: Elemental profiling of the particles retained along the bronchial tree shows two groups with distinct distribution patterns. The proximal bronchial tree is the main site for discrimination of pollution exposure, with an elemental finger print of anthracosis. LCM has also proven to be a useful tool in discriminating areas of interest for elemental analysis of particles retained in the lung when compared to analysis in PS.

L'anévrysme de l'aorte abdominale (AAA) est un problème de Santé Publique qui touche principalement les hommes de plus de 65 ans. L'AAA souvent asymptomatique évolue vers la rupture associée à un taux de mortalité élevé. Parmi les acides ribonucléiques (ARNs) non codants, les microARNs (miARNs), stables dans le tissu et les biofluides, sont des candidats intéressant dans la recherche de biomarqueurs. L'inflammation, la dégradation de la matrice extra-cellulaire (MEC) et la raréfaction de la média participent à l'AAA. De nombreuses cellules inflammatoires sont impliquées dans l'AAA. La raréfaction des cellules musculaires lisses (CML) est secondaire à l'anoïkis, apoptose par détachement cellulaire de la MEC. Une analyse protéomique différentielle de CML en culture, issues de patients porteurs d'AAA, réalisée au laboratoire a montré que la désintégrine et metalloprotéinase avec un motif de thrombospondine 5 (ADAMTS 5) est surexprimée dans les CML de patients présentant un AAA. L'isolement des cellules par la microdissection laser permet de conserver le phénotype des cellules isolées et de mettre en évidence des marqueurs potentiels de l'AAA masqués par l'analyse du tissu global. Mon travail de thèse a consisté à partir des cellules isolées de la paroi anévrysmale de l'aorte abdominale sous-rénale chez l'Homme: à effectuer une analyse globale des miARNS et une analyse ciblée de l'ADAMTS 5, métalloprotéase qui a une action enzymatique sur les protéines de la MEC. Les objectifs de ce travail sont une meilleure compréhension de l'AAA et l'identification de nouveaux biomarqueurs.La distribution des cellules dans la paroi anévrysmale est étudiée par immunohistochimie sur des biopsies anévrysmales et d'aortes saine obtenues chez l'Homme. Les cellules localisées sont isolées par microdissection laser. L'analyse par criblage de l'expression des miARNs des cellules isolées de l'AAA et des CML issues d'aorte saine est réalisée sur puce. L'expression différentielle de miARNs sélectionnés est analysée par PCR quantitative dans des cellules isolées de l'AAA et dans du tissu global. L'expression des miARNs sélectionnés est ensuite comparée dans le plasma des patients présentant un AAA et de patients athérosclérotiques sans AAA par PCR pour identifier de potentiels biomarqueurs. Dans l'AAA, les macrophages M1 proinflammatoires sont retrouvés dans l'adventice et les macrophages M2 anti inflammatoires dans le thrombus intraluminal, les lymphocytes de type B sont retrouvés organisé en organe lymphoïde tertiaire adventitielle ou ATLOs dans 11 échantillons sur 20 analysés. Les CML sont rares et strictement localises au niveau de la média. Sur 850 miARNs testés dans la puce, 205 miARNs sont exprimés dans les cellules isolées. Les miR-29b et let-7f sont augmentés dans le plasma de patients porteurs d'AAA et représentent de potentiel biomarqueurs.L'expression d'ADAMTS 5 dans les CML de la paroi anévrysmale est évaluée par immunohistochimie dans la paroi aortique saine et anévrysmale et quantifiée par Western-blot dans les CML isolées de la paroi aortique saine et anévrysmale.Deux morphotypes de CML anévrysmales ont été identifiés: un morphotype arrondi positif au marqueur de l'apoptose, caspase 3 et un morphotype allongé, similaire aux CML de l'aorte saine. Le profil d'expression des sous-unités d'ADAMTS 5 est diffèrent dans les CML arrondies et les CML allongées anévrysmales et saines. La mise en apoptose des CML a été mise au point in vitro pour étudier les mécanismes impliqués dans les modifications d’ expression d'ADAMTS 5 dans l'AAA et les conséquences sur son action enzymatique.L'approche systématique de l'expression transcriptomique des cellules anévrysmales isolées a identifié des marqueurs potentiels de l'AAA, les miR-29b et let-7f et l'analyse ciblée suggère l'implication d'ADAMTS 5 dans le profil évolutif des CML vers l'anoïkis dans l'AAA. Des études complémentaires permettront une meilleure compréhension de l'AAA. / Abdominal aortic aneurysm (AAA) is a public health problem, which mainly affects men older than 65 year. AAA are usually asymptomatic with a natural evolution towards rupture associated with a high mortality rate. Among non coding ribonucleic acids (RNAs), microRNAs are stable in tissue and biofluids and are interesting candidates for the search of biomarkers. Inflammation, extracellular matrix (ECM) degradation and media rarefaction are involved in AAA. Many inflammatory cells are involved in AAA. Anoikis is an apoptosis secondary to a cell detachment from ECM and is responsible for rarefaction of smooth muscle cells (SMC). Differential proteomic analysis of cultured SMC from AAA patients was performed in the laboratory and highlighted the overexpression of a disintegrin and metalloproteinase with thrombospondin motif of type 5 (ADAMTS 5) in SMC of AAA patients. Isolation of cells with laser microdissection allows to keep their phenotype and to find potential markers that may be masked by global tissue analysis.The aim of my PhD work was to perform a global miRNA screening of cells isolated from human aneurysmal wall and an analysis targeted on ADAMTS 5, a metalloprotease with an enzymatic activity on ECM proteins. The main objectives were a better understanding of AAA and the identification of new biomarkers.The distribution of cells in the aneurysmal wall was studied by immunohistochemistry in human aneurysmal and healthy aortic samples. Once located, the cells were isolated by laser microdissection and screened for miRNAs by microarrays. Differential expression of selected miRNAs was quantified by PCR in the cells isolated by laser microdissection and in whole aortas. They were then compared in plasma of AAA patients and atherosclerotic patients without AAA by quantitative PCR to identify potential biomarkers.In AAA, the M1 proinflammatory macrophages were located in the adventitia and the M2 antiinflammatory macrophages in the intraluminal thrombus; the type B lymphocytes were organized in tertiary lymphoid organs (ATLOs) in 11/20 of analysed samples. SMC were rare and restricted to the media. Among the 850 miRNAs tested on microarray, 205 miRNAs were detected in isolated cells. MiR-29b and let-7f were upregulated in plasma of AAA patients, and thus are potential biomarkers.The expression of ADAMTS 5 in aneurysmal SMC was evaluated by immunohistochemistry of healthy and aneurysmal aortic wall and quantified by Western blot in isolated SMC from healthy and aneurysmal wall.Two aneurysmal SMC morphotypes were identified: a rounded morphotype positive for caspase 3, an apoptotic marker, and a spindle-shaped morphotype similar to the healthy aortic SMC. The expression profile of ADAMTS 5 subunits was different in rounded SMC compared to aneurysmal and healthy spindle-shaped SMC. In vitro induction of apoptosis of SMC was established in order to study the mechanisms involved in ADAMTS 5 expression in AAA and their consequences on enzymatic actions.The global transcriptomic screening of aneurysmal cells isolated by laser microdissection has identified potential markers of AAA, miR-29b and let-7f. The targeted analysis suggested that ADAMTS 5 is involved in the evolution profile of SMC towards anoikis in AAA. Further investigations will allow a better understanding of AAA pathophysiology.

Orientadores: Adriana Franco Paes Leme, Ricardo Della Coletta / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-28T02:02:25Z (GMT). No. of bitstreams: 1 Macedo_CarolinaCarneiroSoares_M.pdf: 3754068 bytes, checksum: 36ddb6796cb6690a37ce3e4e9447009c (MD5) Previous issue date: 2015 / Resumo: O carcinoma espinocelular oral (CEC) é o tipo de neoplasia maligna mais comum em cabeça e pescoço, com alta prevalência e morbidade. O tratamento é baseado em sistemas de classificação pouco precisos e o prognóstico é ruim em muitos casos. Diferentes padrões histológicos já foram descritos na tentativa de melhor compreender o curso da doença, predizer prognóstico e auxiliar no tratamento. As diferentes áreas do tumor apresentam características morfológicas e moleculares distintas resultando em comportamentos clínicos específicos, e estudos recentes apontam a região de invasão tumoral (do inglês invasive front tumor) como área de interesse para análises de perfil molecular e identificação de possíveis marcadores de prognóstico. O padrão de invasão neoplásico tem relação com a agressividade tumoral e a presença de ilhas no fronte invasivo já foi descrita como pior padrão. Assim, o objetivo desta dissertação foi comparar a composição diferencial de proteínas totais de ilhas neoplásicas grandes e pequenas do fronte e do interior do tumor, e correlacionar essas proteínas com o prognóstico. A proteômica foi associada à microdissecção a laser (ML), consideradas juntas como ferramentas de alta robustez para identificação de proteínas em tecidos neoplásicos em regiões específicas. Vinte peças cirúrgicas de CEC oral de língua fixadas em parafina foram submetidas a ML para obtenção das amostras compostas pelas seguintes regiões do tecido: 1) ilhas neoplásicas grandes da região frontal, 2) ilhas neoplásicas pequenas da região frontal, 3) ilhas neoplásicas grandes do interior do tumor e 4) ilhas neoplásicas pequenas do interior do tumor, seguida pela extração de proteínas e análise por cromatografia líquida acoplada à espectrometria de massas (LC-MS/MS). A anotação funcional das proteínas e a correlação aos dados clínico-patológicos dos pacientes foram realizadas. Foram identificadas 1906 proteínas totais, sendo 1480 proteínas comuns entre as quatros regiões estudadas. Duas proteínas foram exclusivas nas ilhas grandes do fronte e sete nas ilhas grandes do interior. Oitenta e cinco proteínas foram diferencialmente expressas entre a região do fronte e o interior tumoral, e destas, 57 foram relacionadas a dados clínico-patológicos importantes para o prognóstico. Os processos biológicos, como desenvolvimento da epiderme, adesão celular, apoptose, ciclo celular, degradação da matriz extracelular e expressão gênica, evidenciados entre as proteínas diferencialmente expressas confirmam as mudanças moleculares associadas à progressão neoplásica. A combinação de ML, MS e bioinformática foi capaz de identificar um painel de proteínas que podem auxiliar a desvendar o curso do CEC oral, predizendo agressividade e prognóstico. Em acréscimo, essa abordagem pode ajudar ainda na compreensão das diferenças e dos mecanismos de sinalização entre diferentes áreas do tecido / Abstract: Oral squamous cell carcinoma (SCC) is the most common type of malignant tumor in head and neck, with high prevalence and morbidity. Treatment is based on inaccurate classification systems and the prognosis is poor in many cases. Different histological patterns have been described in an attempt to better understand the course of the disease, predict prognosis and assist in treatment. Different areas of the tumor have different morphological and molecular characteristics resulting in specific clinical behaviors, and recent studies point to the region of tumor invasion as an area of interest for molecular profile analysis and identification of possible prognostic markers. The pattern of neoplastic invasion is related to tumor aggressiveness and the presence of islands in the invasive front has been described as worst invasion pattern. The objective of this work was to compare the protein differential expression of large and small islands neoplastic from the front and the inner tumor, and to correlate these proteins with prognosis. Proteomics was associated with laser microdissection (ML), and together they are considered robustness tools to identify proteins in neoplastic tissues in specific regions of interest. Twenty surgical specimens of oral tongue SCC fixed in paraffin were subjected to ML to obtain sample composed by the following regions of tissue: 1) large neoplastic frontal islands, 2) small neoplastic islands of the frontal region, 3) large neoplastic islands inside the tumor and 4) small islands within the neoplastic tumor, followed by extraction and analysis of proteins by liquid chromatography coupled to mass spectrometry (LC-MS/MS). The functional annotation of proteins and correlation with clinicopathological data from patients were performed. A total of 1906 proteins were identified, with 1480 common proteins between the four regions studied. Two proteins were exclusives in the large islands of the forehead and seven in large islands in the interior. Eighty-five proteins were differentially expressed between the front region and inner tumor, and of these, 57 were related to clinical and pathological data. The biological processes such as development of the epidermis, cell adhesion, apoptosis, cell cycle, disassembly of the extracellular matrix and gene expression, evidenced among the differentially expressed proteins confirmed the molecular changes associated with neoplastic progression. The combination of ML, MS, and bioinformatics was able to identify a panel of proteins that may help to unravel the course of oral SCC, predicting aggressiveness and prognosis. In addition, this approach may also help in understanding the differences and signaling mechanisms between different areas of the tumor tissue / Mestrado / Patologia / Mestra em Estomatopatologia

ABSTRACT The morphological premalignant changes in prostate epithelium such as high grade prostatic intraepithelial neoplasia (HGPIN) precede invasive prostate cancer (PC) by several decades. The overall aim of this project was to identify patterns of gene expression in HGPIN and early PC which increase our understanding of the early biology of PC and identify genes and pathways that correlate with an aggressive phenotype. A comprehensive tissue cohort of premalignant prostate lesions was collected in a tissue microarray (TMA) platform that was utilised for high-throughput validation of target genes. Using this unique resource, the expression of the tumour suppressor gene PTEN was assessed using immunohistochemistry in an initial candidate gene approach based on mouse models implicating PTEN in carcinogenesis. No significant difference in expression of PTEN was detected in premalignant and benign epithelium. A transcript profiling approach was undertaken by integrating laser capture microdissection, linear RNA amplification and oligonucleotide microarrays to perform a screen of matched patient samples of normal, HGPIN and PC cells. The expression patterns of two genes encoding secreted proteins, neuropeptide Y (NPY) and macrophage inhibitory cytokine (MIC-1) were validated using immunohistochemistry on TMAs representing the progression model of early PC. Increased expression of these proteins in PC was confirmed to occur early in the disease process and altered expression of NPY and MIC-1 was associated with worse clinical outcome. Further analysis of global gene expression patterns using a structured network knowledge base identified a notable aberration in the expression of extracellular matrix and extracellular matrix associated proteins in HGPIN and provided novel evidence for the role of this class of molecules in the development of PC. In summary, contrary to current dogma based on work in animal models, altered PTEN expression is unlikely to represent an important event in the development of malignancy in the human prostate. In contrast, the expression patterns and prognostic value of NPY and MIC-1 in HGPIN support their further evaluation as biomarkers for the development and progression of PC. The aberrant expression of genes and networks of genes detected in HGPIN will assist in further identification of biological pathways which may be targeted in therapeutic strategies against the development and progression of PC.

The tropical warm pool waters surrounding Indonesia are one of the equatorial heat and moisture sources that are considered as a driving force of the global climate system. The climate in Indonesia is dominated by the equatorial monsoon system, and has been linked to El Niño-Southern Oscillation (ENSO) events, which often result in severe droughts or floods over Indonesia with profound societal and economic impacts on the populations living in the world's fourth most populated country. The latest IPCC report states that ENSO will remain the dominant mode in the tropical Pacific with global effects in the 21st century and ENSO-related precipitation extremes will intensify. However, no common agreement exists among climate simulation models for projected change in ENSO and the Australian-Indonesian Monsoon. Exploring high-resolution palaeoclimate archives, like tree rings or varved lake sediments, provide insights into the natural climate variability of the past, and thus helps improving and validating simulations of future climate changes. Centennial tree-ring stable isotope records | Within this doctoral thesis the main goal was to explore the potential of tropical tree rings to record climate signals and to use them as palaeoclimate proxies. In detail, stable carbon (δ13C) and oxygen (δ18O) isotopes were extracted from teak trees in order to establish the first well-replicated centennial (AD 1900-2007) stable isotope records for Java, Indonesia. Furthermore, different climatic variables were tested whether they show significant correlation with tree-ring proxies (ring-width, δ13C, δ18O). Moreover, highly resolved intra-annual oxygen isotope data were established to assess the transfer of the seasonal precipitation signal into the tree rings. Finally, the established oxygen isotope record was used to reveal possible correlations with ENSO events. Methodological achievements | A second goal of this thesis was to assess the applicability of novel techniques which facilitate and optimize high-resolution and high-throughput stable isotope analysis of tree rings. Two different UV-laser-based microscopic dissection systems were evaluated as a novel sampling tool for high-resolution stable isotope analysis. Furthermore, an improved procedure of tree-ring dissection from thin cellulose laths for stable isotope analysis was designed. The most important findings of this thesis are: I) The herein presented novel sampling techniques improve stable isotope analyses for tree-ring studies in terms of precision, efficiency and quality. The UV-laser-based microdissection serve as a valuable tool for sampling plant tissue at ultrahigh-resolution and for unprecedented precision. II) A guideline for a modified method of cellulose extraction from wholewood cross-sections and subsequent tree-ring dissection was established. The novel technique optimizes the stable isotope analysis process in two ways: faster and high-throughput cellulose extraction and precise tree-ring separation at annual to high-resolution scale. III) The centennial tree-ring stable isotope records reveal significant correlation with regional precipitation. High-resolution stable oxygen values, furthermore, allow distinguishing between dry and rainy season rainfall. IV) The δ18O record reveals significant correlation with different ENSO flavors and demonstrates the importance of considering ENSO flavors when interpreting palaeoclimatic data in the tropics. The findings of my dissertation show that seasonally resolved δ18O records from Indonesian teak trees are a valuable proxy for multi-centennial reconstructions of regional precipitation variability (monsoon signals) and large-scale ocean-atmosphere phenomena (ENSO) for the Indo-Pacific region. Furthermore, the novel methodological achievements offer many unexplored avenues for multidisciplinary research in high-resolution palaeoclimatology. / Die tropischen Gewässer um Indonesien sind eine der äquatorialen Wärme- und Feuchtigkeitsquellen, die als treibende Kraft des globalen Klimasystems betrachtet werden können. Das Klima in Indonesien ist geprägt durch das Australisch-Indonesische Monsunsystem. Weiterhin besteht eine Verknüpfung mit El Niño-Southern Oszillation (ENSO) Ereignissen, die oft zu schweren Dürren oder Überschwemmungen in der Region mit tiefgreifenden gesellschaftlichen und wirtschaftlichen Folgen führen. Der neueste IPCC-Bericht legt dar, dass ENSO auch in den nächsten 100 Jahren das vorherrschende Klimaphänomen im tropischen Pazifik bleiben wird. Ferner wird davon ausgegangen, dass sich die ENSO-bezogenen Niederschlagsextrema intensivieren werden. Wenig Übereinstimmung herrscht jedoch bislang zwischen den Klimasimulationsmodellen in Bezug auf die voraussichtlichen Veränderungen von ENSO und dem Australisch-Indonesischen Monsunsystem. Hochaufgelöste Paläoklima-Archive, wie z.B. Jahrringe oder warvierte Seesedimente, geben Auskunft über die natürliche Klimavariabilität der Vergangenheit und können somit dazu beitragen, die Computersimulationen der künftigen Klimaentwicklung zu verbessern und zu validieren. Hundertjährige stabile Jahrring-Isotopenchronologien | Das Hauptziel dieser Doktorarbeit war es, dass Potenzial von tropischen Jahrringen zur Aufzeichnung von Klimasignalen herauszustellen und deren Evaluierung als Paläoklimaproxys. Im Detail wurden stabile Kohlenstoff- (δ13C) und Sauerstoff- (δ18O) Isotopenverhältnisse in Teakbäumen analysiert, und die ersten gut replizierten hundertjährigen (AD 1900-2007) stabilen Isotopenchronologien aus Java (Indonesien) erstellt. Dabei wurden verschiedene klimatische Einflussgrößen getestet, ob diese signifikante Korrelationen mit den Jahrringparametern aufzeigen. Weiterhin wurden hochaufgelöste intra-annuelle Sauerstoffisotopenzeitreihen erstellt, um den Transfer des saisonalen Niederschlagssignals in den jeweiligen Jahrring zu bemessen. Die ermittelte Sauerstoff-Isotopenchronologie wurde anschließend auf mögliche ENSO Signale hin untersucht. Methodische Errungenschaften | Ein zweites Ziel dieser Arbeit war es neue Verfahren zur Analyse stabiler Isotope in Baumjahrringen zu entwickeln und zu optimieren. Zwei verschiedene UV-Lasermikrodissektions-Systeme wurden getestet als neues präzises Präparationswerkzeug für stabile Isotopenstudien. Darüber hinaus wurde eine verbesserte Methode für die Probenaufbereitung stabiler Isotopenmessungen anhand von Zellulose-Dünnschnitten entwickelt. Die wichtigsten Ergebnisse dieser Doktorarbeit sind: I) Die hier vorgestellten neuartigen Techniken zu Probenvorbereitung verbessern die Analyse stabiler Isotope für Jahrringstudien in Hinsicht auf Präzision, Effizienz und Qualität. Es wurde gezeigt, dass die UV-Lasermikrodissektion eine wertvolle Technik ist, um die Beprobung von Pflanzengewebe in höchster Auflösung und beispielloser Präzision durchzuführen. II) Es ist gelungen, einen Leitfaden für ein modifiziertes Verfahren der Zelluloseextraktion an Gesamtholz-Dünnschnitten und der anschließenden Jahrringaufbereitung zu erstellen. Diese neuartige Methode optimiert die Analyse stabiler Isotopenzeitreihen in zweierlei Hinsicht: schnellere und effiziente Zelluloseextraktion und präzise Trennung der Jahrringsequenzen in inter-annueller bis intra-annuelle Auflösung. III) Die hundertjährigen stabilen Jahrring-Isotopenchronologien weisen signifikante Korrelationen mit dem regionalen Niederschlag auf. In den hochaufgelösten stabilen Sauerstoffisotopenwerten spiegelt sich deutlich das Niederschlagssignal der Trocken- und der Regenzeit wieder. IV) Die stabile Sauerstoffisotopenzeitreihe zeigt signifikante Korrelationen mit verschiedenen ENSO Phasen. Dies betont, dass die verschiedenen ENSO Phasen bei der Interpretation von tropischen Paläodaten zu berücksichtigen sind. Die Ergebnisse der Dissertation zeigen, dass saisonal aufgelöste stabile Sauerstoffisotopenchronologien von indonesischen Teakbäumen ein geeigneter Proxy für mehrhundertjährige Rekonstruktionen der regionalen Niederschlagsvariabilität (Monsun-Signale) und großräumiger Ozean-Atmosphären-Systeme (ENSO) für den Indopazifik ist. Darüber hinaus bieten die neuartigen methodischen Errungenschaften viele neue Ansätze für multidisziplinäre hochaufgelöste Studien in der paläoklimatologischen Forschung.