Non-melanoma skin cancer (NMSC) is the most common cancer in the world with a lifetime risk for development as high as 2 in 3 in Queensland, Australia. Mortality is quite low, representing an approximate 360 deaths in Australia annually but cost of treatment is extremely high, estimated at $232 million each year. Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are the two most common forms of NMSC. Although BCC generally do not have the propensity to metastasise, they are highly invasive and can be locally destructive. SCC on the other hand is invasive and has metastatic potential. SCC is generally derived from a precursor lesion, solar keratosis (SK), which is also considered to be a biomarker of BCC, SCC and malignant melanoma. According to one theory, SKs actually represent the first recognisable stage of SCC development and therefore may be indicative of the earliest stage of NMSC development. In addition to these common forms of NMSC, rarer forms such as keratoacanthoma (KA), which spontaneously regress, and SCC in situ, which rarely become invasive, may provide clues into protective mechanisms associated with prevention of development. Like all other cancers, NMSC arises from an accumulation of genetic abnormalities that result in severe cellular dysfunction. A number of genes have been proposed in the development of NMSC, including p53, CDKN2a, Bcl-2 and the Ras family of genes, which are typically associated with proliferative and differentiation processes. Also, a number of genetic disorders that predispose individuals to NMSC have also been identified. Genetic abnormalities in these genes may be a result of somatic mutations that may be promoted by environmental carcinogens. For NMSC, ultraviolet (UV) radiation is the primary environmental stimulus that acts upon skin to generate mutations. UV effects are 2-fold; the first being direct damage produced by UVB radiation and the second being indirect damage as a result of UVA-induced oxidative stress. In addition to mutations of genes that directly result in carcinogenesis, polymorphic variants of genes may also play a role in susceptibility to NMSC. These susceptibility genes may have immunogenic, detoxifying or transcriptional roles that could be involved in increased mutagenesis or activation of cancer causing genes. The purpose of this study was ultimately to identify further molecular based mechanisms associated with the development of non-melanoma skin cancer. Initially, this study aimed to examine the effects of aberrant chromosomal regions on NMSC development and also to identify candidate genes within these regions that may be implicated in the development and progression of NMSC. Also, based on chromosomal and functional implications, a number of candidate genes were assessed using association analysis to determine their involvement in susceptibility to the earliest stages of NMSC development. Implicated susceptibility genes were then further investigated to determine their response to UV radiation. Therefore the methodological approach of these studies was based on three broad technical applications of cytogenetic, association and expression analyses. Previous comparative genomic hybridisation (CGH) studies implicated the 18q chromosomal region in progression of SK to SCC and this region was therefore suspected of harbouring one or more tumour suppressor genes that were associated with a more malignant phenotype. Following on from this analysis, loss of heterozygosity (LOH) analysis was used for further delineation of this region and possibly to implicate candidate genes involved in progression. Additionally, CGH was used to investigate keratoacanthoma to determine aberrant regions that might be involved in progression and also regression of this NMSC. Genes that had potential functional roles in NMSC development and that were located in or near regions implicated by these cytogenetic analyses were further investigated using association analysis. Association analysis was performed using polymerase chain reaction and subsequent restriction enzyme digestion or GeneScan analysis to determine genotype and allele frequencies in an SK affected versus control population for polymorphisms within a number of candidate genes. This population was carefully phenotyped so that not only genotypic factors could be analysed but also their interaction with a number of phenotypic and environmental risk factors. Genes with polymorphisms that did show association with solar keratosis development were then examined functionally. Specifically, gene expression analysis was undertaken to investigate their response to UV radiation. Both UVA only and combined UVA/UVB treatments were used for short term irradiation and also for long term irradiation with recovery to determine differential effects of UV range and dose in human skin. Relative mRNA expression analysis of these genes was performed using quantitative real time reverse transcription polymerase chain reaction to determine if UV radiation imposed gene expression changes in the skin. A combination of these methodologies provided a wide basis for investigation of NMSC. Cytogenetic, association and expression analyses all allow for the identification of molecular risk factors that cause or are associated with NMSC development and progression. These analyses provided diverse results that implicated various molecular mechanisms in the development of NMSC. Cytogenetic analysis is a powerful technique, especially for the identification of a broad range of aberrations throughout the genome. This study employed LOH analysis to investigate an implicated region involved in progression to SCC and to attempt identification of candidate genes that may be involved in this process. LOH analysis was successfully performed on 9 SCCs, 5 SCCs in situ and 2 SKs using 8 microsatellite markers within the 18q region. Polymerase chain reaction (PCR) was used to amplify polymorphic regions of these markers and genotypic composition was determined for normal and cancerous tissue within the specimen. In heterozygote individuals, determined by analysis of normal tissue, the cancerous tissue was examined to determine if alleles within the implicated region had been lost. However, after analysis of multiple different samples, there was no LOH detected in any of the samples examined for this analysis. This does not necessarily reject a role for 18q, or genes within this region, as the localisation of candidate tumour suppressor genes within a small region may indicate a tighter region of involvement than was expected. As such, a more targeted study may further delineate this region and implicate candidate genes in progression of SK to the more malignant phenotype of SCC. Further CGH analysis of keratoacanthoma was also undertaken to identify aberrations associated with development and also regression of this skin cancer. CGH was performed using universal amplification and nick translation to incorporate a fluorescent dye. Differentially labelled normal and tumour DNA were then competitively hybridised to a normal metaphase spread and fluorescence emission indicated either amplification or deletion of specific chromosomal regions. In total, 6 KA samples were analysed, with 2 samples each from evolving, matured and regressing stages of KA development. In general, regressing KAs appeared to be more highly associated with deleted regions than evolving and matured KAs. Specifically, the 15q chromosomal region that was deleted in regressing KAs but amplified in evolving or matured KAs, may be significantly involved in the process of KA regression. Also various candidate genes that were being considered for analysis were located in or near some of these implicated regions, including GSTM1, GSTP1 and SSTR2. As such, these candidate genes were targeted for further investigation. A number of susceptibility genes that were located in or near aberrant regions implicated in NMSC development were investigated using association analysis. These genes included members of the somatostatin receptor family (SSTR1 and SSTR2), members of the glutathione-S-transferase (GST) family (GSTM1, GSTT1, GSTP1 and GSTZ1) and the vitamin D receptor (VDR). Studies detected a number of interesting interactions between genetic, environmental and phenotypic factors in the development of the early stages of non-melanoma skin cancer. Additionally, genes implicated in NMSC development were further investigated using expression analysis to determine response to UV radiation. Association analysis was initially performed on members of the somatostatin receptor family. Somatostatin is a growth inhibiting factor, amongst other things, that mediates its actions through the somatostatin receptors (SSTRs). The presence of these receptors (SSTR1-5) in tumour cells indicates a potential for somatostatin to bind and suppress growth, as well as allowing for therapeutic treatment with somatostatin analogues. Additionally, expression of these receptors in normal tissue, including skin, should allow for potential protection against tumour growth. The genes for SSTR1 and SSTR2 have been shown to contain dinucleotide repeat polymorphisms, and although these polymorphisms may not directly result in altered expression or binding potential, they may be linked to another functional polymorphism that does. Using association analysis the SSTR1 and SSTR2 genes were investigated to determine whether they play a role in the development of solar keratosis. Results showed that there were no significant differences between SSTR1 and SSTR2 polymorphism frequencies in the tested solar keratosis population (P = 0.10 and P = 0.883, respectively) as compared to an unaffected population. Hence, these studies do not support a role for the SSTR1 or SSTR2 genes in solar keratosis development. Further association analysis and subsequent expression analysis was also performed on members of the glutathione-S-transferase family. The GST enzymes play a role in the detoxification of a number of carcinogens and mutagens, including those produced by UV-induced oxidative stress. This study examined the role of GSTM1, GSTT1, GSTP1 and GSTZ1 gene polymorphisms in susceptibility to SK development. Association analysis was performed to detect allele and genotype frequency differences in SK affected and control populations using PCR and restriction enzyme digestion. No significant differences were detected in GSTP1 and GSTZ1 allele or genotype frequencies, however polymorphisms within both genes were found to be in linkage disequilibrium, as previously reported, and a new allelic variant of the GSTZ1 gene was identified. Significant associations between GSTM1 (P = 0.003) and GSTT1 (P = 0.039) genotypes and SK development were detected, with the null variants of both genes conferring an approximate 2-fold increase in risk for solar keratosis development (OR: 2.1; CI: 1.3-3.5 and OR: 2.3; CI: 1.0-5.0 for GSTM1 and GSTT1, respectively). For the GSTM1 gene, this risk was significantly higher in conjunction with high outdoor exposure (OR: 3.4; CI: 1.9-6.3) and although the GSTT1 gene showed a similar trend (OR: 2.9; CI: 1.1-7.7), this did not reach significance. The increased risk of SK development associated with these genes is likely due to a decreased ability of the skin to detoxify mutagenic compounds produced by UV-induced oxidative stress, and hence a decreased ability to protect against carcinogenesis. Implication of the GSTM1 and GSTT1 null variants in solar keratosis development prompted interest in analysis of gene expression changes in response to UV radiation. Due to the high homology of the GSTM1 gene with other GSTM genes, and therefore potential issues with primer specificity, the GSTT1 gene was focussed on for the expression studies. Real time reverse transcription PCR, incorporating SYBR green fluorescence and 18S as a comparative gene, was used to study GSTT1 gene expression changes in response to both UVA and combined UVA/UVB radiation. It was found that only short term UV radiation had an effect on GSTT1 expression changes, whereas no alteration of gene expression was seen after 4 and 12 hours of recovery from long term irradiation between irradiated and matched non-irradiated skin samples. This indicated that changes in gene expression for the GSTT1 gene apparently occur relatively quickly after exposure to UV radiation. Analysis of both UVA only and combined UVA/UVB short term irradiation indicated that an initial decrease in expression, followed by an increase was likely to represent translation into protein and subsequent transcription of mRNA, and in some cases a second decrease indicated further translation. Hence, it appears as though UV radiation does have a significant effect on the expression of at least one GST gene, and that UV radiation in combination with genetic variation of these genes may play a role in the development of NMSC. Finally, association and subsequent expression analysis was also performed on the vitamin D receptor. The hormonal form of vitamin D, 1a25 dihydroxyvitamin D3, has been shown to have numerous cancer-related effects, including antiproliferative, differentiation, proapoptotic and antiangiogenic effects. These effects are mediated through the binding of 1a25 dihydroxyvitamin D3 to the vitamin D receptor and subsequent transcriptional pathways. Polymorphisms within the VDR are known to regulate its transcription and therefore expression, which is linked to the ability of 1a25 dihydroxyvitamin D3 to bind. Association analysis of a 5’ initiation codon variant (Fok I) and two 3’ variants (Apa I and Taq I) was performed in SK affected and control populations. Although the Fok I variant showed no association with SK development, both the Apa I and Taq I variants were found to be associated with SK development (P = 0.043 and P = 0.012, respectively). In particular, the Aa and Tt genotypes were associated with increased risk of SK. These results were however more complicated, as shown by further analysis. This showed that genotypes containing at least one allele that conferred decreased VDR transcription (ie. AA/Aa and Tt/tt) increased risk of SK development by 2-fold in fair skinned individuals (OR: 2.1; CI: 1.2-3.7 and OR: 1.7; CI: 1.1-2.7 for Apa I and Taq I variants, respectively) but also found to decrease the risk of SK development by 2-fold in medium skinned individuals (OR: 0.5; CI: 0.3-1.0 for Apa I variants). Additionally, genotypes containing 2 alleles conferring decreased transcription of the VDR gene were found to further increase the risk for SK development in fair skinned individuals (OR: 2.5; CI: 1.4-4.5 and OR: 2.4; CI: 1.2-5.0 for Apa I and Taq I variants, respectively), indicating a possible additive effect for the alleles. The highly differential association of the VDR gene polymorphisms amongst phenotypes may reflect a combination between the ability of an individual to synthesise 1a25 dihydroxyvitamin D3 with the binding availability of the VDR. To further investigate the role of VDR in NMSC, expression analysis of the VDR gene was undertaken using real time reverse transcription PCR, with SYBR green fluorescence and 18S as a comparative gene, to examine expression pattern changes associated with UV radiation. It was found that short term irradiation, as well as long term irradiation and recovery were associated with gene expression changes. Short term irradiation resulted in patterns indicative of translation and subsequent transcription, whereas long term irradiated samples resulted in reduction of VDR expression that was recovered after an extended period of time. Thus, VDR expression is clearly influenced by UV exposure. It would be very interesting to see more specifically if particular VDR genotypes, which appear to play a role in NMSC risk, also are affected differentially by UV exposure. It is possible that VDR expression is reduced to limit excessive binding of 1a25 dihydroxyvitamin D3, although since both UVA and UVB radiation affect VDR expression, this may not be mediated the effect of 1a25 dihydroxyvitamin D3 but rather a different pathway resulting from a general UV response. In summary, the detection of a number of susceptibility genes involved in SK development and their subsequent expression analysis in response to UV radiation has given further insight into the molecular changes associated with NMSC. In fact, both detoxification genes (GSTM1 and GSTT1) and a transcription related gene (VDR), were found to confer susceptibility to solar keratosis, an early stage skin lesion with tumourigenic potential. This suggests that even the earliest stages of skin cancer are mediated through a wide range of effects. Additionally, expression changes related to these genes indicate that they are associated with the well known environmental carcinogen of UV radiation and that their effects may be mediated through a wide range of pathways. Although implication of the 18q region in SCC progression was not confirmed in this study, it is still likely to play a role in malignant transformation. The implication of this region, as well as the implication of susceptibility genes has vastly increased knowledge into processes associated with NMSC. Although additional analysis can confirm and further implicate these molecular alterations, this study has resulted in a more comprehensive understanding of NMSC that may ultimately be of benefit in terms of prognosis and treatment.

Doctor of Philosophy(PhD) / BCC is the commonest cancer in European-derived populations and Australia has the highest recorded incidence in the world, creating enormous individual and societal cost in management of this disease. The incidence of this cancer has been increasing internationally, with evidence of a 1 to 2% rise in incidence in Australia per year over the last two decades. The main four epidemiological risk factors for the development of BCC are ultraviolet radiation (UVR) exposure, increasing age, male sex, and inability to tan. The pattern and timing of UVR exposure is important to BCC risk, with childhood and intermittent UVR exposure both associated with an increased risk. The complex of inherited characteristics making up an individual’s ‘sun sensitivity’ is also important in determining BCC risk. Very little is known about population genetic susceptibility to BCC outside of the rare genodermatosis Gorlin syndrome. Mutations in the tumour suppressor gene patched (PTCH) are responsible for this BCC predisposition syndrome and the molecular pathway and target genes of this highly conserved pathway are well described. Derangments in this pathway occur in sporadic BCC development, and the PTCH gene is an obvious candidate to contribute to non-syndromic susceptibility to BCC. The melanocortin 1 receptor (MC1R) locus is known to be involved in pigmentary traits and the cutaneous response to UVR, and variants have been associated with skin cancer risk. Many other genes have been considered with respect to population BCC risk and include p53, HPV, GSTs, and HLAs. There is preliminary evidence for specific familial aggregation of BCC, but very little known about the causes. 56 individuals who developed BCC under the age of 40 in the year 2000 were recruited from the Skin and Cancer Foundation of Australia’s database. This represents the youngest 7 – 8% of Australians with BCC from a database that captures approximately 10% of Sydney’s BCCs. 212 of their first degree relatives were also recruited, including 89 parents and 123 siblings of these 56 probands. All subjects were interviewed with respect to their cancer history and all reports of cancer verified with histopathological reports where possible. The oldest unaffected sibling for each proband (where available) was designated as an intra-family control. All cases and control siblings filled out a questionnaire regarding their pigmentary and sun sensitivity factors and underwent a skin examination by a trained examiner. Peripheral blood was collected from these cases and controls for genotyping of PTCH. All the exons of PTCH for which mutations have been documented in Gorlin patients were amplified using PCR. PCR products were screened for mutations using dHPLC, and all detectable variants sequenced. Prevalence of BCC and SCC for the Australian population was estimated from incidence data using a novel statistical approach. Familial aggregation of BCC, SCC and MM occurred within the 56 families studied here. The majority of families with aggregation of skin cancer had a combination of SCC and BCC, however nearly one fifth of families in this study had aggregation of BCC to the exclusion of SCC or MM, suggesting that BCCspecific risk factors are also likely to be at work. Skin cancer risks for first-degree relatives of people with early onset BCC were calculated: sisters and mothers of people with early-onset BCC had a 2-fold increased risk of BCC; brothers had a 5-fold increased risk of BCC; and sisters and fathers of people with early-onset BCC had over four times the prevalence of SCC than that expected. For melanoma, the increased risk was significant for male relatives only, with a 10-fold increased risk for brothers of people with early-onset BCC and 3-fold for fathers. On skin examination of cases and controls, several phenotypic factors were significantly associated with BCC risk. These included increasing risk of BCC with having fair, easyburning skin (ie decreasing skin phototype), and with having signs of cumulative sun damage to the skin in the form of actinic keratoses. Signs reflecting the combination of pigmentary characteristics and sun exposure - in the form of arm freckling and solar lentigines - also gave subjects a significantly increased risk BCC. Constitutive red-green reflectance of the skin was associated with decreased risk of BCC, as measured by spectrophotometery. Other non-significant trends were seen that may become significant in larger studies including associations of BCC with propensity to burn, moderate tanning ability and an inability to tan. No convincing trend for risk of BCC was seen with the pigmentary variables of hair or eye colour, and a non-significant reduced risk of BCC was associated with increasing numbers of seborrhoeic keratoses. Twenty PTCH exons (exons 2, 3, 5 to 18, and 20 to 23) were screened, accounting for 97% of the coding regions with published mutations in PTCH. Nine of these 20 exons were found to harbour single nucleotide polymorphisms (SNPs), seen on dHPLC as variant melting curves and confirmed on direct sequencing. SNPs frequencies were not significantly different to published population frequencies, or to Australian general population frequencies where SNP database population data was unavailable. Assuming a Poisson distribution, and having observed no mutations in a sample of 56, we can be 97.5% confident that if there are any PTCH mutations contributing to early-onset BCC in the Australian population, then their prevalence is less than 5.1%. Overall, this study provides evidence that familial aggregation of BCC is occurring, that first-degree relatives are at increased risk of all three types of skin cancer, and that a combination of environmental and genetic risk factors are likely to be responsible. The PTCH gene is excluded as a major cause of this increased susceptibility to BCC in particular and skin cancer in general. The weaknesses of the study design are explored, the possible clinical relevance of the data is examined, and future directions for research into the genetics of basal cell carcinoma are discussed.

Sonic hedgehog (Shh) is a secreted morphogen involved in patterning a wide range of structures in the developing embryo. When cells receive the Shh signal a cascade of effects begin which in turn regulate downstream target genes. The genes controlled by Sonic hedgehog provide messages instructing cells how to differentiate or when to divide. Disruption of the hedgehog signalling cascade leads to a number of developmental disorders and plays a key role in the formation of a range of human cancers. Patched, the receptor for Shh, acts as a tumour suppressor and is mutated in naevoid basal cell carcinoma syndrome (NBCCS). NBCCS patients display a susceptibility to tumour formation, particularly for basal cell carcinoma (BCC). The discovery of Patched mutations in sporadic BCCs and other tumour types further highlights the importance of this pathway to human cancer. The identification of genes regulated by hedgehog is crucial for understanding how disruption of this pathway leads to neoplastic transformation. It is assumed that the abnormal expression of such genes plays a large role in directing cells to divide at inappropriate times. Only a small number of genes controlled by Shh have been described in vertebrate tissues. In the work presented in this thesis a Sonic hedgehog responsive embryonic mouse cell line, C3H/10T1/2, was used as a model system for hedgehog target gene discovery. Known downstream target genes were profiled to determine their induction kinetics, building up a body of knowledge on the response to Shh for this cell type. During this work, it was discovered that C3H/10T1/2 cells do not become fully competent to respond to Shh stimulation until the cells reach a critical density, a factor that had to be taken into account when determining timepoints of interest for further investigation. Several techniques were employed to identify genes that show expression changes between Shh stimulated and control cells. In one of these techniques, RNA from cell cultures activated with Shh was used to interrogate cDNA microarrays, and this provided many insights into the downstream transcriptional consequences of hedgehog stimulation. Microarrays consist of thousands of spots of DNA of known sequence gridded onto glass slides. Experiments using this technology allow the expression level of thousands of genes to be measured simultaneously. Independent stimulation methods combined with northern blotting were used to investigate individual genes of interest, allowing genuine targets to be confirmed and false positives eliminated. This resulted in the identification of eleven target genes. Seven of these are induced by Sonic hedgehog (Thrombomodulin (Thbd), Glucocorticoid induced leucine zipper (Gilz), Brain factor 2 (Bf2), Nuclear receptor subfamily 4, group A, member 1 (Nr4a1), Insulin-like growth factor 2 (Igf2), Peripheral myelin protein 22 (Pmp22), Lim and SH3 Protein 1 (Lasp1)), and four are repressed (Secreted frizzled related proteins 1 and 2 (Sfrp1 and Sfrp2), Macrophage inflammatory protein-1 gamma (Mip-1?), and Anti-mullerian hormone (Amh)). The majority of these represent novel downstream genes not previously reported as targets of Shh. The new target genes have a diverse range of functions, and include transcriptional regulators and molecules known to be involved in regulating cell growth or apoptosis. The corroboration of genes previously implicated in hedgehog signalling, along with the finding of novel targets, demonstrates both the validity and power of the C3H/10T1/2 system for Shh target gene discovery. The identification of novel Sonic hedgehog responsive genes provides candidates whose abnormal expression may be decisive in initiating tumour formation and future studies will investigate their role in development and disease. It is expected that such findings will provide vital clues to the aetiology of various human cancers, and that an understanding of their roles may ultimately provide greater opportunities in the future design of anti-tumour therapies.

<p>This doctoral thesis examines how modernisation affects and is affected by existing local culture and identity. It is about the relation between the social and mental barriers experienced, expressed and manifested in the social culture of local community, and modernisation’s dynamic powers over time. The thesis deals with different time periods from the 1800’s until today with regard to expressions and consequences of modernity. People during the societal transformation of Sweden in the 19th and 20th centuries are culturally depicted from a micro-perspective.</p><p>An overall perspective for the analysis of modernity uses the concepts of basal and variable modernity, borrowed from the historian of ideas Sven-Eric Liedman. The perspective makes possible the separation between on the one hand the structural modernisation within the fields of economy, technology and natural sciences, and on the other hand the cultural modernity manifested in conceptions of the world, politics, existential viewpoints, aesthetic expressions and social culture. Within the first-mentioned fields, where basal modernity dominates, a uniform and cumulative developmental pattern emerges as well as an almost self-propelled continuity toward the next innovation or stage of development. Within the latter fields, however, a non-uniform pattern emerges, where modernisation is constantly the object of alternative interpretations and attitudes. This variable modernity is characterised by a cultural struggle between conflicting ideologies and strategies in relation to ongoing modernisation. Different individuals and groups position themselves between acceptance and resistance, progressiveness and the critique of civilisation, the preservation of traditions and the will to change. In this course of events new affinities and identifications, but also new dissociations and antagonisms are created in local social contexts. Modernity leads both to the obliteration of boundaries and to the emergence of new social and mental boundaries. This process can also lead to existing geographical borders being charged with a new ideological content so their importance is revitalised.</p>

<p>Mutation of the p53 gene appears to be an early event in skin cancer development. The present study is based on morphology and represents a cellular and genetic investigation of p53 alterations in normal human skin and basal cell cancer.</p><p>Using double immunofluorescent labelling, we have demonstrated an increase in thymine dimers and p53 protein expression in the same keratinocytes following ultraviolet radiation. Large inter-individual differences in the kinetics of thymine dimer repair and subsequent epidermal p53 response were evident in both sunscreen-protected and non-protected skin. The formation of thymine dimers and the epidermal p53 response were partially blocked by topical sunscreen. We have optimized a method to analyze the p53 gene in single cells from frozen tissue sections. In chronically sun-exposed skin there exist clusters of p53 immunoreactive keratinocytes (p53 clones) in addition to scattered p53 immunoreactive cells. Laser assisted microdissection was used to retrieve single keratinocytes from immunostained tissue sections, single cells were amplified and the p53 gene was sequenced. We have shown that p53 mutations are prevalent in normal skin. Furthermore, we detected an epidermal p53 clone which had prevailed despite two months of total protection from ultraviolet light. Loss of heterozygosity in the PTCH and p53 loci as well as in the sequenced p53 gene was determined in basal cell cancer from sporadic cases and in patients with Gorlin syndrome. Allelic loss in the PTCH region was prominent in both sporadic and hereditary tumors, while loss of heterozygosity in the p53 locus was rare in both groups. p53 mutations found in the hereditary tumors differed from the typical mutations found in sporadic cases. In addition, we found genetically linked subclones with partially different p53 and/or PTCH genotypes in individual tumors. Our data show that both genes are important in the development of basal cell cancer.</p>

<p>The aim of this thesis was to gain further insight into the biology of normal human skin and basal cell carcinoma (BCC). Morphology in combination with microdissection was used as primary tool for sampling.</p><p>Using the X-chromosome inactivation assay, we found normal human skin to consist of a mosaic of cells, with either the maternal or the paternal X-chromosome inactivated. We believe that each tile is made up of several epidermal proliferative units with identical X-chromosome inactivation patterns. Using the same method, we found BCC to be a monoclonal neoplasm imbedded in polyclonal stroma. However, one tumor displayed clear evidence of being composed of two intermingled monoclonal tumors.</p><p>To better enable molecular analysis of defined cells from tissue sections, we investigated a zinc-based fixative as alternative to neutral-buffered formalin. Zinc-based fixative preserves good quality of genomic DNA, with only slight impairment of morphology. In addition, it partly abrogates the need for antigen retrieval.</p><p>The patched gene is involved in BCC development. We analyzed the distribution of a coding polymorphism (Pro/Leu) at codon 1315 in populations with different skin types. We found a reduced Pro/Pro genotype frequency in populations with lighter pigmentation. This in combination with genotype analyses of patients with multiple BCCs, showed that failure to lose the Pro allele during a shift towards lighter pigmented skin may be associated with an increased risk of developing BCC.</p><p>We compared the expression profile of BCC cells with putative progenitor cells in the basal layer of epidermis. In addition to discovering several unknown genes, we found the Wnt signaling pathway to upregulated. Furthermore, differentiation markers were downregulated together with proteins important for scavenging of oxygen radicals.</p><p>In conclusion, the combination of morphology, microdissection and subsequent molecular applications provided valid information deepening our understanding of normal skin and BCC.</p>

The period between pollination and fertilization, or the progamic phase, is a critical life history stage in seed plants and innovations in this life history stage are hypothesized to have played an important role in the diversification of flowering plants. Over the course of this dissertation research, I investigated programic phase development in Nymphaeales (water lilies), an ancient angiosperm lineage that diverged from the basalmost or next most basal node of the angiosperm phylogenetic tree and that is represented in the oldest angiosperm fossil record. I used field experiments and microscopy to document pollination biology, breeding system, and reproductive developmental traits in two families of Nymphaeales: Cabombaceae (Brasenia, Cabomba) and Hydatellaceae (Trithuria). Nymphaeales exhibits considerable variation in reproductive traits and true carpel closure, wind-pollination, and a primarily selfing breeding system have arisen independently in the lineage. Pollen tube pathway length, timing of stigma receptivity, and pollen tube growth rates are conspicuous traits that have undergone considerable modification in concert with shifts in pollination biology and breeding system. Post-pollination developmental processes in Nymphaeales appear to experience selective pressures similar to those experienced by more derived angiosperms and to evolve in similar ways. Nymphaeales also exhibits traits, such as accelerated pollen tube growth, callosic pollen tube walls, and the formation of callose plugs, that are almost certainly plesiomorphic in angiosperms and may have facilitated modification of carpel structure and progamic phase ontogenies. The finding that pollen tube traits that underlie developmental flexibility were already in place before the divergence of Nymphaeales supports the hypothesis that innovations in male gametophyte development were instrumental in facilitating early angiosperm diversification.

Seit den Anfängen empirisch-neurowissenschaftlicher Forschung gilt Sprachkompetenz zuvorderst als eine Leistung der Hirnrinde (Kortex), jedoch wurden v. a. im Zuge sich verbessernder bildgebender Verfahren aphasische Syndrome auch nach Läsionen subkortikaler Hirnregionen, insbesondere der Basalganglien und des Thalamus nachgewiesen. Diese Strukturen liegen in der Tiefe des Gehirns und kommunizieren über weit gefächerte Faserverbindungen mit dem Kortex. In erster Linie werden den Basalganglien senso-motorische Kontrollfunktionen zugewiesen. Dementsprechend werden diverse Erkrankungen, die durch Störungen physiologischer Bewegungsabläufe gekennzeichnet sind (z. B. Morbus Parkinson, Chorea Huntington), auf Funktionsdefekte dieser Strukturen zurückgeführt. Der Thalamus wird häufig als Relaisstation des Informationsaustauschs zwischen anatomisch entfernten Arealen des Nervensystems aufgefasst. Basalganglien und Thalamus werden jedoch auch darüber hinausgehende Funktionen, z. B. zur Bereitstellung, Aufrechterhaltung und Auslenkung von Aufmerksamkeit bei der Bearbeitung kognitiver Aufgaben zugesprochen. In der vorliegenden Arbeit wurde mit elektrophysiologischen Methoden untersucht, ob auf der Ebene von Thalamus und Basalganglien kognitive Sprachleistungen, spezifisch der syntaktischen und semantischen Verarbeitung nachgewiesen werden können und inwieweit sich eventuell subkortikale von kortikaler Sprachverarbeitung unterscheidet. Die Untersuchung spezieller Sprachfunktionen der Basalganglien und des Thalamus ist im Rahmen der operativen Behandlung bewegungsgestörter Patienten mit der sog. Tiefenhirnstimulation (DBS = engl. Deep Brain Stimulation) möglich. Hierbei werden Patienten mit Morbus Parkinson Stimulationselektroden in den Nucleus subthalamicus (STN) implantiert. Bei Patienten mit generalisierten Dystonien erfolgt die Implantation in den Globus pallidus internus (GPI) und bei Patienten mit essentiellem Tremor in den Nucleus ventralis intermedius (VIM). STN und GPI sind Kernareale der Basalganglien, der VIM ist Teil des motorischen Systems. Nach der Implantation besteht die Möglichkeit, direkt von diesen Elektroden elektroenzephalographische (EEG)-Signale abzuleiten und diese mit simultan abgeleiteten Oberflächen-EEG zu vergleichen. In dieser Arbeit wurden DBS-Patienten aus allen genannten Gruppen in Bezug auf Sprachverständnisleistungen untersucht. Neben der Präsentation korrekter Sätze hörten die Patienten Sätze mit syntaktischen oder semantischen Fehlern. In verschiedenen Studien wurden an der Skalp-Oberfläche EKP-Komponenten (EKP = ereigniskorrelierte Potentiale) beschrieben, welche mit der Verarbeitung solcher Fehler in Verbindung gebracht werden. So verursachen syntaktische Phrasenstrukturverletzungen eine frühe links-anteriore Negativierung (ELAN). Dieser Komponente folgt eine späte Positivierung (P600), die mit Reanalyse und Reparaturmechanismen in Verbindung gebracht wird. Semantische Verletzungen evozieren eine breite Negativierung um 400ms (N400). In den thalamischen Ableitungen wurden zwei zusätzliche syntaktische fehlerbezogene Komponenten gefunden, die (i) ~ 80ms nach der Skalp-ELAN und (ii) ~ 70ms vor der Skalp-P600 auftraten. Bei semantischen Verletzungen wurde im Thalamus ein fehlerbezogenes Potential nachgewiesen, welches weitgehend parallel mit dem am Skalp gefundenen Muster verläuft. Aus den Ergebnissen der vorliegenden Studie folgt, dass der Thalamus spezifische Sprachfunktionen erfüllt. Komponenten, die Sprachverarbeitungsprozesse reflektieren, konnten in den Basalganglienstrukturen STN und GPI nicht identifiziert werden. Aufgrund der erhobenen Daten werden zwei getrennte Netzwerke für die Verarbeitung syntaktischer bzw. semantischer Fehler angenommen. In diesen Netzwerken scheint der Thalamus spezifische Aufgaben zu übernehmen. In einem ‚Syntaxnetzwerk’ kommunizieren frontale Hirnstrukturen unter Einbeziehung des Thalamus mit parietalen Hirnstrukturen. Dem Thalamus wurde eine Mediationsfunktion in der syntaktischen Reanalyse zugesprochen. In einem ‚Semantiknetzwerk’ waren keine eindeutig zuordenbaren Prozesse auf thalamischer Ebene nachweisbar. Es wurde eine unscharfe, jedoch aber spezifische Aktivierung des Thalamus über den gesamten Zeitraum der kortikalen semantischen Analyse gezeigt, welche als Integration verschiedener Analysemechanismen gewertet wurde. / Since the beginning of empirical neuroscientific research language competence has been primary localized at the brain cortex. Improved functional neuroimaging techniques were able to localize lesions in structures which caused aphasic syndromes. These syndromes were particularly found after lesions of the basal ganglia and the thalamus These structures are located in the depth of the brain and communicate over widespread fiber connections with the cortex. Sensori-motor control functions are primarily assigned to the basal ganglia. Various diseases, which are characterized by disturbances of physiological courses of motion (e.g. Parkinson’s disease, Chorea Huntington) are attributed to function defects of these structures. The thalamus was originally understood as a simple relay station of information exchange between various cortical areas of the nervous system. However, additional functions were assigned to the basal ganglia and thalamus, e.g. maintenance and deflection of attention while processing of cognitive tasks. Within the present investigation thalamic and basal ganglia achievements in language processing were studied with electro-physiological methods to investigate differences between cortical and subcortical processes. The investigation of special language functions of the basal ganglia and the thalamus is possible in the context of the surgical treatment of movement-disordered patients with "Deep Brain Stimulation (DBS)". Stimulation electrodes have been implanted to patients with Parkinson’s disease into the subthalamic nucleus (STN), patients with generalized dystonia into the globus pallidus internus (GPi), and patients suffering from essential tremor into the ventral intermediate nucleus of the thalamus (VIM). STN and GPi are core areas of the basal ganglia, the VIM is part of the motor system. EEG-signals may be derived directly from these implanted electrodes and be compared to the simultaneously derived signals of a surface EEG. In this thesis DBS patients from all groups mentioned above were examined regarding language understanding achievements. The patients listened to sentences, which were either correct or syntactical or semantical incorrect. Different studies described scalp ERP components (ERP = event related potentials) which occurred after different types of errors in sentences. Thus, syntactic phrase structure violations cause an early left anterior negativity (ELAN). A late positivity (P600) follows this component and was hypothesized as a reflection of syntactical reanalysis and/or repair. Semantic violations evoke a broad negativity around 400ms (N400). In the thalamic EEG two additional syntactic components were identified, which were seen (i) ~ 80ms after the scalp ELAN and (ii) ~ 70ms before the scalp-P600. At thalamic level semantic violations caused a negativity, which parallels largely with the negativity found at the scalp. The results of this study suggest, that the thalamus fulfills specific language functions. In the basal ganglia structures (GPI and STN) no language specific components were found. Due to the collected data two separate networks are suggested for the processing of syntactic and/or semantic errors. In these networks the thalamus seems to fulfill specific tasks. In a "syntax network” frontal brain structures communicate with parietale brain structures via the thalamus. The function of the thalamus in this network is the mediation of the syntactic reanalysis. In a "semantic network” no clearly classified processes were proved at thalamic level, because of a similarity of thalamic and scalp signals. However, during the entire period of the cortical analysis activation of the thalamus was apparent. This activation was rated as integration of different analysis mechanisms.

Mutation of the p53 gene appears to be an early event in skin cancer development. The present study is based on morphology and represents a cellular and genetic investigation of p53 alterations in normal human skin and basal cell cancer. Using double immunofluorescent labelling, we have demonstrated an increase in thymine dimers and p53 protein expression in the same keratinocytes following ultraviolet radiation. Large inter-individual differences in the kinetics of thymine dimer repair and subsequent epidermal p53 response were evident in both sunscreen-protected and non-protected skin. The formation of thymine dimers and the epidermal p53 response were partially blocked by topical sunscreen. We have optimized a method to analyze the p53 gene in single cells from frozen tissue sections. In chronically sun-exposed skin there exist clusters of p53 immunoreactive keratinocytes (p53 clones) in addition to scattered p53 immunoreactive cells. Laser assisted microdissection was used to retrieve single keratinocytes from immunostained tissue sections, single cells were amplified and the p53 gene was sequenced. We have shown that p53 mutations are prevalent in normal skin. Furthermore, we detected an epidermal p53 clone which had prevailed despite two months of total protection from ultraviolet light. Loss of heterozygosity in the PTCH and p53 loci as well as in the sequenced p53 gene was determined in basal cell cancer from sporadic cases and in patients with Gorlin syndrome. Allelic loss in the PTCH region was prominent in both sporadic and hereditary tumors, while loss of heterozygosity in the p53 locus was rare in both groups. p53 mutations found in the hereditary tumors differed from the typical mutations found in sporadic cases. In addition, we found genetically linked subclones with partially different p53 and/or PTCH genotypes in individual tumors. Our data show that both genes are important in the development of basal cell cancer.

The aim of this thesis was to gain further insight into the biology of normal human skin and basal cell carcinoma (BCC). Morphology in combination with microdissection was used as primary tool for sampling. Using the X-chromosome inactivation assay, we found normal human skin to consist of a mosaic of cells, with either the maternal or the paternal X-chromosome inactivated. We believe that each tile is made up of several epidermal proliferative units with identical X-chromosome inactivation patterns. Using the same method, we found BCC to be a monoclonal neoplasm imbedded in polyclonal stroma. However, one tumor displayed clear evidence of being composed of two intermingled monoclonal tumors. To better enable molecular analysis of defined cells from tissue sections, we investigated a zinc-based fixative as alternative to neutral-buffered formalin. Zinc-based fixative preserves good quality of genomic DNA, with only slight impairment of morphology. In addition, it partly abrogates the need for antigen retrieval. The patched gene is involved in BCC development. We analyzed the distribution of a coding polymorphism (Pro/Leu) at codon 1315 in populations with different skin types. We found a reduced Pro/Pro genotype frequency in populations with lighter pigmentation. This in combination with genotype analyses of patients with multiple BCCs, showed that failure to lose the Pro allele during a shift towards lighter pigmented skin may be associated with an increased risk of developing BCC. We compared the expression profile of BCC cells with putative progenitor cells in the basal layer of epidermis. In addition to discovering several unknown genes, we found the Wnt signaling pathway to upregulated. Furthermore, differentiation markers were downregulated together with proteins important for scavenging of oxygen radicals. In conclusion, the combination of morphology, microdissection and subsequent molecular applications provided valid information deepening our understanding of normal skin and BCC.