In Australia, up to 80% of individuals diagnosed with childhood cancer are now expected to survive for more than five years after their initial diagnosis. However, survivors of childhood cancer are at risk of developing late sequelae as a consequence of therapies received during childhood. The aim of this study was to determine the incidence of selected late sequelae in a cohort of Australian childhood cancer survivors and identify treatment and genetic factors that may modify the risk of late sequelae in survivors. Our study included 1150 individuals treated for childhood cancer at the Sydney Children??s Hospital between 1962 and 1999, who had remained in remission >3 years and were confirmed to be alive. Rates of mortality and second cancers among survivors were compared against population rates to determine standardised mortality and incidence ratios. Survivors completed a questionnaire on the incidence of adverse health conditions and provided a buccal specimen. Real time PCR was used to detect polymorphisms in genes involved in drug detoxification and transport. Rates of mortality and secondary cancers were found to be 7.5-fold (95%CI 5.4-10.1) and 4.9-fold (95%CI 2.9-8.0) higher among survivors of childhood cancer relative to the general population, respectively, with the highest risks observed for those survivors previously treated for Hodgkin??s disease. Over 60% of survivors reported at least one cardiopulmonary, endocrine or sensory-motor condition following diagnosis of childhood cancer; the most frequently observed conditions included growth hormone deficiency, hypothyroidism, and hypertension. Late sequelae were most frequently reported by females and survivors of brain tumours. Genetic investigations showed that an increased risk of growth hormone deficiency was associated with homozygosity for the GSTM1 null polymorphism, while no gene associations were observed to influence the risk of second cancers among survivors. Our study demonstrates that survivors of childhood cancer are at risk of developing a variety of health conditions as a result of anti-cancer therapies received during childhood. Determining risk factors for late sequelae based on therapy type, lifestyle and genetic predisposition will enable the optimisation of treatment protocols and promote the future well-being of childhood cancer survivors.

Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X7 receptor. These responses include opening of a cation selective channel/pore which allows entry of the fluorescent dye, ethidium+ and activation of a membrane metalloprotease which sheds the adhesion molecule L-selectin. In this thesis, the surface expression of P2X7 receptors was measured in normal leucocytes, platelets and B-CLL lymphocytes and compared with their functional responses. Monocytes showed 4-5 fold greater expression of P2X7 than B-, T- and NK- lymphocytes, while P2X7 expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed surface P2X7 at about the same level as for B-lymphocytes from normal subjects. P2X7 function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes. However, the ATP-induced uptake of ethidium into the malignant B-lymphocytes in 3 patients was low or absent. The lack of P2X7 function in these B-lymphocytes was confirmed by the failure of ATP to induce Ba2+ uptake into their lymphocytes. This lack of function of the P2X7 receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule which directs the recirculation of lymphocytes from blood into the lymph node. To study a possible genetic basis of non-functional P2X7 receptor, we sequenced DNA coding for the carboxyl terminal tail of P2X7. In 33 of 130 normal subjects a heterozygous nucleotide substitution (1513A--C) was found while 3 subject carried the homozygous substitution which codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X7 on lymphocytes was not affected by this 496Glu--Ala polymorphism demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the 496Glu--Ala homozygote subject expressed non-functional receptor while heterozygotes showed P2X7 function which was half that of wild type P2X7. Results of transfection experiments showed the mutant P2X7 receptor was non-functional when expressed at low receptor density but regained function at a high receptor density. This density-dependence of mutant P2X7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma which upregulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with wild type. The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7 receptor. Apart from the 496Glu--Ala polymorphism, three other single nucleotide polymorphisms, 155His--Tyr, 348Ala--Thr and 568Ile--Asn were also found in the P2X7 receptor. The site directed mutant cDNA were generated for all 3 polymorphisms and transfected into HEK293 cells to study the impact of these polymorphisms on P2X7 function. Results suggested that Ile568 is important for P2X7 protein trafficking to cell surface. Further study of these two loss-of-function polymorphisms (496Glu--Ala and 568Ile--Asn) may help better understanding of the functional domains in the P2X7 receptor and its role in CLL, lymphoma and infectious diseases. Conclusions: 1.P2X7 receptor is expressed in human leukocytes, including lymphocytes, natural killer cells as well as monocytes, on both surface and intracellular locations. 2.Both the expression and function of P2X7 are highly variable between in human individuals. Non-functional P2X7 receptors are found in some subjects, including both normal subjects and CLL patients, and are often associated with defects in ATP-induced cytotoxicity and L-selectin shedding. 3.Two single nucleotide polymorphisms (SNPs), 496Glu--Ala and 568Ile--Asn, are found at low frequency in the human population and lead to the loss-of-function of P2X7. Both permeabllity function and the downstream effects mediated by P2X7 are affected by these two SNPs. The mechanisms for the loss-of-function differs between the two polymorphisms.

[Truncated abstract] For the convenience of the reader, detailed figures and tables have been enlarged and compiled in Appendix 2, at the end of this thesis. This thesis is presented as an approach to identify, annotate and detect genomic duplication and polymorphism within large genomic regions. To demonstrate this, I have used as a model, the genomic region known as the Regulators of Complement Activation (RCA). The RCA complex is located on the long arm of chromosome 1 at position 1q32 and is a reservoir of complement regulatory proteins. The genes of the RCA share many similarities implying that all have arisen through multiple complex duplication events. My analysis of this region in the following chapters demonstrates the complexity of this duplication and identifies the many functional units within the RCA. It was my aim at the beginning of these studies to demonstrate an approach that could define the Ancestral Haplotypes (AHs) of the RCA gene cluster. To do this, extensive genomic analysis was required and the ever-increasing availability of genomic sequence has made this thesis possible. Each of the chapters serves to address the following aims set out at the beginning of this thesis: 1. Further characterise the relationship between the genes (Complement Control proteins-CCPs) and domains of the Regulators of Complement Activation (RCA). 2. Identify and examine the duplicated elements within the RCA. - 6 - 3. Examine the effects of retroviruses and other insertions and deletions (indels) in generating the divergence of duplicated genes. 4. Investigate the applicability of the Genomic Matching Technique (GMT) to define AH within the region. 5. Examine association of AHs with CCP implicated diseases. 6. Determine the GMT applicability in non-human species

The specificity of fungal isolates from endemic Western Australian orchid species and hybrids in the tribe Diurideae was investigated using symbiotic seed germination and analysis of the fungal DNA by amplified fragment length polymorphism (AFLP). The distribution of the fungal isolates in the field was also assessed using two different seed baiting techniques. The information from these investigations is essential for developing protocols for reintroduction and translocation of orchid species. Two groups of orchids in the tribe Diurideae were studied. Firstly, a number of Caladenia species, their natural hybrids and close relatives from the southwest of Western Australia were selected because orchid species from the genus Caladenia are considered to have among the most specific mycorrhizal relationships known in the orchid family – an ideal situation for the investigation of mycorrhizal specificity. Secondly, species of Drakaea and close relatives, from the southwest of Western Australia and elsewhere in Australia, which are never common in nature and occur in highly specialised habitats, were selected to investigate the influence of habitat on specificity. Seed from the common species Caladenia arenicola germinated on fungal isolates from adult plants of both C. arenicola and its rare and endangered relative C. huegelii, while seed from C. huegelii only germinated on its own fungal isolates. The AFLP analysis grouped the fungal isolates into three categories: nonefficaceous fungi, C. huegelii type fungi, and C. arenicola type fungi. The group of C. huegelii type fungi included some fungal isolates from C. arenicola. An analysis of the AFLP fingerprints of C. arenicola fungal isolates from different collection locations showed that some, but not all, populations were genetically distinct, and that one population in particular was very variable. Despite being thought to have very specific mycorrhizal relationships, Caladenia species hybridise frequently and prolifically in nature, often forming self-perpetuating hybrid lineages. Five natural hybrids within Caladenia and its closest relatives were investigated. Symbiotic cross-germination studies of parental and hybrid seed on fungi from the species and the naturally occurring hybrids were compared with AFLP analyses of the fungal isolates to answer the question of which fungi the hybrids use. The germination study found that, while hybrid seeds can utilise the fungi from either parental species under laboratory conditions, it is likely that the natural hybrids in situ utilise the fungus of only one parental species. Supporting these observations, the AFLP analyses indicated that while the parental species always possessed genetically distinct fungal strains, the hybrids may share the mycorrhizal fungus of one parental species or possess a genetically distinct fungal strain which is more closely related to the fungus of one parental species than the other. The work on Caladenia hybrids revealed that C. falcata has a broadly compatible fungus that germinated seeds of C. falcata, the hybrid C. falcata x longicauda, and species with different degrees of taxonomic affinity to C. falcata. In general, germination was greater from species that were more closely related to C. falcata: seeds from Caladenia species generally germinated well on most C. falcata isolates; species from same subtribe (Caladeniinae) germinated well to the stage of trichome development on only some of the fungal isolates and rarely developed further; and seeds from species from different subtribes (Diuridinae, Prasophyllinae, Thelymitrinae) or tribes (Orchideae, Cranichideae) either germinated well to the stage of trichome development but did not develop further, or did not germinate at all. The AFLP analysis of the fungal isolates revealed that the fungi from each location were genetically distinct. In situ seed baiting was used to study the introduction, growth and persistence of orchid mycorrhizal fungi. A mycorrhizal fungus from Caladenia arenicola was introduced to sites within an area from which the orchid and fungus were absent, adjacent to a natural population of C. arenicola. In the first growing season, the fungus grew up to 50 cm from its introduction point, usually persisted over the summer drought into the second season and even into the third season, stimulating germination and growth to tuber formation of the seeds in the baits. Watering the inoculated areas significantly increased seed germination. Mycorrhizal relationships in Drakaeinae were less specific than in Caladeniinae. A study of the species Spiculaea ciliata revealed that this species, when germinated symbiotically, develops very rapidly and has photosynthetic protocorms, unlike all other members of the Drakaeinae. An AFLP analysis of the fungal isolates of this species grouped the isolates according to whether they had been isolated from adult plants or reisolated from protocorms produced in vitro. Isolates were genetically distinct when compared before germination and after reisolation. A cross-species symbiotic germination study of seeds of three Drakaea species and one Paracaleana species against fungal isolates from the same species and several other Drakaeinae species revealed lower specificity in this group than previously thought. A number of fungal isolates from Drakaea and Paracaleana species germinated two or more seed types, while all seed types germinated on fungal isolates from other species and the seed of Drakaea thynniphila germinated to some extent on every fungal isolate tested. An AFLP analysis of the Drakaeinae fungal isolates supported this information, revealing little genetic differentiation between the fungi of different orchid species. An ex situ seed baiting technique was used to examine the role of mycorrhizal fungi in microniche specialisation in the narrow endemic Drakaea. Soil samples from within and outside two Drakaea populations were tested for germination of the relevant seed types. In both cases, germination was significantly higher on soil samples from within than outside the populations, suggesting that the relevant mycorrhizal fungi may be restricted to the same microniches as the Drakaea species. The presence of similar fungi at distant, disjunct locations may be related to the extreme age and geological stabilityof the Western Australian landscape. The information from these investigations is essential for developing protocols for reintroduction and translocation of orchid species. It appears that the mycorrhizal relationships in these groups of orchids are not as specific as was previously thought. For reintroduction work, a broad sampling strategy is necessary, as it cannot be assumed that the same orchid species has the same fungus at different locations. A broadly compatible fungus may be of considerable utility in conservation work, such as in situations where a specific fungus appears to have poor saprophytic competence or where soil conditions have been altered. Seed baiting studies provide additional data on fungal distribution in situ. In general, molecular data do not provide information about efficacy or fungal distribution, so research programs that combine symbiotic germination studies with seed baiting investigations and genetic analyses of the fungi will provide the maximum benefit for designing more effective conservation programs.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.