Alzheimer’s disease (AD) is the most prevalent form of dementia. There is considerable evidence that AD is caused by accumulating amyloid beta peptides in the brain, as a result of amyloid precursor protein (APP) cleavage by secretase enzymes. The presenilin proteins are central to the gamma-secretase cleavage of the intramembrane domain of APP. Aberrant splicing and point mutations in the human presenilin genes, PSEN1 and PSEN2, have been linked to familial forms of AD, through aberrant APP cleavage resulting in irregular amyloid beta formation. Paper 1 gives a review of the literature on AD research and how animal models are used to elucidate mechanisms of AD pathogenesis. The zebrafish model is used in this thesis to investigate genes with potential relevance to AD initiation and pathogenesis. Paper 2 demonstrates that lowlevel aberrant splicing of exon 8 in psen1 transcripts in zebrafish embryos produces potent dominant negative effects that increased psen1 transcription, cause a dramatic hydrocephalus phenotype, decreased pigmentation and other developmental defects. Similar effects are also observed after low-level interference with splicing of exon 8 in psen2 transcripts. In paper 3, a microarray analysis was performed to analyse global gene expression changes to illuminate the molecular aetiology of the phenotypic effects described in paper 2. Of the 100 genes that showed greatest dysregulation after psen1 or psen2 manipulation, 12 genes were common to both treatments. Five of these have known function and showed increased expression. Cyclin G1 (ccng1) was of particular interest as the human CCNG1 protein shows increased immunoreactivity in the cytoplasm of neurons in human AD brains. Phylogenetic and conserved synteny analysis confirmed the orthology of zebrafish ccng1 with human CCNG1. Expression of zebrafish ccng1 in developing embryos at 24 hours post fertilization (hpf) was observed in the eye, tectum and somites. Decreased Ccng1 expression does not lead to any developmental defects and also cannot rescue the hydrocephalus or pigmentation phenotypes of embryos with aberrant splicing of psen1 exon 8. An analysis of zebrafish ccng1 function in paper 4 (thesis chapter in the form of a manuscript) indicates that truncation of Ccng1 appears to cause developmental defects in the brain, notochord and somites, however, it does not decrease the level of normal ccng1 transcript. The CCNG1 paralogue, Cyclin G2, (CCNG2), is also expressed in zebrafiish (ccng2). Decreasing the expression of Ccng2 results in similar effects on embryo development as truncating Ccng1. Therefore, the truncated forms of Ccng1 potentially interfere with Ccng2 function in a dominant negative manner. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342482 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Alzheimer’s disease (AD) is the most prevalent form of dementia. There is considerable evidence that AD is caused by accumulating amyloid beta peptides in the brain, as a result of amyloid precursor protein (APP) cleavage by secretase enzymes. The presenilin proteins are central to the gamma-secretase cleavage of the intramembrane domain of APP. Aberrant splicing and point mutations in the human presenilin genes, PSEN1 and PSEN2, have been linked to familial forms of AD, through aberrant APP cleavage resulting in irregular amyloid beta formation. Paper 1 gives a review of the literature on AD research and how animal models are used to elucidate mechanisms of AD pathogenesis. The zebrafish model is used in this thesis to investigate genes with potential relevance to AD initiation and pathogenesis. Paper 2 demonstrates that lowlevel aberrant splicing of exon 8 in psen1 transcripts in zebrafish embryos produces potent dominant negative effects that increased psen1 transcription, cause a dramatic hydrocephalus phenotype, decreased pigmentation and other developmental defects. Similar effects are also observed after low-level interference with splicing of exon 8 in psen2 transcripts. In paper 3, a microarray analysis was performed to analyse global gene expression changes to illuminate the molecular aetiology of the phenotypic effects described in paper 2. Of the 100 genes that showed greatest dysregulation after psen1 or psen2 manipulation, 12 genes were common to both treatments. Five of these have known function and showed increased expression. Cyclin G1 (ccng1) was of particular interest as the human CCNG1 protein shows increased immunoreactivity in the cytoplasm of neurons in human AD brains. Phylogenetic and conserved synteny analysis confirmed the orthology of zebrafish ccng1 with human CCNG1. Expression of zebrafish ccng1 in developing embryos at 24 hours post fertilization (hpf) was observed in the eye, tectum and somites. Decreased Ccng1 expression does not lead to any developmental defects and also cannot rescue the hydrocephalus or pigmentation phenotypes of embryos with aberrant splicing of psen1 exon 8. An analysis of zebrafish ccng1 function in paper 4 (thesis chapter in the form of a manuscript) indicates that truncation of Ccng1 appears to cause developmental defects in the brain, notochord and somites, however, it does not decrease the level of normal ccng1 transcript. The CCNG1 paralogue, Cyclin G2, (CCNG2), is also expressed in zebrafiish (ccng2). Decreasing the expression of Ccng2 results in similar effects on embryo development as truncating Ccng1. Therefore, the truncated forms of Ccng1 potentially interfere with Ccng2 function in a dominant negative manner. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342482 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Several cell cycle events require specific forms of the cyclin-CDK complexes. It has been known for some time that cyclins not only contribute by activating the CDK but also by choosing substrates and/or specifying the location of the CDK holoenzyme. There are several examples of B-type cyclins identifying certain peptide motifs in their specific substrates through a conserved region in their structure. Such interactions were not known for the G1 class of cyclins, which are instrumental in helping the cell decide whether or not to commit to a new cell cycle, a function that is non-redundant with B-type cylins in budding yeast. In this dissertation, I have presented evidence that some G1 cyclins in budding yeast, Cln1/2, specifically identify substrates by interacting with a leucine-proline rich sequence different from the ones used by B-type cyclins. These “LP” type docking motifs determine cyclin specificity, promote phosphorylation of suboptimal CDK sites and multi-site phosphorylation of substrates both in vivo and in vitro. Subsequently, we have discovered the substrate-binding region in Cln2 and further showed that this region is highly conserved amongst a variety of fungal G1 cyclins from budding yeasts to molds and mushrooms, thus suggesting a conserved function across fungal evolution. Interestingly, this region is close to but not same as the one implicated in B-type cyclins to binding substrates. We discovered that the main effect of obliterating this interaction is to delay cell cycle entry in budding yeast, such that cells begin DNA replication and budding only at a larger than normal cell size, possibly resulting from incomplete multi-site phosphorylation of several key substrates. The docking-deficient Cln2 was also defective in promoting polarized bud morphogenesis. Quite interestingly, we found that a CDK inhibitor, Far1, could regulate the Cln2-CDK1 activity partly by inhibiting the Cln2-substrate interaction, thus demonstrating that docking interactions can be targets of regulation. Finally, by studying many fungal cyclins exogenously expressed in budding yeast, we discovered that some have the ability to make the CDK hyper-potent, which suggests that these cyclins confer special properties to the CDK. My work provides mechanistic clues for cyclinspecific events during the cell cycle, demonstrates the usefulness of synthetic strategies in problem solving and also possibly resolves long-standing uncertainties regarding functions of some cell cycle proteins.

Introdução: O Câncer de próstata metastático (mCaP) é uma doença incurável com progressão para o mCaP resistente à castração (mCPRC) após terapia de deprivação androgênica. Os microRNAs (miR) -23b e -27b tem ação antioncogênica e são suprimidos neste contexto. O gene da ciclina G1 (CCNG1) codifica uma quinase dependente de ciclina com potencial de inibição do crescimento e é um dos alvos dos miR-23b/-27b. Objetivos: Estimular os miR-23b/-27b isoladamente e em conjunto para avaliar e comparar o crescimento tumoral e a expressão do gene alvo CCNG1 em relação ao grupo controle em xenenxertos de PC-3M-luc-C6 em camundongos atímicos castrados. Métodos: Xenoenxertos subcutâneos da linhagem celular PC-3M-luc-C6 foram implantados em camundongos machos BALB/c nude. Os animais foram castrados 10 dias após o implante e utilizamos injeções intratumorais para induzir o aumento da expressão dos miR-23b/-27b separadamente e em conjunto através de Pre-miR® específicos. Realizamos avaliações semanais da bioluminescência (BLI) para avaliar o crescimento tumoral após a castração. Utilizamos a reação em cadeia de polimerase reversa em tempo real (qRT-PCR) para analisar a expressão da CCNG1 e os animais foram sacrificados 21 dias após a castração. Dividimos um total de 21 xenoenxertos nos seguintes grupos de tratamento: 4 no grupo controle, 5 no grupo Pró miR-23b, 6 no grupo Pró miR-27 e 6 no grupo Pró miR-23b associado ao Pró miR-27b. Resultados: Confirmamos o sucesso da transfecção dos miRs por qRT-PCR, e apresentamos o achado de superexpressão relativa da CCNG1 em relação ao grupo controle em: 9% (p=0,76), 46% (p=0,05) e 203% (p=0,01) nos grupos Pró miR-23b, Pró miR-27b e Pró miR-23b associado ao Pró miR-27b respectivamente. Comparamos o crescimento proporcional de cada tumor através da BLI, por meio da leitura no momento da castração ao final do experimento. Obtivemos um crescimento de 13,5; 8,69; 5,96 e 9,98 vezes nos grupos: controle, Pró miR-23b, Pró miR-27b e Pró miR-23b associado ao Pró miR-27b respectivamente. Conclusão: Demonstramos um modelo in vivo de CPRC que apresentou supreexpressão da CCNG1 após o tratamento intratumoral que aumentou a expressão dos miRs -23b e -27b. Este conjunto de miRs tem ação antioncogênica descrita no contexto do mCPRC e a sua estimulação neste contexto aumentou a expressão da CCNG1. Nosso estudo sugere que a CCNG1 deve apresentar uma ação pró-apoptótica quando superexpresso pelos miRs-23b/-27 no CPRC / Introduction: Metastatic prostate cancer (mPCa) is an incurable disease that invariably progresses to castration-resistant mPCa (mCRPC) after androgen deprivation therapy. The microRNAs miR-23b/-27b have been reported as tumor suppressors and are underexpressed in this context. The cyclin G1 gene (CCNG1) encodes a cyclin-dependent kinase with potential growth inhibitory activity that is a potential target of miR-23b/-27b. Objectives: We aim to explore a bioluminescent xenograft model of CRPC in castrated mice the effect positive modulation of the miR-23b/-27b on CCNG1 expression and mCRPC growth. Material and Methods: We injected subcutaneous xenografts of PC-3M-luc-C6 PCa cell line in BALB/c nude male mice. We neutered the animals after 10 days and used intratumoral injections up-regulating miR-23b/-27b separately and simultaneously through specific Pre-miRTM. We used weekly bioluminescence imaging (BLI) to assess tumor growth after castration and real-time polymerase chain reaction (qRT-PCR) to analyze the expression of CCNG1. We sacrificed the animals 21 days after castration. We randomized 21 xenografts in experimental groups as follows: n=4 in the negative control group; n=5 in Pro miR-23b group; n=6 in Pro miR-27b group and an n=6 tumors in the Pro miR-23b plus Pro miR-27b. Results: We confirmed successful transfection of both miRNAs with overexpression of CCNG1 of 9% (p=0.76), 46% (p=0.05) and 203% (p=0.01) in the Pro miR-23b, Pro miR-27b and Pro miR-23b plus -27b groups respectively. We compared the fold-change in BLI growth by the end of experiment finding an increase of 13.5-fold, 8.69-fold, 5.96-fold and 9.98-fold in groups Pro miR-negative control, Pro miR-23b, Pro miR-27b and Pro miR-23b plus Pro miR-27b groups respectively. Conclusions: We showed an in vivo model with overexpression of CCNG1 upon artificial upregulation of miR-23b and -27b in CRPC. This cluster of antineoplastic miRNA increased the expression of this cyclin, often described as oncogenic. Our study suggests that CCNG1 has a pro-apoptotic role when up-regulated by miR-23b/-27b in CPRC