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

Arthritis is a debilitating disease of the joints caused by the accelerated breakdown of cartilage, resulting in painful, swollen joints. Cartilage protects the joint by absorbing the shock that would otherwise be transferred directly to the underlying bone. One crucial component of cartilage is a specialised molecule known as aggrecan. Aggrecan consists of a core protein with three globular domains (G1, G2 and G3) and is modified with over one hundred highly sulphated glycosaminoglycan chains. Two types of glycosaminoglycans are substituted along the length of the protein, chondroitin sulphate and keratan sulphate. The glycosaminoglycans impart a highly negative charge to the tissue, giving it the ability to retain water and resist compressive forces. / Aggrecan is lost from cartilage following cleavage by aggrecanases. Too little aggrecan in cartilage destabilises the structural integrity of the tissue and is associated with arthritis. Of the five known aggrecanase cleavage sites, it is cleavage within the interglobular domain (IGD) between the G1 and G2 domains at NITEGE373 - 374ARGSVI that directly contributes to loss of aggrecan function. / The chondroitin sulphate and keratan sulphate located between the G2 and G3 domains is responsible for maintaining the biomechanical properties of aggrecan. The role of keratan sulphate within the G1-G2 domain is unknown, but it is not thought to be essential for aggrecan function. However the literature suggests a possible role of keratan sulphate in facilitating aggrecanase cleavage of NITEGE373 - 374ARGSVI in the IGD. The aim of my project was to examine the role of keratan sulphate in aggrecanase-mediated cleavage of aggrecan in the IGD. Three major goals have been accomplished in this thesis: 1) Identification of a cell type capable of sustained keratan sulphate synthesis. 2) Expression of a recombinant G1-G2 protein substituted with keratan sulphate (rG1-G2). 3) Demonstration that endogenous N-linked keratan sulphate is sufficient to potentiate aggrecanase cleavage of rG1-G2 in the IGD. / Cultured cells do not synthesise keratan sulphate. Therefore identifying a cell type, and culture conditions to maximise keratan sulphate synthesis, was a major undertaking. Conditions were identified which allowed for maximal keratan sulphate synthesis, albeit on a small scale, in primary bovine keratocytes. Using a Vaccinia virus expression system, recombinant G1-G2 was expressed in primary bovine keratocytes. / Analysis of the rG1-G2 revealed that it was substituted with 5 kDa of keratan sulphate. One important aspect of the study was that the keratan sulphate was all N-linked to the core protein. Subsequent aggrecanase digests, comparing substrates before and after removal of keratan sulphate, showed that aggrecanase cleavage was markedly more efficient when keratan sulphate was present. The results contained in this thesis add significantly to the established literature by providing a greater understanding of the mechanisms involved in aggrecanase-mediated cleavage of aggrecan and cartilage destruction. The results suggest that aggrecan substitution with N-linked keratan sulphate potentiates aggrecanase activity. The results from this study identify N-linked keratan sulphate as a possible target for the development of new drugs for the management of arthritis.

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

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 3 uppsatser.

Cellular checkpoints are typically considered to both facilitate the ordered execution of the cell cycle and to act as a barrier to oncogene driven cell cycles and the transmission of unresolved genetic lesions from one phase to the next. Furthermore, these mechanisms are also believed to underpin the responses of cells, both in normal and cancerous tissues, to those therapies that either directly or indirectly generate DNA damage. In recent studies however, it has become clear these checkpoints permit the passage of significant genomic aberrations into subsequent cell cycle phases and even descendant cells, and that heterogeneous responses are apparent amongst genetically identical cells. The consequences of this checkpoint ‘negligence’ remain relatively uncharacterised despite the importance of checkpoints in current models for how genomic instability is avoided in the face of ubiquitous DNA damage. Unresolved DNA damage is presumably inherited by subsequent cell cycle phases and descendant cells yet characterisation of the consequences of this has been relatively limited to date. I therefore utilised microscopy-based lineage tracing of cells expressing genetically encoded fluorescent sensors, particularly the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) probes (Sakaue-Sawano et al., 2008), with semi-automated image analysis to characterise the response of single cells and their descendants to DNA lesions across multiple cell cycle generations. This approach, complemented by generational tracing by flow cytometry, permitted me to characterise the timing of cell fate determination in treated and descendant cells, the non-genetic heterogeneity in checkpoint responses and overall lineage behaviour, correlations between cells (similarly to Sandler et al., 2015) and cell cycle timing dependencies in the response to DNA damaging agents. With these single cell analytical approaches I show that the consequences of DNA damage on descendant cell fate is dramatic, suggesting checkpoint mechanisms may have consequences and even cooperate across phases and generations. U2OS cell lineages traced for three generations following the induction of DNA damage in the form of strand breaks showed greatly induced cell death in the daughters and granddaughters of DNA damaged cells, termed delayed death. Furthermore, lineage behaviour was characterised as highly heterogeneous in when and whether cell death occurred. Complementary flow cytometric approaches validated the findings in U2OS cells and suggested HeLa cells may show similar behaviour. These findings indicate that checkpoint models need to incorporate multigenerational behaviour in order to better describe the response of cells to DNA damage. Understanding the processes governing cell fate determination in descendant cells will impact upon our understanding of the development of genomic instability during carcinogenesis and how DNA-damaging chemotherapeutics drive cells to ‘quit’ the cell cycle.

This dissertation is a contribution to the ethnography of contemporary indigenous reindeer pastoralism in Norway: specifically, to the study of the neglected fields of reindeer killing and slaughtering practice. Its central contention is that in recent decades, the proliferation of human powers vested in the conduct of reindeer slaughter has created new conditions for practice, placing the identities of reindeer and herders at stake in new and still only dimly conceptualized ways. By exploring these, the dissertation aims to broaden existing debates concerning the so-called modernization of pastoral practice in Norway, drawing attention to some of its neglected aspects and inscribing them in a new register. Two principal strands inform the theoretical framework: one, approaches to the social study of knowledge that emphasise its practical, non-verbal and material aspects; and two, Foucauldian concepts of biopower as these may – or may not – be applicable to the human management of animal life.Individual chapters examine, in turn: the local politics of space on the Varanger peninsula, focusing particularly on links between the spatial management and the killing of reindeer; the practices and social relations of slaughter as it is conducted at the round-up corral; the social effects of the introduction of slaughterhouses, and of the regime of which they form a part; controversies surrounding specific slaughtering techniques and instruments, particularly the curved knife; and the politics of animal welfare discourse and practices in their application to reindeer herding. Finally, using the figure of animal sacrifice as a guiding trope, the concluding chapter attempts to situate some key aspects of the modernization of reindeer slaughter in relation to the operation of broader sacrificial economies that regulate the destruction of life at aggregate or populational levels. / Research Council of Norway

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

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