This research studied recombinant DNA-derived protein expression utilising expression vectors containing IRES sequences to link the gene of interest with the gene encoding selectable marker in mammalian cell cultures. Polycistronic expression constructs utilising internal ribosome entry site (IRES) can link unrelated genes under control of a single promoter. Transient study on the IRESlinked gene expression was performed. It was possible to standardise the level of protein expression to plasmid number by determining the number of free plasmids in the cytoplasm. The expression of a selectable marker when downstream of IRES was reduced in comparison to the monocistronic construct. Importantly when IRES was used, there were no negative effects on recombinant gene expression upstream of IRES. Down-regulating the selectable marker gene expression has been shown to enhance the probability of obtaining highly expressing clones. To investigate the effects of down-regulating fusion metallothionein green fluorescent protein (MTGFP), new constructs were created to combine metal inducible M2.6 promoter to drive the expression of human growth hormone linked to MTGFP by an attenuated IRES. This resulted in less MTGFP expression, reduced survivability and mean fluorescence in the presence of heavy metal. The increased metal sensitivity lengthened the initial selection period using reduced metal concentration in comparison to cells transfected with wildtype MTGFP. FACS can be used to select for resistance conferred by MTGFP despite reduced expression. FACS enrichment and sorting increased the hGH expression, which was correlated with mean fluorescence of the population; therefore fluorescence can be used as an indication of the final recombinant protein expression. Different approaches to isolate suitable clones were also investigated. It is preferable to select the transfected pool in low metal concentration for two weeks, sort for cells of high-fluorescence, and allow for recovery and proliferation. It is then possible to amplify gene expression by culturing the clones in increasing metal, resulting in further improvement of recombinant protein expression.

Promoter hypermethylation mediated gene silencing is a frequent epigenetic finding in many cancers that affects genes known to have important roles in several aspects of cell biology. Hematological malignancies have been reported to harbor multiple genes aberrantly silenced by promoter hypermethylation and as a result, cytosine analogs known to inhibit the DNA methylation machinery are currently being evaluated in clinical trials. As such, the general goal of this thesis was to identify genes silenced by promoter hypermethylation in human acute myeloid leukemia (AML) and to study the mechanism of promoter hypermethylation mediated gene silencing. Interestingly, the cyclin dependent kinase inhibitor p15 was found to be methylated at a high frequency in AML patients and cell lines in association with a lack of detectable p15 mRNA. Treatment with the cytosine analog 5-Aza-2-deoxycytidine (5-Aza-dC) in vitro resulted in promoter demethylation and p15 mRNA re-expression, which was associated with a release of a transcriptionally repressive complex at the p15 promoter. Importantly, 5-Aza-dC treatment also reversed specific histone amino-terminal modifications at the p15 promoter which are normally associated with transcriptionally inactive chromatin regions, implicating chromatin remodeling in promoter hypermethylation mediated gene silencing. The recently discovered DNA methylation inhibitor, zebularine considered more stable than 5-Aza-dC was also able to reconstitute p15 mRNA in vitro in association with promoter demethylation, regional enrichment of histone acetylation, and growth inhibition. To identify novel genes silenced by promoter hypermethylation in AML, cDNA microarray analysis was employed following in vitro pharmacological inhibition of DNA methylation and histone deacetylation. Of note, four genes from the metallothionein family of cysteine rich small molecules were consistently upregulated following drug treatment and further evaluation identified the gene MT1H to be hypermethylated at a high frequency in AML patients and cell lines. Taken together, the data suggests that aberrant promoter hypermethylation mediated gene silencing occurs in multiple genes from different gene families during the molecular pathogenesis of human AML. Furthermore, the mechanism of promoter methylation mediated transcriptional silencing acts in concert with specific histone modifications which, importantly, can be reversed by treatment with pharmacological inhibitors of DNA methylation.

Promoter hypermethylation mediated gene silencing is a frequent epigenetic finding in many cancers that affects genes known to have important roles in several aspects of cell biology. Hematological malignancies have been reported to harbor multiple genes aberrantly silenced by promoter hypermethylation and as a result, cytosine analogs known to inhibit the DNA methylation machinery are currently being evaluated in clinical trials. As such, the general goal of this thesis was to identify genes silenced by promoter hypermethylation in human acute myeloid leukemia (AML) and to study the mechanism of promoter hypermethylation mediated gene silencing. Interestingly, the cyclin dependent kinase inhibitor p15 was found to be methylated at a high frequency in AML patients and cell lines in association with a lack of detectable p15 mRNA. Treatment with the cytosine analog 5-Aza-2-deoxycytidine (5-Aza-dC) in vitro resulted in promoter demethylation and p15 mRNA re-expression, which was associated with a release of a transcriptionally repressive complex at the p15 promoter. Importantly, 5-Aza-dC treatment also reversed specific histone amino-terminal modifications at the p15 promoter which are normally associated with transcriptionally inactive chromatin regions, implicating chromatin remodeling in promoter hypermethylation mediated gene silencing. The recently discovered DNA methylation inhibitor, zebularine considered more stable than 5-Aza-dC was also able to reconstitute p15 mRNA in vitro in association with promoter demethylation, regional enrichment of histone acetylation, and growth inhibition. To identify novel genes silenced by promoter hypermethylation in AML, cDNA microarray analysis was employed following in vitro pharmacological inhibition of DNA methylation and histone deacetylation. Of note, four genes from the metallothionein family of cysteine rich small molecules were consistently upregulated following drug treatment and further evaluation identified the gene MT1H to be hypermethylated at a high frequency in AML patients and cell lines. Taken together, the data suggests that aberrant promoter hypermethylation mediated gene silencing occurs in multiple genes from different gene families during the molecular pathogenesis of human AML. Furthermore, the mechanism of promoter methylation mediated transcriptional silencing acts in concert with specific histone modifications which, importantly, can be reversed by treatment with pharmacological inhibitors of DNA methylation.

It is hypothesized that exposing endothelial cells to steady or non-reversing pulsatile shear stress produces a healthy, anti-atherogenic endothelium, whereas a reversing pulsatile shear stress promotes an unhealthy, pro-atherogenic endothelium. To further investigate this hypothesis, a novel parallel plate flow chamber system was used to expose human endothelial cells to a pro-atherogenic reversing shear stress waveform designed to simulate the wall shear stress at the carotid sinus, a region prone to atherosclerosis. Cells exposed to this reversing shear stress were compared to cells exposed to high levels of steady shear stress (15 dynes/cm²), low steady shear stress (1 dyne/cm², the time-average of the carotid shear stress), and static culture conditions. Functional analysis confirmed previous findings that reversing shear stress increases cell proliferation and monocyte adhesion. Microarray results indicate that although there are unique sets of genes controlled by both low average shear stress and by reversing flow, more genes were controlled by low average shear stress. We propose that low-time average shear stress, and not fluid reversal/oscillation, may be the more significant mechanical force. The reversing shear stress system was also used to investigate two shear stress-responsive genes, CYP1A1 and CYP1B1. Both were maximally up-regulated at arterial steady shear stresses of at least 15 dynes/cm² and reversing pulsatile shear stress attenuated expression of both genes. Furthermore, AhR nuclear localization and CYP1A1 protein expression correlate with the flow patterns in the mouse aortic arch. The data strongly suggest that the AhR/CYP1 pathway promotes an anti-atherogenic phenotype in the endothelium. Changes in free zinc were measured under different shear stresses. High steady shear stress dramatically increases the levels of free zinc in endothelial cells as compared to cells grown in static culture. This increase in free zinc is attenuated under reversing shear stress and low steady shear stress, which correlates with an increase in zinc-binding metallothinein proteins and zinc exporter Znt-1. Overall, the findings provide further insight into endothelial responses to mechanical forces and may be important in understanding mechanisms of atherosclerotic development and localization to regions of disturbed flow.

Thesis (Ph.D.)--York University, 2007. Graduate Programme in Chemistry. / Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR39012

Maternal exposure to viral and bacterial infection during pregnancy is associated with fetal dysmorphology and neurodevelopmental disorders including schizophrenia, cerebral palsy, autism and mental retardation. Previous studies in our laboratory using an established mouse model of endotoxin-induced fetal dysmorphology have led to the hypothesis that birth defects caused by infections during pregnancy are the result of fetal zinc deficiency resulting from the induction of a zinc-binding protein, metallothionein (MT) in the maternal liver as part of the maternal inflammatory response. Thus, we predicted that zinc deficiency would exacerbate the negative fetal outcomes caused by bacterial endotoxin lipopolysaccharide (LPS) and that zinc supplementation would protect against LPS-mediated teratogenicity. This premise was investigated herein and was extended to investigate underlying molecular mechanism, including the identification of markers of neurodevelopmental damage following LPS administration in early and late pregnancy, and to determine the influence of zinc treatment on any changes in expression of these markers. In Chapter 2 it was demonstrated that prenatal exposure to LPS on gestational day (GD) 8 resulted in the development of physical birth defects including exencephaly, microcephaly, cleft lip and or palate, and micrognathia in GD 18 fetuses. Dietary zinc supplementation throughout pregnancy was found to prevent the LPS-related abnormalities. Furthermore, low dietary zinc and LPS exposure were found to be synergistic on teratogenicity. In addition, an inverse linear relationship was observed between the concentration of zinc in the diet and teratogenicity with a reduction in the incidence of birth defects observed with increasing concentration of dietary zinc, a finding suggesting that even small increments of zinc above normal dietary intake are likely to have a beneficial impact on teratogenicity. Maternal infection during late pregnancy has also been linked with prenatal brain damage. A major causal link underpinning this relationship is thought to be the cytokines released following a maternal inflammatory response to infection. In Chapter 3, the presence of cytokines released in response to LPS given on GD 16 was demonstrated by an increased number of tumour necrosis factor-alpha (TNF-!)-reactive cells and astrogliosis accompanied by extensive apoptotic cell death in GD 18 fetal brain. Recently our laboratory has reported that dietary zinc supplementation throughout pregnancy, prevented impairments in object recognition memory in offspring from dams exposed to prenatal LPS on GD 8. The question arises as to whether zinc is protective against LPS-exposure in late pregnancy. In Chapter 3, it is further demonstrated that LPS-induced brain injury was prevented by concurrent zinc treatment at the time of LPS exposure. In Chapter 4, the expression of activity-dependent neuroprotective protein (ADNP) mRNA was identified as a marker of changes occurring in the fetus as a result of LPS exposure in early pregnancy. ADNP has been found to be essential for organogenesis and is a sensitive indicator of brain injury. Here it was demonstrated that LPS caused a rapid increase in embryonic ADNP expression, which was highly significant 24 hours after exposure. Whether the elevation in ADNP expression is in response to inflammatory damage or is induced by cytokines released by the maternal inflammatory response is not clear. However, a major finding of the study is that concomitant zinc treatment prevented the LPS-induced increase in ADNP activity. The mechanism of protection by zinc is presumed to be centred on preventing the fall in plasma zinc and associated fetal zinc deficiency caused by LPS induction of MT, but may also include MT-independent actions of zinc including prevention of apoptosis and oxidative damage, or enhance tissue repair processes. Taken together the findings in this thesis support earlier evidence that maternal MTmediated transient fetal zinc deficiency in early pregnancy underpins LPS-induced teratogenicity. This is the first study to demonstrate that this mechanism may also apply to LPS-induced neurodevelopmental damage in early and late pregnancy. However, further studies are warranted to discriminate between the influence of MT and that of other inflammatory reactants (e.g. cytokines) on LPS-mediated damage late in pregnancy. The major finding of the thesis is that zinc treatment (either given subcutaneously with LPS or as dietary zinc supplementation throughout pregnancy) prevents the negative fetal outcomes including neurodevelopmental damage caused by prenatal exposure to LPS. This finding highlights the importance of zinc nutrition in pregnancy and the benefits that might be gained as a potential prophylactic treatment to minimise fetal damage caused by infections during pregnancy. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Maternal exposure to viral and bacterial infection during pregnancy is associated with fetal dysmorphology and neurodevelopmental disorders including schizophrenia, cerebral palsy, autism and mental retardation. Previous studies in our laboratory using an established mouse model of endotoxin-induced fetal dysmorphology have led to the hypothesis that birth defects caused by infections during pregnancy are the result of fetal zinc deficiency resulting from the induction of a zinc-binding protein, metallothionein (MT) in the maternal liver as part of the maternal inflammatory response. Thus, we predicted that zinc deficiency would exacerbate the negative fetal outcomes caused by bacterial endotoxin lipopolysaccharide (LPS) and that zinc supplementation would protect against LPS-mediated teratogenicity. This premise was investigated herein and was extended to investigate underlying molecular mechanism, including the identification of markers of neurodevelopmental damage following LPS administration in early and late pregnancy, and to determine the influence of zinc treatment on any changes in expression of these markers. In Chapter 2 it was demonstrated that prenatal exposure to LPS on gestational day (GD) 8 resulted in the development of physical birth defects including exencephaly, microcephaly, cleft lip and or palate, and micrognathia in GD 18 fetuses. Dietary zinc supplementation throughout pregnancy was found to prevent the LPS-related abnormalities. Furthermore, low dietary zinc and LPS exposure were found to be synergistic on teratogenicity. In addition, an inverse linear relationship was observed between the concentration of zinc in the diet and teratogenicity with a reduction in the incidence of birth defects observed with increasing concentration of dietary zinc, a finding suggesting that even small increments of zinc above normal dietary intake are likely to have a beneficial impact on teratogenicity. Maternal infection during late pregnancy has also been linked with prenatal brain damage. A major causal link underpinning this relationship is thought to be the cytokines released following a maternal inflammatory response to infection. In Chapter 3, the presence of cytokines released in response to LPS given on GD 16 was demonstrated by an increased number of tumour necrosis factor-alpha (TNF-!)-reactive cells and astrogliosis accompanied by extensive apoptotic cell death in GD 18 fetal brain. Recently our laboratory has reported that dietary zinc supplementation throughout pregnancy, prevented impairments in object recognition memory in offspring from dams exposed to prenatal LPS on GD 8. The question arises as to whether zinc is protective against LPS-exposure in late pregnancy. In Chapter 3, it is further demonstrated that LPS-induced brain injury was prevented by concurrent zinc treatment at the time of LPS exposure. In Chapter 4, the expression of activity-dependent neuroprotective protein (ADNP) mRNA was identified as a marker of changes occurring in the fetus as a result of LPS exposure in early pregnancy. ADNP has been found to be essential for organogenesis and is a sensitive indicator of brain injury. Here it was demonstrated that LPS caused a rapid increase in embryonic ADNP expression, which was highly significant 24 hours after exposure. Whether the elevation in ADNP expression is in response to inflammatory damage or is induced by cytokines released by the maternal inflammatory response is not clear. However, a major finding of the study is that concomitant zinc treatment prevented the LPS-induced increase in ADNP activity. The mechanism of protection by zinc is presumed to be centred on preventing the fall in plasma zinc and associated fetal zinc deficiency caused by LPS induction of MT, but may also include MT-independent actions of zinc including prevention of apoptosis and oxidative damage, or enhance tissue repair processes. Taken together the findings in this thesis support earlier evidence that maternal MTmediated transient fetal zinc deficiency in early pregnancy underpins LPS-induced teratogenicity. This is the first study to demonstrate that this mechanism may also apply to LPS-induced neurodevelopmental damage in early and late pregnancy. However, further studies are warranted to discriminate between the influence of MT and that of other inflammatory reactants (e.g. cytokines) on LPS-mediated damage late in pregnancy. The major finding of the thesis is that zinc treatment (either given subcutaneously with LPS or as dietary zinc supplementation throughout pregnancy) prevents the negative fetal outcomes including neurodevelopmental damage caused by prenatal exposure to LPS. This finding highlights the importance of zinc nutrition in pregnancy and the benefits that might be gained as a potential prophylactic treatment to minimise fetal damage caused by infections during pregnancy. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

[Truncated abstract] During development of the visual system, topographic connections between the retina and the superior colliculus are established using guidance molecules. The EphA family of tyrosine kinase receptors and their ephrin-A ligands are important for establishing topography between the temporo-nasal axis of the retina and the rostro-caudal axis of the superior colliculus. After injury to the visual system via unilateral optic nerve transection, adult mammalian retinal ganglion cells fail to regenerate axons spontaneously to their main visual centre, which in rodents, is the superior colliculus. The EphA5 gradient is down-regulated from a temporalhigh to nasallow gradient to a uniform low level in the few surviving retinal ganglion cells, but ephrin-A2 is upregulated back to a significant rostrallow to caudalhigh gradient in the superior colliculus, similar to that seen during development. In this thesis, a number of experiments have been undertaken to investigate further how EphA5 and ephrin-A2 are regulated after injury and how they may play a role once regeneration has been encouraged through surgical intervention. In the first study, targeted unilateral retinal laser lesions were used to ablate either dorso-nasal or ventro-temporal quadrants of the retina. ... Surviving and regenerating retinal ganglion cells in the retina, and axons in the optic nerve, were analysed. The data suggest that metallothionein-I/II increases axonal regeneration through the optic nerve injury site but, at the dose administered, had no neuroprotective effects on retinal ganglion cells. This thesis provides further insight into the response of guidance molecules to injury, and the potential of metallothionein-I/II as a neuroregenerative factor in the adult mammalian visual system. The regulation of both EphA5 and ephrin-A2 through transsynaptic connections may be a response common to other guidance molecules. Such connectivity now needs to be studied further to understand how it may impact on various treatments designed to increase re-connectivity after other brain injuries, including stroke. The ectopic expression of ephrin-A2 at the insertion site of a peripheral nerve graft in the superior colliculus, implicate this guidance molecule in the glial scar for the first time. Therefore, to overcome inhibition by the glial scar, axons must also overcome ephrin-A2 mediated inhibition, potentially by the addition of EphA5 fusion proteins. Metallothionein-I/II?s effect of increasing axonal regeneration through the optic nerve injury site suggests that it could be used to increase the number of regenerating axons reaching their target. Such strategies to increase the absolute number of regenerated axons should enable these axons to better use the EphA5 and ephrin-A2 topographic gradients to optimize regenerative success.

É bem conhecido que muitos efeitos tóxicos do cádmio (Cd) resultem da ação da interação com metais essenciais, incluindo zinco (Zn). Sendo um poluente ambiental, a exposição à Cd conduz a distúrbios no conteúdo e atividade de Zn no organismo, representando importante via para o desenvolvimento de sua toxicidade. Evidências suportam que Zn pode reduzir os efeitos do Cd, prevendo ou reduzindo a ação tóxica deste metal, enquanto que a deficiência de Zn pode intensificar a toxicidade de Cd. Com base nisto, este trabalho buscou investigar (1) o efeito da interação Zn-Cd sobre o tecido hepático de ratos adultos, por representar um importante alvo biológico à ação dos metais e (2) se estes efeitos são estendidos ao SNC mesmo protegido pela barreira hemato-encefálica. Através da avaliação de parâmetros bioquímicos, como [delta]-aminolevulinato-desidratase e metalotioneína, os resultados encontrados suportam a sobreposição do efeito tóxico de Cd sobre as funções essenciais de Zn, entretanto existe diferença entre os tecidos quanto ao mecanismo protetivo exercido sobre a toxicidade de Cd. Além disso, obtemos dados sobre os níveis de MT que contrapõem suas funções benéficas previamente descritas. Em resumo, essa dissertação reforça a ação tóxica de Cd por vias biológicas comuns ao Zn e expõe diferenças entre o tecido hepático e o SNC quanto ao mecanismo interativo. / It is well known that many toxic effects of cadmium (Cd) results from the action of the interaction with essential metals, for example zinc (Zn). Cd is an environmental pollutant, which exposure leads to disturbance in the content and atividade of Zn in the body. Evidences indicate that Zn can reduce the effects of Cd for provide or reduce the toxic action of this metal, whereas Zn deficiency can intensify the toxicity of Cd. On account of this, our study aimed investigate (1) the effect of Zn-Cd interaction on the liver tissue of adult rats, which represents important biological target for action of the metals and (2) if these effects are extended to the CNS even protected by the blood-brain barrier. Through the evaluation of biochemical parameters, as [delta]-aminolevulinate-dehydratase ([delta]-ALA-D) and metallothionein (MT), the results support the overlap of the toxic effect of Cd on the essential functions of Zn; however there is difference between the tissues on the protective mechanism exerted on the toxicity of Cd. Furthermore, we obtain data on the levels of MT that contrast to their beneficial functions previously described. In summary, this work reinforces the toxic effect of Cd by biological pathways common to the Zn and explains differences between the liver tissue and CNS in relation to the interactive mechanism.

É bem conhecido que muitos efeitos tóxicos do cádmio (Cd) resultem da ação da interação com metais essenciais, incluindo zinco (Zn). Sendo um poluente ambiental, a exposição à Cd conduz a distúrbios no conteúdo e atividade de Zn no organismo, representando importante via para o desenvolvimento de sua toxicidade. Evidências suportam que Zn pode reduzir os efeitos do Cd, prevendo ou reduzindo a ação tóxica deste metal, enquanto que a deficiência de Zn pode intensificar a toxicidade de Cd. Com base nisto, este trabalho buscou investigar (1) o efeito da interação Zn-Cd sobre o tecido hepático de ratos adultos, por representar um importante alvo biológico à ação dos metais e (2) se estes efeitos são estendidos ao SNC mesmo protegido pela barreira hemato-encefálica. Através da avaliação de parâmetros bioquímicos, como [delta]-aminolevulinato-desidratase e metalotioneína, os resultados encontrados suportam a sobreposição do efeito tóxico de Cd sobre as funções essenciais de Zn, entretanto existe diferença entre os tecidos quanto ao mecanismo protetivo exercido sobre a toxicidade de Cd. Além disso, obtemos dados sobre os níveis de MT que contrapõem suas funções benéficas previamente descritas. Em resumo, essa dissertação reforça a ação tóxica de Cd por vias biológicas comuns ao Zn e expõe diferenças entre o tecido hepático e o SNC quanto ao mecanismo interativo. / It is well known that many toxic effects of cadmium (Cd) results from the action of the interaction with essential metals, for example zinc (Zn). Cd is an environmental pollutant, which exposure leads to disturbance in the content and atividade of Zn in the body. Evidences indicate that Zn can reduce the effects of Cd for provide or reduce the toxic action of this metal, whereas Zn deficiency can intensify the toxicity of Cd. On account of this, our study aimed investigate (1) the effect of Zn-Cd interaction on the liver tissue of adult rats, which represents important biological target for action of the metals and (2) if these effects are extended to the CNS even protected by the blood-brain barrier. Through the evaluation of biochemical parameters, as [delta]-aminolevulinate-dehydratase ([delta]-ALA-D) and metallothionein (MT), the results support the overlap of the toxic effect of Cd on the essential functions of Zn; however there is difference between the tissues on the protective mechanism exerted on the toxicity of Cd. Furthermore, we obtain data on the levels of MT that contrast to their beneficial functions previously described. In summary, this work reinforces the toxic effect of Cd by biological pathways common to the Zn and explains differences between the liver tissue and CNS in relation to the interactive mechanism.