S100A8 is a highly-expressed calcium-binding protein in neutrophils and activated macrophages, and has proposed roles in myeloid cell differentiation and host defense. Functions of S100A8 are not fully understood, partly because of difficulties in generating S100A8 knockout mice. Attempts to silence S100A8 gene expression in activated macrophages and fibroblasts using RNA interference (RNAi) technology were unsuccessful. Despite establishing validated small interfering RNA (siRNA) systems, enzymaticallysynthesized siRNA targeted to S100A8 suppressed mRNA levels by only 40% in fibroblasts activated with FGF-2+heparin, whereas chemically-synthesized siRNAs suppressed S100A8 driven by an S100A8-expression vector by ~75% in fibroblasts. Suppression of the gene in activated macrophages/fibroblasts was low, and some enzymatically-synthesized siRNAs to S100A8, and unrelated siRNA to GAPDH, induced/enhanced S100A8 expression in macrophages. This indicated that S100A8 may be upregulated by type-1 interferon (IFN). IFN-β enhanced expression, but did not directly induce S100A8. Poly (I:C), a synthetic dsRNA, directly induced S100A8 through IL-10 and IFN-dependent pathways. Induction by dsRNA was dependent on RNA-dependent protein kinase (PKR), but not cyclooxygenase-2, suggesting divergent pathways in LPS- and dsRNA-induced responses. New mechanisms of S100A8 gene regulation are presented, that suggest functions in anti-viral defense. S100A8 expression was confirmed in lungs from influenza virus-infected mice and from a patient with severe acute respiratory syndrome (SARS). Multiple pathways via mitochondria mediated S100A8 induction in LPS-activated macrophages; Generation of reactive oxygen species via the mitochondrial electron transport chain and de novo synthesis of ATP may be involved. This pathway also regulated IL-10 production, possibly via PKR. Extracellular ATP and its metabolites enhanced S100A8 induction. Results support involvement of cell stress, such as transfection, in S100A8 expression. A breast tumor cell line (MCF-7) in which the S100A8 gene was silenced, was established using micro RNA technology; S100A8 induction by oncostatin M was reduced by >90% in stably-transfected cells. This did not alter MCF-7 growth. The new approach to investigate the role of S100A8 in a human tumor cell line may assist in exploring its functions and lead to new studies concerning its role in cancer.

[Truncated abstract] Mitochondria are semi-autonomous organelles found in almost all eukaryotic cells to contain more than 1000 different proteins. The majority of these proteins are encoded in the nucleus, translated in the cytosol and imported into mitochondria. The overall aim of this study was to characterise the regulation of nuclear-encoded mitochondrial proteins (NEMP). This was carried out in the plant, Arabidopsis thaliana, using the alternative oxidase (AOX) as a model. Specifically, the aims were to i) determine how regulation of NEMP interact with known regulatory pathways/mechanisms; ii) determine if the pattern of coexpression observed for NEMP are due to co-regulation, and iii) to determine whether mitochondrial retrograde regulatory pathways interact with known chloroplast regulatory pathways. AOX1c is one of five genes encoding AOX in Arabidopsis. It is expressed in a variety of organs and is not induced by stress. Thus, its regulation was characterised in order to gain insight into the regulation of NEMP under normal growth conditions. Analysis of the promoter of AOX1c revealed cis-acting regulatory elements (CAREs) common to both AOX1c from Arabidopsis and AOX2b from soybean. Additionally, Site II elements, previously shown to be involved in the regulation of the proliferating cell nuclear antigen, are present in the upstream promoter region of AtAOX1c and were shown to be strong negative regulators of AtAOX1c expression. AOX1a is a gene encoding AOX that is induced at a transcript level, by many stress treatments. BA signalling and provide evidence of at least one common factor between chloroplastic and mitochondrial retrograde regulatory pathways, i.e. ABI4. ... The above results reveal that the regulation of NEMP are integrated with the mainstream regulatory pathways that control gene expression for a variety of proteins in various locations. Although this is not unexpected, it does raise the question of how mitochondrial function impacts, or feeds back, to alter these pathways, i.e. how mitochondrial retrograde signals affects the regulation of genes encoding proteins in a variety of locations. The observed interaction of mitochondrial and plastid retrograde regulatory pathways at the level of ABI4, suggests that mitochondrial signals have the potential to act as a powerful regulators of many cellular functions. Although interaction between mitochondrial and other organelles at a cellular level has been known for some time, there is still much work left to be done to define the network of molecular interactions that exists to regulate and integrate the expression of NEMP with all other proteins in the cell. This study reveals that interactions also occur at regulatory steps that have to potential to regulate many function in organelles, even if no direct metabolic link exists. However, this study has only begun to uncover these interactions at a molecular level.

Mitochondria are the main sites for adenosine triphosphate (ATP) generation within most cells. Structural and functional alterations of mitochondria due to genetic abnormalities of mitochondria can cause respiratory chain dysfunction. In this study, the important role of mitochondria in energy metabolism was determined by comparing the effect of mitochondrial DNA (mtDNA) mutations on growth patterns and oxidative phosphorylation (OXPHOS) enzyme activities of six isolated clones (B5, B12, D4, D9, E1 and E8); as well as the effect of ATP supplement to culture using the slowest growing clone. The isolated clones had shown distinct growth pattern and morphology. The difference in proliferation rates among the clones was ascertained by the doubling times (B5=26.4h. B12=43.2h. D4=25.7h. D9=33.6h. E1=26.9h and E8=28.8h). The clone's slow growth rate was likely the result of mitochondrial mutations in the 16S rRNA gene, ND1, ND4, ND6 and COX III. Five heteroplasmic mutations were found in clone B12 (G2480T, C2513G, A2520T, C9527T and C14263G), one heteroplasmic mutation in clone D9 (A4137G) and one homoplasmic mutation in clone D4 (C11496). The mutations in clone B12 appeared to be deleterious to the cell by disrupting mitochondrial OXPHOS activities and reducing energy output. Additionally, extracellular ATP supplement to OXPHOS deficient clone B12 facilitated cell growth and enhances the gene expression. Increased expression of mtDNA-encoded respiratory chain complexes observed in clone B12 compared to clone D4 may reflect mitochondrial genomic adaptation to perturbations in cellular energy requirements. The stimulation of mitochondrial biogenesis may be a cellular response in compensation for defects in OXPHOS associated with mtDNA mutations. My data support the hypothesis that the variability in functional manifestations of mtDNA is attributed to the nature of the mutation, number of mutation and the gene specifically affected. These results will help to further our understanding of the relationship between mitochondrial mutation and cellular function.

Thesis (Ph.D.)--McMaster University, 2006. / Supervisor: Jianping Xu). Includes bibliographical references.

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

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 104-114).

Thesis (M.Sc.) - Carleton University, 2005. / Includes bibliographical references (p.103-107 ). Also available in electronic format on the Internet.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 3 uppsatser.

Thesis (M.Sc. (Tropical Medicine))--Mahidol University, 2003.