Reduction of cardiac myocyte loss and repair of the vasculature post myocardial infarction are important therapeutic goals because the potential for intrinsic repair is limited. Preclinical and limited clinical data support the possibility that bone marrow-derived mesenchymal stem cells may be a suitable cell type for cellular therapy. The goal of this research was to determine the effectiveness of using MSCs from aged mice in cellular therapy for the treatment of AMI. The central hypothesis for this research was that therapeutic potential of mesenchymal stem cells decreases with age. This research utilized global gene expression analysis to investigate molecular differences in MSCs harvested from three different age groups of mice. Microarray analysis was performed to investigate changes in gene expression with respect to aging. Furthermore, both in vitro and in vivo experiments were completed to analyze the functional and molecular characteristics of the MSCs. The data identified age-related defects in mouse MSCs as well as determined the molecular basis for these deficiencies. This study indicates that MSCs from 26m mice are severely deficient in the induction of angiogenesis and cardiac repair due to defective paracrine factor secretion caused by decreased expression of growth factor/cytokine genes. Hypoxia attenuates the deficiency in the aged mice, whereas in young mice low oxygen promotes secretion of paracrine growth factors. It was determined a dysfunction in HIF-1 alpha signaling was present in MSCs from 26m mice and is regulated by the PI3K/Akt signaling in MSCs. Furthermore, two novel and important and novel aspects of this study were the discovery that cell cycle regulation gene expression decreases with age and MSCs have increased insulin resistance with age. Increased insulin resistance in this cell type with aging is likely to have profound effects on the clinical outcomes of using these cells therapeutically. Likewise, loss of cell cycle regulation during proliferation could also lead to undesirable clinical effects. Gaining insight to the repair potential of these cells with respect to age will help to better define future trials of autologous stem cells not only for heart disease but for all of the many applications proposed for these cells.

The mitochondrial control region (MCR) has played an important role as a population genetic marker in many taxa but sequencing of complete eukaryotic genomes has revealed that nuclear integrations of mitochondrial DNA (numts) are abundant and widespread across many taxa. If left undetected, numts can inflate mitochondrial diversity and mislead interpretation of phylogenetic relationships. Comparative analyses of complete genomes in humans, orangutans and chimpanzees, and preliminary studies in gorillas have revealed high numt prevalence in great apes, but rigorous comparative analyses across taxa have been lacking. The present study aimed to systematically compare the evolutionary dynamics of MCR numts in great apes. Firstly, an inventory numts derived from the region containing the MCR subdomains was carried out by genomic BLAST searches. Secondly, presence/absence of each candidate numt was determined in great ape taxa to estimate numt insertion rate. Thirdly, alternative mechanisms of numt insertion, either through direct mitochondrial integration or post-insertional duplications, were also assessed. Fourthly, the effect of nuclear and mitochondrial environment on patterns of nucleotide composition and substitution was assessed through sequence comparisons of nuclear and mitochondrial paralogous sequences. Finally, numts in the gorilla genome were identified through two experimental methods and their use as polymorphic genetic markers was then evaluated in a sample of captive gorillas from U.S. zoos. A deficit of MCR numts covering two particular mitochondrial subdomains was detected in all three apes examined, and is largely attributed to rapid loss of mitochondrial and nuclear sequence identity in the mitochondrial genome. Insertion rates have varied during the great ape evolution and exhibit substantial differences even between related taxa. The most likely mechanism of numt insertion is direct mitochondrial integration through Non-Homologous-End-Joining Repair. Transition/transversion ratios differed significantly between both mitochondrial and nuclear sequences and between numts from coding and non-coding mitochondrial regions. A previously documented upward bias in the GC content of the primate mitochondrial genome was confirmed and the extent of this bias relative to the corresponding numt sequences increased with numt age. Five gorilla-specific numts were isolated, including three exhibiting insertional polymorphisms that will be used in future population genetic studies in free-range gorilla.

In this study, we sought to define an underlying molecular mechanism of how inflammation induces cancer initiation. Cancer-associated inflammation is marked by the presence of inflammatory cells and mediators including cytokines, chemokines, and reactive oxygen species. There is a growing body of evidence establishing the link between chronic inflammation and cancer. Twenty percent of cancers have been linked to chronic infections. For instance, bacterial and viral infections induce inflammation which is a known risk factor for cancer. During inflammation, Ml macrophages' production of pro-inflammatory cytokines and reactive oxygen species (ROS) drives their function as anti-microbial. Likewise, the transcription factor nuclear factor kappa B (NF-KB) is known to induce a variety of stimulators, including ROS, to contribute to the inflammatory process. Therefore, we sought to explore the relationship between Ml macrophages and NF-KB, suggesting that Ml macrophage mediates cancer initiation via a NF-KB-dependent pathway, which collectively contributes to a metastatic phenotype.

Student Number : 9906751K - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / In addition to its crucial role in cell adhesion, β-catenin is also known to augment gene expression by forming a complex with lymphoid enhancer factor/T-cell factor in the nucleus. Unregulated β-catenin expression and/or its increased nuclear presence can lead to abnormal cell proliferation, tumour invasion and metastasis. Pertinent is the fact that the actin cytoskeleton is central to the translocation of several nuclear proteins. This study investigated whether the actin cytoskeleton influences the nuclear translocation of β-catenin in human oesophageal squamous cell carcinoma (HOSCC), a metastatic disease of common occurrence in South Africa. Disruption of the actin cytoskeleton of five moderately differentiated HOSCC cell lines, with cytochalasin D (cytoD), showed that the nuclear β-catenin level was unaltered in SNO, WHCO1 and WHCO5, but decreased in WHCO3 and WHCO6. CytoD treatment did not affect the cytoplasmic/membrane β-catenin level in these cell lines. Further examination of the possible association between the actin cytoskeleton and nuclear β-catenin translocation, required the design and stable transfection, of a vector containing full-length human β-catenin cDNA into one of the HOSCC lines. Stimulation of exogenous β-catenin expression in transfected WHCO1 cells did not increase cellular β-catenin level, nor did the stimulation of endogenous β-catenin expression with DMSO. In most cases (SNO, WHCO1 and WHCO5) the nuclear distribution of β-catenin in HOSCC is independent of a functional actin cytoskeleton, nonetheless there are some exceptions (WHCO3 and WHCO6). The observed variation within the HOSCC lines is possibly due to specific underlying event/s particular to the cell line. The stable level of β-catenin expression could be a consequence of regulatory pathways in WHCO1 compensating for the induced imbalance of β-catenin expression.

Caveolae are small plasma membrane invaginations present in many different cell types, which have been linked to diverse cellular functions, including cell signalling, membrane rearrangements and lipid regulation. The caveolae markers, members of the caveolin family of proteins, are essential for caveola formation and function. Recently, however, a protein named PTRF (Polymerase I and Transcript Release Factor) or cavin, originally identified as a nuclear factor that regulates transcription in vitro, was shown to be associated with caveolae in adipocytes. In the first chapter of this thesis, I have used the zebrafish Danio rerio to investigate the relation of PTRF/cavin to caveolae as well as caveola function in vivo. During zebrafish development, PTRF/cavin was highly expressed in the notochord in 18 h, 24 h and 35 h post-fertilization embryos, as detected by in situ hybrydization. Analysis of later development stages showed that PTRF/cavin is also present in the otic vesicle, brachial arches, and periderm. Disruption of PTRF/cavin expression, via morpholino-mediated inhibition, caused severely defective development of the notochord as well as heart edema, in a dose-dependent manner. PTRF/cavin knockdown embryos had curved notochords and were shorter than the controls. Examination of the notochord by electron microscopy showed that the number of caveolae was greatly reduced in PTRF/cavin-morpholino-injected embryos. Similar effects were observed when caveolin-1, the major protein of caveolae in non-muscle cells, was down-regulated. Altogether, these results indicated a role for PTRF/cavin during formation and/or stabilization of caveolae as well as an essential role for caveolae during zebrafish embryo development. Combined with results obtained in mammalian cells, these findings identify PTRF/cavin as the first component of a caveolar coat, required for caveola formation and function (Hill et al., 2008). We subsequently identified a family of PTRF/cavin-related proteins, the cavins, that all associate with caveolae. Using biochemistry, light microscopy, and FRET-based approaches we characterised PTRF/cavin and the new members of this family of proteins SDR/cavin-2, SRBC/cavin-3 and MURC/cavin-4. We have shown that the four members of the cavin family form a multi-protein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and then associate with caveolin at the plasma membrane caveolae; interestingly, caveolin is essential for the plasma membrane translocation of the cavin complex, and in caveolin-1 knockout cells the four cavin proteins are restricted to the cytosol. PTRF/cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The four cavin proteins present distinct patterns of tissue expression, which suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. MURC/cavin-4 is expressed predominantly in muscle and its distribution is perturbed in human muscle disease associated with caveolin-3 dysfunction, identifying MURC/cavin-4 as a novel muscle disease candidate caveolar protein. To functionally investigate the relation of cavins and caveolae, we explored a caveolar function in mechanosensation. Through the use of hypo-osmotic media, we induced membrane-stretch and showed that the increased membrane tension leads to dissociation of the caveolin-cavin module and caveola disassembly as observed by immunofluorescence and FLIM/FRET techniques. Once released from caveolae, caveolin was seen internalized in late endosomes and lysosomes. Cavin-1, on the other hand, was found to be diffused in the cytosol and from there it was translocated to the nuclear compartment. The nuclear translocation was observed in several different cell types, which suggests a universal role for nuclear cavin-1, and was independent of caveolin expression. Analysis of live cells using real-time FLIM/FRET showed that cells quickly respond to variations in membrane tension by dissociation/re-association of caveolin and cavin-1. Altogether, in the course of this project, I was able to show that cavin-1 is an essential regulator of caveola biogenesis in cultured cells and in vivo. Cavin-1 and the other members of the PTRF/Cavin family form a multiprotein complex that is recruited to caveolae by caveolin and coats plasma membrane caveolae. The association between cavin-1 and caveolin is crucial for caveolae assembly and this interaction has a role in the cellular sensation of plasma membrane tension. Under high membrane tensions, caveolin and cavin-1 dissociate with the consequent flattening of caveolae. Under these circumstances, caveolin is internalized into enlarged endosomes and lysosomes while cavin-1 is translocated to the nucleus, identifying for the first time a caveola- to nucleus signalling pathway. The exact role of nuclear cavin-1 under plasma membrane stretch is now amenable to analysis.

No description available.

Yes / The Forkhead transcription factor, FoxO3a induces genomic death responses in neurones following translocation from the cytosol to the nucleus. Nuclear translocation of FoxO3a is triggered by trophic factor withdrawal, oxidative stress and the stimulation of extrasynaptic NMDA receptors. Receptor activation of phosphatidylinositol 3-kinase (PI3K)-Akt signalling pathways retains FoxO3a in the cytoplasm, thereby inhibiting the transcriptional activation of death-promoting genes. We hypothesized that phenolic antioxidants such as tert-Butylhydroquinone (tBHQ), which is known to stimulate PI3K-Akt signalling, would inhibit FoxO3a translocation and activity. Treatment of cultured cortical neurones with NMDA increased the nuclear localization of FoxO3a, reduced the phosphorylation of FoxO3a, increased caspase activity and up-regulated Fas ligand expression. In contrast the phenolic antioxidant, tBHQ, caused retention of FoxO3a in the cytosol coincident with enhanced PI3K- dependent phosphorylation of FoxO3a. tBHQ-induced nuclear exclusion of FoxO3a was associated with reduced FoxO-mediated transcriptional activity. Exposure of neurones to tBHQ inhibited NMDA-induced nuclear translocation of FoxO3a, prevented NMDA-induced up-regulation of FoxO-mediated transcriptional activity, blocked caspase activation and protected neurones from NMDA-induced excitotoxic death. Collectively, these data suggest that phenolic antioxidants such as tBHQ oppose stress-induced activation of FoxO3a and therefore have potential neuroprotective utility in neurodegeneration.

Activities of the DEAD box (Asp-Glu-Ala-Asp) family of proteins- including RNA-dependent ATPase and RNA helicase- function in all organisms to sculpt RNA-RNA duplex and RNA-protein complexes, ensuring that necessary rearrangements are rapidly and properly resolved during genetic information processing. Identified as a prototypic member of the DEAD box family and documented as an ATPase and RNA helicase, p68 plays essential and diverse functions in the control of gene expression ranging from pre-mRNA/rRNA processing and mRNA decay/stability to transcriptional activation and initiation. Despite the early implied roles in organ maturation and tumor progression, the functional contributions of p68 to growth/differentiation regulation and cancer development remain undefined. Here, we show c-Abl-dependent phosphorylation of p68 markedly associates with abnormal cell growth and cancer development. Importantly, we characterize an unanticipated signaling module through which p68 functionally contributes to Epithelial-Mesenchymal Transition (EMT) and cell proliferation. p68, which appears to be phosphorylated by c-Abl at tyrosine 593, consequently promotes an EMT through its ability to recruit â-catenin into cell nucleus via a canonic Wnt/â-catenin axis independent way; accordingly, phosphor-p68 (phosphorylated at tyrosine 593 residue) also stimulates tumor cell growth, which requires the ATPase activity of the protein. These findings define a potential mechanism whereby phosphor-p68 recruits â-catenin into cell nucleus in ATP hydrolysis driven fashion and cooperatively regulates transcriptional programs that control an EMT. The dissertation thus demonstrates a tight coordination between DEAD box RNA helicase and cancer development.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a promiscuous protein serving as a required dimerization partner for the aryl hydrocarbon receptor (AhR) and hypoxia inducible factor-1α (HIF-1α) transcription factors. Additionally it serves as a potent co-activator for estrogen receptor (ER) signaling. We sought to take advantage of these cross-talk mechanisms by designing an AhR construct that can influence the regulation of these pathways by sequestering ARNT. CΔ553 is a truncated form of the AhR lacking the C-terminal 553 amino acids which harbors the complete transactivation (TAD) and significant portions of the ligand binding (LBD) domains. Altering the LBD allows CΔ553 to become constitutively active and has been shown to associate with ARNT and bind DNA. Without the TAD, CΔ553 cannot recruit co-activators to the promoter so that no activation of gene transcription may occur. Transient transfection studies using a corresponding luciferase reporter plasmid in MCF-7 cells showed that CΔ553 effectively suppressed the AhR, HIF-1α, and ER signaling pathways. RT/real-time QPCR data showed that CΔ553 blocked the up-regulation of the target genes controlled by AhR ( CYP1A1 ), HIF-1α ( VEGF, aldolase C , and LDH-A ), and ER ( GREB1 ) in breast cancer cells. Since both HIF-1α and ER are highly active in ER-positive breast cancers, CΔ553 has the potential to be developed as a protein drug to treat breast cancer by blocking these two signaling pathways. Seeking to determine if complete suppression of genes is possible with CΔ553, a tetracycline regulated retroviral expression system is investigated along with the possibilities for cellular administration via the HIV-1 Tat protein transduction domain. Since ARNT dimerizes with both AhR and HIF-1α, we present further studies looking into the role protein factors play in the activation of each system. Previously within our lab p23 and Cyp40, two components of the hsp90 chaperon complex, were found to facilitate the formation of the AhR•ARNT•DNA binding complex. Analysis of these proteins within the hypoxia signaling pathway found that only p23 was capable of generating the HIF-1α•ARNT•DNA binding complex.

No description available.