No description available.

>Magister Scientiae - MSc / Methamphetamine (MA), also known as ‘Tik’, has detrimental short- and long-term psychological and morphological effects on the central nervous system (CNS). The lipophilic nature of MA allows it to cross the blood-brain barrier (BBB) which normally plays a protective role in limiting solute exchange (including narcotics) into the neuronal tissue. Numerous studies have indicated that MA not only crosses the BBB but is implicated in distorting its crucial role in that it increases the permeability of the endothelial cells and thereby compromises its core homeostatic function. The speculated mechanism by which MA elicits its effects involves elevated ROS production which may be reversed by antioxidant treatment. Rooibos herbal tea (Aspalathus linearis) which is well documented for its antioxidative properties and ROS scavenging abilities may therefore be the ideal candidate to reverse the harmful ROS-induced effects of MA. The aim of the study was to investigate the in vitro ameliorating potential of fermented rooibos (Rf) against the MA-induced effects on mouse brain endothelial (bEnd5) cells by utilizing various assays (trypan blue exclusion and XTT viability assays) and physiological parameters (cell numbers, viability, monolayer permeability and cell cycle phases) over a period of 96 hrs. Statistical analysis was performed using the Wilcoxon rank sum test with P<0.05 denoted as significant. Once-off exposure to physiological MA concentrations and Rf resulted in % viability similar to controls by 96 hrs with suppression observed only when the cells were exposed to daily MA (0.1-1000 μM) (P≤0.0063). Exposure to supraphysiological concentrations (≥100 μM) of MA greatly suppressed viability (P≤0.0463). Both daily and once-off treatment to the combinations initially resulted in increased viability however by 96 hrs was similar to- or exceeding the controls (P≤0.0180). MA exposure also resulted in decreased live cell numbers (P≤0.0339) with no effect when exposed to Rf by 96 hrs. The combinations resulted in cell numbers comparable to controls. Dose-dependent increases in electrical resistance were observed in response to singular MA and Rf treatment with lower MA concentrations displaying significant decreases (P≤0.0064). Similar trends were observed with combinations however greater resistance was observed. Increased G1-phase populations (P≤0.0495) in response to singular MA and Rf exposure was noted followed by decreased S-phase fractions (P≤0.0356). While MA decreased G2-M phase cells (P≤0.0498) it was unaffected by Rf. In contrast, the combination of MA and Rf decreased events in the G1-phase (P≤0.0483), with an increased S-phase population (P≤0.0415). In conclusion, the single compounds displayed mirroring effects, decreasing the cells’ permeability and causing G1-phase arrest. The modulatory effects of Rf in combination with MA was illustrated with the restoration of viability and live cell numbers comparable to that of controls, and a more restrictive monolayer as well as reversal of the G1-phase arrest. Findings suggest that Rf may reverse the adverse effects of MA on the BBB.

Thesis (Ph. D.)--Colorado State University, 2004. / Includes bibliographical references.

How do precursor cells know when it is time to stop dividing and differentiate? The phenomenon of lineage-specific progenitor cells undergoing a limited period of proliferation prior to terminal differentiation is a common theme in multicellular development. Despite this, little is understood about how these two events are co-ordinated during the normal schedule of development. I have studied the question of how proliferation and differentiation are co-regulated in the oligodendrocyte lineage in the rodent optic nerve. Oligodendrocytes are post-mitotic cells that myelinate axons in the vertebrate central nervous system. They develop from precursor cells whose maturation is controlled by a timer, which is an intrinsic property of the cells, that limits proliferation. The timer seems not to control the number of divisions the cell can undergo but rather the length of time during which divisions can occur. Significant effort has been devoted to understanding how the intracellular timer regulates oligodendrocyte development. The timer consists of two components that are modulated by distinct kinds of extracellular signals. Mitogens drive a timing component whose value increases as precursor cells continue to divide. Once this value exceeds a critical threshold, it signals that the proliferative period has elapsed, and hydrophobic signalling molecules trigger an effector component that elicits cell-cycle arrest and differentiation. The value of the timing component is determined by several intracellular molecules whose activities change as the timer runs. One of these molecules is the cell-cycle inhibitor p27: it accumulates in oligodendrocyte precursor cells as they proliferate in culture. When p27 expression is high the precursor cells are more likely to stop dividing and differentiate than when it is low. In oligodendrocyte precursor cells derived from mice that lack p27, the timer runs aberrantly and cell-cycle arrest and terminal differentiation are delayed. It is not understood how the molecular mechanics of the timer control oligodendrocyte development. Does the timer serve to arrest the cell-cycle, with differentiation following by default, or is cell-cycle arrest subordinate to the programme of terminal differentiation? These questions remain unanswered, largely because of a persistent inability to experimentally manipulate the genome of oligodendrocyte precursor cells. The present study was an attempt to overcome these problems and had two aims - first, to devise a reliable system for transfecting oligodendrocyte precursor cells and second, to determine whether the timer primarily controls the timing of cell-cycle arrest. I developed a new retroviral vector that co-expresses p27 and green fluorescent protein (GFP) in precursor cells. The use of GFP allows the identification of living precursor cells that over-express p27, which can then be followed over many days in culture. My findings support previous work showing that p27 plays a role in governing the timing of oligodendrocyte differentiation. They show that over-expression of p27 promotes oligodendrocyte differentiation by advancing the value of the timing component, although it does not promote differentiation if the effector component is inoperative. The cell-cycle time of precursor cells that over-express p27 is dramatically extended, but not stopped. It appears that a firm cell-cycle arrest and entry into a quiescent state may be required to elicit terminal differentiation.

No description available.

p16INK4a (p16) traps the cell at the restriction point of the cell cycle by binding to cyclin-dependent kinase 4/6 thus preventing the phosphorylation of the retinoblastoma protein (pRB). As p16 accumulates the cell stops dividing and becomes senescent. This study investigates the modulation of p16 function by the putative membrane protein ABCB5 and a group of five putative oncogenic microRNAs (oncomiRs). ABCB5 is a poorly characterised member of the B-subfamily of human ATP Binding Cassette transporters. ABCB5 is reportedly transcribed into four transcripts, one of which could potentially encode a full-length transporter (ABCB5fl) whilst a second could encode a half-transporter (ABCB5β). The other two transcripts (ABCB5α and ABCB5γ) could only encode short polypeptides. Exogenous expression of ABCB5fl and ABCB5β was achieved in HEK293T cells, but the recombinant protein expressed poorly and localised to the endoplasmic reticulum. Point mutations introduced into the ATP catalytic domain failed to improve expression levels suggesting that protein function was not deleterious to the cell. Exogenous expression in HEK293T cells also allowed commercial antibodies purportedly raised against ABCB5 isoforms to be tested. Several were found not to recognise ABCB5 necessitating re-interpretation of published data. However, one antibody recognised both ABCB5fl and ABCB5β, and was subsequently used to evaluate protein expression levels in other cell types.siRNA knockdown of ABCB5 in human mammary epithelial cells (HMECs) caused a concomitant reduction in p16 expression and an increase in cellular proliferation. Differential siRNAs and RT-qPCR analyses demonstrated ABCB5β to be the relevant transcript with respect to the reduction in p16 expression; however, no native ABCB5β protein was detected in HMECs. Together these data lead to the hypothesis that the ABCB5β transcript may act as a long noncoding RNA to regulate p16. Exogenous expression of each of five distinct putative oncomiRs in HMECs was found to increase cellular proliferation and, surprisingly, increase p16 expression. These results mirror a phenotype commonly observed in p16-positive basal-like breast cancer (BLBC), an aggressive form of breast cancer with poor prognosis and few treatment options. Bioinformatic analysis of the predicted target genes for these oncomiRs identified multiple transcriptional regulators of pRB. These predictions, together with the work performed in a cellular model of p16-positive BLBC, suggest that the oncomiRs may cause unrestricted cell proliferation by indirectly reducing transcription of the pRB gene, RB1. In the absence of pRB, p16 expression is induced via a previously reported oncogeneinduced senescence-like positive feedback loop. These data, and previously published observations, suggest that a similar mechanism may explain the basis of p16-positive BLBC.

Durante o desenvolvimento deste projeto adotou-se como estratégia o sequenciamento de ESTs, com a finalidade de encontrar mensagens relacionadas com desenvolvimento, metabolismo e principalmente amplificação/politenização em glândulas salivares de Rhynchosciara americana, um díptero (Sciarídeo) que apresenta cromossomos politênicos e amplificação gênica rigidamente regulada ao longo do desenvolvimento larval, tanto neste tecido quanto em outros. Um total de 8193 ESTs foi gerado, estas foram anotadas e categorizadas segundo os termos do Gene Ontology Consortium, proporcionando uma visão geral do status metabólico, como em um Northern eletrônico, de um ponto importante no desenvolvimento desta espécie, quando surgem amplificações gênicas específicas e a glândula salivar necessita secretar as proteínas do casulo. Outros frutos deste seqüenciamento foram a determinação de 91 polimorfismos e a criação de uma tabela de códon usage. Diversos ESTs foram identificados com potencial envolvimento com os endociclos observados neste tecido, destes, RaDup e RaMCM5 foram selecionados para estudo. Suas regiões genômicas foram isoladas e suas localizações cromossômicas foram identificadas, em relação a RaDup, toda a porção codificante de seu mensageiro e 12kb de DNA genômico contendo seu gene foram seqüenciados, revelando sua estrutura gênica. Anticorpos foram produzidos para detectar esta proteína, gerando evidências de sua participação tanto na replicação mitótica como nos endociclos presentes nas glândulas salivares. A localização cromossômica de RaDup é um dado muito interessante, pois pela primeira vez um pufe amplificado é relacionado com um gene regulatório. / In this work EST sequencing was used as strategy to find messages related to development, metabolism and polyteny/amplification in salivary glands of Rhynchosciara americana, a dipteran (Sciaridae) that shows in this tissue giant polytene chromosomes and gene amplification tightly regulated throughout development of the larvae. A total of 8193 EST sequences were generated, annotated and categorized using Gene Ontology Consortium terms, providing a general view of the metabolic status, like an electronic Northern, of an important point in development of the larvae, that shows where specific genes are amplified and the salivary gland needs to secrete the proteins to form the cocoon. Other data include determination of 91 SNPs and a statistic of codon usage. Several ESTs were identified with potential connection to endocicles, from these RaDup and RaMCM5 were selected for further studies. Both chromosomal loci were identified and genomic regions isolated, for RaDup the coding region of its mRNA and 12kb of genomic region were completely sequenced, revealing its gene structure, and antibodies were raised against this protein, making evident data about its involvement in replication in mitotic cells and in endocicles in salivary glands. About the chromosomal locus of RaDup, it becomes very interesting, because for the first time one amplified puff can be related to a regulatory gene.

Durante o desenvolvimento deste projeto adotou-se como estratégia o sequenciamento de ESTs, com a finalidade de encontrar mensagens relacionadas com desenvolvimento, metabolismo e principalmente amplificação/politenização em glândulas salivares de Rhynchosciara americana, um díptero (Sciarídeo) que apresenta cromossomos politênicos e amplificação gênica rigidamente regulada ao longo do desenvolvimento larval, tanto neste tecido quanto em outros. Um total de 8193 ESTs foi gerado, estas foram anotadas e categorizadas segundo os termos do Gene Ontology Consortium, proporcionando uma visão geral do status metabólico, como em um Northern eletrônico, de um ponto importante no desenvolvimento desta espécie, quando surgem amplificações gênicas específicas e a glândula salivar necessita secretar as proteínas do casulo. Outros frutos deste seqüenciamento foram a determinação de 91 polimorfismos e a criação de uma tabela de códon usage. Diversos ESTs foram identificados com potencial envolvimento com os endociclos observados neste tecido, destes, RaDup e RaMCM5 foram selecionados para estudo. Suas regiões genômicas foram isoladas e suas localizações cromossômicas foram identificadas, em relação a RaDup, toda a porção codificante de seu mensageiro e 12kb de DNA genômico contendo seu gene foram seqüenciados, revelando sua estrutura gênica. Anticorpos foram produzidos para detectar esta proteína, gerando evidências de sua participação tanto na replicação mitótica como nos endociclos presentes nas glândulas salivares. A localização cromossômica de RaDup é um dado muito interessante, pois pela primeira vez um pufe amplificado é relacionado com um gene regulatório. / In this work EST sequencing was used as strategy to find messages related to development, metabolism and polyteny/amplification in salivary glands of Rhynchosciara americana, a dipteran (Sciaridae) that shows in this tissue giant polytene chromosomes and gene amplification tightly regulated throughout development of the larvae. A total of 8193 EST sequences were generated, annotated and categorized using Gene Ontology Consortium terms, providing a general view of the metabolic status, like an electronic Northern, of an important point in development of the larvae, that shows where specific genes are amplified and the salivary gland needs to secrete the proteins to form the cocoon. Other data include determination of 91 SNPs and a statistic of codon usage. Several ESTs were identified with potential connection to endocicles, from these RaDup and RaMCM5 were selected for further studies. Both chromosomal loci were identified and genomic regions isolated, for RaDup the coding region of its mRNA and 12kb of genomic region were completely sequenced, revealing its gene structure, and antibodies were raised against this protein, making evident data about its involvement in replication in mitotic cells and in endocicles in salivary glands. About the chromosomal locus of RaDup, it becomes very interesting, because for the first time one amplified puff can be related to a regulatory gene.