published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

TSPYL2 (Testis-specific Y-encoded-like protein 2) is a member of the Nucleosome Assembly Protein (NAP) superfamily. It is a nuclear protein expressed in the cerebral cortex and the hippocampus. Our group has generated Tspyl2 knockout (Tspyl2m) mice, which are deficit in hippocampal long-term potentiation (LTP) with downregulation of Nr2a and Nr2b. Since Nr2a and Nr2b, subunits of the N-Methyl-D-Aspartate receptors, and hippocampal LTP are important in learning and memory, our Tspyl2m mice are likely to have behavioral deficits particularly in those related to memory. TSPYL2 could also affect LTP via CREB-dependent gene expression, since other NAP members have shown interaction with CBP/p300 - transcriptional co-activators of CREB which are well-known to be involved in memory formation. Furthermore, TSPYL2 may be linked to X-linked mental retardation (XLMR), since it is located at Xp11.2, a region with a high density of XLMR genes; and one of its interacting partners, CASK, is a XLMR gene. This thesis examines the three issues mentioned above. First, to characterize the behavior of our Tspyl2m mice, a behavioral test battery including open-field with amphetamine challenge, social interaction, prepulse inhibition and fear conditioning were conducted. Second, to examine the role of TSPYL2 on CREB-dependent gene expression, I first examined the subcellular localization of HA-TSPYL2 and endogenous CBP, p300 and pCREB in HEK293 cells. Then the interactions between TSPYL2 and CBP were tested by mammalian two-hybrid assay and co-immunoprecipitation. Thereafter, luciferase assay was used to measure CRE-luc activity in HEK293 and NG108-15 cells with overexpression and knockdown of TSPYL2. Third, to investigate the potential role of TSPYL2 on XLMR, a mutation analysis on the TSPYL2 gene was conducted with a cohort of 82 male patients with unexplained mental retardation. The analysis included examining the methylation on the TSPYL2 upstream sequence, DNA sequencing of the TSPYL2 exons, and in silico splice site analysis of the identified sequence variants. In the behavioral test battery, our Tspyl2m mice were normal in social ability, but showed enhanced hyperlocomotion after amphetamine injection, and deficit in prepulse inhibition and cued fear conditioning. When expressed in HEK293 cells, HA-TSPYL2 colocalized completely with endogenous CBP, but not with p300 and pCREB. In mammalian two-hybrid assay, pVP16(AD)-TSPYL2 interacted with GAL4(DBD)-CBP; however, HA-TSPYL2 did not immunoprecipitate with CBP. The luciferase assay data indicated that TSPYL2 suppressed the transcription of CREB-target genes. Lastly, no methylation was detected in the target sites in the TSPYL2 upstream sequence. Seven TSPYL2 sequence variations being identified were not deleterious as predicted by splice site analysis. To sum up, our Tspyl2m mice were deficit in cued fear memory, a form of associative memory. Moreover, they resembled the glutamatergic antagonist-induced schizophrenic rodent models in having enhanced hyperlocomotion after amphetamine injection, and deficit in prepulse inhibition. TSPYL2 interacted with CBP and suppressed the CRE-luc activity. The importance of TSPYL2 in XLMR has yet to be determined by larger studies. I propose that TSPYL2 represses CREB-dependent gene expression via sequestration of CBP as one of the possible mechanisms of how TSPYL2 causes various behavioral phenotypes. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

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Arabinogalactan proteins (AGPs) are highly glycosylated cell wall proteins specific to plants. AGPs have been implicated in almost all aspects of plant development and defense responses, nevertheless, most of such studies are correlative. To define the specific functions of individual AGPs, direct evidence from analyses of genetic knockout mutants of individual AGPs is required. Up to now, only a few AGPs have been demonstrated to have defined functions by mutant analyses. This dissertation identified a non-classical AGP (AGP31), described its expression and characterized the null mutant of AGP31 in Arabidopsis. In agp31 mutant, microarray analyses revealed that the expressions of genes encoding a subset of seed storage proteins (SSP): CRU3, CRA1 and OLEOSIN2 were induced. Further analysis showed that induction by agp31 knockout was specific to these three SSP genes, indicating a novel pathway to regulate the SSP gene expression. Comprehensive characterizations of AGP31 were carried out. Yariv reagent staining and monosaccharide analysis of purified AGP31 showed that AGP31 was a bona fide galactose-rich AGP. The cell wall localization of AGP31 was confirmed by expression of an AGP31::eGFP fusion protein. AGP31 promoter-GUS reporter gene analysis showed that AGP31 was expressed in the vascular bundle throughout the plant, except in the flower. In the flower, it was expressed throughout the pistil except in the stigma. Detailed analysis showed that GUS staining occurred in all cell types in the vascular bundle of roots, while GUS staining was restricted to phloem cells in the inflorescence stem. AGP31 mRNA was down-regulated by several stress treatments, including wounding, methyl jasmonic acid (MeJA) and abscisic acid (ABA). In response to MeJA treatment of whole seedlings, AGP31 mRNA level decreased to about 30% of its original level within 8 hr and almost returned to its original level after 24 hr. Nuclei run-on assay showed that the down-regulation of AGP31 mRNA upon MeJA treatment was due to reduced transcription. The strong preferential expression in vascular tissues and negative regulations by MeJA and ABA suggest that AGP31 may be involved in vascular tissue function both during development and the defense response.

The current democratization developments and the need to adequately protect freedom of expression and media freedom in Africa have ushered in significant constitutional and legislative changes in the continent. As such, the adoption of the African Charter on Human and Peoples' Rights constitutes significant machinery for entrenching the liberty of the individuals and a trend in the fight against human rights violations, but how effective are these changes, at the regional and local levels? This thesis critically analyses these developments with a view to finding new constitutional and legislative provisions that facilitate freedom of expression and media freedom within the context of Article 9 of the Africa Charter. While tracing the history of human rights in Africa, the thesis articulates the importance of freedom of expression and consensus democracy in Africa's political and cultural traditions, the early socio-political and ideological history, and its colonial past. Furthermore, the thesis highlights Africa's entrenched political and cultural traditions that promote human rights in general and freedom of expression in particular, although some African communitarian principles might be considered anachronistic and anti-democratic. This research is concerned about the impact of African governments' criminal penalties for defamatory statements and policies restricting the legitimate exercise of the right to freedom of expression. These coercive measures in light of the intolerant culture in African politics are often used to deprive citizens and the media their right to freedom of expression and valuable information which merited legitimate public concern. While we acknowledge that media activities and freedom of expression may be restricted in order to protect reputation, private life, public order, public health, public morality, public safety and national security, as enshrined in Article 27 (2) of the African Charter, the thesis is, however, concerned at the broad reach of several of the restrictive measures that have been enacted by arbitrary governments in which there is a very limited basis of public debate or competitive politics to buttress the freedom of the media. On the attainment of political independence, most of the African states adopted constitutions enshrining bills of rights justiciable by the courts. Despite these bills of rights, many of the African states have been guilty of gargantuan crimes and human rights violations. Despite the ratification of international and regional instruments dealing with the right to freedom of expression and information, African political leaders continue to misapply and misinterpret Article 9 (2) of the ACHPR because of its vagueness and continue to rely on criminal defamation statutes and the like to suppress critical and dissenting views. These broadly phrased prohibitions encouraged by the poor drafting of Article 9 (2) of the Charter, criminalise the legitimate exercise of freedom of expression and have a real “chilling effect” on debate on matters of public interest. The main conclusion of the thesis is that while most recent African constitutional developments offer promises of free expression and democratic accountability, there are persistent problems that need to be addressed as part of the overall democratization process. For example, there is the need to abrogate anachronistic criminal defamation statutes and insult laws in many African countries, put an end to the use of extralegal and coercive measures in the state's relation with the media, as well as a need for opposition political parties' contribution in parliament without government interference. Judicial independence, awareness of international standards on freedom of expression and information, equitable access to government media, journalistic morality and professionalism are other problems highlighted in the thesis.

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published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy