Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 187-217) Also available in print.

Mospd3, a gene located on mouse chromosome 5, was identified in a gene trap screen in ES cells. The gene trap vector integration in multiple copies into the putative promoter of the gene, resulted in a loss of expression of Mospd3 at the trapped allele. In mice generated from ES cells carrying the vector integration it was found that the lack of Mospd3 expression resulted in the death of a proportion of the homozygote mutants within the first day after birth. Homozygote neonates exhibited a thinning of the right ventricular free heart wall which resembles other mouse mutant phenotypes as well as human congenital heart defects caused by a loss of desmosome and adherens junction mediated cell adhesion between cardiomyocytes. The protein encoded by Mospd3, contains an N-terminal Major Sperm Protein (MSP) domain implicated as a mediator of protein- protein interactions, as well as a two C-terminal transmembrane domains. Both, protein structure and phenotypic similarities with defects in desmosomal and adherens junction proteins suggests that Mospd proteins might play a role in cell adhesion and maintaining the structural integrity of the heart. The phenotype of Mospd3 mutants was highly dependent on genetic background, which led us to speculate that there might be genetic redundancy between Mospd3 and its closest family member the X-linked Mospd1. The aims of this thesis were to generate tools to better understand the function of the Mospd gene family in cardiac development as well as assessing genetic redundancy between Mospd1 and Mospd3. A conditional gene targeting strategy was designed for both Mospd genes. Large genomic regions of the Mospd1 and Mospd3 loci were subcloned from bacterial artificial chromosomes (BACs) and using a recombineering approach, loxP sites and a drug selection cassette (neomycin) were placed in precise locations surrounding the MSP domain of both genes. The conditional targeting vectors were electroporated into both CGR8 and E14 ES cells and homologous recombinant clones were identified at a frequency of 2% and 1.3% for Mospd1 and Mospd3 respectively. Five euploid targeted clones for both Mospd1 and Mospd3 have been generated. Transient expression of Cre recombinase in ES cells carrying the conditional Mospd1 allele was used to delete the one copy of this X-linked gene. Phenotypic characterisation of this null ES cell line revealed that Mospd1 is neither essential for ES cell viability and self-renewal, nor for the early differentiation of these cells towards a cardiac fate. In order to investigate the mechanism of action of Mospd proteins, specific polyclonal antibodies were generated to detect either Mospd1 or Mospd3. These antibodies were purified and tested by western blotting using COS7 cells overexpressing either Mospd protein as well as mouse tissue lysates. Whilst the antibodies were found to detect the proteins and differentiate between Mospd1 and Mospd3, they showed insufficient purification to be used in co-localisation and co-immunoprecipitation experiments to identify interacting proteins and determine whether Mospd proteins are involved in cell adhesion complexes. Monoclonal antibodies were subsequently generated and initial western blotting experiments showed promising results, indicating that these antibodies may be better suited for immunohistochemical analysis of Mospd proteins.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Paediatrics / Master / Master of Philosophy

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

The cannabinoid system is involved in many functions of mammalian brain, such as learning and memory, pain perception and 'locomotion. The "brain type" cannabinoid receptor CB 1 is one of the key elements of the cannabinoid system. In this Thesis, some aspects of the neurobiology of mouse CB 1 are described. CB 1 mRNA distribution was analysed by single and double in situ hybridization (ISH), revealing the expression of the receptor in specific neuronal subpopulations. This expression pattern suggests many putative functional cross-talks between the cannabinoid system and other signalling molecules in the brain, such as glutamate, GABA, cholecystokinin and nitric oxide (NO). The putative functional interactions of the cannabinoid system with the NO pathway was studied by pharmacological treatment of neuronal NO synthase (nNOS) mutant mice with the CBI agonist A9-tetrahydrocannabinol (A9-THC). The results showed that nNOS is necessary for some central effects of A9-THC. Moreover, ISH analysis revealed. that nNOS-deficient mice contain levels of CBI lower than normal in selected brain regions. A "conditional" targeting approach was developed to gain insights into the specific functions of CB 1 in mouse brain. By gene targeting experiments, two mutant lines were obtained. The "Flox CB 1" mouse line, containing the whole open reading frame of CB I flanked by two loxP sites will be the key tool for the generation of mouse mutants with a spatiotemporal-restricted deletion of CB I. The "CBN" mice, carrying a "null" mutation of CB 1, were used for a study aimed to clarify some aspects of the in vitro neuroprotective activity of cannabinoids and, in particular, the involvement of CB 1. In vitro oxidative stress assays were performed on cell lines and on primary neuronal cultures derived from homozygous CBN/CBN mice and wild type littermates. The results indicate a differential protective activity of cannabinoids on cell lines and primary cultures. However, CBI does not appear to be involved in the in vitro leuroprotective effects of cannabinoids.

The Lactococcus lactis Ll.LtrB group II intron retrohomes by reverse splicing into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it as a primer for reverse transcription of the inserted intron RNA. The protein and intron RNA function in a ribonucleoprotein particle, with much of the DNA target sequence recognized by base pairing of the intron RNA. Consequently, Ll.LtrB introns can be reprogrammed to insert into specific or random DNA sites by substituting specific or random nucleotide residues in the intron RNA. Here, I show that an Escherichia coli gene disruption library obtained using randomly inserted Ll.LtrB introns contains most viable E. coli gene disruptions. Further, each inserted intron is targeted to a specific site by its unique base-pairing regions, and in most cases, could be recovered by PCR and used unmodified to obtain the desired single disruptant. I also demonstrate that Ll.LtrB introns can be used for efficient gene targeting in a variety of Gram-negative and positive bacteria, including E. coli, Pseudomonas aeruginosa, Agrobacterium tumefaciens, Bacillus subtilis, and Staphylococcus aureus. Ll.LtrB introns expressed from a broad-host-range vector or an E. coli-S. aureus shuttle vector yielded targeted disruptions in a variety of test genes in these organisms at frequencies of 1-100% without selection. By using an Ll.LtrB intron that integrates in the sense orientation relative to target gene transcription and thus could be removed by RNA splicing, I disrupted the essential gene hsa in S. aureus. Because the splicing of the Ll.LtrB intron by the intron-encoded protein is temperature-sensitive, this method yields a conditional hsa disruptant that grows at 32oC, but not at 43oC. Finally, I developed high-throughput screens to identify E. coli genes that affect either the splicing or retrohoming of the Ll.LtrB intron. By using these screens, I identified fourteen mutants in a variety of genes that have decreased intron retrohoming efficiencies and additional mutants that have increased intron retrohoming efficiencies, in some cases apparently resulting from increased stability of the intron RNA. / text

Non-viral vectors have been used to deliver therapeutic genes to treat different diseases. There are a variety of non-viral vectors such as liposomes, cationic polymers and peptides. Among all, pH responsive peptides showed excellent DNA transfection efficiency in many types of cell. These peptides are capable of changing their structural conformation as pH decreases, adopting a disordered structure which can destabilize endosomal membrane and therefore enhancing the release of DNA from endosomes into cytosol. Traditional pH responsive histidine-rich peptides showed good DNA transfection efficiency and low toxicity to the cells when compared with other non-viral vectors. However, their low pKa value restricted these peptides to be protonated only at late endosomal stage, in which DNA is extremely susceptible to endosomal degradation. This hindered the DNA to be released to the cytosol efficiently and therefore reduced DNA transfection efficiency. In response to this, it is of great interest to probe into the insertion of either 2,3-diaminopropionic acid (Dap) or methylated-2,3-diaminopropionic acid Dap(Me) to the peptide as alternative pH sensitive components. The pKa values for both Dap and Dap(Me) peptides are higher than that of histidine. It is anticipated that the higher pKa value, the protonation of peptide could be happened at an earlier stage of endosomal maturation. Such protonation of peptide destabilizes the endosome membrane rapidly, causing the release of DNA to the cytosol effectively and hence improving DNA transfection efficiency. In this experiment, LADap(Me)4-L1 peptide was the optimal candidate within the series. It showed good DNA transfection efficiency and cell viability in A549 cells among all Dap and Dap(Me) peptides. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

Spermatogenesis is regulated by steroid hormones which induce expression of various genes responsible for the growth, proliferation and differentiation of spermatogonia to form mature haploid spermatozoa. The surrounding somatic cells including Leydig and Sertoli cells support the whole process in vivo. Previously, we used the post-vitamin A treated vitamin-A-deficient (PVA-VAD) rat model to study spermatogenesis, and identified 24 genes that are differentially up-regulated after retinol treatment. Vad1.2 is one of the up-regulated transcripts expressed in the rat testis from postnatal day 25. Vad1.2 transcript is localized to the round and elongating spermatids in the adult mouse testis. In silico analyses showed that Vad1.2 transcript is down-regulated in patients with teratozoospermia and non-obstructive azoospermia, suggesting that Vad1.2 may have important roles in spermatogenesis. However, how Vad1.2 affects spermatogenesis remains unclear. Therefore, the present study was designed to study the functional roles of Vad1.2 protein in mice using gene targeting approach and investigate the molecular changes in mice with Vad1.2 deficiency. Vad1.2 polyclonal antibody was raised against the full-length mouse Vad1.2 recombination protein and affinity purified. Vad1.2 protein was localized to the cytoplasm and flagellum of condensing spermatids, specifically to the fibrous sheath (FS) in cauda epididymal spermatozoa. Vad1.2 conditional knockout vector was constructed and used to generate Vad1.2 null mice. Vad1.2-/- male mice developed normally but were subfertile with reduced sperm count and motility. Vad1.2-/- male mice had smaller testis and higher incident of sloughing of immature germ cells into the seminiferous lumens when compared to the wild-type. Yet, the rates of germ cell proliferation and apoptosis were similar between the wild-type and the mutant testis. Interestingly approximately 50% of the mutant cauda epididymal spermatozoa showed deformed flagella and demonstrated structural defects typically associated with bending of flagellum at the principal piece or at the midpiece/principal piece junctions. The acrosome, nucleus and mitochondrial sheath of these spermatozoa appeared normal, while the flagellum displayed structural abnormalities including deformation of the two longitudinal columns of the FS and disruption of a portion of FS, suggesting that Vad1.2 might be involved in the biogenesis of FS in spermatogenesis. Furthermore, Vad1.2 interacted with Akap4 in vivo, and the two proteins were co-immunoprecipitated from the testis or cauda epididymal spermatozoa lysates. Akap4 and Vad1.2 were localized to the tail region of the testicular spermatids and cauda epididymal spermatozoa. The expression levels of pro- and mature Akap4 in Vad1.2-/- testes were markedly increased when compared with the wild-type mice. However, a significant decrease of Akap4 was found in the mutant cauda epididymal spermatozoa, suggesting that most of the mature Akap4 failed to incorporate into the FS. Taken together, Vad1.2 plays an important role in spermatogenesis and Vad1.2 deficiency leads to subfertility in mice with the deformed flagella in mature spermatozoa. Further studies on the regulation of FS formation may uncover the underlying molecular changes associated with Vad1.2 deficiency, and may provide fundamental information for treatment of infertile patients with FS defect in the spermatozoa. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy