Thesis (M.Res.(Med.))--University of Hong Kong, 2009. / Includes bibliographical references (p. 105-124).

Mutations in the beta-globin (β-globin) gene cause beta-thalassaemia (β-thalassaemia).The screening strategy for β-thalassaemiais based on the value of mean corpuscular volume (MCV) from the complete blood count (CBC) data. Current laboratory practice considers blood samples with MCV higher than 80fL as normal. No further assessment will be done on these samples. However, there are clinically silent β-globin gene mutations with MCV higher than 80fL, for example, heterozygous haemoglobin E (HbE). The importance of finding out this kind of mutations is due to the serious outcome when they occur together with classic β thalassaemia mutations in compound heterozygous states, which may produce a condition mimicking β thalassaemia major. The method used to recognize the presence of clinically silent β-globin gene mutations should be robust and with high sensitivity. High resolution melting (HRM) is a suitable technique to screen gene mutations. It is fast and convenient. The process is completed in a closed system without any post PCR manipulation. The sensitivity is up to a single nucleotide change. Using HRM for mutations screening followed by confirmation with sequencing can reduce time and cost of testing clinically silent β-globin gene mutations on a large scale. This study first shows the ability of HRM in detecting various types of β-globin gene mutations. The technique is then applied to detect clinically silent β-globin gene mutations in a group of high school students with normal CBC data. Mutations with different clinically significance were found. The frequency of mutation found in the samples of the study suggests that screening for β-globin gene mutation may be worthwhile in subjects with MCV higher than 80fL. / published_or_final_version / Pathology / Master / Master of Medical Sciences

The thalassaemias are the commonest monogenic disorders in the world population. They occur at a particularly high frequency in Mediterranean regions and Southeast Asia, which cause a massive public health problem. In Hong Kong, the prevalence of heterozygous carriers of α or β thalassaemia mutations is approximately 8% [1]. Thalassaemia is characterized by the reduced synthesis of one or more normal globin chains. This causes globin chain imbalance and finally leads to hypochromic microcytic anaemia. Different types of thalassaemia are named according to the under-produced chains. The majority of thalassaemia can be diagnosed by basic haematologic profiles and simple phenotypic techniques. However, in some cases of thalassaemia the diagnosis are not apparent after routine laboratory investigations. To arrive at a diagnosis which is important for antenatal diagnosis and genetic counseling, it is necessary to use molecular approaches. In this study, 25 patients with microcytosis, normal phenotypic haemoglobin study results and without iron deficiency were analyzed retrospectively. This cohort of patients was suspected to have occult or masked thalassaemia. DNA was extracted from archive samples and further investigated by alpha multiplex gap polymerase chain reaction (α multiplex gap-PCR), alpha amplification refractory mutation system (α ARMS) and direct nucleotide sequencing of globin genes for the detection of possible underlying globin gene mutations. Results indicated that 60% of these cases with microcytosis were occult and silent αthalassaemia caused by deletional or non-deletional mutations. Maskedβthalassaemia due to co-existing δ thalassaemia or variants or normal Hb A2 β thalassaemia due to mild β globin gene mutations were not detected in the cohort. Forty percent of these cases of microcytosis remained unexplained, which await further molecular testing. / published_or_final_version / Pathology / Master / Master of Medical Sciences

The two key concepts of Neo-Darwinian evolution theory are genotype and phenotype. Genotype is defined as the genetic constitution of an organism and phenotype refers to the observable characteristics of that organism. Schematically the relationship between genotype and phenotype can be settled as Genotype + Environment + Random Variation \(\underrightarrow{\text{yields}}\) Phenotype. This schematic representation has led to the fundamental problem of given the interactions of the genes and environment, up to what extent is possible to establish a relationship between gene structure and function to the phenotype (Weatherall, D. J., et. al., (2001)). Since R. A. Fisher establishing the basis of quantitative trait loci up to the work of Subramanian, et. al., (1995) gene set enrichment analysis, several statistical methods have been devoted to answer this question, some with more success and scientific repercussion than others. In this work we attempt to answer to this question by delineating the biological mechanisms driven by the genes that are characterize the differences and actions of the phenotypes of interest. Our contribution resides on two pillars: we present an alternative way to conceive gene expression measurements and the use of functional gene set annotation systems as guided prior knowledge of the biological mechanisms that drive the phenotype of interest. Based on these two pillars we propose a method to infer the Functional Network Inference and an alternative method to perform expression Quantitative Trait Loci analysis. (eQTL) From the Functional Network Inference method we are able to identify what mechanisms describe the behavior of most of the, there fore establishing its importance. The alternative method to perform eQTL analysis that we present, is more direct way to associated variations at a sequence level and the biological mechanisms it affects. With this proposal we attempt to address two important issues of traditional eQTL analysis: statistical power and biological implications.

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Molecular Biology / Doctoral / Doctor of Philosophy

Changes in the pituitary content of prolactin (PRL) and in the ratio of immunoreactive forms of PRL were measured during a reproductive cycle in turkey hens. Low levels of PRL were observed in pituitary glands from sexually immature, out-of-lay and moulting hens. Higher levels were present during the egg laying period and the highest levels were detected in hens expressing incubating behaviour. Two immunoreactive bands of apparent molecular weights of 24 and 27 kDa were visualised on western blots, corresponding to the non-glycosylated (NG-) and glycosylated (G-) forms of PRL, respectively. The percentage of G-PRL was about 60 in sexually immature and egg laying hens. In pituitaries from incubating, out-of-lay and moulting hens the percentage of G-PRL was about 70, 38 and 33, respectively. Thus, higher percentages of G-isoforms (27 kDa) were associated with high levels of total PRL and Iower percentages were associated with low levels of PRL content in the pituitary gland. Digestion of the isoforms with N-glycosidase F resulted in a single band with an apparent molecular weight of 24 kDa. Partial deglycosylation was achieved using neuraminidase, whereas digestion with O-glycosidase had no apparent effect on the isoforms. Thus, G-PRL has N-linked carbohydrates containing sialic acid. In order to study the in vitro release of the PRL isoforms, pituitary glands from turkeys at various physiological stages were stimulated by cVIP in a perifusion system. Total PRL content and the ratio of immunoreactive PRL isoforms in the perifusate were monitored. All the perifused pituitaries responded to cVIP stimulation by increasing the release of PRL. Two immunoreactive bands corresponding to the ones detected in pituitary extracts were detected by western blotting. The G-PRL (27 kDa) was predominant in samples from egg laying and incubating hens and the NG-PRL (24 kDa) was predominant in samples from out-of-lay and moulting hens. No change in the ratio of isoforms released was / A competitive reverse transcription polymerase chain reaction assay was developed in order to semi-quantify the PRL mRNA in individual pituitary glands from turkey embryos and poults. Pituitary content and plasma levels of PRL were also monitored, and the PRL isoforms present in the pituitary gland were detected. The levels of PRL mRNA remained low until five days before hatching, increased until the day of hatch, plateaued during the first three days of age and significantly increased at two weeks of age. Similar changes were observed in pituitary content and plasma concentrations of PRL which were highly correlated. Two immunoreactive bands corresponding to the NG- and G-PRL detected in adult pituitary gland were visualised on western blots. The percentage of G-PRL in pituitary glands were 31.5, 48.6, 48.0 and 56.2 at 22 and 27 days of incubation and at I and 7 days of age, respectively. Thus, higher percentages of G-PRL (27 kDa) were associated with higher levels of total PRL in the pituitary gland.

TCP genes comprise a large family of genes that have been implicated in a diverse range of plant developmental pathways ranging from lateral branching (Doebley et al, 1997) to organ symmetry (Luo et al, 1999) and leaf curvature (Nath et al, 2003; Palatnik et al, 2003). I studied three closely related Arabidopsis TCP genes, one of which was recovered in an enhancer trap screen to identify downstream targets of the regulator of inflorescence architecture, BREVIPEDICELLUS (Douglas and Riggs, 2005). The enhancer trap marker line served as a reporter for TCP15 expression. Data mining has revealed a possible link between TCP15 and the hormone auxin. Using the DR5::GUS molecular reporter for auxin accumulation I found that TCP15 and the related TCP14 genes limit auxin maxima in seedling and reproductive tissues and that auxin transport is necessary for correct TCP15 expression. The closely related TCP8 gene was found to regulate leaf shape as demonstrated by decreased leaf index values. The rounder leaves of tcp8 plants also exhibited increased adaxial trichome and stomatal densities resulting in significantly decreased spacing between adjacent cells. tcp8 leaves showed increased serration density suggesting that TCP8 limits marginal outgrowth. Vein patterning was also perturbed in the mutants. Vein loops were rounder and smaller, and decreased loop subdivision indicated that vein patterning is retarded in the mutant. TCP8 evokes organ-specific effects on vascular patterning as mutant rosette leaves showed increased vascular complexity, whereas mutant cauline leaves showed decreased vein complexity. These results suggest that TCP8 is necessary for correct leaf development. The Arabidopsis genome contains 24 TCP genes, many of which have not been characterized. Studies of these genes will lead to the identification of additional factors necessary to control plant architecture and enable us to optimize plant growth and yield using genetic engineering.

A principal concern in crop agriculture is yield, and a key factor for crop growth is the availability of nitrogen. The large amount of nitrogen fertilizer required by plants is a major cost to farmers. Moreover, environmental issues such as groundwater pollution arise from the utilization of nitrogen fertilizers. Therefore, improvement in the nitrogen use efficiency (NUE) of plants is of urgent importance for sustainable and efficient agriculture. Although hybrid varieties have increased crop yields in low N conditions, the molecular mechanism of plant adaptation to N stress is not completely understood. Herein, the study of responses to N limitations in the natural signalling pathways of model plants facilitates the understanding of complex responses in plants to N stress, and this information can be used to further improve NUE. In this research, the transcriptomes of three model plants Arabidopsis, maize, and rice were compared under diverse N growth conditions. An evaluation of the response of the three plants to varying N levels was also conducted. From a statistical point of view, three distinct methods of detecting differential expression were utilized to reduce the likelihood of false positives due to the tens of thousands of genes simultaneously studied. Furthermore, the performance of three statistical approaches was compared during detection of the N-responsive genes. Finally, a clustering analysis (agglomerative hierarchical clustering) was performed on the genes that significantly responded to N levels as identified by a more biologically intuitive method called Rank Products (RP).

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