Phagocytosis is an innate immune response that is paramount in the clearance of pathogenic particles. Recognition of target particles by phagocytic receptors expressed on phagocytes induces modifications in the underlying actin cytoskeleton to form pseudopods that encircle and internalize the target particle into a membrane bound organelle called the phagosome. The nascent phagosome undergoes a maturation sequence that is characterized by substantial remodeling of the membrane and its luminal contents through interactions with components of the endocytic pathway, culminating in an acidic and hydrolytic organelle capable of digesting and elminating pathogens. Phagosome maturation is a complicated pathway that involves many protein and lipid signaling molecules. Several factors that influence phagosome maturation particularly the participation of pH, lysosome-associated membrane proteins-1 and –2, cholesterol, in addition to the survival and escape mechanisms used by, Burkholderica cenocepacia were explored. All three tenets are essential for phagosome maturation, although each factor has different mechanistic consequences. Acidification alters Rab5 activation, while ablation of LAMPs and accumulation of cholesterol interferes with various aspects of Rab 7 turnover in phagosomes and/or endosome membranes. Moreover, Burkholderia cenocepacia, an intracellular pathogen, inactivates Rab7 on phagosome membranes from within the vacuole lumen. Herein, mechanisms that govern phagosome maturation are explored and several molecules are added to the long list of essential players in this complicated pathway.

Phagocytosis is an innate immune response that is paramount in the clearance of pathogenic particles. Recognition of target particles by phagocytic receptors expressed on phagocytes induces modifications in the underlying actin cytoskeleton to form pseudopods that encircle and internalize the target particle into a membrane bound organelle called the phagosome. The nascent phagosome undergoes a maturation sequence that is characterized by substantial remodeling of the membrane and its luminal contents through interactions with components of the endocytic pathway, culminating in an acidic and hydrolytic organelle capable of digesting and elminating pathogens. Phagosome maturation is a complicated pathway that involves many protein and lipid signaling molecules. Several factors that influence phagosome maturation particularly the participation of pH, lysosome-associated membrane proteins-1 and –2, cholesterol, in addition to the survival and escape mechanisms used by, Burkholderica cenocepacia were explored. All three tenets are essential for phagosome maturation, although each factor has different mechanistic consequences. Acidification alters Rab5 activation, while ablation of LAMPs and accumulation of cholesterol interferes with various aspects of Rab 7 turnover in phagosomes and/or endosome membranes. Moreover, Burkholderia cenocepacia, an intracellular pathogen, inactivates Rab7 on phagosome membranes from within the vacuole lumen. Herein, mechanisms that govern phagosome maturation are explored and several molecules are added to the long list of essential players in this complicated pathway.

Most bacteriophage escape the confines of the host bacterium by compromising the integrity of its cell wall, an event that results in rupture (lysis) of the cell. The lysis strategy of bacteriophage Qβ is inhibition of cell wall biosynthesis while the cell is growing. To elicit lysis, the maturation protein (A2) of Qβ inhibits the catalytic activity of MurA, an essential, induced fit enzyme in the cell wall biosynthetic pathway. Consequent lysis releases progeny phage into the environment. The research in this dissertation addresses how lysis timing is integrated into Qβ’s life cycle and discerns the molecular basis of the lytic event. Working off the notion that, as displayed by the mature virion, A2 inhibits MurA, we developed an in vivo bioassay to resolve the amount of inhibitory A2 during infection. We found that the amount of free A2 is vastly greater than the amount of virion-associated A2 and that both forms inhibit MurA. Additionally, the amount of A2 correlates to lysis time and the burst size, as mutant Qβ with upregulated expression of A2 (Qβpor) elicit host cell lysis faster and release fewer mature virions than with the wildtype level of A2. This further suggests that protection from Qβ lysis afforded by MurAL138Q is due to perturbed affinity between A2 and MurA. Yeast two-hybrid analysis supports that A2 and MurAL138Q interact with weaker affinity by rendering small colonies compared to yeast containing interacting A2-MurAwt. Scanning mutagenesis of MurA’s surface near L138 identified residues that may be important for A2 contact in the inhibitory complex. Potentially important residues map to a contiguous area on the surface of MurA that spans both lobes on the flexible loop face of the enzyme, suggesting that A2 prevents the induced fit mechanism of MurA in an uncompetitive manner. Subsequent truncation analysis reveals that the aminoterminal half of A2 is sufficient to mediate host cell lysis. Together, these findings insinuate a model in which the amino-terminus of free A2 interacts with, and inhibits MurA. Then, when the infected cell initiates division, septal catastrophe ensues causing the cell to lyse and liberate progeny bacteriophage Qβ.

Prenatal mortality in the swine ranges from 30%~40%. Little is known about genes that involve in the preovulation events that the initiation of swine oocyte development. The main objective of this study was to utilize suppression subtractive hybridization¡]SSH¡^to delineate the differential gene expression between in vitro culture mature and antral-follicle oocytes of swine development. The knowledge of genes and their accumulated mRNA is essential to better understand the mechanisms involved in the oocyte maturation and the survival of the in vitro produced embryos. Porcine ovaries obtained from the slaughterhouse were used to collect oocytes from follicle with a diameter ≥ 3 mm. After in vitro mature for two days, oocytes with first polar body were subjected to as the testers and were lysed for mRNA extraction. Pools of 26 denuded oocytes without culture were submitted to suppressive subtraction hybridization (SSH) as the drivers. Forward and reverse subtractions were performed to identify candidate genes differentially expressed between in vitro culture mature and primordial-follicle oocytes. A total of one hundred and thirty-five differential expressed plasmid clones were sequenced, and each was analyzed by BLAST programs. Of these transcripts, 40 clones were subjected to differential screening by a dot blot cDNA array. We identified three genes like zona pellucida glycoprotein (ZP1), 1-aminocyclopropane-1-carboxylate synthase (ACS), and death associated protein 5¡]DAP 5¡^while other numerous clones remain novel. The in vivo functions of the genes remain further investigation.

The newly discovered oocyte-derived growth factor BMP15, like GDF9, is a member of the transforming growth factor-£] (TGF-£]) superfamily. To our knowledge, however, the expression and function of BMP15 in ovarian tissues has not yet been studied in the pig. We asked whether the relative abundance (RA) of BMP15 mRNA changes in oocytes and follicular cells during pre-ovulatory period or culture of cumulus-oocyte-complexs (COCs) in vitro. Denuded oocytes, cumulus cells and COCs were isolated from growing and pre-ovulatory follicles. Total RNA was extracted from the cells, and reverse transcriptase polymerase chain (RT-PCR) was carried out using specific oligo-nucleotide primers. Expansion of COCs was firstly observed at 18 h, when this period we found BMP15 mRNA expression obviously. But when we culture of denuded oocytes instead of COCs, BMP15 mRNA didn¡¦t express throughout the period. We also detected GDF9 and BMP15 mRNA in pig embryonic stage and in differentiation stage of pig stem cell. GDF9 mRNA level continued to express after fertilization, but BMP15 mRNA didn¡¦t appear. We also added BMP15 antibody against Expanding of COCs. The present results support the concept that BMP15 is a key mediator of oocyte-enabled cumulus expansion in pig.

The objectives of this study were to investigate the characteristics and mechanism of gonad maturation including identified maturation inducing steroids and its physiological role in the final oocyte maturation (FOM) in yellowfin porgy. yellowfin porgy, Acanthopagrus latus are functional male for the first year of life but begin to change sex during the second year. Only 30~50% of cultured yellowfin porgy are change into females in 2-year-old, while 80% fish become females at 3-year-old. Vitellogenesis were observe between August and November and have the highest gonadosomatic index (GSI %) at 6.88 % and 14.35 % in 2 and 3-year-old female yellowfin porgy respectively. Highest serum levels of T occurred simultaneously during spawning season in one and two years of age in functional male, while highest level concentration E2 were observed at the same time in two and three years old female fish. GSI increased 1.5-fold during FOM and reach 2-fold at germinal vesicle breakdown stage (GVBD). Oogenesis in 3-year-old female yellowfin porgy was divided into three stages¡F(1) previtellogenic stage from February to July with the primary oocytes¡F(2) vitellogenic stage from August to October and with full growth oocyte of diameter at 400

Thesis (Ph. D.)--University of Hong Kong, 1984.

No description available.

This retrospective cohort study of 336 lateral cephalometric radiographs from 62 children (34 males and 28 females) with non-syndromic complete unilateral cleft lip and palate from the Hospital for Sick Children and 50 non-cleft children (25 females and 25 males) from the Burlington Growth Centre. Cervical vertebral maturation stages at age 10, 12 and 14 were determined. The cervical vertebral maturation (CVM) was established using the 6-stage method described by Baccetti and coworkers. The reproducibility of classifying CVM stages was high, with an inter-rater reliability (ICC) with the standard (Baccetti et al, 2005) of 80% and intra-rater reliability of 85%. The Cervical vertebral maturation stage for both males and females with UCLP was significantly later than children without a cleft at age 10, 12 and 14. The results suggest that patients with UCLP show delayed skeletal maturation in comparison to non-cleft patients.

This retrospective cohort study of 336 lateral cephalometric radiographs from 62 children (34 males and 28 females) with non-syndromic complete unilateral cleft lip and palate from the Hospital for Sick Children and 50 non-cleft children (25 females and 25 males) from the Burlington Growth Centre. Cervical vertebral maturation stages at age 10, 12 and 14 were determined. The cervical vertebral maturation (CVM) was established using the 6-stage method described by Baccetti and coworkers. The reproducibility of classifying CVM stages was high, with an inter-rater reliability (ICC) with the standard (Baccetti et al, 2005) of 80% and intra-rater reliability of 85%. The Cervical vertebral maturation stage for both males and females with UCLP was significantly later than children without a cleft at age 10, 12 and 14. The results suggest that patients with UCLP show delayed skeletal maturation in comparison to non-cleft patients.