High plasma levels of lipoprotein(a) [Lp(a)] are an independent risk factor for cardiovascular disease. Currently, no drugs have proven successful at lowering Lp(a) levels. Lp(a) is comprised of a LDL particle which is covalently bound to an additional glycoprotein, apolipoprotein(a) [apo(a)]. One approach is to develop inhibitors of Lp(a) assembly. It is therefore worth investigating inhibitors for the interaction between apo(a) and apoB-100 for their therapeutic potential to reduce plasma Lp(a) levels. A synthetic α-helical peptide, apoB₄₃₇₂₋₉₂ peptide, has been shown to be an effective inhibitor of Lp(a) assembly in vitro (IC₅₀= 40 [mu]M). Complexing of the apoB₄₃₇₂₋₉₂ peptide with dimyristoylphosphatidylcholine (DMPC) in 1:45 molar ratio increased its inhibitory activity to 4 [mu]M. Since the peptide:DMPC molar ratio previously used in vitro could not be obtained for in vivo study, lower molar ratios of the apoB₄₃₇₂₋₉₂ peptide-DMPC complex were assessed for their inhibitory activities on Lp(a) assembly in vitro and the Lp(a) lowering effect in vivo. A version of the apoB peptide modified to increase stability with DMPC under physiological conditions was also assessed. The aim of this project was to investigate the Lp(a) lowering effect of this peptide-DMPC complex in vivo and to determine its possible mechanism of action. Studies showed that a 1:20 ratio of the apoB₄₃₇₂₋₉₂ peptide-DMPC complex exhibited an effective inhibition of Lp(a) assembly in vitro (IC₅₀= 9 [mu]M). Intravenous injection of this peptide-DMPC complex (1.3 � 0.2 mg peptide/mouse) significantly decreased Lp(a) levels at 1, 2 and 4 hours after injection compared with DMPC (p< 0.01). However, a lethal effect occurred in some animals after treatment. This effect is likely associated with the aggregation of the apoB₄₃₇₂₋₉₂ peptide-DMPC complex under physiological conditions. In order to develop a more stable peptide, a synthetic peptide, apoB[4365-4396K] based on the apoB4365-4396 sequence and substituted with extra lysines was analyzed and shown to be α-helical in the absence and presence of DMPC. Intrinsic fluorescence studies showed that the apoB[4365-96K] peptide interacted with DMPC under physiological conditions. Also, the apoB[4365-96K] peptide-DMPC complex was shown to form small stable particles by dynamic light scattering. The apoB[4365-96K] peptide proved to be an effective inhibitor of Lp(a) assembly in vitro (IC₅₀= 38 [mu]M). Complexing the apoB[4365-96K] peptide to DMPC increased its inhibitory activity to 16 [mu]M. Intravenous injection of the apoB[4365-96K] peptide-DMPC complex decreased the Lp(a) levels at 1 hour and 2 hours after injection compared to DMPC (p< 0.05). The apoB[4365-96K] peptide was present in circulation prominently at 1 hour after intravenous injection and this coincided with a decrease in Lp(a) levels. Neither the apoB[4365-96K] peptide-DMPC complex nor DMPC had a significant effect on Lp(a) levels after intraperitoneal injection. The mechanism by which the apoB[4365-96K] peptide-DMPC complex lowers Lp(a) levels was further investigated. The apoB[4365-96K] peptide was found to associate with plasma lipoproteins through ultrafiltration and agarose gel shift assays. The association of the peptide with lipoproteins was dependent on α-helical structure and enhanced by complexing with DMPC. The association of the apoB[4365-96K] peptide with lipoproteins was also observed in plasma samples from mice treated with the apoB[436S-96K] peptide-DMPC complex intravenously. The association of the peptide-DMPC complex with plasma lipoproteins was most prominent at 1 hour after injection and this coincided with a maximum decrease in Lp(a) levels. These results suggest that the lipoprotein association of the apoB[4365-96K] peptide-DMPC complex played a role in lowering Lp(a) levels in vivo. The work presented in this thesis has shown that apoB peptides containing apoB4372-4392 sequence have inhibitory effect on Lp(a) assembly in vitro and lower Lp(a) levels in vivo. The α-helical structure of the peptide is important for its lipid binding ability and its inhibitory activity on Lp(a) assembly. Furthermore, the association of peptide with lipoproteins appears to play a role in its ability to inhibit Lp(a) assembly in vitro and lower Lp(a) levels in vivo. The studies undertaken in this thesis suggest that altering lipoprotein charges and conformation may reduce the efficiency of Lp(a) assembly. The knowledge gained in this study may provide a basis for future design of inhibitors of Lp(a) assembly.

Graduation date: 1997

Increasing the odds of hit identification in screening is of significance for drug discovery. The odds for finding a hit are closely related either to the diversity of libraries or to the availability of focused libraries. There are no truly diverse libraries and it is difficult to design focused libraries without sufficient information. Hence it is helpful to consider alternative approaches that can enhance the odds using existing libraries. Multiple members of a protein family have been considered collectively in inhibitor design, on the basis of the correlation between protein families and ligands derived from specific compound classes. Such a correlation has been exploited in various drug discovery studies and a general receptor-homolog-based screening scheme may be devised. The feasibility of such a scheme in enhancing the odds of hit identification is discussed. / Singapore-MIT Alliance (SMA)

The cyclin-dependent kinase (CDK) inhibitors have been demonstrated to be an important component in the regulation of plant cell cycle. Although they share a conserved CDK inhibitory region with a family of CDK inhibitors in mammals, the plant CDK inhibitors are very different from the animal and yeast CDK inhibitors. Thus studies of the plant CDK inhibitors could provide insight on the molecular mechanisms regulating the cell cycle in plants as well as the differences between plants and animals. The research described in this thesis investigated the seven Arabidopsis CDK inhibitors ICKs in terms of transgenic expression, phosphorylation, stability and interactions with other proteins. <p>ICKs were expressed in transgenic Arabidopsis plants as fusion proteins with the green fluorescent protein (GFP). Consistent with the previous studies on ICK1, ICK2 and ICK4, overexpression of all seven ICKs inhibited plant growth and resulted in plants with serrated leaves and flowers with altered morphology. A Survey based on large a number of independent transformants showed that GFP-ICK3 and GFP-ICK4 had weaker phenotypic effects compared to other GFP-ICKs. The Western blotting results showed that all GFP-ICKs were expressed at a low level in general. The levels of GFP-ICK3 and GFP-ICK4 were the lowest, suggesting that the weaker effects for ICK3 and ICK4 may partly be due to low protein levels. Treatments with MG132, an inhibitor of the proteasome, resulted in moderate but clear accumulation of fusion proteins for ICK1, ICK5, ICK6 and ICK7 in plants, suggesting that the proteasome is involved in the degradation of these proteins. <p>To study the state of protein phosphorylation, the proteins extracted from the plants were treated with calf intestinal phosphatase (CIP). The CIP treatment caused a faster migration of the GFP fusion protein for ICK1, ICK2, ICK5, ICK6 and ICK7, while the effect was not observed for control GFP and other non-specific proteins, indicating that these proteins can be phosphorylated in plants. The shift also differed among ICKs. Interestingly, dephosphorylation of ICK7 might have rendered it less stable. The protein pulldown experiments using p13Suc1-conjugated agarose beads showed that GFP-ICK4, GFP-ICK5 and GFP-ICK6 could associate with the CDK complex, similar to what has been shown for ICK1 and ICK2. CIP treatments of the p13Suc1 affinity-purified proteins also showed that ICK1, ICK2, ICK5 and ICK6 associated with the CDK complex were phosphorylated. <p>Attempts were also made to isolate peptide aptamers that are able to interact with ICKs for the purpose for expressing such an aptamer in plants. However, an aptamer that has a strong ability to interact with ICKs in two of yeast two-hybrid systems was not identified. In addition, the analysis of Arabidopsis CYCD3;1 for its interaction with ICK1 using a series of deletion mutants showed that the removal of both the N-terminal and C-terminal regions of CYCD3;1 greatly reduced or abolished the interaction with ICK1. <p> In summary, transgenic Arabidopsis plants have been obtained for expressing each of the seven Arabidopsis CDK inhibitors fused to GFP. The results confirmed and extended previous finding that overexpression of a CDK inhibitor inhibits plant growth as well as changes plant morphology. The observation that the ICK fusion proteins were generally at low and often undetectable levels, in comparison to much higher levels of the GFP protein, suggests that ICKs are unstable in the cell. Results from the MG132 experiments indicate that the 26S proteasome may play a role in the degradation of ICK1, ICK5, ICK6 and ICK7. Results from CIP treatments further show that most ICKs, particularly ICK1, ICK2, ICK5, ICK6 and ICK7, can be phosphorylated in vivo. Interestingly, ICK7 stability may depend on the status of protein phosphorylation. This study provides new understanding on how the family of proteins is regulated at the post-transcriptional level and the differences among Arabidopsis CDK inhibitors.

The cyclin-dependent kinase (CDK) inhibitors have been demonstrated to be an important component in the regulation of plant cell cycle. Although they share a conserved CDK inhibitory region with a family of CDK inhibitors in mammals, the plant CDK inhibitors are very different from the animal and yeast CDK inhibitors. Thus studies of the plant CDK inhibitors could provide insight on the molecular mechanisms regulating the cell cycle in plants as well as the differences between plants and animals. The research described in this thesis investigated the seven Arabidopsis CDK inhibitors ICKs in terms of transgenic expression, phosphorylation, stability and interactions with other proteins. <p>ICKs were expressed in transgenic Arabidopsis plants as fusion proteins with the green fluorescent protein (GFP). Consistent with the previous studies on ICK1, ICK2 and ICK4, overexpression of all seven ICKs inhibited plant growth and resulted in plants with serrated leaves and flowers with altered morphology. A Survey based on large a number of independent transformants showed that GFP-ICK3 and GFP-ICK4 had weaker phenotypic effects compared to other GFP-ICKs. The Western blotting results showed that all GFP-ICKs were expressed at a low level in general. The levels of GFP-ICK3 and GFP-ICK4 were the lowest, suggesting that the weaker effects for ICK3 and ICK4 may partly be due to low protein levels. Treatments with MG132, an inhibitor of the proteasome, resulted in moderate but clear accumulation of fusion proteins for ICK1, ICK5, ICK6 and ICK7 in plants, suggesting that the proteasome is involved in the degradation of these proteins. <p>To study the state of protein phosphorylation, the proteins extracted from the plants were treated with calf intestinal phosphatase (CIP). The CIP treatment caused a faster migration of the GFP fusion protein for ICK1, ICK2, ICK5, ICK6 and ICK7, while the effect was not observed for control GFP and other non-specific proteins, indicating that these proteins can be phosphorylated in plants. The shift also differed among ICKs. Interestingly, dephosphorylation of ICK7 might have rendered it less stable. The protein pulldown experiments using p13Suc1-conjugated agarose beads showed that GFP-ICK4, GFP-ICK5 and GFP-ICK6 could associate with the CDK complex, similar to what has been shown for ICK1 and ICK2. CIP treatments of the p13Suc1 affinity-purified proteins also showed that ICK1, ICK2, ICK5 and ICK6 associated with the CDK complex were phosphorylated. <p>Attempts were also made to isolate peptide aptamers that are able to interact with ICKs for the purpose for expressing such an aptamer in plants. However, an aptamer that has a strong ability to interact with ICKs in two of yeast two-hybrid systems was not identified. In addition, the analysis of Arabidopsis CYCD3;1 for its interaction with ICK1 using a series of deletion mutants showed that the removal of both the N-terminal and C-terminal regions of CYCD3;1 greatly reduced or abolished the interaction with ICK1. <p> In summary, transgenic Arabidopsis plants have been obtained for expressing each of the seven Arabidopsis CDK inhibitors fused to GFP. The results confirmed and extended previous finding that overexpression of a CDK inhibitor inhibits plant growth as well as changes plant morphology. The observation that the ICK fusion proteins were generally at low and often undetectable levels, in comparison to much higher levels of the GFP protein, suggests that ICKs are unstable in the cell. Results from the MG132 experiments indicate that the 26S proteasome may play a role in the degradation of ICK1, ICK5, ICK6 and ICK7. Results from CIP treatments further show that most ICKs, particularly ICK1, ICK2, ICK5, ICK6 and ICK7, can be phosphorylated in vivo. Interestingly, ICK7 stability may depend on the status of protein phosphorylation. This study provides new understanding on how the family of proteins is regulated at the post-transcriptional level and the differences among Arabidopsis CDK inhibitors.

Abstract Two kinds of unsaturated polyesters¡¦ prepolymers were synthesized via two steps. For the hard segment, isophthalic acid was reacted with 1,2-propanediol first, then maleic anhydride was added to yield a copolyester with Tg around 13

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (M. Phil.)--University of Hong Kong, 1978. / Xeorx copy of typescript.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Influenza A virus is one of the most common human pathogens which caused considerable disease burdens through annual epidemics and occasional pandemics. The consequences vary from mild to severe or even fatal. What are the host and viral elements which determine the consequence of infection? In the past 15 years, several avian influenza A viruses including H5N1, H9N2, H7N7 and H7N9 subtypes were found to cross host barrier and infect humans. Question about how avian influenza A viruses gained the ability to replicate in human cells remains unanswered. Studies on host factors associated with virus replication would provide important information for understanding host restriction, virus pathogenesis and for antiviral drug development. Nuclear factor 90 (NF90) is a host protein identified in our previous study to inhibit influenza A virus replication. Antiviral activity of NF90 was also found for other viruses. However, detailed mechanisms for the antiviral function of NF90 remains largely unknown. This study is focused on NF90’s antiviral functions through exploring its relationship with PKR activation and stress granules formation using influenza A virus as a model. I characterized the interaction between NF90 and PKR, and showed the C-terminal of NF90 interacts with PKR in an RNA-binding dependent manner. Using transient and stable NF90 knockdown cells, I found that NF90 is required for PKR activation upon stimulation by dsRNA or infection with a NS1 mutated virus. PKR activation leads to the formation of stress granules and stall of protein translation. I found that NF90 is a core component of stress granules, which may underlie the mechanism for the antiviral activity of NF90. However, NF90 may also complete with PKR for RNA binding and regulate PKR activation. To further delineate the interaction between NF90 and PKR by using influenza A virus, my study constructed a panel of NS1 mutant viruses which were attenuated in antagonizing specific host antiviral pathways. I characterized the NS1 123-127 mutant virus which is unable to inhibit PKR phosphorylation but retained other functions unaffected. It was demonstrated that NF90 mediates PKR-dependent antiviral pathway since NS1 123-127 mutant virus replicated to a comparable level as wild type virus in the NF90 knockdown but not scramble knockdown 293T cells or in the interferon deficient Vero cells. This study for the first time found NF90 serves as a regulator of PKR antiviral pathway. To understand the mechanism for NF90 inhibition of influenza A virus replication, I found that NP, but not the other polymerase subunits, of influenza A virus was targeted to the stress granules. Since NF90 interacts with NP, it is reasonable to postulate that NF90 mediates the localization of NP, and possibly viral mRNA, to the stress granules in order to inhibit influenza A virus replication through regulation of proteins synthesis. In summary, my study provided comprehensive evidence to support a novel NF90-PKR antiviral pathway and suggests that NF90 may play critical roles to balance PKR phosphorylation in response to virus infection in cells. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy