Doctor of Philosophy

dissertationThe self-assembly of multiple protein subunits via noncovalent interactions provides a diverse collection of nanoarchitectures. Polyhedral capsids represent a particularly interesting type of structure in which the protein forms a closed three-dimensional surface that can function as a molecular container. Understanding capsid self-assembly could benefit further development of nanomaterials for many applications, such as drug delivery and biocatalysis. In this thesis, Aquifex aeolicus lumazine synthase (AaLS) is used as a model for investigating capsid self-assembly. The 60-subunit capsids formed by AaLS in vivo can be viewed as dodecamers-of-pentamers. Currently, methods are lacking for controlling AaLS assembly in vitro, which imposes important limitations on cargo loading. Interestingly, the diverse quaternary structures in the lumazine synthase family, which includes capsids and pentamers, imply the possibility of exchanging assembly states in vitro. To better understand AaLS capsid assembly, the dodecahedral capsid was converted to pentamers via a strategy involving rational design and sitedirected mutagenesis. Biophysical characterizations confirm that simultaneous substitution of three interfacial residues can yield stable pentamers. A pentameric AaLS variant that possesses a unique, strategically placed cysteine residue was engineered to enable capsid formation in vitro. This cysteine was modified with a thiophenol group by sequential thiol-disulfide exchange reactions. The increase in nonpolar surface area upon modification of this cysteine allows for assembly of the pentamers into capsids that resemble wild-type AaLS, presumably by recapitulating hydrophobic interactions present at the pentamer-pentamer interface. In an alternative approach, a pH-dependent switch for AaLS capsid disassembly was developed. For this switch, the ability to change assembly state relies on the presence of three engineered histidine residues per subunit, located near the three-fold symmetric interface of the capsid. These histidines minimally interfere with the capsid structure at high pH where their side chains are neutral. However, at lower pH, histidine protonation can trigger the dissociation of the capsid into pentamers, presumably due to charge repulsion. Further, this switch is reversible, as 60-subunit capsids can reform upon raising the pH. These studies of interconverting AaLS quaternary structures open the door to the development of improved encapsulation systems for use in medicine or nanobiomaterials

Women with endometriosis have higher comorbidities: Analysis of domestic data in Taiwan

AbstractEndometriosis, defined by the presence of viable extrauterine endometrial glands and stroma, can grow or bleed cyclically, and possesses characteristics including a destructive, invasive, and metastatic nature. Since endometriosis may result in pelvic inflammation, adhesion, chronic pain, and infertility, and can progress to biologically malignant tumors, it is a long-term major health issue in women of reproductive age. In this review, we analyze the Taiwan domestic research addressing associations between endometriosis and other diseases. Concerning malignant tumors, we identified four studies on the links between endometriosis and ovarian cancer, one on breast cancer, two on endometrial cancer, one on colorectal cancer, and one on other malignancies, as well as one on associations between endometriosis and irritable bowel syndrome, one on links with migraine headache, three on links with pelvic inflammatory diseases, four on links with infertility, four on links with obesity, four on links with chronic liver disease, four on links with rheumatoid arthritis, four on links with chronic renal disease, five on links with diabetes mellitus, and five on links with cardiovascular diseases (hypertension, hyperlipidemia, etc.). The data available to date support that women with endometriosis might be at risk of some chronic illnesses and certain malignancies, although we consider the evidence for some comorbidities to be of low quality, for example, the association between colon cancer and adenomyosis/endometriosis. We still believe that the risk of comorbidity might be higher in women with endometriosis than that we supposed before. More research is needed to determine whether women with endometriosis are really at risk of these comorbidities

Conversion of a Dodecahedral Protein Capsid into Pentamers via Minimal Point Mutations

Protein self-assembly relies upon the formation of stabilizing noncovalent interactions across subunit interfaces. Identifying the determinants of self-assembly is crucial for understanding structure–function relationships in symmetric protein complexes and for engineering responsive nanoscale architectures for applications in medicine and biotechnology. Lumazine synthases (LS’s) comprise a protein family that forms diverse quaternary structures, including pentamers and 60-subunit dodecahedral capsids. To improve our understanding of the basis for this difference in assembly, we attempted to convert the capsid-forming LS from <i>Aquifex aeolicus</i> (AaLS) into pentamers through a small number of rationally designed amino acid substitutions. Our mutations targeted side chains at ionic (R40), hydrogen bonding (H41), and hydrophobic (L121 and I125) interaction sites along the interfaces between pentamers. We found that substitutions at two or three of these positions could reliably generate pentameric variants of AaLS. Biophysical characterization indicates that this quaternary structure change is not accompanied by substantial changes in secondary or tertiary structure. Interestingly, previous homology-based studies of the assembly determinants in LS’s had identified only one of these four positions. The ability to control assembly state in protein capsids such as AaLS could aid efforts in the development of new systems for drug delivery, biocatalysis, or materials synthesis

Synthesis of Pyranmycin Derivatives with N-1 and O-6 Modifications

Continuing from our ongoing effort in modifying aminoglycoside antibiotics with the goal of counteracting drug resistant bacteria, we have further derivatized pyranmycin, a neomycin class aminoglycoside antibiotic, with modifications at O-6 and N-1 positions. The revealed SAR results demonstrated that the antibacterial activity of pyranmycin can be modulated by different acylic substituents at O-6. Among these results, the 6-O-aminoethyl derivative, JT050, showed effective activity against resistant strain Escherichia coli (pTZ19U-3) and E. coli (pSF815), which provides insight into further structural modifications

Investigation of the Regioselectivity for the Staudinger Reaction and Its Application for the Synthesis of Aminoglycosides with N-1 Modification

The criteria for controlling the regioselectivity of Staudinger reduction of azides have been investigated. These findings enable a convenient direct N-1 modification of the perazidoneamine and perazidoribostamycin resulting in the synthesis of aminoglycoside antibiotics with activity against drug-resistant bacteria

Sonication-Assisted Library Synthesis of Oxazolidinone−Carbohydrate Conjugates

Oxazolidinone derivatives have attracted growing interest in their pharmacological applications

Maternal pregnancy-induced hypertension increases the subsequent risk of neonatal candidiasis: A nationwide population-based cohort study

Objective: Neonatal candidiasis is a leading infectious cause of significant morbidity and mortality in premature birth mainly due to impaired physical barriers and immature immune system of fetus. Maternal pregnancy-induced hypertension (PIH) has been reported to be able to disturb the neonatal immune system, which could cause the increased possibility of neonatal infection. Therefore, we hypothesized that maternal PIH may increase the risk of neonatal candidiasis. The aim of this study was to evaluate whether PIH increased the risk of neonatal candidiasis and identify the predictive risk factors. Materials and methods: Patients with newly diagnosed PIH between January 1, 2000, and December 31, 2013 were selected from the Taiwan National Health Insurance Research Database (NHIRD). For each patient in the PIH cohort, 4 subjects without PIH, matched for age and year of delivery, were randomly selected as the comparison cohort. A Cox proportional regression model was used to estimate the risks of neonatal candidiasis in both cohorts. Results: Among the 23.3 million individuals registered in the NHIRD, 29,013 patients with PIH and 116,052 matched controls were identified. Patients with PIH had a higher incidence of neonatal candidiasis than did those without PIH. According to the multivariate analysis, PIH (odds ratio [OR] = 2.08, 95% confidence interval [CI] = 1.11–3.19, p < 0.0228), single parity (OR = 1.91, 95% CI = 1.00–3.65, p < 0.0499), and preterm birth (OR = 3.57, 95% CI = 1.84–6.93, p = 0.0002) were independent risk factors for the development of neonatal candidiasis. Conclusion: Patients who had a history of PIH was associated with an increased risk of having infants who develop neonatal candidiasis compared with those without PIH. Additionally, preterm birth was an independent risk factor for the development of neonatal candidiasis. Keywords: Gestational hypertension, Hypertension in pregnancy, Neonatal candidiasis, Preeclampsia, Pregnancy-induced hypertensio

In-Depth Comparison of Lysine-Based Antibody-Drug Conjugates Prepared on Solid Support Versus in Solution

Antibody drug conjugates are a rapidly growing form of targeted chemotherapeutics. As companies and researchers move to develop new antibody–drug conjugate (ADC) candidates, high-throughput methods will become increasingly common. Here we use advanced characterization techniques to assess two trastuzumab-DM1 (T-DM1) ADCs; one produced using Protein A immobilization and the other produced in solution. Following determination of payload site and distribution with liquid chromatography-mass spectrometry (LC/MS), thermal stability, heat-induced aggregation, tertiary structure, and binding affinity were characterized using differential scanning calorimetry (DSC), dynamic light scattering (DLS), Raman spectroscopy, and isothermal titration calorimetry (ITC), respectively. Small differences in the thermal stability of the CH2 domain of the antibody as well as aggregation onset temperatures were observed from DSC and DLS, respectively. However, no significant differences in secondary and tertiary structure were observed with Raman spectroscopy, or binding affinity as measured by ITC. Lysine-based ADC conjugation produces an innately heterogeneous population that can generate significant variability in the results of sensitive characterization techniques. Characterization of these ADCs indicated nominal differences in thermal stability but not in tertiary structure or binding affinity. Our results lead us to conclude that lysine-based ADCs synthesized following Protein A immobilization, common in small-scale conjugations, are highly similar to equivalent ADCs produced in larger scale, solution-based methods