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This dissertation is a comprehensive analysis of voice constructions in Kanakanavu, a critically endangered Formosan language (Austronesian language of Taiwan) spoken in the Namasia District of Kaohsiung, southern Taiwan. The data considered for the analysis are mainly drawn from a corpus of spontaneous speech produced by contemporary speakers, which are complemented by elicited data and a verb database constructed by the author. The Kanakanavu voice system has been treated in various previous studies, but there is a general lack of consensus regarding several of its fundamental properties. The language has been analyzed as exhibiting a two-way, three-way or even four-way/Philippine-type voice distinction; it also remains unclear how voice interacts with tense-aspect-mood marking on the one hand, and transitivity and grammatical relations on the other. By investigating the morphosyntactic, semantic and discourse-pragmatic properties of voice, this dissertation (i) establishes a framework for describing and representing Kanakanavu natural discourse data, (ii) analyzes the morphological and syntactic foundations of Kanakanavu voice and (iii) explores the functional-typological implications of Kanakanavu voice constructions both within and beyond the Austronesian language family.It is argued that Kanakanavu exhibits a binary voice distinction in its verbal-clause morphosyntax.The structural opposition --- between what are labelled "agent voice" and "patient voice" --- is evidenced in two specific patterns of interaction between voice marking and tense-aspect-mood marking: while agent-voice verbs always involve separate exponents for voice and tense-aspect-mood, patient-voice verbs are consistently marked by voice markers that also serve a tense-aspect-marking or mood-marking function. The finding regarding the number of voice distinctions is in line with those found in some recent analyses of Kanakanavu voice, but it is motivated by typologically informed analyses of verbal tense-aspect-mood marking in the language based on detailed examinations of how different verb forms are used in spontaneous speech.At the syntactic level, voice alternation in Kanakanavu is argued to be essentially a transitivity-alternation phenomenon, leading to the analysis of the agent-voice construction as the intransitive construction (involving only one core argument), and the patient-voice construction as the transitive construction (involving two core arguments). The analysis is based on the observation from natural discourse that the patient-voice construction is always used for expressing transitive situations, while the agent-voice construction is the default construction for forming (i) basic intransitive clauses and (ii) clauses involving syntactically demoted and discourse-functionally backgrounded patients. The finding suggests that the Kanakanavu verbal clause exhibits ergativity, which is in line with many analyses of Formosan and Philippine languages where voice interacts closely with transitivity. It is, however, further shown that Kanakanavu exhibits differential (pronominal) agent marking in its patient-voice construction. This is a typologically and areally unusual phenomenon within the larger Western Austronesian context, which has been generally overlooked in previous studies on the language despite having been identified early on by Tsuchida (1976).

Abstract: Small interfering ribonucleic acids (siRNA) effectively downregulate the specific gene expression from cleaving the associated messenger RNA (mRNA) in the cytoplasm, which is a promising strategy in cancer management as one of the origins of cancer is the unregulated expression of the oncogene. Over decades, nanotechnological research has focused on enhancing siRNA delivery to the cytoplasm of the target cell since the large molecular weight and anionic nature of siRNA impede the siRNA from permeating the cellular membrane, and the blood circulation time of free siRNA is really short. However, even after a tremendous amount of research on cationic polymers, a safe and effective non-viral vector is not yet available. Obstacles, such as opsonization, non-specific delivery, low cellular uptake, endosomal trap, intracellular release of siRNA and cytotoxicity, further make the development of gene therapy in cancer management more challenging. In this thesis, we report the development of three low toxic, nano-sized polymeric siRNA carriers with cytoplasmic triggered siRNA release capability. We also investigated their efficiency in mediating the knockdown of epidermal growth factor receptor (EGFR) in HeLa cells to determine their potential advantages in siRNA delivery. First, several acid degradable, galactose-based, cationic, and hyperbranched polymers with varying molecular weights (10 to 20 kDa) (MW) and compositions with 2-lactobioamidoethyl methacrylamide [LAEMA] and 2-aminoethyl methacrylamide hydrochloride [AEMA] at different ratios (2.0, 1.0, and 0.5) were prepared via reversible addition-fragmentation chain-transfer (RAFT) polymerization. Subsequently, the polymers were used to form polyplexes with siRNA, and the EGFR knockdown efficiency in cervical carcinoma was determined. By quantifying the EGFR expression for each treatment group by Western blot assays, 10 kDa polymer, which has a LAEMA:AEMA (L/A) ratio of 2.0, demonstrated a superior EGFR knockdown efficiency (~60%) than the others, and low toxicity levels. In addition, the polyplexes demonstrated to have excellent stability under physiological conditions for up to 2 days. Next, novel thermo-responsive and cationic hyperbranched polymers were prepared from (AEMA) and di(ethylene glycol) methyl ether methacrylate (DEGMA) via the RAFT polymerization for siRNA delivery. Thermo-responsiveness of polymers allows the formation of stable polyplexes in the human body environment. Non-degradable and acid-degradable hyperbranched polymers were synthesized using N,N′-methylene bis(acrylamide) (MBAm) and 2,2-dimethacroyloxy-1-ethoxypropane (DEP) cross-linkers, respectively. Both types of polymers were capable of forming stable nanosized polyplexes with siRNA. Epidermal growth factor receptor (EGFR) silencing of 95% was achieved with a cationic hyperbranched polymer that incorporated an acid-trigger release strategy, and no significant cytotoxicity was observed. Our results confirmed the high potency of using such hyperbranched polymers for the efficient protection and delivery of siRNA. Finally, redox-responsive galactose-based hyperbranched polymers (HRRP) were synthesized via RAFT polymerization from redox-responsive cross-linker N,N′-bis(methacryloyl)cystamine, LAEMA(L), and AEMA (A) with molecular weights of 10 and 20 kDa and L/A ratios of 1.5 and 1.0 were prepared. Disulfide-based cross-linker was chosen to selectively release the siRNA into the cytoplasm of the cancer cells by the trigger of elevating the level of glutathione (GSH) concentration in the tumor environment. 85% EGFR knockdown efficiency was achieved using 10 kDa HRRP with L/A ratio of 1.5 without triggering significant cellular death (around 95% cell viability). All proposed polymers showed enhanced EGFR knockdown efficiency (60% with the least efficient one) while maintaining a low toxicity level with cancer (HeLa) and normal (MRC-5) cell lines in vitro studies. All the polymers were capable of forming stable nano-sized polyplexes in the growth medium, even in the presence of serum. By employing passive targeting strategy, enhanced permeation and retention (EPR) effect, and extending the blood circulation time from having stealth property to escape reticuloendothelial system and renal clearance, the potential for success in vivo studies is noteworthy high. Before studying these materials in clinical trials, more in vivo studies will be required. Furthermore, we intend to use these polymers to deliver other kinds of therapeutic nucleic acid to treat additional cancer types, such as hepatocytes, which would potentially unfold the potential of these polymers as a new nanotechnological platform for nucleic acid delivery.

The Diels-Alder reaction which generates two carbon-carbon bonds during a [4 + 2] cycloaddition is biochemically and pharmacologically significant. In order to expand and explore the utility and the application of this reaction to the assembly of complex natural products, an in vitro SELEX approach was used to obtain RNA biocatalysts of this transformation. A DA22⁄96 was prepared by the sequence truncation from the parent DA22. An essential requirement for catalytic activity was the incorporation of 5-(4- pyridylmethyl) carboxamide-uridine in place of uridine during the transcription of the DA22⁄96. My research was interested in finding the secondary structure of the DA22⁄96. This particular ribozyme exhibited catalytic activity similar to an enzyme. Mutual information and covariation analysis of 50 different catalytic sequences were used to identify the potential secondary structural elements. The comparisons between the MFold program derived secondary structures and mutual information analysis plots suggests that there were several conserved structural elements in all active DA22⁄96. RNase mapping method was used to validate the predicted secondary structure of DA22⁄96. The mapping data is largely consistent with the predicted secondary structure of DA22⁄96. Diels-Alderase activity assay experiment proved that DA22⁄96 had true enzyme activity. In addition, results showed that the modified DA22⁄96 had the better turn over rate when copper was presented.