Secretory granule biogenesis describes the events leading up to the budding of a nascent granule from the trans Golgi network. Literature surrounding secretory granule biogenesis is conflicting and has generated much debate. This thesis aims to address the important issues of this debate by utilizing the insulin-producing liver cell line HUH7-ins. This cell line has been shown to synthesize, store and secrete mature insulin in response to glucose via the possession of secretory granules. Using microarray technology the gene expression profile of HUH7-ins cells was compared with parental HUH7 cells, hoping to identify possible candidate genes contributing to secretory granule biogenesis. 164 genes were shown to be differentially expressed although no known granulogenic protein exhibited a change in expression. The data did suggest a nervous system differentiation event and implicates myosin Vc in the regulated secretion of insulin. HUH7-ins cells express a number of granulogenic protein mRNAs and while chromogranin B (CgB) protein level remained constant upon insulin expression, a significant increase in the level of chromogranin A (CgA) was observed, though the significance of this increase in expression is unknown. The over-expression of CgA in a clone of HUH7-ins that did not possess the regulated secretory pathway was unable to rescue the regulated secretory pathway, suggesting that CgA expression alone is unable to form secretory granules in our model. To determine if the secretory granule biogenesis seen in HUH7-ins cells was specific to insulin, three prohormones of different neuroendocrine origin were over-expressed in HUH7 cells; amylin ( cell), pancreatic polypeptide (pancreatic islet) and proopiomelanocortin (pituitary). None of these prohormones were able to form structures in the cytoplasm that resembled secretory granules by immunofluorescent microscopy, nor did they induce the expression of CgA. No prohormone was detected in cell lysates or conditioned media, raising the possibility that these exogenous prohormone aggregates are trafficked to the lysosomal/endosomal system for degradation. This study provides significant information regarding the genome-wide expression changes induced upon secretory granule biogenesis in a liver cell line, describes the lack of effect of CgA in this event and suggests that secretory granule biogenesis in this liver cell line is specific to insulin.

This dissertation provides a rebuttal to the claims of Richard Dawkins in explaining the origin of life. The bulk of the ensuing analysis challenges his philosophical assumptions as it notes his vacillation between several models over time. The study further details Dawkins's multifaceted approach to the problem as it points out a number of errors permeating his general methodology and reasoning.

The question about the origins of life often appears as a difficult question to answer. A more reliable candidate molecule for the chemical origins of life would be a molecule that is capable of making copies of itself from simple precursors. With the finding of the catalytic activities of RNA molecules by Thomas Cech and Sid Altman in late 1980s, the term ribozyme was introduced to define an RNA molecule with catalytic activity. The RNA World is a conceptual period in the early stages in the development of life because RNA simultaneously possesses evolvability and catalytic function. An RNA molecule that could evolve in such a fashion is likely to have been one of the Earth's first life forms. The most important problem facing the RNA World is the difficulty of prebiotic synthesis of RNA. Different prebiotic environments could provide the right reaction conditions for synthesis of catalytically active RNA molecules. Most importantly, these environments can support new ways to assemble monomers into polymers. In order to understand and demonstrate how small inactive RNA oligomers can self-assemble into an autocatalytic ribozyme molecule, here I have used the Azoarcus Group I intron. First, the fragmentation and the self-assembly reactions were done using the natural IGS-tag combination of the Azoarcus group I intron, which is GUG/CAU. The main purpose was to experimentally demonstrate that the ribozyme can be broken down into five or more shorter RNA fragments and these fragments can self-assemble into a catalytically active covalent full-length molecule. Then, with the successful demonstration that five inactive RNA fragments can self-assemble, the next step to test the other possible breaking locations with mutated IGS/tag combinations. A new IGS/tag pair GCG-CGU also successfully demonstrated the five-piece self-assembly reaction. Finally, we tested these reactions in different Mg2+ concentrations to optimize the self-assembly reactions. By focusing not only one single reaction but on a collection of different sequence requirement combinations and with the development of evaporation and subsequent rehydration by spinning down methods this study successfully illustrates that covalent self-assembly from inactive RNA oligomers is possible. Therefore, this thesis work focuses on a more broader aspect of intermolecular interactions in the study of the RNA World, as illustrated in following chapters.

C-type cytochromes are ubiquitous proteins with crucial functions in organisms, which include electron transfer and apoptotic signalling. In eukaryotic organisms, mitochondrial cytochrome c is located in the intermembrane space, and it is a component of the electron transport chain; it is responsible for transferring electrons from Complex III to Complex IV. The regulated release of cytochrome c from mitochondria results in the activation of a signal transduction pathway leading to controlled cell death, or apoptosis. In mitochondrial c-type cytochromes, the heme is bound to both cysteines of a CXXCH motif located near the N-terminus. The covalent heme attachment in c-type cytochromes, the final step in its biosynthesis, is achieved by different cytochrome c biogenesis systems in different organisms. Out of these systems, System III, found in many eukaryotes, has a single component - holocytochrome c synthase (HCCS) which is the enzyme responsible for the catalysis of heme binding to cytochrome c. HCCS recognises apocytochrome c as a substrate upon the import of the apocytochrome from the cytosolic space to the mitochondrial intermembrane space. The requirements of amino acid sequence for HCCS recognition had remained an intriguing question, despite the relatively long period since the discovery of the enzyme. Thus, HCCS in System III and its substrate recognition is the subject of this thesis. This thesis describes the experiments showing that the N-terminal region of the mitochondrial cytochrome c protein is important for substrate recognition, as well as further characterisation of this sequence by mutagenesis. Out of several highly conserved residues in the N-terminus, a phenylalanine residue in the N-terminus is identified to be critical for heme attachment by HCCS. The role of this phenylalanine residue in the interaction between the two proteins was probed by substituting it with a range of residues. Furthermore, the importance of the spacing between the key phenylalanine residue and the CXXCH motif was investigated. A single-cysteine variant of the mitochondrial cytochrome c with a single bond to the heme is produced by HCCS, but heme attachment only occurs if histidine is present as an axial ligand to the heme iron. Replacement of the histidine with other potential iron-ligating residues abolished heme attachment. These results bring insight into the critical features in amino acid sequence of cytochrome c for the substrate recognition specificity of HCCS. Sequence analysis on the N-terminal region of mitochondrial cytochromes c in a variety of organisms reveals evolutionary implications for cytochrome c biogenesis systems. It also attempts to explain the reason for negative results in previous chapters for the analysis of the N-terminal region of cytochrome c. An improved method for human HCCS production is also described in this thesis, for the exploitation of purification and characterisation in future studies of HCCS.

We describe the initial realization of behavior in the biosphere, which we term behavioral chemistry. If molecules are complex enough to attain a stochastic element to their structural conformation in such a way as to radically affect their function in a biological (evolvable) setting, then they have the capacity to behave. This circumstance is described here as behavioral chemistry, unique in its definition from the colloquial chemical behavior. This transition between chemical behavior and behavioral chemistry need be explicit when discussing the root cause of behavior, which itself lies squarely at the origins of life and is the foundation of choice. RNA polymers of sufficient length meet the criteria for behavioral chemistry and therefore are capable of making a choice. We test these theoretical findings with a empirical systems; since RNA simultaneously possesses evolvability and catalytic function we set to construct a model system. Firstly, during in vitro transcription of the Chlorella PBCV-1 pdg intron, we observed multiple RNAs of slower electrophoretic mobility along with the expected 98-nucleotide transcript. The preferences observed for TI or linear RNA for the pdg intron are manifestations of alternative phenotypic states and represent evidence of behavior at the chemical level. Secondly, we provide a basis for the marriage between empirical findings and the philosophical studies of biosemiotics. As well, provide a basis for the minimal criteria for an interpretative response and suggest that interpretation is evolvable. The studies hold relevance to the origins of life showing a minimal system capable of carrying out a choice based on the environment. We have shown this phenomenon is not an intrinsic characteristic to all RNAs and that the Tetrahymena ribozyme recognizes the presence of the object through the sensing of a sign.

The study of extrasolar planets, or exoplanets for short, has developed rapidly over the last decade. While we have spent much effort building both ground-based and space telescopes to search for exoplanets, it is even more important that we use the observational data wisely to understand them. Exoplanets are of great interest to both astronomers and the general public because they have shown varieties of characteristics that we couldn't have anticipated from planets within our Solar System. To properly analyze the exoplanet populations, we need the tools of statistics. Therefore, in Chapter 1, I describe the science background as well as the statistical methods which will be applied in this thesis. In Chapter 2, I discuss how to train a hierarchical Bayesian model in detail to fit the relationship between masses and radii of exoplanets and categorize exoplanets based on that. A natural application that comes with the model is to use it for future observations of mass/radius and predict the other measurement. Thus I will show two application cases in Chapter 3. Composition of an exoplanet is also very much constrained by its mass and radius. I will show an easy way to constrain the composition of exoplanets in Chapter 4 and discuss how more complicated methods can be applied in future works. Of even greater interest is whether there is life elsewhere in the Universe. Although the future discovery of extraterrestrial life might be totally a fluke, a clear sketched plan always gives us some directions. Research in this area is still very preliminary. Fortunately, besides directly searching for extraterrestrial life, we can also apply statistical reasoning to first estimate the rate of abiogenesis, which will give us some clue on the question of whether there is extraterrestrial life in a probabilistic way. In Chapter 5, I will discuss how different methods can constrain the abiogenesis rate in an informatics perspective. Finally I will give a brief summary in Chapter 6.

The RNA World hypothesis proposes a period of time during the origins of life in which RNA molecules were the only source of both genotypes and phenotypes. Although a vast amount of evidence has been obtained in support of this hypothesis, a few critical demonstrations are lacking. A most crucial one is a demonstration of self-replication of RNA molecule from prebiotic soup. Previously in the Lehman laboratory, it has been demonstrated that a 198-nucleotide molecule derived from the Azoarcus group I intron can self-assemble from up to four fragments of RNA via recombination. Furthermore, the covalent full-length molecules are catalytically active and can make copies of themselves from the remaining pieces in the solution leading to their autocatalytic growth. I was able to demonstrate how this recombination system can overcome different obstacles and evolve to be an efficient replicating system. I discovered the ability of a single RNA fragment to be multifunctional in a single reaction pathway during RNA recombination events that avoids the necessity of multiple genotypes. I also confirmed the capacity of self-replicating ribozymes to form cooperative catalytic cycles and networks that would potentially prevent informational decay. Finally, I have discovered a recycling phenomenon in the RNA recombination system that exploits dynamic covalent chemistry. Recycling provides the earliest replicating system with adequate concentrations of reagents and ability to explore sequence space. Together these findings have improved our understanding of RNA recombination and bolstered the plausibility of the RNA World.

A brief introduction to the current state of research in the Origins of Life field is given in Part I of this work. Part II covers original research performed by the author and co-workers. Layered Double Hydroxide (LDH) systems are anion-exchanging clays that have the general formula M(II)xM(III)(OH)(2x+2)Y, where M(II) and M(III) are any divalent and trivalent metals, respectively. Y can be nearly any anion, although modern naturally occuring LDH systems incorporate carbonate (CO32-), chloride (Cl-), or sulfate (SO42-) anions. Intercalated cobalticyanide anion shows a small yet observable deviation from local Oh symmetry causing small differences between its oriented and non-oriented infrared spectra. Nitroprusside is shown to intercalate into 2:1 Mg:Al LDH with decomposition to form intercalated ferrocyanide and nitrosyl groups of an unidentified nature. The [Ru(CN)6]4- anion is shown to intercalate into layered double hydroxides in the same manner as other hexacyano anions, such as ferrocyanide and cobalticyanide, with its three-fold rotational axis perpendicular to the hydroxide sheets. The square-planar tetracyano-nickelate(II), -palladate(II), and platinate(II) anions were intercalated into both 2:1 and 3:1 Mg:Al layered double hydroxides (LDH). The basal spacings in the 2:1 hosts are approximately 11 Å, indicating that the anions are inclined approximately 75 degrees relative to the hydroxide layers, while in the 3:1 hosts the square-planar anions have enough space to lie more nearly parallel to the LDH cation layers, giving basal spacings of approximately 8 Å. It has been found that the LDH Mg2Al(OH)6Cl catalyzes the self-addition of cyanide, to give in a one-pot reaction at low concentrations an increased yield of diaminomaleonitrile and in addition, at higher ($0.1M) concentrations, a purple-pink material that adheres to the LDH. We are investigating whether this reaction also occurs with hydrotalcite itself, what is the minimum effective concentration of cyanide, and what can be learned about the products and how they compare with those reported at high HCN concentrations in the absence of catalyst.

The RNA World hypothesis suggests that RNA, or a proto-RNA, existed in an early form of life that had not yet developed the ability to synthesize protein enzymes. This hypothesis, by some interpretations, implies that nucleic acid polymers were the first polymers of life, and must have therefore spontaneously formed from simple molecular building blocks in the "prebiotic soup." Although prebiotic chemists have searched for decades for a process by which RNA can be made from plausible prebiotic reactions, numerous problems persist that stand in the way of a chemically-sound model for the spontaneous generation of an RNA World (e.g., strand-cyclization, heterogeneous backbones, non-selective ligation of activated nucleotides). The Molecular Midwife hypothesis, proposed by Hud and Anet in 2000, provides a possible solution to several problems associated with the assembly of the first nucleic acids. In this hypothesis, nucleic acid base pairs are assembled by small, planar molecules that resemble molecules which are known today to intercalate the base pairs of nucleic acid duplexes. Thus, the validity and merits of the Molecular Midwife hypothesis can be, to some extent, explored by studying the effects of intercalation on the non-covalent assembly of nucleic acids. In this thesis, I explore the role of the sugar-phosphate backbone in dictating the structure and thermodynamics of nucleic acid intercalation by using 2′,5′-linked RNA intercalation as a model system of non-natural nucleic acid intercalation. The solution structure of an intercalator-bound 2′,5′ RNA duplex reveals structural and thermodynamic aspects of intercalation that provide insight into the origin of the nearest-neighbor exclusion principle, a principle that is uniformly obeyed upon the intercalation of natural (i.e. 3′,5′-linked) RNA and DNA. I also demonstrate the ability of intercalator-mediated assembly to circumvent the strand-cyclization problem, a problem that otherwise greatly limits the polymerization of short oligonucleotides into long polymers. Together, the data presented in this thesis illustrate the important role that the nucleic acid backbone plays in governing the thermodynamics of intercalation, and provide support for the proposed role of intercalator-mediated assembly in the prebiotic formation of nucleic acids.

Here, for the first time, the formation of adenine, hypoxanthine, and guanine from formamide solutions with heating only to 130 degrees C and UV-irradiation in the absence of minerals or inorganic salts is shown using LC-MS/MS as the analysis technique. The thesis goes on to demonstrate that the product distributions change drastically when the temperature is increased to 160 degrees C from 130 degrees C, specifically that the amount of hypoxanthine increases with the addition of UV light, and the amount of adenine increases with an increase in temperature. Along with showing the formation of purines in these reactions, the identification of pyrimidines was also achieved by GCxGC-MS. GCxGC-MS was also used to study additional samples, specifically bio-oils, where the type of compounds in the samples were easily identifiable, which allowed for a direct comparison between different types of bio-oils (e.g. Douglas-fir bark, Southern pine bark, and a Southern pine bark-wood mixture).