Ciliates are model organisms for studying programmed genome rearrangement because each cell houses two distinct genomes. During postzygotic development, the somatic genome rearranges from a copy of the germline genome via extensive genome remodeling, including DNA elimination, religation and sometimes translocation or inversion of genomic regions. Previous studies of this process were restricted to a few model ciliates including Tetrahymena thermophila, Paramecium tetraurelia and Oxytricha trifallax. Oxytricha diverged from Tetrahymena and Paramecium over one billion years ago, and it possesses a massively fragmented and scrambled germline genome. My thesis compares Oxytricha to more closely related ciliates to address the evolutionary origin of genome complexity. Chapter 1 provides a general introduction to genome architecture, comparison of well-studied ciliate genomes and challenges of studying genome rearrangement in non-model ciliates. Chapter 2 describes a computational pipeline, SIGAR (Split-read Inference of Genome Architecture and Rearrangements), which infers genome rearrangement features without a germline genome assembly. We validated the pipeline using a published Oxytricha dataset, and also applied it to six diverse ciliate species including Ichthyophthirius multifiliis, a fish pathogen. This pipeline enables pilot surveys or exploration of chromosomal rearrangement in ciliates with limited germline DNA access, thereby providing new insights into the evolution of DNA rearrangement. Chapter 3 presents a comparative genomic study of three ciliate species including Oxytricha trifallax, Tetmemena sp. and Euplotes woodruffi. Collaborating with my colleagues, I assembled and annotated germline genomes in Tetmemena and E. woodruffi, as well as E. woodruffi’s somatic genome. We identified scrambled genes in all three species, especially the earlier-diverged E. woodruffi, though at a lower level (7.3% of gene loci) compared to Oxytricha (15.6%) and Tetmemena (13.6%). E. woodruffi may therefore represent an intermediate between the nonscrambled ancestral genome and more massively scrambled genomes as can be seen in Oxytricha and Tetmemena. We also found that scrambled genes tend to have more paralogs or have partial MDS duplications, suggesting that local duplications might play a role in the evolutionary origin of scrambled genes. Chapter 4 reports a new genetic code identified in a basal spirotrich ciliate, Licnophora macfarlandi. Ciliates have been a hot spot for the evolution of alternative genetic codes. All variant genetic codes in ciliates reassign canonical stop codons to amino acids, and in most cases the UAA and UAG are reassigned to the same amino acid, or are both used as stop codons. The codon usage analysis in Licnophora revealed an unprecedented genetic code that translates the UAA to glutamic acid and the UAG to glutamine. We also detected candidate tRNAs from the somatic genome which can recognize the UAA and UAG. Chapter 5 describes possible future directions to understand the genome complexity of ciliates.

The sequence of 1360 base pairs of the P22 PL operon was determined, linking a continuous sequence from PL through abc2. P22 mutants bearing deletions in the sequenced region were constructed and tested for their phenotypes. Plasmids were constructed to express PL operon genes singly and in combinations from PlacUV5. Two previously known genes, 17 and c3, are located within this sequence. In addition, three new genes have been identified: ral, kil and arf. Genes ral and c3 are homologous, as well as functionally analogous, to λ ral and cIII, respectively. P22 kil, like λ kil, kills the host cell when it is expressed. The two kil genes, although analogous in cell killing and map location, have no apparent sequence homology. The functions of the P22 and λ kil genes are unknown; however, P22 kil is essential for lytic growth in the absence of abc. Gene arf (accessory recombination function) is located just upstream of erf; it is essential for P22 growth in the absence of kil or other genes upstream in PL. The growth defect of P22 bearing a deletion that removes arf is complemented by expression of either arf or the λ red genes from plasmids. P22 sequences that include the stop codon for 17 potentially form a small stem-loop structure; these sequences are nearly identical to λ sequences that contain the stop codon for ssb. In λ this potential stem-loop structure occupies a map position near the terminator tL2b. Plasmids that include the potential P22 structure negatively regulate kil gene expression in cis.

Thesis advisor: Abhisheck Chatterjee / The genetic codon expansion (GCE) is a technique that uses an orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pair to incorporate noncanonical amino acids (ncAA) into proteins, to enable more protein-based chemistry. In the past two decades, more than 200 ncAAs have been site-specifically introduced into proteins in E. coli, and facilitated studies of protein structures, functions and interaction with other molecules. Although a large variety of ncAAs are available for incorporation in the bacterial systems, significantly fewer ncAAs are accessible for incorporation in eukaryotic cells. An expanded GCE toolbox will be beneficial for numerous applications in eukaryotic systems. Currently, introducing ncAAs in eukaryotes predominantly relies on the archaeal pyrrolysyl tRNA/aaRS pair. Such a strong dependence on a single platform has precluded genetic encoding of many desirable ncAAs, including structural mimics of many important post-translational modifications. The work presented in this thesis first developed an engineered E. coli leucyl tRNA/aaRS pair to enable site-specific incorporation of citrulline, an important PTM, into proteins expressed in mammalian cells. This technology was used to reveal the role of citrullination on site R372 and R374 of PAD4. Additionally, aiming at genetically encoding more diverse ncAAs, all 20 E. coli derived tRNA/aaRS pairs were screened for their ability to suppress TAG and TGA in mammalian cells. This study revealed several tRNA/aaRS pairs that are suitable for ncAA incorporation in mammalian cells, including those selective for phenylalanine, lysine, arginine, serine and glutamine. Efforts are currently under way to engineer these pairs to genetically encode new structural classes of ncAAs. / Thesis (PhD) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Catechol 1,2-Dioxygenase from Rhodococcus opacus is a type of intradiol dioxygenase enzyme that catalyzes the conversion of catechol to cis, cis muconic acid. This enzymatic conversion has the potential to be useful in a number of different applications such as treating wastewater contaminated with aromatic compounds to creating a greener method to produce cis, cis muconic acid which can be used to make a number of industrially important base chemicals. However, for enzymes to be used in industrial conditions, they must be highly stable. The experimental chapters in this thesis explore whether this enzyme can be stabilized to meet industrial requirements while minimizing any loss in catalytic activity. Through the studies described in Chapter 2, a mutant enzyme was generated through disulfide bond engineering with significantly improved thermostability. However overall catalytic activity was reduced. Toward addressing this loss of catalytic activity, in Chapter 3, attempts were made to implement state-of-the-art genetic code expansion strategies to increase catalytic activity of the enzymes. However, these attempts were unsuccessful. Finally, Chapter 4 describes how future stability engineering could be optimized using design pipelines similar to the one developed in this study. Additionally, it describes possible additional optimizations toward making the application of these enzymes cost effective in the near future.

Over the past years, small RNAs (smRNAs) have been identified as important molecular regulators of gene expression and specifically eukaryotic messenger RNAs (mRNAs). Small RNAs including small-interfering RNAs (siRNAs) and microRNAs (miRNAs) take part in the RNA silencing pathway and regulate various pathways in the cell including transcription, genome integrity, chromatin structure, mRNA stability, and translation. siRNAs are usually from exogenously derived molecules, while miRNAs are expressed endogenously by the genome. The RNA silencing pathway is highly conserved between organisms and plays a critical part in maintaining homeostasis, host-pathogen interaction, and disease progression. Thus, a better understanding of the RNA silencing pathway and probing of the molecules involved in the process is instrumental in developing tools that can better regulate the expression of specific genes. The viral suppressor of RNA silencing (VSRS) p19, is a 19 kDa protein that is expressed by tombusviruses and exhibits the highest reported affinity to small RNAs, including siRNA and miRNA. Further engineering of this protein acts as an interesting means to control the RNA silencing pathway and provides a platform to design novel tools to further modulate the activity of smRNAs in living systems. The ability to incorporate new and useful chemical functionality into proteins within living organisms has been greatly enhanced by technologies that expand the genetic code. These usually involve bioorthogonal transfer RNA (tRNA) /aminoacyl-tRNA synthetase (aaRS) pairs that can selectively incorporate an unnatural amino acid (UAA) site specifically into ribosomally synthesized proteins. Site-specificity is coded for by using a rare codon such as the amber stop codon. In Chapter 2, we demonstrate the engineering of p19 for the development of a Förster resonance energy transfer (FRET) reporter system for the visualization of RNA delivery and release in cells using UAAs and bioorthogonal click chemistry, which was done by incorporating azidophenylalanine (AzF). In Chapter 3, by incorporating UAAs into p19’s binding pocket, we were able to enhance its smRNA suppressing activity by covalently trapping the bound substrates. We have demonstrated the engineering of a molecular switch that contains photo-crosslinking groups that covalently trap smRNAs. In Chapter 4, incorporating a metal-ion chelating UAA (2,2′-bipyridin-5-yl) alanine (BpyAla) into p19’s binding pocket has successfully led to site-specific cleavage of small RNAs including siRNAs and endogenous miRNAs. The genetic introduction of BpyAla provides a unique method of introducing catalytic activity into proteins of interest. The developed unnatural enzyme provides a new tool for catalytic suppression of the RNA silencing pathway. These results demonstrate the power of adding new chemistries to proteins using UAAs to achieve possible, diverse applications in therapy and biotechnology.

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Thesis advisor: Abhishek Chatterjee / Thesis advisor: Jia Niu / Directed Evolution has emerged as an invaluable tool for advancing protein functions in both research and industry. Our lab has pioneered a directed evolution platform in mammalian cells, utilizing an AAV delivery vector to package a DNA library and linking the biomolecule of interest to AAV production. During my tenure in Prof. Chatterjee's lab, I focused on harnessing our lab’s directed evolution platform, known as Virus-Assisted Directed Evolution of tRNA (VADER), to develop highly efficient tRNAs for genetic code expansion. Additionally, I contributed to the development of the AAV-based selection platform, termed Virus-Assisted Mutational Profiling (VAMP), as a profiling tool. Through the utilization of VAMP, I conducted comprehensive profiling of tRNA and RNA polymerase III promoter sequences. This enabled me to gain insights into regions of flexibility and evolution, ultimately leading to the construction of improved constructs with enhanced activity relative to the starting sequence. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Le code génétique est la table de correspondance entre codons (unité structurelle d'un gène) et acides aminés (brique élémentaire des protéines). Le code génétique est (1) universel, tous les êtres vivants ou presque partagent le même code; (2) univoque, chaque codon spécifie un seul acide aminé et (3) dégénéré, les acides aminés peuvent être codés par plusieurs codons. Ce code dégénéré est donc utilisé par l'ensemble du vivant mais pas de la même manière, certains codons synonymes étant utilisés préférentiellement chez des espèces et pas d'autres. Pour comprendre l'émergence des biais d'usage du code (BUC) génétique entre espèces, je me place dans un contexte évolutif.Dans ce manuscrit, je présente mes travaux de recherche en quatre parties. La première partie introductive décrit la mise en évidence et les propriétés du code génétique, son biais d'usage et les diverses caractéristiques de précédents modèles de codons. La deuxième partie présente le modèle d'évolution de codons SENCA pour Sites Evolution at the Nucleotides, Codons and Amino-acids layers que j'ai développé durant ma thèse. SENCA prend en compte la structure du code génétique. Je valide sa paramétrisation par des simulations numériques et une étude sur des espèces bactériennes ou archées. La partie suivante décrit deux extensions de SENCA qui modélisent plusieurs hypothèses d'origines évolutives du BUC et une application de SENCA sur les conséquences génomiques d'adaptations environnementales. La dernière partie étudie les origines de variations de BUC le long du génome humain par une approche de génomique comparative / In this manuscript, I introduce my doctoral research in four parts. The first introductive part highlights the properties of the genetic code and its usage bias but also the caracteristics of previous published codons models. The second part presents an evolutionary codons models named SENCA for Sites Evolution at the Nucleotides, Codons and Amino-acids layers that I developped. SENCA takes into account the genetic code structure. I perform simulations and study prokaryotes species to confirm its parametrization. The following part provides two extensions of SENCA to test the hypotheses concerning the evolutive origins of CUB and an application of SENCA to study the genomic consequences of an environmental adaptation. The last part studies the origins of CUB variation within the human genome using a comparative genomic strategy

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