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Eukaryotic ribosome synthesis is a complex process that consumes a lot of energy and involves several hundreds of trans-acting factors that transiently associate with nascent ribosomes. Biogenesis of ribosomal subunits (the small 40S and the large 60S) starts with transcription of a long precursor ribosomal RNA (pre-rRNA) by RNA polymerase I (Pol I) in the nucleolus. This is a key step that globally controls yeast ribosome synthesis. The pre-rRNA, ‘the 35S transcript’, encodes the mature sequence (18S, 5.8S, and 25S) rRNA constituents of both the 40S and 60S subunits. The 35S transcript is subsequently modified, cleaved (processed) and assembled with numerous structural ribosomal proteins and ribosome synthesis factors (trans-acting factors) to form various ribosomal particles (pre-ribosomes, precursors to the 40S and 60S subunits) along ribosome assembly pathway. In the budding yeast Saccharomyces cerevisiae, it has been reported recently that the processing of the 35S nascent transcript and the assembly of pre-ribosomes occur concomitantly with Pol I transcription in the nucleolus. In this process, the growing Pol I transcript gradually assembles with pre-40S structural ribosomal proteins and ribosomal synthesis factors to form the so-called ‘SSU-processome’ or ‘90S pre-ribosome’, the earliest precursor of the 40S subunit. The SSU-processome/90S pre-ribosome localizes to the nucleolus and consists of the 35S pre-rRNA, the U3 small nucleolar (sno) RNA, about a dozen of 40S ribosomal proteins and more than forty ribosome synthesis factors. The U3 snoRNA and pre-40S ribosome synthesis factors are all implicated in the processing of the 35S precursor (at sites A0, A1 and A2) and therefore in the synthesis of the 18S rRNA component of the 40S subunit. Significantly, the association of the U3 snoRNA with the growing 35S transcript is important for pre-40S assembly, whereas its dissociation from the processed transcript (following cleavage at sites A0-A2) is crucial for the overall structural remodeling of the 18S rRNA and for the formation of pre-40S ribosomes from the earliest precursor 90S particles. This thesis mostly addresses the identification and functional characterization of Esf2 and Bfr2, two novel 40S synthesis factors, components of the SSU-processome/90S pre-ribosome in yeast. Both proteins localize to the nucleolus and their genetic depletions lead to failure in the production of 40S subunits. In the absence of either factor, the 35S pre-rRNA is not processed at sites A0-A2 and the 18S rRNA is not synthesized. Also, pre-ribosome assembly is affected and pre-40S ribosomes fail to mature properly. Strikingly, in the absence of either factor, the U3 snoRNA remains associated with unprocessed 35S transcript within pre-ribosomes indicating that Esf2 and Bfr2 are required to dissociate U3 from pre-ribosomes. This process also involves RNP (ribonucleoprotein particle) unwinding activities of the putative RNA helicase Dbp8. La biogenèse du ribosome eucaryote est un processus complexe qui consomme beaucoup d’énergie et implique plusieurs centaines de facteurs trans qui s’associent de manière transitoire avec les pré-ribosomes en cours de formation. La biogenèse des sous-unités ribosomiques (la petite sous-unité 40S et la grande sous-unité 60S) débute dans le nucléole par la synthèse d’un long précurseur d’ARN ribosomique (le pré-ARNr, dit 35S chez la levure Saccharomyces cerevisiae) par l’ARN Polymérase I (Pol I). Ceci constitue une étape clé dans le contrôle global de la synthèse du ribosome chez la levure. Le pré-ARNr 35S renferme les séquences des ARNr matures 18S (ARNr de la sous-unité 40S) et 5.8S et 25S (deux des trois ARNr de la sous-unité 60S). Le pré-ARNr 35S subit un long processus de maturation et d’assemblage au cours duquel il est modifié, clivé (on parle du « processing » du pré-ARNr) et s’assemble avec des protéines ribosomiques (« RP », composants structuraux des sous-unités ribosomiques matures) et de nombreux facteurs de synthèse (facteurs trans) pour former différentes particules pré-ribosomiques (précurseurs des sous-unités 40S et 60S). Chez la levure S. cerevisiae, il a récemment été montré que le processing du pré-ARNr 35S et l’assemblage des pré-ribosomes se produisent de manière concomminante avec la transcription Pol I dans le nucléole. Ainsi, le transcrit Pol I en cours de synthèse s’assemble progressivement avec des facteurs de synthèse ainsi que des RP pour former le « SSU processome » ou « pré-ribosome 90S », tout premier précurseur de la petite sous-unité 40S. Le SSU processome/pré-ribosome 90S est localisé dans le nucléole et est consisté du pré-ARNr 35S naissant, du petit ARN nucléolaire (snoRNA) U3, d’une douzaine de RP de la petite sous-unité 40S et de plus de 40 facteurs de synthèse. Le snoRNA U3 et ces facteurs de synthèse sont tous impliqués dans les clivages du pré-ARNr 35S aux sites A0, A1 et A2, et donc dans la biogenèse de l’ARNr 18S. L’association du snoRNA U3 avec le pré-ARNr 35S naissant est importante pour l’assemblage du SSU processome/pré-ribosome 90S. Par ailleurs, sa dissociation après les clivages aux sites A0-A2 permet un remodelage structural général de l’ARNr 18S et la formation du « pré-ribosome 40S » à partir de la particule précoce 90S. Au cours de cette thèse, nous avons identifié et caractérisé fonctionnelement chez la levure deux nouveaux facteurs de synthèse de la petite sous-unité 40S et composants du SSU processome/pré-ribosome 90S: Esf2 et Bfr2. Ces deux protéines sont localisées dans le nucléole. Leur déplétion entraîne une incapacité à produire la sous-unité ribosomique 40S. En l’absence d’Esf2 ou Bfr2, le pré-ARNr 35S n’est plus clivé aux sites A0-A2 et l’ARNr 18S mature n’est plus produit. L’assemblage des pré-ribosomes est aussi affecté, notamment la formation du pré-ribosome 40S. De manière importante, en l’absence de l’un ou l’autre de ces facteurs, le snoRNA U3 reste associé au pré-ARNr 35S non clivé au sein des pré-ribosomes, indiquant qu’Esf2 et Bfr2 sont requises pour la dissociation d’U3 des pré-ribosomes. Ce processus implique aussi Dbp8, une hélicase à ARN présumée.

The anaerobic intestinal spirochaete Brachyspira pilosicoli is the causative agent of “intestinal spirochaetosis” (IS), a disease of humans and a number of animal species. IS has been reported in adults and children worldwide but the prevalence in people living in poor hygienic conditions, indigenous populations, homosexual males, and in immunocompromised people is much higher than in other populations. IS is also widespread in pigs and chickens, and causes significant economic impact in the associated industries. To date attempts to develop a vaccine against B. pilosicoli to protect humans and animals have not been successful. In this study a reverse vaccinology approach was used, in which 24 putative open reading frames (ORFs) derived from a partial genome sequence of B. pilosicoli were subjected to in silico and laboratory screening processes to identify potential efficacious vaccine antigens. In silico analysis of the ORFs using a range of bioinformatics algorithms assigned 12 ORF products as periplasmic, outer membrane, or secretory proteins, and these were given a high priority as potential vaccine candidates. The 12 selected ORFs were amplified from a human strain of B. pilosicoli (Wes-B) and cloned. Products from nine ORFS were successfully over-expressed in an Escherichia coli expression system, and then purified using affinity chromatography. In an in vitro immunogenicity trial all the recombinant proteins except for NAV-P27 were strongly recognised in Western immunoblots by a mouse serum raised against B. pilosicoli strain WesB, and by a subset of convalescent sera from pigs naturally and experimentally infected with B. pilosicoli. In an analysis of in vivo immunogenicity, the post-immunisation mouse sera raised against each recombinant protein reacted strongly with each specific proteins, and also recognised the native protein in extracts of B. pilosicoli strain WesB. Sequence analysis of four randomly selected ORFs showed that these were highly conserved amongst the genomes of different human and swine strains of B. pilosicoli. Evaluation of all the data obtained in the reverse vaccinology approach resulted in selection of four ORF products (NAV-P3, NAV-13, NAV-22 and NAV-31) as being potentially protective antigens to be analysed for their further efficacy. These four recombinant proteins were assessed for their efficacy as vaccine components in a mouse model of IS, where the animals were challenged with a human strain of B. pilosicoli. The proteins all induced systemic and local antibody responses, and tended to reduce spirochaete colonisation following experimental infection. These proteins used individually or in combination now have the potential to be further developed into a new vaccine to prevent B. pilosicoli infections.

S-palmitoylation is a reversible post-translational lipid modification of proteins by adding a 16-carbon palmitate onto a cysteine residue. Palmitoylation has been shown to control the trafficking and function of many signalling proteins including ion channels. In this Thesis, palmitoylation is shown to control both the plasma membrane expression and gating properties of large conductance calcium- and voltage- dependent potassium (BK) channels. The BK channel is assembled from four pore-forming α-subunits. Each α-subunit contains seven transmembrane domains (S0-S6), with an extracellular N-terminus and a large intracellular C-terminus. BK channel α-subunit is encoded by a single gene Kcnma1 that undergoes extensive pre mRNA splicing at various splice sites, thus there are a number of alternatively spliced variants of α-subunits. Using quantitative imaging assays, palmitoylation of the intracellular S0-S1 loop controlled trafficking of full length ZERO variant BK channels to the plasma membrane in HEK293 cells as well as neuronal N2a cells. Importantly, all four α-subunits need to be palmitoylated for robust surface expression. Thus, palmitoylation of the S0-S1 loop of the α-subunit is important for surface expression of BK channels. The BK channel may also assemble with auxiliary β-subunits (β1-4) that regulate surface expression and gating properties of BK channels. The N-terminus of the β1- subunit and the C-terminus of the β4-subunit were shown to be palmitoylated using [3H]-palmitate incorporation, respectively. However, mutation of the palmitoylated cysteine (C18 in β1 and C193 in β4) to alanine to generate depalmitoylated β- subunits had no significant effects on the electrophysiological properties resulting from co-expression with the ZERO variant of the BK channel. However, although palmitoylation of the S0-S1 loop does not affect the electrophysiological properties of the ZERO channels alone, it is important for the shift in the V0.5max of ZERO channel when co-expressed with the β1-subunit, but not β4-subunit. These data suggest that palmitoylation of the S0-S1 loop controls the functional coupling between the ZERO α-subunit and β1-subunit. Although palmitoylation of C18 in the N-terminus of the β1-subunit was not required for functional coupling to α-subunits, we identified other critical residues within the short intracellular N-terminus of the β1-subunit that are essential. The functional coupling between BK α- and β1-subunit was predicted to be controlled by the interaction between a non-classic amphipathic α-helix in the β1 subunit N-terminus and the plasma membrane. Deletion, or mutations predicted to disrupt the interaction significantly decreased the β1-subunit induced left shift in the BK channel V0.5max. This suggests that the amphipathic in-plane anchor is critical for functional coupling of β1-subunits with BK channel α-subunits. In this Thesis, we demonstrated: i) palmitoylation of the α-subunit S0-S1 loop controls surface membrane expression of BK channels, and also controls functional regulation by β1, but not β4-subunits; and ii) a potential non-classical amphipathic in-plane anchor in the β1 N-terminus is essential for functional coupling with α- subunits. These studies help us further understand the regulation of BK channels and suggest potential therapeutic targets for various diseases related to dysfunctional BK channels, such as hypertension.

Zooxanthellae are very important for the coral reef ecosystem. The diversity of coral hosts is high in the Indo-Pacific, but the diversity of zooxanthellae has not been broadly investigated. Southern Taiwan and Penghu Islands are coral reef and non-reefal communities, respectively. These localities were chosen as the sampling sites for this study to maximize the opportunity of surveying this region in the Indo-Pacific. Zooxanthellae diversity was investigated in 40 host species including 32 species of Scleractinia, 4 species of Actiniaria, 3 species of Milleporina and 1 species of Helioporacea using polymerase chain reaction (PCR) of the ssrRNA gene and restriction fragment length polymorphism (RFLP) patterns. The phylogenetic relationship of partial and complete sequences of the ssrRNA gene were also analysed. Aiptasia puchella harbors clade B; Oulastrea crispata only harbors clade E; while Acropora palifera and Montipora cactus harbor both clades C and E. Zooxanthellae isolated from all except the above 4 host species are identified as "clade C" sensu Rowan and Powers (1991a). Therefore, the clade C is the dominant type in the Indo-Pacific. Phylogenetic analyses based on partial and complete sequences obtained in this study and also from the GenBank data base demonstrate 4 clades (A, B, C and E) in the genus Symbiodinium. Clade E, classed as D3 RFLP type in previous studies, is a distinct clade differing from A, B and C by RFLP and sequencing data. Clade E has only been found in Scleractinia host species collected in shallow-water habitats in the Pacific. The composition of zooxanthellae clades and ecological pattern of polymorphic symbiosis is not consistent with the irradiance adaptation hypothesis in the Caribbean. A literature survey of zooxanthellae in Scleractinian hosts indicates a significant difference between the Caribbean and the Pacific. The documented biogeography of zooxanthellae clades and the ecological pattern of polymorphic symbiosis are also differ between the Caribbean and the Indo-Pacific.

Ribosomes are universally conserved macromolecular machines that translate cellular genetic information into proteins. All ribosomes are com- posed of two ribonucleoprotein subunits. In eukaryotes these are called 40S (small) and 60S (large) subunits. Biogenesis of both subunits begins from a common precursor ribosomal RNA (rRNA) transcript in the nucleolus. The 18S rRNA of the small subunit is encoded in the 5ʹ end of the precursor transcript. U3 snoRNA and about 70 accessory factors associate with the 50 end of the pre-rRNA, to form the SSU processome or 90S pre-ribosome, which can be observed as terminal knobs in electron micrographs. After the initial processing and folding, the pre-rRNA is cleaved at site A2 to release the pre--40S. This event is dependent on the formation of the central pseudoknot, a structure that maintains the integrity of 40S architecture. Bud23 is the methyltransferase responsible for modification of the base G1575 in the P-site of the small subunit. Work presented here demonstrates that the in vivo stability, and thus function, of Bud23 is dependent on the presence of Trm112, a novel ribosome biogenesis factor identified in this work. Analysis of rRNA processing and strong negative genetic interactions with RNaseMRP mutants, provide strong evidence for that BUD23 is required for A2 cleavage. Extragenic suppressors of bud23 [delta] were identified in UTP14, UTP2, IMP4 and ECM16, coding for SSU processome components. Bud23 and the RNA helicase Ecm16 interact physically as well as genetically. Most fascinatingly, using ecm16 enzymatic mutants, this work provides compelling evidence that Ecm16 facilitates removal of U3 snoRNA from pre-rRNA, a prerequisite for central pseudoknot formation and 90S to pre--40S transition. These findings suggest a model in which binding of Bud23 monitors the status of 40S assembly, triggering Ecm16 activity to promote release of the pre--40S from 90S only after the critical folding of the small subunit rRNA. / text

Voltage-gated calcium channels (CaV) are key regulators of cellular excitability; they translate electrical information into biochemical responses in excitable cells such as nerve and muscle cells. CaV are separated in three families: CaV1, CaV2 and CaV3. CaV1 and CaV2 typically comprise a pore-forming alpha1 with auxiliary β and alpha2delta subunits. The alpha2delta enhances surface expression and modulates the biophysical properties of CaV. It has been implicated in pain and epilepsy, and the target for anti-epileptic and anti-nociceptive gabapentinoid drugs. Despite its clinical significance, the relationship between the structure and function of this subunit remains poorly understood. Fitzgerald and co-workers recently showed that the N-terminal region of alpha2delta-1, termed the R domain (Rd), is both necessary and sufficient to replicate the effects of full-length alpha2delta on CaV2.2 channels. In order to understand the functional role(s) of Rd and the regions downstream of it, the biochemical and cell biological properties of alpha2delta were explored producing a set of alpha2delta-truncated proteins, in which the delta protein was inserted into an inert type-1 transmembrane reporter protein (PIN-G). The construct was then extended towards the N-terminal of the alpha2delta-1 (C- to N- PIN-constructs). Other sets of constructs, lacking the delta protein, were prepared after successive additions of stop codons (TGA) in the alpha2delta (N- to C- PIN-constructs). The MIDAS motif within the VWA domain of alpha2delta-1/-2 has been suggested to be critical for trafficking of alpha2delta to the cell surface. Whilst the present study supports a role for MIDAS in surface expression of alpha2delta, it is the Rd that appears essential. Mutation of MIDAS reduced expression, whereas the removal of Rd completely abolished the presence of alpha2delta at the cell surface. Examination of the electrophysiological effects of N- to C- terminal truncated constructs (PIN-Rd, PIN-Rd-VWA and PIN-alpha2) on CaV2.2/β1b channels revealed that, in contrast to the full functionality of Rd alone, extension to the end of the VWA domain, or the alpha2 region, abolished typical alpha2delta-mediated current enhancement. Nevertheless, both constructs increased rate of voltage-dependent inactivation, indicating that they interact with the channel via Rd. Thus, Rd appears to contain all the machinery required to support the electrophysiological and trafficking effects of alpha2delta. Preliminary work has generated tools that could be used to conduct competition-based assays to identify the extracellular loops of the CaV2.2 alpha1 subunit that interact with the Rd. Such an approach could be applied to other alpha1 subtypes to determine discrete alpha2-Rd interactions, information that is critical for further therapeutic exploitation of alpha2delta. Finally, the data from this thesis and the existing literature have been used to propose a revised model of how alpha2delta interacts with CaV.

The aim of his study was to investigate the role of Dctn2 in mouse preimplantation embryonic development, specifically its effect on the first cell fate decision, when the number of cells increases from eight to sixteen.

abstract: The primary carbon fixing enzyme Rubisco maintains its activity through release of trapped inhibitors by Rubisco activase (Rca). Very little is known about the interaction, but binding has been proposed to be weak and transient. Extensive effort was made to develop Förster resonance energy transfer (FRET) based assays to understand the physical interaction between Rubisco and Rca, as well as understand subunit exchange in Rca. Preparations of labeled Rubisco and Rca were utilized in a FRET-based binding assay. Although initial data looked promising, this approach was not fruitful, as no true FRET signal was observed. One possibility is that under the conditions tested, Rca is not able to undergo the structural reorganizations necessary to achieve binding-competent conformations. Rca may also be asymmetric, leading to less stable binding of an already weak interaction. To better understand the structural adjustments of Rca, subunit exchange between different oligomeric species was examined. It was discovered that subunit exchange is nucleotide dependent, with ADP giving the fastest exchange, ATP giving slower exchange and ATPS inhibiting exchange. Manganese, like ADP, destabilizes subunit-subunit interactions for rapid and facile exchange between oligomers. Three different types of assemblies were deduced from the rates of subunit exchange: rigid types with extremely slow dissociation of individual protomers, tight assemblies with the physiological substrate ATP, and loose assemblies that provide fast exchange due to high ADP. Information gained about Rca subunit exchange can be used to reexamine the physical interaction between Rubisco and Rca using the FRET-binding assay. These binding assays will provide insight into Rca states able to interact with Rubisco, as well as define conditions to generate bound states for structural analysis. In combination with assembly assays, subunit exchange assays and reactivation studies will provide critical information about the structure/function relationship of Rca in the presence of different nucleotides. Together, these FRET-based assays will help to characterize the Rca regulation mechanism and provide valuable insight into the Rubisco reactivation mechanism. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2017

Calcium entry through voltage-gated calcium (CaV) channels is important in diverse cellular processes including neurotransmitter release, gene expression and cardiac pacemaker activity. CaV channels auxiliary CaVβ subunits enhance plasma membrane expression and modify the biophysical properties of CaVα1 subunits. Due to their multi-domain structures - including a conserved SH3-GK 'core' and hypervariable N- and C- terminal domains - CaVβs are also considered to be members of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins, and may therefore act as molecular scaffolds both within and outside the CaV channel complex. This project studied the roles of CaVβ N- and C-terminal hypervariable domains in contributing to isoform-specific differences in CaVβ functions both in a) CaV channel complex expression and distribution, and b) interactions with non channel proteins. To analyse such contributions a series of molecular tools were developed to assess the distributions of CaVβs (both within and outside the CaV channel complex) and their interactions with novel potential partner proteins. This involved systematically testing fluorophore- and epitope-tagged CaVβs for co-localisation with both fluorophore-tagged CaV2.2 and a range of myc-tagged potential interaction partners (as quantified either by a 'Membrane Localisation Index' developed during this project or Intensity Correlation Analysis). This approach uncovered much detail about relative isoform specificities of CaVβ non-channel complex protein-protein interactions, however one particularly striking interaction was discovered between CaVβ1b/CaVβ4 and the nuclear protein Heterochromatin 1 γ (HP1γ), where nuclear translocation of CaVβ1b or CaVβ4 was induced upon association with HP1γ. Given the similarity of CaVβ1b and CaVβ4 N termini, a series of CaVβ1b N-terminal chimeras were then created, where the N terminus was exchanged with that of CaVβ3 (which did not interact with HP1γ). Subsequent imaging studies using these chimeras then confirmed that the CaVβ1b N terminus is necessary for co-localisation with HP1γ and subsequent HP1γ mediated CaVβ nuclear uptake. Given that an interaction between the CaVβ3 isoform and Pax6(S) - another nuclear protein - have been reported, where the CaVβ3-Pax6(S) interaction also induces nuclear translocation of both proteins, the CaVβ1b/CaVβ4-HP1γ interaction may represent one of a range of as-yet undiscovered CaVβ1b/gene regulatory protein interactions. As interaction with CaVβ3 suppresses the transcriptional activity of Pax6(S), nuclear targeting may be an important means by which CaVβs modulate gene expression - which in the case of HP1γ interactions may occur via de-repression.