Thesis (M.B.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / 2031-01-01

Chloroplast biogenesis relies on the import of thousands of nuclear encoded proteins into the organelle and proper sorting to their sub-organellar compartment. The majority of nucleus-encoded chloroplast proteins are synthesized in the cytoplasm and imported into the organelle via the Toc-Tic translocation systems of the chloroplast envelope. In many cases, these proteins are further targeted to subcompartments of the organelle (e.g. the thylakoid membrane and lumen or inner envelope membrane) by additional targeting systems that function downstream of the import apparatus. The inner envelope membrane (IEM) plays key roles in controlling metabolite transport between the organelle and cytoplasm, and is the major site of lipid and membrane biogenesis within the organelle. In contrast to the protein import and thylakoid targeting systems, our knowledge of the pathways and molecular mechanisms of protein targeting and integration at the IEM are very limited. Previous reports have led to the conclusion that IEM proteins are transferred to the IEM during protein import via a stop-transfer mechanism. Recent studies have shown that at least two components of the Tic machinery (AtTic40 and AtTic110) are completely imported into the stroma and then re-inserted into the IEM in a post-import mechanism. This led me to investigate the mechanisms and pathways involved in the integration of chloroplast IEM proteins in more detail. I selected candidates (AtTic40 for post-import and IEP37 for stop-transfer) that are predicted to have only one membranespanning helix and adopt the same IEM topology to facilitate my analysis. My studies confirm the existence of both stop-transfer and post-import mechanisms of IEM protein targeting. Furthermore, I conclude that the IEP37 transmembrane domain (TMD) is a stop-transfer signal and is able of diverting AtTic40 to this pathway in the absence of AtTic40 IEM targeting information. Moreover, the IEP37 TMD also functions as a topology determinant. I also show that the AtTic40 targeting signals are context dependent, with evidence that in the absence of specific information in the appropriate context, the AtTic40 TMD behaves as a stop-transfer signal. This is an indication that the stop-transfer pathway is the default mechanism of protein insertion in the IEM.

Historically, our perception of the nuclear envelope has evolved from a simple barrier isolating the genome from the rest of a cell to a complex system that regulates functions including transcription, splicing, DNA replication and repair and development. Several recent proteomic studies uncovered a great variety of nuclear envelope transmembrane proteins (NETs). Diseases associated with several nuclear envelope proteins, mostly NETs, affect many tissues e.g. muscle, adipose tissue, skin, bones. Many NETs of the inner nuclear membrane have been shown to interact with chromatin, suggesting that their influencing gene expression might explain NET roles in disease. This work is focused on finding novel interactions of NETs with chromatin. First, SUN2 post-translational modifications were analysed and the effect of phosphomimetic and phospho-null mutants on heterochromatin and the cytoskeleton was tested by overexpression. However, no obvious changes were found. Second, several tissue-preferential NETs were tested in an adipocyte differentiation system. NET29 changed chromosome 6 position in pre-adipocytes. This matched changes in chromosome positioning that occur during adipocyte differentiation when NET29 is normally induced. Post-translational modifications of NET29 are likely to play a vital role in this process because a phospho-null mutant dominantly blocked chromosome repositioning. The effect of over-expression and down-regulation of NET29 on transcription was tested and results suggest that NET29 negatively regulates expression of myogenic genes during adipogenesis. This thesis is split into six chapters. Chapter I is an overview of the nuclear envelope, adipogenesis and chromatin remodelling, Chapter II is a detailed description of methods used in this study. Chapter III focuses on post-translational modifications of SUN2, as well as trials to identify novel partners of SUN2. Chapter IV and V deal with a novel nuclear envelope transmembrane protein and its role in adipogenesis. Finally, the last chapter includes a discussion and recommended future directions.

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The nuclear envelope is a complex double membrane system that separates the activities of the nuclear and cytoplasmic compartments. A recent explosion in the number of proteins associated with this subnuclear organelle together with it now being linked to over 2 dozen diseases indicates the importance of better understanding its functional organisation. This thesis addresses two important questions for this: how do integral proteins of the nuclear envelope get to their sites of function and do any of these proteins direct genome organisation? To address the first question I used FRAP and photoactivation methods to find that different proteins use at least 4 distinct mechanisms to reach the inner nuclear membrane. Some appeared to be translocated by simple unaided lateral diffusion in the membrane while others needed Ran GTPase activity, others ATP, some others were aided by phenylalanine/glycines (FGs). Both Ran and FG mechanisms required the nucleoporin Nup35, albeit the mechanisms appeared to be completely independent of one another. To investigate the role of the nuclear envelope in genome organization, I screened for nuclear envelope proteins that reposition particular chromosomes to the nuclear periphery, finding five with this function. Interestingly, all of the proteins with this effect are tissuespecific. Depletion of two liver-specific nuclear envelope proteins reversed their effects on a specific chromosome for positioning with respect to the nuclear periphery. Finally, exogenous expression of these proteins in tissue culture cells caused induction of genes involved in differentiation pathways.

Microtubules are essential components of eukaryotic cytoskeleton and play a crucial role in variety of cell activities, such as cell mobility, intracellular transportation, cell division and organelle spatial organization. The initiation of microtubule nucleation is an important event to trigger the microtubule growth from different microtubule organizing centres (MTOCs) during the cell cycle in fission yeast. In interphase, the major MTOCs in the cells are nuclear envelope (NE) and microtubules existing in the cytoplasm. During mitosis, spindle pole body (SPB) that is the centrosome equivalent is the MTOC where astral and spindle microtubules initiate from. Once cells enter anaphase, the post-anaphase array (PAA) of microtubules will initiate from the equatorial MTOC (eMTOC) at the cell division site. To initiate microtubule nucleation, γ-tubulin small complex (γ-TuSC) will be recruited to the MTOC to form the “lock-washer” like γ-tubulin ring complex (γ- TuRC) as the scaffold of the microtubule. Fission yeast γ-TuSC is composed of two molecules of γ-tubulin and one each of GCP2 and GCP3 homologue, Alp4 and Alp6, respectively. Apart from Alp4 and Alp6, the homologue of human GCP4, GCP5 and GCP6, named Ghf1, Mod21 and Alp16 were identified as independent components of γ-TuRC in fission yeast. Protein Mto1 form a complex with its partner Mto2 and the complex directly interacts with γ-TuSC at all the MTOCs through the cell cycle. Mto1 is a large coiled-coil protein composed of 1115 amino acids. It has three main functional domains, including an N-terminal ~60 amino acids region termed CM1 motif that is required for the recruitment γ-TuSC to MTOCs, a central region that is required for the interaction with Mto2 at all cytoplasmic MTOCs and a ~44 amino acids region close to the C-terminus (named MASC) which is required for the binding of Mto1 to SPBs and eMTOC. The C-terminal truncation for Mto1 shows Mto1[1-549]-GFP mainly localizes on the NE and this Mto1 mutant is still functional for microtubule nucleation. It is referred as “Mto1[NE]”. In addition, truncation of 1-130 amino acids region for Mto1[1-549]-GFP creates the smallest Mto1 mutant that is able to initiate the microtubule nucleation in cytoplasm in a random manner. This Mto1[131-549]-GFP mutant fails to localize at any MTOCs, including NE. It is then referred as “Mto1[bonsai]”. To understand the mechanism that how is Mto1[NE] recruited to the NE, I performed two-step purification and mass spectrometry for both Mto1[1-9A1-549] and Mto1[131-9A1-549] strains, which form more significant puncta on both NE and cytoplasm respectively, to identify the potential receptor(s) of Mto1[1-9A1-549] on the nuclear envelope by comparing the different interactomes of Mto1[1-9A1-549] and Mto1[131-9A1-549]. Here, I show both exportin Crm1 and nucleoporin Nup146 are essential for the binding of Mto1 to the NE. I find the Localization of Mto1[1-9A1-549] GFP on the NE depends on binding of Mto1 to Crm1 via a nuclear export signal (NES)-like sequence within the N-terminus of Mto1. Further, I figure out that Spi1GTP (RanGTP in fission yeast) is involved in the formation of Crm1-Spi1GTP-Mto1 complex and required for the interaction of Mto1 to the NE. In addition, I also find that the FG repeats of Nup146 are essential for the binding of Mto1 to the nuclear envelope. It’s likely Nup146 anchors Crm1-Spi1GTP-Mto1 complex to the NE through the interaction between its FG repeats and Crm1. Last, my data are consistent with previous findings that the association of Mto1 to the NE is important for the microtubule nucleation from the NE.

Improving the energy efficiency of our existing building stock is attainable by upgrading the building envelope through carrying out various retrofit measures. The objective of this thesis is to evaluate the life-cycle cost implications of energy retrofits for existing buildings. Measures examined include improving insulation and air-tightness with overcladding strategies. The life-cycle costs of the upgrades are determined for an existing building and compared with model energy performance. A life-cycle cost evaluation for the building envelope upgrades is provided, together with the payback period and the projected return on investment (ROI) for two energy escalation rate scenarios. A costbenefit matrix for various over-cladding strategies is provided to facilitate the evaluation of each option. Further, this thesis presents a simplified ROI algorithm to enable owners, architects and engineers to evaluate the cost-benefit of their building envelope retrofit options.

Improving the energy efficiency of our existing building stock is attainable by upgrading the building envelope through carrying out various retrofit measures. The objective of this thesis is to evaluate the life-cycle cost implications of energy retrofits for existing buildings. Measures examined include improving insulation and air-tightness with overcladding strategies. The life-cycle costs of the upgrades are determined for an existing building and compared with model energy performance. A life-cycle cost evaluation for the building envelope upgrades is provided, together with the payback period and the projected return on investment (ROI) for two energy escalation rate scenarios. A costbenefit matrix for various over-cladding strategies is provided to facilitate the evaluation of each option. Further, this thesis presents a simplified ROI algorithm to enable owners, architects and engineers to evaluate the cost-benefit of their building envelope retrofit options.

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Pathology. Includes bibliographical references. Available online via OhioLINK's ETD Center.