There have been many efforts to improve efficiency of transmitter while meeting stringent linearity requirement of modern communication system. Among the technology to enhance efficiency of linear transmitter, the out-phasing technologies, also called the linear amplification with nonlinear components (LINC), is considered as a promising technology. LINC has been studied long times, since it provides excellent linearity with high efficiency by allowing adopt high efficient switch-mode power amplifiers. However, The LINC transmitter has some technical challenges: linearity degradation due to amplitude and phase mismatches, efficiency degradation due to poor combining efficiency, and narrow frequency bandwidth due to output matching network of switching power amplifier. In this thesis, some state-of-the-art techniques for solving the problems of LINC transmitters are presented. An unbalanced phase calibration technique compensates amplitude/phase mismatches between two parallel paths in the LINC system, and multi-level LINC (MLINC) and an uneven multi-level LINC (UMLINC) structure improve the overall power efficiency. And the reconfigurable Class-D switching PA enables multi-band operation with high efficiency and good linearity. With these techniques, the new multi-band out-phasing transmitter improves the efficiency without sacrificing the linearity performance.

In response to the scant studies comparing the language content of the employment-related units in Australia's Adult Migrant English Program (AMEP) and Canada's Language Instruction for Newcomers to Canada (LINC) program, the present study examines the curriculum guidelines and the selected instructional materials of AMEP's Certificate in Spoken and Written English (CSWE) III and LINC level 4 and investigates four instructors' implementation of the curricula (two instructors from each program). Through qualitatively analysis using NVivo, the results show that both LINC 4 and CSWE III exhibit strengths and limitations, and also alignment and misalignment, as compared to their theoretical frameworks. While LINC 4 covers more components in the framework, CSWE III demonstrates greater depth in implementing its theoretical foundation. Further, LINC 4 instructors mostly implement a task-based approach; the CSWE III instructors incorporate multiple approaches, while the curriculum claims text-based teaching as its main approach. This study suggests that in teaching English for employment purposes, more content that introduces the different functions of language use and communication strategies would be beneficial, and authentic texts and learner experience can also be valuable. The incorporation of different teaching approaches may be advantageous. Future research can further examine newcomer language program outcomes by comparing language production data from learners of similar programs in different contexts, in order to evaluate the impact of language training on learners’ ability to engage in employment-purposed communication. / Graduate

This manuscript is dedicated to researching the link between language acquisition and cultural integration. As this has overtime become a glaring gap in multiple federal integration policy instruments, we carried out both theoretical reviews as well as fieldwork to answer this question. In so far as fieldwork goes, we recruited two contrasting participants twenty-two and thirty-five years old respectively, male and female, from different cultural groups but both sharing the overall goal of integration in Canada and enrolled in the Language Instruction for Newcomers to Canada (LINC) program. We carried out semi-structured interviews by way of a theory-based protocol and subsequently processed the data via thematic analysis techniques to arrive at our results. Empirically speaking, we synthesized our participants’ lived experiences and perceptions and found that language plays four distinct roles related to culture and cultural integration. First, it is a tool with which to transmit cultural information directly (the referential function). Second, it is the carrier of a second wave of pragmatic (e.g. body language, prosody) from which cultural norms and conventions can be inferred. Third, language is a tool for group differentiation on the basis of which prototypical members (i.e. native-speakers both in the source and destination culture) at times ostracize learners based on linguistic markers. Lastly, we find that it is precisely the experience of loss of membership, disembeddedness, and lack of belonging in previous and future speech groups which then drives newcomers to cultural integration patterns which are less than additive in nature such as intersection and compartmentalization.

Thesis advisor: Eric S. Folker / Thesis advisor: David R. Burgess / Skeletal muscle is a multinucleated cell type in which the many nuclei are precisely positioned to maximize the distance between adjacent nuclei. In order to reach this final positioning, nuclei undergo an elaborate set of movements during muscle development. The disruption of this process is evident throughout muscular dystrophies and myopathies. However, the contribution of aberrant nuclear positioning toward disease progression is unclear and the mechanisms regulating nuclear movement and positioning are poorly defined. The goal of this thesis is to determine the contribution of disease-linked genes to the regulation of nuclear movement and positioning and how these mechanisms are coordinated in skeletal muscle. In this thesis, we utilize Drosophila melanogaster skeletal muscle as an in vivo model system to investigate nuclear positioning throughout muscle development and correlate aberrant nuclear positioning with a decrease in muscle function. We provide the first evidence of distinct mechanisms that are independently regulated by genes that are associated with two different muscle diseases, Emery-Dreifuss muscular dystrophy and Centronuclear myopathy (Chapter 2). We also provide evidence that Emerin-dependent regulation of the LINC complex is a critical determinant of nuclear positioning and for the first time demonstrate a division of Emerin functions among the two Drosophila emerin homologs, bocksbeutel and otefin (Chapter 3). Finally, we conduct a proof-of-concept screen to identify novel regulators of muscle development and function (Chapter 4). Together, the work presented in this thesis provides a framework to further our understanding of the mechanisms regulating nuclear movement and positioning as well as muscle development as a whole. Using the tools and techniques developed throughout this thesis, we provide novel insight into the mechanisms regulating nuclear movement and positioning and strengthen Drosophila as an in vivo model for investigating muscle development and function. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

With the explosion of protein tools as popular platforms for discovery and therapeutics, we see greater need for regulator systems that work congruently within these frameworks, especially safe and effective tools that can be implemented in humans. To this end, we endeavor to create orthogonal, precise and flexible protein modulators that can be easily employed to control protein tools with little need to iterate design for novel contexts. Hepatitis C NS3 protease is employed as a stabilizable linker between protein domains, enabling control over protein localization with FDA approved anti-viral drugs. The power of this tool is demonstrated by controlling gene expression through the controlled tether and release of a transcription factor. Inteins have already been employed to modulate proteins in synthetic contexts, however we observe that natural systems lack the avenues of control necessary to make them indispensable. We employ existing protein tools to construct a system of modular protein association, as well as drug and light inducible schema that reveal gaps in our knowledge of how to repurpose inteins in vivo. Despite this, we use inteins in the construction of a novel cargo delivery platform based on the fusogenic properties of the viral envelope glycoprotein from Vesicular stomatitis virus (VSV-G). We confirm reduced tropism of cargo delivery based on an intein lock-and-key mechanism that has implications for both biosafety as well as targeted delivery in vivo of natively folded proteins to target cells. / 2024-05-23T00:00:00Z

Le complexe linker of nucleoskeleton and cytoskeleton (LINC) est un complexe protéique conservé au cours de l’évolution, reliant les compartiments cytoplasmiques et nucléaires au travers la membrane nucléaire. Bien que les données récentes montrent une de ce complexe dans la régulation de la morphologie nucléaire et de la méiose, son implication dans l’organisation de la chromatine a été moins étudié chez les plantes. Le premier objectif de ce travail était de développer un plugin NucleusJ ImageJ dédié à la caractérisation de la morphologie nucléaire et de l’organisation de la chromatine en 3D. NucleusJ calcul 15 paramètres, y compris la forme et la taille des noyaux ainsi que des objets intra-nuclaires et leur position dans le noyau. Une documentation pour ce programme est disponible pour son utilisation, ainsi que des qu’un jeu données de noyaux pour tester ce programme. Plusieurs améliorations sont en cours pour développer une nouvelle version de ce plugin. Dans une deuxième partie de ce travail, des méthodes d’imagerie 3D ont été utilisées pour étudier la morphologie nucléaire et l’organisation de la chromatine dans les noyaux interphasiques chez Arabidopsis thaliana dans lequel les domaines d’htrochromatique sont groupés en régions detectable appelés chromocentres. Ces chromocentres forment un environnement répressif contribuant la rpression transcriptionnelle de séquences répétées permettant la stabilité du génome. Des mesures quantitatives de la position 3D de chromocentres dans le noyau montrent que la plupart chromocentres sont situés proximité de la périphérie du noyau, mais que cette distance peut être modifiée par le volume nucléaire ou dans certains mutants affectant le complexe LINC. Ce complexe LINC est proposé pour contribuer l’organisation de la chromatine et à son positionnement, de plus la mutation de ce complexe est associée une dérégulation l’inactivation de la transcription, ainsi qu’a une décompaction des séquences hétérochromatiques. La dernière partie de ce travail tire profit de séquences gnomiques disponibles et les données de RNA-seq pour explorer l’évolution des protines de la NE chez les plantes. Au Final, le travail réalisé durant cette thèse associe la génétique, la biologie moléculaire, la bioinformatique et de l’imagerie afin de mieux comprendre la contribution de l’enveloppe nucléaire dans l’organisation de la morphologie du noyaux et de la chromatine et suggère l’implication fonctionnelle du complexe LINC dans ces processus. / The linker of nucleoskeleton and cytoskeleton (LINC) complex is an evolutionarily well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data supports its function in nuclear morphology and meiosis, its implication for chromatin organisation has been less studied in plants. The first aim of this work was to develop NucleusJ a simple and user-friendly ImageJ plugin dedicated to the characterisation of nuclear morphology and chromatin organisation in 3D. NucleusJ quantifies 15 parameters including shape and size of nuclei as well as intra-nuclear objects and their position within the nucleus. A step-by-step documentation is available for self-training, together with data sets of nuclei with different nuclear organisation. Several improvements are ongoing to release a new version of this plugin. In a second part of this work, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the plant model Arabidopsis thaliana in which heterochromatin domains cluster in conspicuous chromatin regions called chromocentres. Chromocentres form a repressive chromatin environment contributing to the transcriptional silencing of repeated sequences a general mechanism needed for genome stability. Quantitative measurements of 3D position of chromocentres in the nucleus indicate that most chromocentres are situated in close proximity to the periphery of the nucleus but that this distance can be altered according to nuclear volume or in specific mutants affecting the LINC complex. Finally, the LINC complex is proposed to contribute at the proper chromatin organisation and positioning since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences. The last part of this work takes advantage of available genomic sequences and RNA-seq data to explore the evolution of NE proteins in plants and propose a minimal requirement to built the simplest functional nuclear envelope. Altogether, work achieved in this thesis associate genetics, molecular biology, bioinformatics and imaging to better understand the contribution of the nuclear envelope in nuclear morphology and chromatin organisation and suggests the functional implication of the LINC complex in these processes.

While a large body of work has focused on the transcriptional regulation of cellular identity, the role of the mechanical properties of cells and the importance of their physical interactions with the local environment remains less well understood. In this project, we explored the impact of cytoskeleton-generated forces exerted on the nucleus in the context of early embryonic stem (ES) cell fate decisions. We chose to perturb force generating components in the cytoskeleton – notably the molecular motor non-muscle myosin II - and key structural and chromatin binding proteins in the nuclear envelope, notably, the lamins (LMNA), Lamin B receptor (LBR) and components of the LINC complex (nesprins/KASH). The structural proteins in the nuclear envelope regulate both the mechanical response of the nucleus to force and the stabilization of peripheral heterochromatin (repressed genes). Our hypothesis is that reducing forces transmitted directly to chromatin or increasing tethering of peripheral heterochromatin to the nuclear envelope would restrict access to lineage specific genes sequestered at the nuclear lamina and thereby either impair, or delay, differentiation. We found phenotypes in the capacity of mouse ES cells to specify to the neural lineage following our perturbations: overexpression of LMNA, LBR and KASH proteins resulted in a significant fraction of cells that did not express the neuroectoderm marker Sox1 after four days of differentiation, while inhibiting non-muscle myosin II delayed Sox1 expression in the entire population. Overexpression of LMNA and LBR did not affect the ability of the cells to exit the naive pluripotent state, which raises the possibility that the perturbations are halting the cells in a formative phase prior to lineage specification. Future work will focus on looking at genome-wide transcriptional changes accompanying differentiation combined with an analysis of spatial information of differentially regulated genes.

There is a large body of evidence supporting the theory that cell physiology largely depends on the mechanical properties of its surroundings or micro-environment. More recently studies have shown that changes to intra-cellular mechanical properties can also have a meaningful impact on cell function and in some cases lead to the progression of ailments or disease. For example, small changes to the protein sequence of a structural nuclear envelope protein called lamin-A is known to cause a variety of neurological and musculoskeletal diseases referred to as laminopathies. Currently, there is little incite into how these mutations lead to disease progression due in part to an inability to measure protein-specific mechanical changes and how these alterations may relate to disruptions in intra-cellular signaling or function. \par To improve upon the ability to measure mechanical properties inside living cells, a previously validated, genetically-encoded resonant energy transfer (FRET)-force biosensor was modified to localize to the nuclear envelope. This biosensor integrated into the nuclear envelope protein Nesprin-2G and senses small deformations that are resolved by indirect measurements of spectroscopic fluctuations in the fluorescent emission of the sensor. To accurately measure these changes, a new spectral-imaging technique named SensorFRET was developed which can resolve small changes in the FRET sensor under varying levels of fluorescent intensity and with known absolute precision. Using SensorFRET, the Nesprin-2G biosensor (Nesprin-TS) reported changes in actomyosin contractility, nuclear shape, and nuclear deformation. Using Nesprin-TS, fibroblasts derived from patients with Hutchinson-Gilford progeria syndrome (HGPS) reported less force on Nesprin-2G molecules relative to healthy fibroblasts on average.\par To demonstrate how intra-cellular forces on the nucleus may impact normal cell physiology, bone-marrow derived mesenchymal stem cells (MSCs) were genetically modified such that the cytoskeleton was decoupled from the nucleus by saturating KASH binding proteins with a non-functional truncated protein called DN-KASH. MSCs treated with DN-KASH preferentially differentiated into osteocytes (bone cells) at a higher rate than MSCs exposed to osteogenic growth factors. This osteogenic preference after DN-KASH treatment was independent of the cell substrate topology and did not significantly alter integrin expression. However, this tendency to differentiate into osteocytes was dependent on substrate stiffness. Overall, the data imply that an intra-cellular force-dependent mechanism connected to the cell nucleus strongly influences MSC differentiation.

Nucleus is the largest cellular organelle in animal cells. Due to its bulky nature and the stiffness of nuclear lamina the nucleus constitutes the substantial problem for migrating cells where nucleus has to move. The actomyosin generated forces and LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, that is composed of SUN and nesprin proteins, play key role in nuclear movement. LINC complex mechanically couples nuclear lamina to the cytoskeleton and allows the forces exerted by the cytoskeleton to move the nucleus. Perinuclear actin fibers, also termed actin cap, mechanically link focal adhesions with nucleus and they may generate forces that position the nucleus in a way that is optimal for cellular movement. However, molecular mechanism of how perinuclear actin fibers and LINC complex orchestrate the nuclear movement and functional significance of this process remain poorly understood. The specific aim was to determine the mechanisms by which perinuclear actin fibers are formed and how are these mechanisms employed to facilitate cell migration. The role of LPA-RhoA signaling axis and LINC complex in the formation of perinuclear actin fibers was also examined. It was confirmed that LPA is essencial stimulus during actin cap formation. On the other hand, FAK kinase was found necessary for...

Le noyau est une innovation évolutive majeure caractéristique des organismes eucaryotes. Ces dernières années de nombreux travaux se sont intéressés à l’organisation de la chromatine dans l’espace nucléaire lors de l’interphase. Les protéines associées à la périphérie nucléaire ou ancrées dans la membrane nucléaire interne ont suscité un intérêt majeur due à leur contribution dans l’organisation spatiale de la chromatine. Chez les animaux, les lamines qui forment des filaments à la périphérie nucléaire et le complexe LINC, un complexe protéique reliant la membrane externe et interne du noyau sont connues pour interagir avec la chromatine, influencer l’organisation de cette dernière et moduler la régulation transcriptionnelle. Chez la plante modèle Arabidopsis thaliana utilisée dans ce travail, le complexe LINC est conservé, par contre les lamines ne le sont pas et seraient remplacées par d’autres acteurs spécifiques du règne végétal. Le travail détaillé dans ce manuscrit porte sur la mise en évidence d’un nouveau réseau d’interaction protéique localisé à la périphérie nucléaire et sur l’impact de ces protéines dans la morphologie du noyau et l’organisation de la chromatine. Mes travaux se sont concentrés sur les protéines à domaine SUN, l’une des composantes du complexe LINC et sur les protéines CRWN et KAKU4 présentes à la périphérie du noyau. Des cribles double hybride chez la levure m’ont permis d’identifier 24 partenaires protéiques potentiels dont plus d’un tiers sont des facteurs de transcription L’étude plus précise du facteur de transcription MaMYB pour lequel nous avons créé un allèle nul par la méthode CRISPR montre qu’il joue un rôle plus spécifique dans la formation des racines. L’étude de mutants combinatoires pour les gènes SUN, CRWN et KAKU4 montre des anomalies développementales notamment des tissus reproductifs. Enfin, une étude plus détaillée de la protéine KAKU4 suggère sa participation au maintien de la morphologie du noyau et au rapprochement de l’hétérochromatine vers la périphérie nucléaire. En résumé, mes travaux ont mis en évidence l’existence d’un réseau de facteurs de transcription recrutés à la périphérie nucléaire par les protéines SUN, CRWN et KAKU4. Ce réseau d’interaction protéine-protéine participerait à un mécanisme de séquestration de certains facteurs de transcription et/ou d'un rapprochement à la périphérie nucléaire de certains domaines de chromatine afin d’activer ou de réprimer leur transcription. / The nucleus is a major evolutionary innovation characteristic of eukaryotic organisms. In recent years, numerous studies have focused on the organization of chromatin in nuclear space during interphase. Proteins associated with the nuclear periphery or anchored in the inner nuclear membrane have been particularly studied for their contribution to the spatial organization of chromatin. In animals, the lamina that forms filaments at the nuclear periphery and the LINC complex, a protein complex linking the outer and inner membrane of the nucleus, are known to interact with chromatin, to influence its organization and to modulate transcriptional regulation. In the model plant Arabidopsis thaliana used in this work, the LINC complex is conserved, but not the lamina constituents, which are replaced by other specific actors of the plant kingdom. The work detailed in this manuscript identified a new protein interaction network located on the nuclear periphery and studied the impact of these proteins on nuclear morphology and chromatin organization. My work focused on SUN-domain proteins, one of the components of the LINC complex, and on the CRWN and KAKU4 proteins at the periphery of the nucleus. Double hybrid screens in yeast allowed me to identify 24 potential protein partners, more than a third of which are transcription factors. The more precise study of the transcription factor MaMYB for which we created a null allele using the CRISPR method, shows that it plays a more specific role in root formation. The study of mutant combinations for SUN, CRWN and KAKU4 genes reveals developmental abnormalities, particularly in reproductive tissue. Finally, a more detailed study of the role of the KAKU4 protein suggests that it contributes to the morphology of the nucleus in maintaining heterochromatin at the nuclear periphery. In summary, we propose the existence of a transcription factor network recruited to the nuclear periphery by SUN, CRWN and KAKU4 proteins. This protein-protein interaction network would participate in the sequestration of certain transcription factors and/or the localization of certain chromatin domains to the nuclear periphery in order to activate or suppress their transcription.