171 |
Reading Heart of Darkness in the ESL/EFL Classroom : A Case Study in Student Response to Literary Didactic Methodologies Designed to Enhance Aesthetic and Efferent Reading of a Literary Text in Language InstructionBrinkley, Steven January 2015 (has links)
The purpose of this degree project has been to examine the implications of the provision of certain methodological support mechanisms, what has often been referred to as "instructional scaffolding" in literary didactics, to assist students in the ESL/EFL classroom in their interaction with the various literary texts into which they come into contact during their English language education at the upper secondary level in Sweden. My primary interest has been to gauge the response of the students involved in this study to the particular types of literary didactic methods utilized, for example, regarding their effectiveness in aiding the learning process as well as their impact on the literary, or aesthetic, experience itself. An analysis of student responses to a literature instruction module based on a reading of Joseph Conrad's Heart of Darkness will demonstrate that certain forms of literary didactic methods in general, and significantly, particular forms of what can be conceptualized as instructional scaffolding, play a crucial role for both the learning process and the student's aesthetic experience of literature.
|
172 |
Renal proximal tubular handling of nucleosides by human nucleoside transporter proteinsElwi, Adam 11 1900 (has links)
Human cells possess multiple nucleoside transporters (NTs) that belong to either the human equilibrative or concentrative NT (hENT: hENT1/2/3/4; hCNT: CNT1/2/3) families. In the kidney, coupling of apical hCNT3 activities to basolateral hENT1/2 activities is hypothesized to mediate renal nucleoside proximal tubular absorption while apical ENT1 may have a role in secretion. The overall aim of this research was to increase understanding of the roles of hENTs and hCNTs in renal handling of physiological nucleosides and anti-cancer nucleoside analog drugs. This was achieved by investigating the distribution of hENTs and hCNTs in human kidney tissue and the function of hENTs and hCNTs in cellular uptake and transepithelial fluxes of nucleosides in cultured human renal proximal tubule cells (hRPTCs).
Immunolocalization of hCNT3 and hENT1 in human kidney tissue revealed that hENT and hCNT3 were present in apical membranes of proximal tubules. Production and characterization of adherent hRPTC cultures demonstrated endogenous hCNT3, hENT1, and hENT2 activities. These results provided evidence for the involvement of hCNT3, hENT1, and hENT2 in renal handling of nucleosides.
Comparison of adherent hRPTC cultures derived from kidneys from different individuals demonstrated that hCNT3 activities varied between cultures. Also, the extent of cellular uptake of fludarabine, an anti-cancer nucleoside drug, and degree of cytotoxicity was reflected in the different hCNT3 activities observed between cultures. These results suggested that hCNT3 plays an important role in fludarabine renal handling and is a determinant of potential renal toxicities.
Production of polarized monolayer cultures of hRPTCs on transwell permeable inserts enabled the functional localization of hCNT3 and hENT1 to apical membranes and hENT2 to basolateral membranes. Transepithelial flux studies demonstrated that (i) apical-to-basolateral fluxes of adenosine were mediated by apical hCNT3 and basolateral hENT2, (ii) basolateral-to-apical fluxes of 2′-deoxyadenosine were mediated, in part, by apical hENT1 and basolateral hOATs, and (iii) apical-to-basolateral fluxes of fludarabine, cladribine, and clofarabine were mediated by apical hCNT3.
These studies showed that coupling of apical hCNT3 to basolateral hENT2 mediates proximal tubular nucleoside reabsorption, that coupling of basolateral human organic anion transporters (hOATs) to apical hENT1 mediates proximal tubular nucleoside secretion, and that hCNT3 is a key determinant of fludarabine proximal tubular reabsorption and cytoxicity.
|
173 |
Developing a common understanding and a mutual meaning structure of early childhood practices between trainees and educarers and children in child care settingsShore, Margaret Ellen Unknown Date (has links)
This thesis explores the effectiveness of a professional education model that was specifically developed for on-the-job training to assist seven child care trainees in six Child Care Centres in Queensland, Australia, to develop the competencies, motives, strategies and processes required to become expert educarers of pre-school age children. The intervention, which took place over 65 days, was implemented by six expert educarers who had previously been trained in using the Zone of Proximal Development (socio-cultural theory) to extend both the adult and the child learners development. The educarers were asked to assist the trainees demonstration of motives, strategies, and processes during daily activities, and the educarers and trainees were asked to assist the childrens demonstration of competencies in daily activities through the Zone of Proximal Development. As mass training for child care workers in group child care is still a relatively new phenomenon in Australia, and as little research has addressed both an adult and child learners demonstration of competencies in the workplace with a permanent work-based instructor interacting through the Zone of Proximal Development, qualitative and quantitative approaches to analysing the data were chosen. These approaches present both a descriptive and a measured analysis of the day-to-day operations in which the training occurred. The study aimed to generate substantive theoretical ideas by extending socio-cultural theory (Tharp & Gallimore, 1988) with Activity Theory (Leontev, 1979), Legitimate Peripheral Participation (LPP) (Lave & Wenger, 1991) and Cognitive Apprenticeship (Brown, Collins & Duguid, 1989). Data collection occurred through questionnaires at three periods of the study, through pre- and post-training Semantic Differentials, through the trainees daily journals and the professional tutors comments, and the educarers weekly and monthly reports. The study provides a triangulated perspective of two foci. The primary focus was on the changing relationship of the trainees with the educarers during the training programme, and whether this led to a converging understanding and the development of an evolving, mutual meaning structure. The perspectives are the trainees, the educarers, and their joint interactions. The personal experiences of the trainees as observers and participants in the day to day activities of the centre are interwoven with the modelling and assistance of the educarers and demonstration of the children. The results revealed that some of the trainees relationships with some of the educarers changed over three phases. These changes demonstrated that, in Phase I, the trainees observed and practiced activities under the supervision of the educarers who instructed them what to do and why, and discussed parents, as they started to develop a mutual meaning structure. Their relationship reflected that of a novice-expert. In Phase II the seven trainees acted independently, planning and implementing minor activities, still under the educarers supervision however. In this phase the educarers modelled, observed, and commented on the trainees performance, as an evolving mutual meaning structure that reflected an aspiring expert-expert relationship developed. Three trainees attained only Phase II, while the other four continued into Phase III. In Phase III, the four trainees planned and implemented major activities independently while the educarers observed and commented on their performance and engaged jointly in tasks. Their relationships, which evidenced a developing and evolving mutual meaning structure reflecting a converging understanding, became collaborative for one dyad, one educarer perceived the relationship was collaborative and the trainee perceived it to be an aspiring expert-expert, while the other two dyads demonstrated an expert-expert relationship. It appears that the trainees, who experienced education motives with various strategies and processes implemented by the educarers through the ZPD in the training programme, demonstrated the greatest change in the relationships. For these trainees the peripherality of participation changed from Phase I, operating at the outer peripherality of the child care practice to operating closer to the centre of the practice in Phase II. The four trainees who transitioned to the centre of the practice in Phase III experienced educarers who exhibited consistent motives with flexible strategies and processes combining a wide variety of interactions through the ZPD. Emerging from the data, a dominant authentic activity, and a secondary aspect of this study was classroom management in general and morally acceptable behaviour (MAB) in particular. The dominance of this activity reinforces Tharp and Gallimores (1988) finding that classroom management was the most stressful activity for first year teachers. The trainees appropriation of the motives, strategies and processes in episodes of morally acceptable behaviour as modelled by the educarers, showed, over the study, a convergence of understanding and the development of an evolving mutual meaning structure between the dyads. The second focus of the study provides a triangulated perspective on the changes in the relationship between the trainees and children during the training programme. The trainees and childrens demonstration of competencies in morally acceptable behaviour is also addressed in this section: the perspectives are the trainees, the educarers and joint trainee-child interactions. The results reveal that the trainees relationship with children changed over the three Phases of the training programme. With a minor focus on children in Phase I, the trainees observed and practiced implementing activities demonstrating a novice-novice relationship. In Phase II, increasing their focus on children, the trainees took responsibility in minor activities demonstrating an aspiring expert-novice relationship. However in Phase III, with a dominant focus on children, expert-novice relationships were apparent. The four competencies developed for trainees to facilitate the childrens demonstration of competencies in morally acceptable behaviour, discussing feelings and discussing actions, and showing verbal and non-verbal empathy, were acquired and used by the trainees who experienced education motives in the training programme, in Phases II and III. Concurrently, the children developed and increased their competencies in verbal and non-verbal acceptance of the interventions, and showed increased understanding of cause and effect and the ability to formulate hypotheses. Changes were also evidenced in the trainees conceptual understanding of educaring children as they integrated the concepts of discipline and control with three other essentials of educaring (caring, teaching and learning), into their conceptual understanding of educaring over the study. The development of an evolving, mutual meaning structure of the authentic activities that were practiced in the child care centres, showed a converging understanding was being established between educarers, trainees and children. There was also evidence of reciprocal assistance. Influence, assistance and teaching were not a one-way process. They did not flow in one direction only, from professional tutor to educarer to trainee to child. The child in turn influenced the trainee who influenced the educarer who influenced the professional tutor.
|
174 |
Regulation of iNOS expression : in response to pressure in proximal tubule epithelial cells /Broadbelt, Nalini V. January 2008 (has links)
Thesis (Ph. D.)--Cornell University, August, 2008. / Vita. Includes bibliographical references (leaves 116-140).
|
175 |
Sexual dimorphism at the proximal tibia: a geometric morphometric analysisToon, Celena 12 March 2016 (has links)
In the past few decades, an area of skeletal research focusing on shape analyses has gained popularity in the field of physical anthropology, and subsequently forensic anthropology. Known as geometric morphometrics, this type of analysis allows the researcher to place the morphological shape of bones into a statistical framework to answer questions on a variety of topics, including sexual dimorphism. Sex assessment from the long bones has been traditionally conducted using traditional morphometric methods (Iscan and Miller-Shaivitz 1984; Steyn and Iscan 1997), and as a result, relies mainly on size differences and has not considered how joint morphology and shape affect sex. For this project, a geometric morphometric analysis of the proximal tibia in a modern Caucasian American population was conducted using a sample of 100 male and 100 female tibiae from the William M. Bass Donated Skeletal Collection at the University of Tennessee at Knoxville. The proximal tibia's effectiveness as an indicator of sex in a modern American population was evaluated via generalized Procrustes, principal components, and discriminant function analyses. Principal components revealed a lack of separation between males and females in terms of proximal tibia shape. The discriminant function analysis was successful at discriminating males from females, but cross-validation yielded a low total accuracy rate of 58%. The shape of the proximal tibia contributes to sexual dimorphism in a Caucasian American population, but is only slightly useful in a discriminant function. Further research should be conducted on different populations and using different skeletal landmarks.
|
176 |
Development of circulatory microRNAs as markers of organ injury and mediators of inter-organ signallingMorrison, Emma Elisabeth January 2018 (has links)
Plasma contains small, non-protein coding RNA species, microRNAs (miRNAs). Circulating miRNAs originate from tissues throughout the body and circulate in the blood bound to proteins or encapsulated in extracellular vesicles (EVs). The pattern of circulating miRNAs changes in different pathological states, leading to the hypothesis that they could act as biomarkers or mediators of inter-organ signalling. Acute kidney injury (AKI) is associated with high morbidity worldwide. Recent work has highlighted a potential role for EV signalling in the delivery of functional exogenous miRNA into kidney cells, which may contribute to the pathogenesis of AKI. The studies described in this thesis investigate the effects of circulating miRNAs on renal proximal tubular (PT) cells. Utilising next generation sequencing technology, circulating miRNA profiles were demonstrated to change significantly following myocardial injury. These findings were translated from humans into a mouse model of myocardial injury. Investigation of EV cell signalling, using flow cytometry and nanoparticle tracking analysis, demonstrated that PT cell EV uptake was not affected by known physiological agonists. By contrast, EV release from PT cells was regulated by purinergic P2Y1 and dopamine D1 receptors. Toxic cisplatin injury of PT cells resulted in increased EV release and reduced EV uptake in a dose-dependent manner. Cisplatin toxicity in PT cells was unaffected by EVs from mice with myocardial injury, but toxicity was reduced by EVs from mice with drug-induced liver injury (DILI). Circulating EVs from mice with DILI transferred the liver specific miRNA, miR-122, into PT cells in both in vivo and in vitro models. The consequence of miR-122 transfer was modulation of downstream target genes including Foxo3 which has been implicated in cell injury by apoptosis. These findings therefore show that circulatory miRNA profiles change in different models of organ injury and suggest miRNAs can be transferred to PT cells in vivo and in vitro. The improved viability of injured PT cells following co-incubation with DILI EVs, and subsequent transcriptomic work, suggests this may be as a consequence of miRNA transfer. In conclusion, circulatory miRNAs may act as mediators of inter-organ signalling and could play a crucial role in the propagation of systemic illness.
|
177 |
Tecnologias digitais e emergência de zonas de desenvolvimento proximal na sala de aula / Digital technologies and development of areas of emergency proximal in classroomARRUDA, Juliana Silva January 2016 (has links)
ARRUDA, Juliana Silva. Tecnologias digitais e emergência de zonas de desenvolvimento proximal em sala de aula. 2016. 105f. – Dissertação (Mestrado) – Universidade Federal do Ceará, Programa de Pós-graduação em Educação Brasileira, Fortaleza (CE), 2016. / Submitted by Gustavo Daher (gdaherufc@hotmail.com) on 2016-09-22T13:22:51Z
No. of bitstreams: 1
2016_dis_jsarruda.pdf: 3571578 bytes, checksum: bf4966a1777257a5f02840cc145943e2 (MD5) / Approved for entry into archive by Márcia Araújo (marcia_m_bezerra@yahoo.com.br) on 2016-09-22T16:34:13Z (GMT) No. of bitstreams: 1
2016_dis_jsarruda.pdf: 3571578 bytes, checksum: bf4966a1777257a5f02840cc145943e2 (MD5) / Made available in DSpace on 2016-09-22T16:34:13Z (GMT). No. of bitstreams: 1
2016_dis_jsarruda.pdf: 3571578 bytes, checksum: bf4966a1777257a5f02840cc145943e2 (MD5)
Previous issue date: 2016 / Brazil is experiencing a period of technological immersion in all areas of society and every day, technologies are created that power the various human activities. The school should monitor the resulting changes of technological resources and be open to new educational possibilities to develop contemporary practices, from the inclusion of computers in school activities. Computers are features that enable interaction in group activities, the exchange of ideas and opinions; or the research of other realities. Thus, this study aims to analyze how the computer through digital resources used in activities collaboratively, can emerge Proximal Development Zone (ZDP). This research was developed in a municipal school in Aquiraz / CE, in groups of two or more learners. Qualitative methodology with interpretative character was used. The research technique involved participant observation, with micro genetic analysis and as tools for collecting videos and daily data field. Planned activities included the curriculum subjects, the use of computer and some Internet resources. The methodological procedures were considered from a micro genetic approach, and defined with the statement of contemporary researchers, as Meira and Lerman (2009) and Colaco et al. (2007) which expanded and interpreted new concepts of the ZDP Vygotsky (1994). The results were analyzed according to categories at the time of data analysis. In the first category, interaction among peers, was detailed as group activities, and peer contribution contributed to the development of the zone. In the second category, interaction with someone more experienced, involved contributions and assistance from someone more experienced, contributing to facilitating learning. The next third, technology support, has detailed the use of technology by students and contributions of this resource for teaching and learning processes. In the last category, student Protagonism discussed how students managed to overcome their level of development and can act as author of knowledge itself, and observed two characteristics: Activism and autonomy. The results highlighted times when resources combined with peer interaction promote and facilitate the emergence of ZPD, resulting in learning, promoting performances and authoring postures of these learners in the process. / O Brasil vivencia um período de imersão tecnológica em todas as áreas da sociedade, e a cada dia são criadas tecnologias que potencializam as diversas atividades humanas. A escola deve acompanhar as mudanças advindas dos avanços tecnológicos e ficar atenta às novas possibilidades educacionais para desenvolver práticas contemporâneas, a partir da inserção de computadores em atividades curriculares. Estes recursos possibilitam a interação nas atividades em grupo, na troca de ideias e opiniões; ou para a pesquisa de outras realidades. Dessa forma, este estudo tem por objetivo analisar como o computador, por meio de recursos digitais, utilizados em atividades realizadas colaborativamente, pode fazer emergir Zonas de Desenvolvimento Proximal (ZDP). Esta pesquisa foi desenvolvida em uma escola pública municipal de Aquiraz/CE, em grupos de dois ou mais aprendentes. Foi utilizada a metodologia qualitativa, com caráter interpretativo. A técnica de pesquisa envolveu a observação participante, com análise microgenética, e, como instrumentos de coleta de dados, vídeos e diários de campo. As atividades planejadas envolveram as disciplinas curriculares, o uso do computador e alguns recursos da Internet. Os procedimentos metodológicos foram considerados a partir de uma abordagem microgenética, e definidos com a fundamentação de pesquisadores contemporâneos, como Colaço et al. (2007) e Meira e Lerman (2009), que ampliaram e interpretaram novos conceitos da ZDP de Vygotsky (1994). Os resultados foram analisados de acordo com categorias, no momento da análise dos dados. A primeira categoria – Interação entre pares – foi detalhada como as atividades em grupo, e a participação dos pares contribuiu no desenvolvimento de ZDP. A segunda categoria – Interação com alguém mais experiente – envolveu contribuições e assistência de alguém mais experiente, contribuindo para facilitação da aprendizagem. Na categoria seguinte – Suporte da tecnologia – foram detalhados o uso da tecnologia pelos alunos e as contribuições desse recurso para os processos de ensino e aprendizagem. Na última categoria – Protagonismo estudantil – foi discutida a maneira como os alunos conseguiram ultrapassar seu nível de desenvolvimento, podendo atuar como autores do próprio conhecimento, sendo observadas duas características: Ativismo e Autonomia. Os resultados destacaram momentos em que os recursos, aliados à interação entre pares, promovem e facilitam a emergência de ZDP, acarretando aprendizagem e promovendo atuações e posturas de autoria desses aprendentes em seu processo.
|
178 |
O trabalho pedagógico e a zona de desenvolvimento proximal na aprendizagem da linguagem escritaBassan, Larissa Helyne [UNESP] 11 February 2008 (has links) (PDF)
Made available in DSpace on 2014-06-11T19:25:16Z (GMT). No. of bitstreams: 0
Previous issue date: 2008-02-11Bitstream added on 2014-06-13T20:32:38Z : No. of bitstreams: 1
bassan_lh_me_mar.pdf: 1542580 bytes, checksum: 2f369a8af799029aa6560531a59e7408 (MD5) / Vigotski, um dos maiores representantes da Teoria Histórico-Cultural, definiu a categoria zona de desenvolvimento proximal (ZDP) como sendo o nível de desenvolvimento em que a criança é capaz de realizar suas ações mediante a colaboração de alguém culturalmente mais experiente. Para ele, o bom ensino é aquele que se adianta ao desenvolvimento da criança, e isso é possível, acreditamos, desde que o ensino incida em sua zona de desenvolvimento proximal. Coerente com essa orientação, a presente pesquisa teve por objetivo geral estabelecer as possíveis relações entre o trabalho pedagógico do professor e a zona de desenvolvimento proximal do aluno no processo de ensino e de aprendizagem da linguagem escrita. Esta pesquisa foi realizada com oito professoras e dezesseis alunos das 1ª e 2ª séries do ensino fundamental da rede municipal de ensino da cidade de Marília. Os dados foram coletados por meio de entrevistas semi-estruturadas, observações e registros de episódios da interação professor-aluno nos estritos limites das situações de produção textual escrita, além de análise de textos dos alunos, coletados durante o período pesquisado. Os resultados obtidos indicam que o professor que desenvolve ações mais potencializadoras organiza o ensino de modo a lidar com as possibilidades de aprendizagem dos alunos, fornecendo-lhes as ajudas necessárias à apropriação dos conteúdos culturais com os quais trabalham, propicia-lhes o seu desenvolvimento, ou seja, potencializa-o. Durante essas ações, o professor se relaciona de maneira envolvente com seus alunos, permitindo que eles objetivem suas necessidades e interesses, além de trabalhar a linguagem escrita pela função social e prover ajudas diferenciadas no momento da produção textual. / Vigotski, one of the greatest representatives of the Historical-Cultural Theory, defined the zone of proximal development (ZPD) category, as the level of development in which the child is capable of performing its actions by means of the involvement with someone culturally more experienced. For him, good instruction is the one that makes the development of the child advance, and that is possible, we believe, once the teaching process works with the possibilities in the area of proximal development. Consistent with this guidance, this search aimed to establish the possible relations between the pedagogical work of the teacher and the zone of proximal development of the students in the teaching and learning processes of the written language. This search was conducted with eight teachers and sixteen students of the first and second series of the elementary school of the municipal system of education of the city of Marília. The data were collected by means of semi-structured interviews, observations and records of episodes of the teacher-student interaction in the strict limits of the situations of textual production. Besides, we analyzed the students’ texts, gathered during the search. The results indicate that the teacher who develops strongly effective actions organizes the instruction in order to deal with the possibilities of students learning capacity by providing them the necessary assistance to the appropriation of cultural contents they work with, makes them progress in their development, giving potency to it. While she develops her work, the teacher interacts with her students, allowing them objectify their needs and interests, besides dealing with the written language by its social function and providing differentiated aid in the moment of textual production. This kind of action has implications in the learning process.
|
179 |
Optimisation déterministe et stochastique pour des problèmes de traitement d'images en grande dimension / Deterministic and stochastic optimization for solving large size inverse problems in image processingVu, Thi Thanh Xuan 13 November 2017 (has links)
Dans cette thèse on s’intéresse au problème des décompositions canoniques polyadiques de tenseurs d’ordre $N$ potentiellement grands et sous différentes contraintes (non-négativité, aspect creux lié à une possible surestimation du rang du tenseur). Pour traiter ce problème, nous proposons trois nouvelles approches itératives différentes: deux approches déterministes dont une approche proximale, et une approche stochastique. La première approche étend les travaux de thèse de J-P. Royer au cas de tenseurs de dimension $N$. Dans l’approche stochastique, nous considérons pour la première fois dans le domaine des décompositions tensorielles, des algorithmes génétiques (mimétiques) dont principe général repose sur l’évolution d’une population de candidats. Dans le dernier type d’approche, nous avons considéré un algorithme proximal pré-conditionné (le Block-Coordinate Variable Metric Forward-Backward), algorithme fonctionnant par blocs de données avec une matrice de pré-conditionnement liée à chaque bloc et fondé sur deux étapes successives principales : une étape de gradient et une étape proximale. Finalement, les différentes méthodes suggérées sont comparées entre elles et avec d’autres algorithmes classiques de la littérature sur des données synthétiques (à la fois aléatoires ou proches des données observées en spectroscopie de fluorescence) et sur des données expérimentales réelles correspondant à une campagne de surveillance des eaux d’une rivière et visant à la détection d’apparition de polluants. / In this PhD thesis, we consider the problem of the Canonical Polyadic Decomposition (CPD) of potentially large $N$-th order tensors under different constraints (non-negativity, sparsity due to a possible overestimation of the tensor rank, etc.). To tackle such a problem, we propose three new iterative methods: a standard gradient based deterministic approach, a stochastic approach (memetic) and finally a proximal approach (Block-Coordinate Variable Metric Forward-Backward). The first approach extends J-P. Royer's works to the case of non-negative N-th order tensors. In the stochastic approach, genetic (memetic) methods are considered for the first time to solve the CPD problem. Their general principle is based on the evolution of a family of candidates. In the third type of approaches, a proximal algorithm namely the Block-Coordinate Variable Metric Forward-Backward is presented. The algorithm relies on two main steps: a gradient step and a proximal step. The blocks of coordinates naturally correspond to latent matrices. We propose a majorant function as well as a preconditioner with regard to each block. All methods are compared with other popular algorithms of the literature on synthetic (fluorescence spectroscopy like or random) data and on real experimental data corresponding to a water monitoring campaign aiming at detecting the appearance of pollutants.
|
180 |
Structural Studies of the Transmembrane and Membrane Proximal Domains of HIV-1 gp41 by X-Ray CrystallographyJanuary 2014 (has links)
abstract: The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR, residues 649-683) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain (residues 684-705) of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the pre-fusion and post-fusion conformations, most of which could not react with the broadly neutralizing antibodies 2F5 and 4E10. Structural information on the TM domain of gp41 is scant and at low resolution.
This thesis describes the structural studies of MPR-TM (residues 649-705) of HIV-1 gp41 by X-ray crystallography. MPR-TM was fused with different fusion proteins to improve the membrane protein overexpression. The expression level of MPR-TM was improved by fusion to the C-terminus of the Mistic protein, yielding ∼1 mg of pure MPR-TM protein per liter cell culture. The fusion partner Mistic was removed for final crystallization. The isolated MPR-TM protein was biophysically characterized and is a monodisperse candidate for crystallization. However, no crystal with diffraction quality was obtained even after extensive crystallization screens. A novel construct was designed to overexpress MPR-TM as a maltose binding protein (MBP) fusion. About 60 mg of MBP/MPR-TM recombinant protein was obtained from 1 liter of cell culture. Crystals of MBP/MPR-TM recombinant protein could not be obtained when MBP and MPR-TM were separated by a 42 amino acid (aa)-long linker but were obtained after changing the linker to three alanine residues. The crystals diffracted to 2.5 Å after crystallization optimization. Further analysis of the diffraction data indicated that the crystals are twinned. The final structure demonstrated that MBP crystallized as a dimer of trimers, but the electron density did not extend beyond the linker region. We determined by SDS-PAGE and MALDI-TOF MS that the crystals contained MBP only. The MPR-TM of gp41 might be cleaved during or after the process of crystallization. Comparison of the MBP trimer reported here with published trimeric MBP fusion structures indicated that MBP might form such a trimeric conformation under the effect of MPR-TM. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2014
|
Page generated in 0.0364 seconds