基于ROV援潜救生自主作业方法研究

利用ROV代替潜水员进行潜艇供排气管对接作业是援潜救生的发展趋势，其中供排气管对接技术是为失事艇员提供生命保障的关键技术。由于水下作业环境比较复杂以及水下作业难度大等特点，人工遥控ROV进行援潜对接供排空气管作业过程中，还停留在只是能够打开潜艇供排气系统花甲板盖的工作上，还没有实现对类似接通供排空气管精确作业的工作。受到海流、风浪、失事潜艇周围较大涡流、供排气管的拖曳阻力、水下可视条件以及高精度定位等因素的限制，作业任务越复杂，要求ROV系统上机械手精确定位精度越高，操纵的难度也越大，由人工操作很难实现。在现有ROV人工操作的基础上，使ROV系统具有自主作业功能，更能发挥其在对接空气管作业中的能力，提高作业效率，缩短救援时间，因此本文在现有ROV基础上扩展预编程自主控制驾驶功能模块即ROV-A系统，借助预编程技术ROV-A系统在对接空气管作业中的一些特殊任务时能够实现自主作业功能。 本文结合中国科学院沈阳自动化研究所水下机器人中心未来援潜救生关键技术研究内容，针对为失事潜艇接通空气管作业中的一些关键技术，开展基于具有自主作业能力的ROV-A系统自主作业方法研究，深入研究了具有自主作业能力ROV-A系统的运动规划与协调控制技术以及水下作业力控制技术，以实现高精度的机械手末端位置控制与期望力跟踪；针对潜艇供排气系统的已知结构研究了水下目标定位技术，为对接空气管的特定需求发展具有自主作业能力的水下作业技术提供理论依据。重点研究针对系统特点的运动规划与多性能指标实现归一化问题；研究基于系统动力学模型误差的系统位姿控制问题；研究基于阻抗力控制水下作业目标定位问题；在系统运动规划与控制研究的基础上，研究能实现机械手末端精确轨迹跟踪的控制策略问题以及力控制问题。本论文研究内容如下： （1）根据ROV-A系统特点，从描述系统的空间位置和姿态着手，研究了ROV-A系统的空间运动学与基于Kane方法的动力学，得出载体基座自由运动模式的系统空间运动数学模型，为论文后续研究工作奠定了基础。 （2）针对对接空气管作业中的一些高精度和复杂的作业，研究了系统作业时的运动规划问题。针对系统运动学冗余、作业规划约束性能指标多，例如机械手可操作性，关节限制，载体的姿态，避障等等，结合梯度投影法和最小范数伪逆矩阵法以及任务优先逆运动学方法，引入模糊控制技术，将模糊控制的定性知识表达与任务优先逆运动学算法相结合，以水下作业末端位置控制及轨迹跟踪为前提，对系统运动分配、关节限位、避免奇异有无海流下的系统性能优化等运动规划进行了研究，仿真证明运动规划算法的有效性。 （3）研究了基于动力学模型的系统基本控制问题。针对系统动力学模型的复杂性和不精确性，在基于载体输出反馈控制的基础上，设计了基于输出反馈自适应控制算法，通过自适应学习的方式直接逼近系统动力学方程状态量之间的非线性关系，在外界不停扰动下逐渐提高系统的控制精度；基于Lyapunov稳定性理论，证明了存在外界干扰和自适应逼近误差条件下ROV-A系统控制器的闭环稳定性；通过仿真实验验证控制系统的有效性，为系统的控制提供了一种新思路。同时在基于位置运动控制的基础上引入了力控制，通过对常规PID外环力控制器基础上的改进，在系统离线规划的前提下实现力与位置控制的解耦，通过仿真验证期望力的稳定跟踪。 （4）研究了基于ROV-A系统阻抗力控制的水下矩形围壁环境约束的位置定位方法。通过在对具有先验知识的矩形围壁环境约束的学习理解基础上，应用外部阻抗力控制环包容内部位置运动控制环的控制策略。利用力传感器的反馈信息变化确定系统末端执行器与约束环境的接触特征点，根据环境的先验知识推理出环境定位信息。通过计算机仿真实验验证了水下具有先验知识环境的定位方法和ROV-A系统的控制策略的有效性。仿真结果证明定位方法和控制策略是可行的。 （5）结合系统控制，分析了系统在为失事潜艇对接空气管作业中的恒定和时变两种期望力输入作业模式，提出了基于在线运动规划下外部力控制环包容内部位置环控制方法，利用ROV-A系统完成了为失事潜艇自主对接空气管中两个典型作业。介绍了整个控制方法的组成和执行过程，分析了综合控制策略，最后通过仿真实验分析了综合力控制方法的性能，包括恒力和时变期望力输入进行了深入研究。

东喜马拉雅构造结新生代碰撞变形与岩石圈演化

The Eastern Himalayan Syntaxis (EHS) is one of the strongest deformation area along the Himalayan belt resulted from the collision between Indian plate and the Eurasian Plate since the 50～60Ma, and has sensitivity tracked and preserved the whole collisional processes. It should depend on the detail geological investigations to establish the deformational accommodate mode, and the uplift history, to elucidate the deep structure and the crust-mantle interaction of the Tibet Plateau of the EHS. The deep-seated (Main Mantle Thrusts) structures were exhumed in the EHS. The MMT juxtapose the Gangdese metamorphic basement and some relic of Gangdese mantle on the high Himalayan crystalline series. The Namjagbawa group which is 1200～1500Ma dated by U/Pb age of zircon and the Namla group which is 550Ma dated by U/Pb age of zircon is belong to High Himalayan crystalline series and Gangdese basement respectively. There is some ophiolitic relic along the MMT, such as metamorphic ocean mantle peridotite and metamorphic tholeiite of the upper part of ocean-crust. The metamorphic ocean mantle peridotites (spinel-orthopyroxene peridotite) show U type REE patterns. The ~(87)Sr/~(86)Sr ratios were, 0.709314～0.720788, and the ~(143)Nd/~(144)Nd ratios were 0.512073～0.512395, plotting in the forth quadrant on the ~(87)Sr/~(86)Sr-~(143)Nd/~(144)Nd isotope diagram. Some metamorphic basalt (garnet amphibolite) enclosures have been found in the HP garnet-kynite granulite. The garnet amphibolites can be divided two groups, the first group is deplete of LREE, and the second group is flat or rich LREE, and their ~(87)Sr/~(86)Sr, ~(143)Nd/~(144)Nd ratios were 0.70563～0.705381 and 0.512468～0.51263 respectively. Trace element and isotopic characteristics of the garnet amphibolites display that they formed in the E-MORB environment. Some phlogolite amphibole harzburgites, which exhibit extensive replacement by Phl, Amp, Tc and Dol etc, were exhumed along the MMT. The Phl-Amp harzburgites are rich in LREE and LILE, such as Rb, K etc, and depletes Eu (Eu~* = 0.36 ～ 0.68) and HFSE, such as Nb, Ta, Zr, Hf, P, Ti etc. The trace element indicate that the Phl-Amp harzburgites have island arc signature. Their ~(87)Sr/~(86)Sr are varied from 0.708912 to 0.879839, ~(143)Nd/~(144)Nd from 0.511993 to 0.512164, ε Nd from- 9.2 to - 12.6. Rb/Sr isochrone age of the phlogolite amphibole harzburgite shows the metasomatism took place at 41Ma, and the Amp ~(40)Ar/~(39)Ar cooling age indcate the Phl-Amp harzburgite raising at 16Ma. There is an intense crust shortening resulted from the thrust faults and folds in the Cayu block which is shortened more 120km than that of the Lasha block in 35～90Ma. With the NE corner of the India plate squash into the Gangdese arc, the sinistral Pai shear fault and the dextral Aniqiao shear fault on the both sides of the Great bent of Yalun Zangbu river come into active in 21～26Ma. On the other hand, the right-lateral Gongrigabu strike-slip faults come into activity at the same period, a lower age bound for the Gongrigabu strike-slip fault is estimated to be 23～24Ma from zircon of ion-probe U/Pb thermochronology. The Gongrigabu strike-slip faults connect with the Lhari strike-slip fault in the northwestern direction and with the Saganing strike-slip at the southeastern direction. Another important structure in the EHS is the Gangdese detachment fault system (GDS) which occurs between the sedimental cover and the metamorphic basement. The lower age of the GDS is to be 16Ma from the preliminary 40Ar/39Ar thermochronology of white mica. The GDS is thought to be related to the reverse of the subducted Indian crust and the fast uplift of the EHS. Structural and thermochronology investigation of the EHS suggest that the eastern Tibet and the western Yunnan rotated clockwise around the EHS in the period of 35～60Ma. Later, the large-scale strike-slip faults (RRD, Gaoligong and Saganing fault) prolongate into the EHS, and connect with the Guyu fault and Gongrigabu fault, which suggest that the Indianchia block escape along these faults. Two kind of magmatic rocks in the EHS have been investigated, one is the mantle-derived amphibole gabbro, dioposide diorite and amphibole diorite, another is crust origin biotit-garnet adamellite, biotit-garnet granodiorite and garnet-amphibole-biotite granite. The amphibole gabbro dioposite diorite and amphibole diorite are rich in LREE, and LILE, such as Ba, Rb, Th, K, Sr etc, depleted in HFSE, such as Nb, Ta, Zr, Hf, Ti etc. The ratio of ~(87)Sr/~(86)Sr are from 0.7044 to 0.7048, ~(143)Nd/~(144)Nd are from 0.5126 to 0.5127. The age of the mantle origin magamatic rocks, which result from the partial melt of the raising and decompression anthenosphere, is 8Ma by ~(40)Ar/~(39)Ar dating of amphibole from the diorite. The later crust origin biotite-garnet adamellite, biotite-garnet granodiorite and garnet-amphibole-biotite granite are characterized by aboudance in LREE, and strong depletion of Eu. The ratios of ~(87)Sr-~(86)Sr are from 0.795035 to 0.812028, ~(143)Nd/~(144)Nd from 0.51187 to 0.511901. The ~(40)Ar/~(39)Ar plateau age of the amphibole from the garnet-amphibole-biotite granite is 17.5±0.3Ma, and the isochrone age is 16.8±0.6Ma. Their geochemical characteristics show that the crust-derived magmatic rocks formed from partial melting of the lower curst in the post-collisional environment. A group of high-pressure kaynite-garnet granulites and enclave of high-pressure garnet-clinopyroxene grnulites and calc-silicate grnulites are outcroped along the MMT. The peak metamorphic condition of the high-pressure granulites yields T=800～960 ℃, P=1.4～1.8Gpa, corresponding the condition of 60km depth. The retrograde assemblages of the high-pressure grnulites occur at the condition of T=772.3～803.3 ℃, P=0.63～0.64Gpa. The age of the peak metamorphic assemblages are 45 ～ 69Ma indicated by the zircon U/Pb ion-plobe thermochronology, and the retrograde assemblage ages are 13～26Ma by U/Pb, ~(40)Ar/~(39)Ar thermochronology. The ITD paths of the high-pressure granulites show that they were generated during the tectonic thickening and more rapid tectonic exhumation caused by the subducting of the Indian plate and subsequent break-off of the subducted slab. A great deal of apatite, zircon and sphene fission-track ages, isotopic thermochronology of the rocks in the EHS show that its rapid raising processes of the EHS can be divided into three main periods. There are 35～60Ma, 13～25Ma, 0～3Ma. 3Ma is a turn in the course of raising in the EHS which is characterized by abruptly acceleration of uplifting. The uplift ratios are lower than 1mm .a~(-1) before 3Ma, and higher than 1mm .a~(-1) with a maximum ratio of 30mm .a~(-1) since 3Ma. The bottom (knick point) of the partial anneal belt is 3.8km above sea level in the EHS, and correspond to age of 3Ma determined by fission-track age of apatite. The average uplift ratio is about 1.4 mm .a~(-1) below the knick point. The EHS has raised 4.3km from the surface of 2.36km above sea level since 3Ma estimated by the fossil partial anneal belt of the EHS. We propose a two-stage subduction model (B+A model) basing on Structural, thermochronological, magmatical, metamorphic and geophysical investigations of the EHS. The first stage is the subduction of the Indian continental margin following after the subduction of the Tethys Ocean crust and subsequent collision with the Gangdese arc, and the second stage is the Indian crust injecting into the lower crust and upper mantle of the Tibet plateau. Slab break-off seems to be occurred between these two stages.

新第三纪红河盆地特征及红河断裂活动对其形成与发育的控制

Based on geodynamic analysis of sedimentary basins, combined sedimentology with structural geology and other methods, the author studied the Honghe basin located in Yunnan province of Southwestern China. Sandstone slice grain size analysis, combined with field geology and indoors study indicate that a set of inland alluvial fan diposits, fluvial deposites, delta deposits and some lacustrine sediments are in Honghe basin. Studying on shape of the Honghe basin, sedimentary and structural characteristic and distribution of different kinds of conglomerate and its structural significance, we hold the idea that the formation and evolution of Honghe basin are controlled by the activity of Red River faut. Correlation of lithostratic cross section in Honghe basin and studying on activity of Red River fault indicate that Honghe basin was formed in two stages. It is a complex basin constitutes of the first-stage trans-releasing basin and the second-stage trans-downfaulted basin. Due to the uplift of Qinghai-Xizang plateau and deformation of orogeny, the western Yunnan and adjacent area move to SE direction as a result of Tectonic Escape. Right lateral strike slip occurred along Red River fault, trans-releasing basin formed at the bend part of the fault due to stress relexation. As the block escaping, it moves away from the other block of the Red River fault, the upper block move down obliquely and trans-downfaulted basin formed. Combined the age of phytolite and regional structural events, we think the first-stage transreleasing basin was formed in late Miocene, on the other words, the dextral strike slip of Red River fault may began in late Miocene (10-7Ma). The second-stage trans-downfaulted basin may be formed in early stage of Pliocene (about 4.7Ma). Subsequently, the bilateral faults dipping to the inside of the plateau and thrusting outwards occurred in the marginal region of Qinghai-Xizang plateau during its uplifting as a fan-shaped mountain body, this results in the uplift of the strata to the east of Red River fault and supply large quantity of provenance for the Honghe basin. In last Pliocene (about 3Ma), strong uplift of Qinghai-Xizang plateau leads to massive clastic sediment entered Honghe basin and causes its closure. As a kind of trans-tentional basin, trans-releasing basin is different to pull-apart basin. The author compared the Mosha trans-releasing basin with Jinggu pull-apart basin in SW Yunan, China, and described their character correspondingly. Otherwise, the author combined the predecessors' studding with conclusion of own study, discussed the kinematics of Ailaoshan-Red River belt in Cenozoic, and the relationship between the formation of Honghe basin and uplifting of Qinghai-Xizang plateau.

藏东南迦巴瓦变质岩楔入体东边界阿尼桥断裂带构造变形性质、年代学与构造意义

The Namche Barwa metamorphic rock indenter is a part of the Indian plate. The Aniqiao fault, a northeastern striking shear zone, is the eastern boundary of the Namche Barwa metamorphic rock indenter. The activities of the Aniqiao fualt reflects the history of structure deformation and uplift of the Namche Barwa metamorphic rock indenter. In this dissertation, studied the history of activities of the Aniqiao fault, I study the deformation of the Namche Barwa metamorphic rock indenter based on which, I try to discuss the history of action and deformation of the eastern Tibet. The Aniqiao fault composes of mica quartz schist. With observing in the field and by the microscope, there are at least two stages of deformation. The earlier is right lateral striking, the later is normal striking. The biotite, in the hornblende biotitic mylonite in western footwall, the muscovite and sericite, in the mica quartz schist in eastern hangingwall, show 4 plateau and isochron ages: 3.7-3.3Ma, 6.8-6.4Ma, 13.4-13.2Ma, 23.9Ma, by ~(40)Ar/~(39)Ar. Combine the characteristics of kinematics with the characteristics of isotopic ages, this dissertation figured three stages of deformation: in 23.9Ma and 13.4-13.2 Ma, the Aniqiao fault undertook twice strike-slip deformation; in 6.8Ma-6.4Ma, the Aniqiao fault occurred normal strike deformation; in 3.7-3.3Ma, there was another thermal case which maybe relating to uplift. Combine the deformation of the Aniqiao fault and the deformation of the western boundary fault of the Namche Barwa metamorphic rock indenter, this dissertation considers that the Namche Barwa metamorphic rock indenter has occurred three defomational cases during the period of Oligocene and Quaternary: in 23Ma and 13Ma, the Namche Barwa metamorphic rock indenter wedged into the Gangdisi granite zone; from 6-7Ma, the Namche Barwa metamorphic rock indenter begins to uplift. From 6-7Ma, the Namche Barwa metamorphic rock indenter must has been occurred multi-stage uplifting. The indentation of the Namche Barwa metamorphic rock indenter is correspond to the structure escape of the Chuanxi, Dianxi blocks. In the surface deformation, the movement of these blocks are very harmonious.

Manejo da resistência de insetos-praga a inseticidas, com ênfase em Spodoptera frugiperda (Smith).

Detecção da resistência; Mecanismos da resistência de insetos a inseticidas; Base genética da resistência; Perspectiva genética no manejo da resistência; Aumento da dose do inseticida até que a resistência seja recessiva ou neutralizada; Uso de compostos que confiram menores níveis de resistência; Tratamento visando os estágios de vida do insetos mais vulneráveis; Uso de sinergismo para suprimir os mecanismos de resistência; Utilização de misturas de inseticidas; Decréscimo na dose de aplicação utilizando uma que mata a maioria dos insetos suscetíveis; Aplicação menos freqüente de inseticidas, de tal modo que os insetos suscetíveis tenham a oportunidade de reproduzir-se e, portanto, diluir a resistência pelo cruzamento com indivíduos resistentes; Uso de produtos químicos de curto período residual, evitando uso de formulações de liberações controladas dos ingredientes ativos, de modo que somente a população-alvo seja eliminada; ou seja, evitar que os colonizadores subseqüentes sejam afetados; Propiciar refúgios para o escape dos indivíduos suscetíveis; Fazer rotação dos pesticidas de modo que nem todas as gerações sejam expostas ao mesmo produto, evitando, porém, o tratamento espacial (em mosaico); Manejo da resistência de Spodoptera frugiperda a inseticidas; Táticas de manejo.

Ben Bowen (Writers of Wales)

Chapman, T. R. Ben Bowen (Writers of Wales)(University of Wales Press, 2003) RAE2008

The ecological effects of exotic disease resistance genes introgressed into British gooseberries

Warren, J. and James, P. (2006). The ecological effects of exotic disease resistance genes introgressed into British gooseberries. Oecologia 147(1),69-75. RAE2008

Wilno-Vilnius. Polskie i litewskie reprezentacje miasta po roku 1990

Wydział Filologii Polskiej i Klasycznej: Instytut Filologii Polskiej

No Muslims Allowed? An analysis of the State of Pluralism in the West

Dissertação de Mestrado apresentada à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Relações Internacionais com o Mundo Árabe e Islâmico.

O discurso de O Portuguez e a Corte portuguesa no Brasil

Relatório apresentado à Universidade Fernando Pessoa como parte dos requisitos para cumprimento do programa de pós-doutoramento em Ciências da Comunicação, vertente Jornalismo

The operation of multiquantum-well barriers

The multiquantum barrier (MQB), proposed by Iga et al in 1986, has been shown by several researchers to be an effective structure for improving the operating characteristics of laser diodes. These improvements include a reduction in the laser threshold current and increased characteristic temperatures. The operation of the MQB has been described as providing an increased barrier to electron overflow by reflecting high energy electrons trying to escape from the active region of the laser.This is achieved in a manner analogous to a Bragg reflector in optics. This thesis presents an investigation of the effectiveness of the MQB as an electron reflector. Numerical models have been developed for calculating the electron reflection due to MQB. Novel optical and electrical characterisation techniques have been used to try to measure an increase in barrier height due to the MQB in AlGaInP.It has been shown that the inclusion of MQB structures in bulk double heterostructure visible laser diodes can halve the threshold current above room temperature and the characteristic temperature of these lasers can be increased by up to 20K.These improvements are shown to occur in visible laser diodes even with the inclusion of theoretically ineffective MQB structures, hence the observed improvement in the characteristics of the laser diodes described above cannot be uniquely attributed to an increased barrier height due to enhance electron reflection. It is proposed here that the MQB improves the performance of laser diodes by proventing the diffusion of zinc into the active region of the laser. It is also proposed that the trapped zinc in the MQB region of the laser diode locally increases the p-type doping bringing the quasi-Fermi level for holes closer to the valence band edge thus increasing the barrier to electron overflow in the conduction band.

An investigation of innovation and knowledge creation in virtual worlds

The Internet and World Wide Web have had, and continue to have, an incredible impact on our civilization. These technologies have radically influenced the way that society is organised and the manner in which people around the world communicate and interact. The structure and function of individual, social, organisational, economic and political life begin to resemble the digital network architectures upon which they are increasingly reliant. It is increasingly difficult to imagine how our ‘offline’ world would look or function without the ‘online’ world; it is becoming less meaningful to distinguish between the ‘actual’ and the ‘virtual’. Thus, the major architectural project of the twenty-first century is to “imagine, build, and enhance an interactive and ever changing cyberspace” (Lévy, 1997, p. 10). Virtual worlds are at the forefront of this evolving digital landscape. Virtual worlds have “critical implications for business, education, social sciences, and our society at large” (Messinger et al., 2009, p. 204). This study focuses on the possibilities of virtual worlds in terms of communication, collaboration, innovation and creativity. The concept of knowledge creation is at the core of this research. The study shows that scholars increasingly recognise that knowledge creation, as a socially enacted process, goes to the very heart of innovation. However, efforts to build upon these insights have struggled to escape the influence of the information processing paradigm of old and have failed to move beyond the persistent but problematic conceptualisation of knowledge creation in terms of tacit and explicit knowledge. Based on these insights, the study leverages extant research to develop the conceptual apparatus necessary to carry out an investigation of innovation and knowledge creation in virtual worlds. The study derives and articulates a set of definitions (of virtual worlds, innovation, knowledge and knowledge creation) to guide research. The study also leverages a number of extant theories in order to develop a preliminary framework to model knowledge creation in virtual worlds. Using a combination of participant observation and six case studies of innovative educational projects in Second Life, the study yields a range of insights into the process of knowledge creation in virtual worlds and into the factors that affect it. The study’s contributions to theory are expressed as a series of propositions and findings and are represented as a revised and empirically grounded theoretical framework of knowledge creation in virtual worlds. These findings highlight the importance of prior related knowledge and intrinsic motivation in terms of shaping and stimulating knowledge creation in virtual worlds. At the same time, they highlight the importance of meta-knowledge (knowledge about knowledge) in terms of guiding the knowledge creation process whilst revealing the diversity of behavioural approaches actually used to create knowledge in virtual worlds and. This theoretical framework is itself one of the chief contributions of the study and the analysis explores how it can be used to guide further research in virtual worlds and on knowledge creation. The study’s contributions to practice are presented as actionable guide to simulate knowledge creation in virtual worlds. This guide utilises a theoretically based classification of four knowledge-creator archetypes (the sage, the lore master, the artisan, and the apprentice) and derives an actionable set of behavioural prescriptions for each archetype. The study concludes with a discussion of the study’s implications in terms of future research.

Women's contribution to the development of the English novel: 1621-1818

Women's contribution to literature is no arbitrary or artificial distinction. However much the reformer may welcome, or the conservative lament, the growth of a harmonious sharing of ideals between men and women, that growth has been a hard-fought struggle. It has been an escape from a prison, which, when it did not entirely shut out the greater world, at least enclosed a little world of education meant for women, literature adapted to the supposed limitations of their intellect, and a course of action prescribed by the other sex. To show how the literary efforts of women developed and justified their claims to free activity is the purpose of this thesis.

Characterisation of bacteriophage-host interactions in Lactococcus lactis

Lactococcus lactis is used extensively world-wide for the production of fermented dairy products. Bacteriophages (phages) infecting L. lactis can result in slow or incomplete fermentations, or may even cause total fermentation failure. Therefore, bacteriophages disrupting L. lactis fermentation are of economic concern. This thesis employed a multifaceted approach to investigate various molecular aspects of phage-host interaction in L. lactis. The genome sequence of an Irish dairy starter strain, the prophage-cured L. lactis subsp. cremoris UC509.9, was studied. The 2,250,427 bp circular chromosome represents the smallest among its sequenced lactococcal equivalents. The genome displays clear genetic adaptation to the dairy niche in the form of extensive reductive evolution. Gene prediction identified 2066 protein-encoding genes, including 104 which showed significant homology to transposase-specifying genes. Over 9 % of the identified genes appear to be inactivated through stop codons or frame shift mutations. Many pseudogenes were found in genes that are assigned to carbohydrate and amino acid transport and metabolism orthologous groups, reflecting L. lactis UC509.9’s adaptation to the lactose and casein-rich dairy environment. Sequence analysis of the eight plasmids of L. lactis revealed extensive adaptation to the dairy environment. Key industrial phenotypes were mapped and novel lactococcal plasmid-associated genes highlighted. In addition to chromosomally-encoded bacteriophage resistance systems, six functional such systems were identified, including two abortive infection systems, AbiB and AbiD1, explaining the observed phage resistance of L. lactis UC509.9 Molecular analysis suggests that the constitutive expression of AbiB is not lethal to cells, suggesting the protein is expressed in an un/inactivated form. Analysis of 936 species phage sk1-escape mutants of AbiB revealed that all such mutants harbour mutations in orf6, which encodes the major capsid protein. Results suggest that the major capsid protein is required for activation of the AbiB system, although this requires furrther investigations. Temporal transcriptomes of L. lactis UC509.9 undergoing lytic infection with either one of two distinct bacteriophages, Tuc2009 and c2, was determined and compared to the transcriptome of uninfected UC509.9 cells. Whole genome microarrays performed at various time-points post-infection demonstrated a rather modest impact on host transcription. Alterations in the UC509.9 transcriptome during lytic infection appear phage-specific, with a relatively small number of differentially transcribed genes shared between infection with either Tuc2009 or c2. Transcriptional profiles of both bacteriophages during lytic infection was shown to generally correlate with previous studies and allowed the confirmation of previously predicted promoter sequences. Bioinformatic analysis of genomic regions encoding the presumed cell wall polysaccharide (CW PS) biosynthesis gene cluster of several strains of L. lactis was performed. Results demonstrate the presence of three dominant genetic types of this gene cluster, termed type A, B and C. These regions were used for the development of a multiplex PCR to identify CW PS genotype of various lactococcal strains. Analysis of 936 species phage receptor binding protein phylogeny (RBP) and CW PS genotype revealed an apparent correlation between RBP phylogeny and CW PS type, thereby providing a partial explanation for the observed narrow host range of 936 phages. Further analysis of the genetic locus encompassing the presumed CW PS biosynthesis operon of eight strains identified as belonging to the CW PS C (geno)type, revealed the presence of a variable region among the examined strains. The obtained comparative analysis allowed for the identification of five subgroups of the C type, named C1 to C5. We purified an acidic polysaccharide from the cell wall of L. lactis 3107 (C2 subtype) and confirmed that it is structurally different from the CW PS of the C1 subtype L. lactis MG1363. Combinations of genes from the variable region of C2 subtype were amplified from L. lactis 3107 and introduced into a mutant of the C1 subtype L. lactis NZ9000 (a direct derivative of MG1363) deficient in CW PS biosynthesis. The resulting recombinant mutant synthesized a CW PS with a composition characteristic for that of the C2 subtype L. lactis 3107 and not the wildtype C1 L. lactis NZ9000. The recombinant mutant exhibited a changed phage resistance/sensitivity profile consistent with that of L. lactis 3107, which unambiguously demonstrated that L. lactis 3107 CW PS is the host cell surface receptor of two bacteriophages belonging to the P335 species as well as phages that are member of the 936 species. The research presented in this thesis has significantly advanced our understanding of L. lactis bacteriophage-host interactions in several ways. Firstly, the examination of plasmidencoded bacteriophage resistance systems has allowed inferences to be made regarding the mode of action of AbiB, thereby providing a platform for further elucidation of the molecular trigger of this system. Secondly, the phage infection transcriptome data presented, in addition to previous work, has made L. lactis a model organism in terms of transcriptomic studies of bacteriophage-host interactions. And finally, the research described in this thesis has for the first time explicitly revealed the nature of a carbohydrate bacteriophage receptor in L. lactis, while also providing a logical explanation for the observed narrow host ranges exhibited by 936 and P335 phages. Future research in discerning the structures of other L. lactis CW PS, combined with the determination of the molecular interplay between receptor binding proteins of these phages and CW PS will allow an in depth understanding of the mechanism by which the most prevalent lactococcal phages identify and adsorb to their specific host.

Molecular and cellular aspects of the SREBP pathway

The SREBP (sterol response element binding proteins) transcription factors are central to regulating de novo biosynthesis of cholesterol and fatty acids. The SREBPs are regulated by retention or escape from the ER to the Golgi where they are proteolytically cleaved into active forms. The SREBP cleavage activating protein (SCAP) and the INSIG proteins are essential in this regulatory process. The aim of this thesis is to further characterise the molecular and cellular aspects surrounding regulation of SREBP processing. SREBP and SCAP are known to interact via their carboxy-terminal regulatory domains (CTDs) but this interaction is poorly characterised. Significant steps were achieved in this thesis towards specific mapping of the interaction site. These included cloning and over expression and partial purification of tagged SREBP1 and SREBP2 CTDs and probing of a SCAP peptide array with the CTDs. Results from the SREBP2 probing were difficult to interpret due to insolubility issues with the protein, however, probing with SREBP1 revealed five potential binding sites which were detected reproducibly. Further research is necessary to overcome SREBP2 insolubility issues and to confirm the identified SREBP1 interaction site(s) on SCAP. INSIG1 has a central role in regulating SREBP processing and in regulating stability of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a rate limiting enzyme in cholesterol biosynthesis. There are two protein isoforms of human INSIG1 produced through the use of two in-frame alternative start sites. Bioinformatic analysis indicated that the presence of two in-frame start sites within the 5-prime region of INSIG1 mRNA is highly conserved and that production of two isoforms of INSIG1is likely a conserved event. Functional differences between these two isoforms were explored. No difference in either the regulation of SREBP processing or HMGCR degradation between the INSIG1 isoforms was observed and the functional significance of the two isoforms is as yet unclear. The final part of this thesis focused on enhancing the cytotoxicity of statins by targeted inhibition of SREBP processing by oxysterols. Statins have significant potential as anti-cancer agents as they inhibit the activity of HMGCR leading to a deficiency in mevalonate which is essential for cell survival. The levels of HMGCR fluctuate widely due to cholesterol feedback of SREBP processing. The relationship between sterol feedback and statin mediated cell death was investigated in depth in HeLa cells. Down regulation of SREBP processing by sterols significantly enhanced the efficacy of statin mediated cell death. Investigation of sterol feedback in additional cancer cell lines showed that sterol feedback was absent in cell lines A- 498, DU-145, MCF-7 and MeWo but was present in cell lines HT-29, HepG2 and KYSE-70. In the latter inhibition of SREBP processing using oxysterols significantly enhanced statin cytotoxicity. The results indicate that this approach is valid to enhance statin cytotoxicity in cancer cells, but may be limited by deregulation of SREBP processing and off target effects of statins, which were observed for some of the cancer cell lines screened.