Rab8 and Rab8-interacting proteins as players in cell polarization

Rab8 and its interacting proteins as regulators of cell polarization During the development of a multi-cellular organism, progenitor cells have to divide and migrate appropriately as well as organize their differentiation with one another, in order to produce a viable embryo. To divide, differentiate and migrate cells have to undergo polarization, a process where internal and external components such as actin, microtubules and adhesion receptors are reorganized to produce a cell that is asymmetric, with functionally different surfaces. Also in the adult organism there is a continuous need for these processes, as cells need to migrate in response to tissue damage and to fight infection. Improper regulation of cell proliferation and migration can conversely lead to disease such as cancer. GTP-binding proteins function as molecular switches by cycling between a GTP-bound (active) conformation and a GDP-bound (inactive) conformation. The Ras super-family of small GTPases are found in all eukaryotic cells. They can be functionally divided into five subfamilies. The Ras family members mainly regulate gene expression, controlling cell proliferation and differentiation. Ras was in fact the first human oncogene to be characterized, and as much as 30% of all human tumors may be directly or indirectly caused by mutations of Ras molecules The Rho family members mainly regulate cytoskeletal reorganization. Arf proteins are known to regulate vesicle budding and Rab proteins regulate vesicular transport. Ran regulates nuclear transport as well as microtubule organization during mitosis. The focus of the thesis of Katarina Hattula, is on Rab8, a small GTPase of the Rab family. Activated Rab8 has previously been shown to induce the formation of new surface extensions, reorganizing both actin and microtubules, and to have a role in directed membrane transport to cell surfaces. However, the exact membrane route it regulates has remained elusive. In the thesis three novel interactors of Rab8 are presented. Rabin8 is a Rab8-specific GEF that localizes to vesicles where it presumably recruits and activates its target Rab8. Its expression in cells leads to remodelling of actin and the formation of polarized cell surface domains. Optineurin, known to be associated with a leading cause of blindness in humans (open-angle glaucoma), is shown to interact specifically with GTP-bound Rab8. Rab8 binds to an amino-terminal region and interestingly, the Huntingtin protein binds a carboxy-terminal region of optineurin. (Aberrant Huntingtin protein is known to be the cause Huntington s disease in humans.) Co-expression of Huntingtin and optineurin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures. Furthermore, optineurin promoted cell polarization in a similar way to Rab8. A third novel interactor of Rab8 presented in this thesis is JFC1, a member of the synaptogamin-like protein (Slp) family. JFC1 interacts with Rab8 specifically in its GTP-bound form, co-localizes with endogenous Rab8 on tubular and vesicular structures, and is probably involved in controlling Rab8 membrane dynamics. Rab8 is in this thesis work clearly shown to have a strong effect on cell shape. Blocking Rab8 activity by expression of Rab8 RNAi, or by expressing the dominant negative Rab8 (T22N) mutant leads to loss of cell polarity. Conversely, cells expressing the constitutively active Rab8 (Q67L) mutant exhibit a strongly polarized phenotype. Experiments in live cells show that Rab8 is associated with macropinosomes generated at ruffling areas of the membrane. These macropinosomes fuse with or transform into tubules that move toward the cell centre, from where they are recycled back to the leading edge to participate in protrusion formation. The biogenesis of these tubules is shown to be dependent on both actin and microtubule dynamics. The Rab8-specific membrane route studied contained several markers known to be internalized and recycled (1 integrin, transferrin, transferrin receptor, cholera toxin B subunit (CTxB), and major histocompatibility complex class I protein (MHCI)). Co-expression studies revealed that Rab8 localization overlaps with that of Rab11 and Arf6. Rab8 is furthermore clearly functionally linked to Arf6. The data presented in this thesis strongly suggests a role for Rab8 as a regulator for a recycling compartment, which is involved in providing structural and regulatory components to the leading edge to participate in protrusion formation.

Mu in vitro transposition applications in protein engineering

Transposons, mobile genetic elements that are ubiquitous in all living organisms have been used as tools in molecular biology for decades. They have the ability to move into discrete DNA locations with no apparent homology to the target site. The utility of transposons as molecular tools is based on their ability to integrate into various DNA sequences efficiently, producing extensive mutant clone libraries that can be used in various molecular biology applications. Bacteriophage Mu is one of the most useful transposons due to its well-characterized and simple in vitro transposition reaction. This study establishes the properties of the Mu in vitro transposition system as a versatile multipurpose tool in molecular biology. In addition, this study describes Mu-based applications for engineering proteins by random insertional transposon mutagenesis in order to study structure-function relationships in proteins. We initially characterized the properties of the minimal Mu in vitro transposition system. We showed that the Mu transposition system works efficiently and accurately and produces insertions into a wide spectrum of target sites in different DNA molecules. Then, we developed a pentapeptide insertion mutagenesis strategy for inserting random five amino acid cassettes into proteins. These protein variants can be used especially for screening important sites for protein-protein interactions. Also, the system may produce temperature-sensitive variants of the protein of interest. Furthermore, we developed an efficient screening system for high-resolution mapping of protein-protein interfaces with the pentapeptide insertion mutagenesis. This was accomplished by combining the mutagenesis with subsequent yeast two-hybrid screening and PCR-based genetic footprinting. This combination allows the analysis of the whole mutant library en masse, without the need for producing or isolating separate mutant clones, and the protein-protein interfaces can be determined at amino acid accuracy. The system was validated by analysing the interacting region of JFC1 with Rab8A, and we show that the interaction is mediated via the JFC1 Slp homology domain. In addition, we developed a procedure for the production of nested sets of N- and C-terminal deletion variants of proteins with the Mu system. These variants are useful in many functional studies of proteins, especially in mapping regions involved in protein-protein interactions. This methodology was validated by analysing the region in yeast Mso1 involved in an interaction with Sec1. The results of this study show that the Mu in vitro transposition system is versatile for various applicational purposes and can efficiently be adapted to random protein engineering applications for functional studies of proteins.

Quasi-periodic, global oscillations in sea level pressure on intraseasonal timescales

The sea level pressure (SLP) variability in 30-60 day intraseasonal timescales is investigated using 25 years of reanalysis data addressing two issues. The first concerns the non-zero zonal mean component of SLP near the equator and its meridional connections, and the second concerns the fast eastward propagation (EP) speed of SLP compared to that of zonal wind. It is shown that the entire globe resonates with high amplitude wave activity during some periods which may last for few to several months, followed by lull periods of varying duration. SLP variations in the tropical belt are highly coherent from 25A degrees S to 25A degrees N, uncorrelated with variations in mid latitudes and again significantly correlated but with opposite phase around 60A degrees S and 65A degrees N. Near the equator (8A degrees S-8A degrees N), the zonal mean contributes significantly to the total variance in SLP, and after its removal, SLP shows a dominant zonal wavenumber one structure having a periodicity of 40 days and EP speeds comparable to that of zonal winds in the Indian Ocean. SLP from many of the atmospheric and coupled general circulation models show similar behaviour in the meridional direction although their propagation characteristics in the tropical belt differ widely.

Tree-ring records as indicators of air-sea interaction in the northeast Pacific sector

Climate conditions in land areas of the Pacific Northwest are strongly influenced by atmosphere/ocean variability, including fluctuations in the Aleutian Low, Pacific-North American (PNA) atmospheric circulation modes, and the El Niño-Southern Oscillation (ENSO). It thus seems likely that climatically sensitive tree-ring data from these coastal land areas would likewise reflect such climatic parameters. In this paper, tree-ring width and maximum lakewood density chronologies from northwestern Washington State and near Vancouver Island, British Columbia, are compared to surface air temperature and precipitation from nearby coastal and near-coastal land stations and to monthly sea surface temperature (SST) and sea level pressure (SLP) data from the northeast Pacific sector. Results show much promise for eventual reconstruction of these parameters, potentially extending available instrumental records for the northeastern Pacific by several hundred years or more.

Influence of climate on environmental factors associated with long-term changes in chlorophyll production for the Sacramento-San Joaquin delta and Suisun Bay, California

Long-term changes in chlorophyll production were predicted from environmental variables for the Sacramento and San Joaquin rivers and Suisun Bay using Box-Jenkins transfer function models. Data used for the analyses were collected semimonthly or monthly between 1971 and 1987. Transfer function models developed to describe changes in chlorophyll production over time as a function of environmental variables were characterized by lagged responses and described between 39 and 51 percent of the data variation. Significant correlations between environmental variables and the California climate index (CA SLP) were used to develop a conceptual model of the link between regional climate and estuarine production.

普适环境下服务发现的语义支持机制

随着计算能力和通信能力的增强，计算设备所占用的体积也越来越小，各种新形态的传感器、移动设备及无线网络设备日趋普及。这极大地促进了以无线、移动和嵌入式设备为基础的普适计算模式的形成和发展。普适环境下的服务发现机制可使得用户能够使用各种移动设备无缝的、随时随地的共享和访问各种服务信息。 普适环境中的网络异构性和动态性，以及服务的多样性和异构性，均对服务发现提出了新的挑战。目前学术界和工业界在服务发现方面进行了不少的探索和研究，当前的一些服务发现协议或系统比如SLP、UPnP、INS等，一般基于语法的方法描述服务，主要关注服务的功能性需求，但基于关键字来匹配请求和服务描述，在普适环境下常常会导致较差的匹配结果。 针对服务发现在普适环境中的新需求，本文基于XML定义了一种轻量级的服务语义建模语言SML，SML定义了丰富的数据类型，以模板和属性的方式定义各领域的实体，可以表达丰富的语义知识；同时，本文以轻量级的推理引擎Jess为依托，将用服务建模语言定义的各应用领域的服务模板和语义知识自动转换成Jess的推理规则和事实。本文定义了一种类似XPath的服务查询语言规范，并在支持精确匹配的基础上，提出了服务的近似匹配策略，提供了各种的近似计算规则。鉴于不同用户对服务的各属性有不同的偏好程度，还提出了基于用户偏好的服务匹配策略。动态的上下文信息是服务匹配过程的重要考虑因素。本文以Jess规则来匹配服务和用户的上下文，选择适合于用户当前情况的服务。对服务各种QoS的描述支持也是服务语义建模语言提供的功能之一，为此，本文还提出了一种基于Pareto最优的服务选择策略，根据服务的QoS以及服务与请求的匹配程度来选择Pareto最优的服务。本文的上述工作已实现到服务发现系统Service CatalogNet Extended中。

多服务发现协议的互操作框架研究

服务发现协议（Service Discovery Protocol）是支持程序和设备自动发现并访问网络中所需服务的协议，是分布式环境特别是普适环境下进行资源共享、数据集成、流程协作的前提。目前学术界和工业界已经提出了多种服务发现协议，如UPnP SSDP、SLP、Salutation、Bluetooth SDP。这些服务发现协议提供了类似的功能，但在适用网络类型、描述模型、发现机制、服务使用方式等方面存在着高度的异构性，导致使用不同服务发现协议的设备之间无法直接进行交互，从而限制了服务使用者和服务提供者之间的交互范围及交互能力。同时这其中的一些协议已经成为事实上的工业标准，但目前并没有一个能够适用于所有场景并占据整个市场，并且在很长的一段时间内不可能出现一个通用的协议来取而代之，同时，要求服务请求者和服务提供者同时支持多种服务发现协议是不现实的，因此迫切需要一种互操作框架来实现各种服务发现协议之间的互操作，从而允许服务使用者能够以其自有的方式在网络上发现并使用所需的各种服务，而不用考虑服务发布所采用的具体服务发现协议及调用方式的不同。 目前学术界和工业界在服务发现协议的互操作方面进行了不少探索和研究，但在互操作对象的可扩展性及互操作的透明性、范围方面仍存在不少问题尚未得到有效地解决。 本文主要研究跨网络环境下的服务发现协议的互操作。本文基于抽象语义信息的互操作模型，综合考虑了上下文信息，采用分阶段事件驱动的技术处理并发请求，设计并实现了具有透明性、可扩展性、高效性的服务发现协议互操作框架（Service CatlogNet Interoperable Framework，SCNIF），本文设计与实现了三种服务发现协议（UPnP SSDP、SLP、SCN）的互操作插件，还给出了新加入服务发现协议的互操作插件的实现方案。

Analysis of the sea-level variability along the Chinese coast and estimation of the impact of a CO2-perturbed atmospheric circulation

The relationship between monthly sea-level data measured at stations located along the Chinese coast and concurrent large-scale atmospheric forcing in the period 1960-1990 is examined. It is found that sea-level varies quite coherently along the whole coast, despite the geographical extension of the station set. A canonical correlation analysis between sea-level and sea-level pressure (SLP) indicates that a great part of the sea-level variability can be explained by the action of the wind stress on the ocean surface. The relationship between sea-level and sea-level pressure is analyzed separately for the summer and winter half-years. In winter, one factor affecting sea-level variability at all stations is the SLP contrast between the continent and the Pacific Ocean, hence the intensity of the winter Monsoon circulation. Another factor that affects coherently all stations is the intensity of the zonal circulation at mid-latitudes. In the summer half year, on the other hand, the influence of SLP on sea-level is spatially less coherent: the stations in the Yellow Sea are affected by a more localized circulation anomaly pattern, whereas the rest of the stations is more directly connected to the intensity of the zonal circulation. Based on this analysis, statistical models (different for summer and winter) to hindcast coastal sealevel anomalies from the large-scale SLP field are formulated. These models have been tested by fitting their internal parameters in a test period and reproducing reasonably the sea-level evolution in an independent period. These statistical models are also used to estimate the contribution of the changes of the atmospheric circulation on sea-level along the Chinese coast in an altered climate. For this purpose the ouput of 150 year-long experiment with the coupled ocean-atmosphere model ECHAM1-LSG has been analyzed, in which the atmospheric concentration of greenhouse gases was continuously increased from 1940 until 2090, according to the Scenario A projection of the Intergovermental Panel on Climate Change. In this experiment the meridional (zonal) circulation relevant for sea-level tends to become weaker (stronger) in the winter half year and stronger (weaker) in summer. The estimated contribution of this atmospheric circulation changes to coastal sea-level is of the order of a few centimeters at the end of the integration, being in winter negative in the Yellow Sea and positive in the China Sea with opposite signs in the summer half-year.

台风海面风场的动力分析、四维同化及数值试验

台风是最具破坏性的海洋大气系统。台风及其引起的沿海强风暴潮和海上巨浪不仅给沿海地区人民的生命财产造成严重的损失，而且对海上交通运输、军事活动、海洋工程和渔业都带来严重影响。准确的台风海面风场是获得比较真实的海浪、风暴潮等海洋现象的关键所在。由于不准确的海面初始场和台风海面风场数值模式中物理过程不完善等问题，模拟预报台风海面风场的精度达不到要求，极大的影响了海浪、风暴潮等海洋过程的模拟预报精度。因此获得准确真实的台风海面风场不仅对国民经济的顺利健康发展起到至关重要的作用，而且会很好地促进海浪、风暴湖等过程的研究。中尺度大气数值模式MM5在灾害性天气的预报研究方面具有重要的地位，并在暴雨研究方面取得了成功。在本论文中，我们结合气象工作者和海洋学者在台风研究中的不同侧重点，耦合自由大气和边界层过程，考察海气相互作用对台风海面风场的作用，首次将MM5应用于台风海面风场的研究之中，这对台风海面风场的研究来说是一个新的起点。本论文在MM5中，将中国国家气象中心中期预报谱模式T63／T106分析和预报场与常规地面和探空观测资料相结合，合理调整模式参数(海面粗糙度等参数)，建立了一套适用于中国区域的台风海面风场的模拟、预报系统。本论文利用改造过的中尺度大气数值模式MM5对两个台风个例：9608号台风(HERB)和9711号台风(WINNIE)进行数值试验。对海面风场而言，海气热通量交换对海面风场发挥着重要作用。因此本论文研究海面热量通量交换对9608号台风的强度和中心位置的影响，这也是以前国内台风数值模式中疏于考虑的方面。最后将模式模拟风场用于波浪的模拟研究之中，考察模拟海面风场在海洋环境研究中应用的有效性；并与目前国内较先进的LAGFD-WIN海面动力风场模式对比，检验MM5在台风海面风场研究中的性能。研究结果表明，MM5较好的反映出台风特性，并且优于LAGFD-WIN海面动力风场模式结果。在考察台风不稳定扰动特征时，我们区别于以往的本征值方法，利用中尺度二维扰动模式，由MM5提供9711号台风数值试验结果作为扰动模式的初始场，从动力学角度研究台风过程的扰动特征。初始资料问题对台风模式的预报模拟精度来说是一个最大、最重要的问题。因此改善、弥补模式初始场所造成的不足是本研究论文的重点工作之一。目前，在国内关于MM5中的四维资料同化(FDDA)方案试验刚刚起步。在本论文中，我们成功的启动MM5的四维资料同化方案：分析逼近和观测逼近。我们改造模式，同时启动这两种逼近方法，充分发挥探空资料和地面观测资料各自的优越性，I探索不同尺度、不同类型的资料在台风海面风场的应用。尝试卫星高度计风场资料溶于MM5中的同化方案中，这是本研究论文的另一个创新之处。本文的主要结论是(1)对两个台风个例：9608号台风(HERB)和9711号台风(WINNIE)进行了控制试验(CNTL)研究，分析台风模拟过程的温度距平、海平面气压及海面风场分布特点。研究表明，两个台风过程分别在不同高度上具有暖中心结构；模拟出的两个台风环流都呈现出台风的非对称及涡旋型态，模拟出的台风强度令人满意。对移动路径来说，对9608号台风过程，12h模拟台风路径误差为1.3个纬度，0.7个经度，而24h的模拟误差则为0.4个纬度，0.2个纬度；而对于9711号台风过程，其12h模拟台风路径误差为1.2个纬度，1.6个经度；24h路径误差为1.9个纬度，1．5个经度；36h模拟路径误差为3.2个纬度，2.8个经度。我们认为，由于9711号台风过程不准确的初始场，9711号台风模拟路径精度逊色于9608号台风过程的结果。(2)9608号台风过程中感热通量和潜热通量交换，在台风中心附近，热通量较小，但在台风周围，存在热通量的高值压，随着台风强度的减弱，热量通量交换也随之减小。在海面上始终是海水给大气提供热量。海面潜热通量总为正通量，并且都大于相应时刻的感热通量，表明水汽通量对台风系统比感热通量更重要。在分析热通量在不同下垫面演变特征时发现，在下午2h左右，在水陆交界处的次之，上最大，陆地上最小。感热通量在陆地下垫面上最大，其峰值出现而在水域上的最小，对于潜热通量则是水域(3)应用9608号台风的模拟海面风场到海浪的数值模拟研究中，发现由于台风海面风场分布的不对称性(在台风前进方向的右侧风速最大)，也因此造成了海浪有效波高分布的不对称性，同时在台风影响的右前方也传播得较远。随着台风海面风场减弱，风浪能量也逐渐消耗，有效波高也逐渐降低。(4)在台风发展的动力学特征研究方面，首先从不稳定扰动理论角度出发，讨论了台风发展的条件，得到了和天气学分析的台风形成发展条件一致的判据；在台风不稳定扰动的数值试验方面，针对9711号台风过程进行了数值试验，数值试验结果表明：扰动环流型以倾斜的扰动特征出现，并略向西倾斜，这是发展型的扰动型态；促使中尺度扰动发展和维持的能源主要来自于基本流场的有效位能和扰动场的有效位能。(5)利用标准探空资料和卫星高度计风场资料，在MM5中对9608号台风进行了七个试验：一个控制试验，三个分析逼近试验(AW，AWTQ，ATQ)，两个观测逼近试验(IWT，IW)和一个同时进行分析和观测逼近的试验(AWTQ+IWT)。七个试验中海平面气压场都形成了闭合低压中心，海面风场具有台风的气旋型态，再次证明MM5对台风的模拟能力；在定量的描述上，AWTQ+IWT试验较好地模拟出台风的中心位置和强度。因此联合有相对好的垂直分辨率的探空资料以及相对于探空资料有好的时间和水平分辨率的地面资料，能有效地提高台风海面风场的模拟预报精度。由于没有同化SLP的观测资料到模式中，SLP成为确定FDDA有效程度的有力手段。我们发现同时同化风场和质量场(温度场和湿度场)的试验AWTQ对风场和质量场均有一致的正面影响；对由强烈动力作用和大尺度强迫的台风过程，风场优于质量场。试验AWTQ，AW和ATQ的结果表明在风场中只有非常少的质量场诱导信息。试验ATQ，IWT对风场，海平面气压场具有负面影响。而利用包含常规气象观测资料无法拥有的海面风场信息的高度计风场资料，仅进行风场同化的IW试验，对海面气压场有非常好的效果。结果表明而AW，AWTQ，IW，AWTQ+IWT在24h模拟期间，则对SLP的平均RMS有明显的减小，分别约为3％，9％，12％和8％。

Live-Cell Tracking Using SIFT Features in DIC Microscopic Videos

In this paper, a novel motion-tracking scheme using scale-invariant features is proposed for automatic cell motility analysis in gray-scale microscopic videos, particularly for the live-cell tracking in low-contrast differential interference contrast (DIC) microscopy. In the proposed approach, scale-invariant feature transform (SIFT) points around live cells in the microscopic image are detected, and a structure locality preservation (SLP) scheme using Laplacian Eigenmap is proposed to track the SIFT feature points along successive frames of low-contrast DIC videos. Experiments on low-contrast DIC microscopic videos of various live-cell lines shows that in comparison with principal component analysis (PCA) based SIFT tracking, the proposed Laplacian-SIFT can significantly reduce the error rate of SIFT feature tracking. With this enhancement, further experimental results demonstrate that the proposed scheme is a robust and accurate approach to tackling the challenge of live-cell tracking in DIC microscopy.

The quality of reporting of diagnostic accuracy studies in glaucoma using scanning laser polarimetry

PURPOSE: Scanning laser polarimetry (SLP) has been proposed as a useful diagnostic test for glaucoma. This study was conducted to evaluate the quality of reporting of published studies using the SLP for diagnosing glaucoma. METHODS: A validated Medline and hand search of English-language articles reporting on measures of diagnostic accuracy of the SLP for glaucoma was performed. Two reviewers independently selected and appraised the manuscripts. The Standards for Reporting of Diagnostic Accuracy (STARD) checklist was used to evaluate the quality of each publication. RESULTS: A total of 47 papers were identified of which the first 10 (from 1997 to 2000) and the last 10 articles (from 2004 to 2005) were appraised. Interobserver rating agreement of STARD items was high (85.5% agreement, ?=0.796). The number of STARD items properly reported ranged from 3/25 to 19/25. Only a quarter of studies (5/20) explicitly reported more than half of the STARD items. Important aspects of the methodology were often missing such as participant sampling (reported in 40% of manuscripts), masking of the readers of the index test and reference standard (reported in 20% of manuscripts), and estimation of uncertainty (eg, 95% confidence intervals, reported in 25% of manuscripts). There was a slight increase in the number of STARD items reported with time. CONCLUSIONS: The quality of reporting of diagnostic accuracy tests for glaucoma with SLP is suboptimal. The STARD initiative may be a useful tool for appraising the strengths and weaknesses of diagnostic accuracy studies. © 2007 Lippincott Williams & Wilkins, Inc.

Study of novel energy metabolism pathways in anaerobic bacteria

Energy conservation in chemotrophic anaerobic bacteria is achieved by two possible processes, substrate level phosphorylation (SLP) and electron transfer phosphorylation (ETP). This second mechanism, also known as respiration, involves chemiosmotic coupling. However, a third mechanism for energy coupling was recently proposed: the flavin-based electron bifurcation (FBEB). (...)

Élaboration d’un anticorps chimère anti-gp350 comme traitement prophylactique éventuel des syndromes lymphoprolifératifs B chez les greffés

Le virus Epstein-Barr (VEB) est fortement associé au développement de syndromes lymphoprolifératifs (SLP) en greffe pédiatrique. Ce virus a la capacité d’immortaliser les lymphocytes B et de provoquer leur prolifération incontrôlée chez l’hôte immunodéprimé. Plusieurs études démontrent que le cycle lytique du virus jouerait un rôle primordial dans la genèse des SLP en produisant des particules virales pouvant infecter les cellules B adjacentes. Chez un individu immunodéprimé, ces cellules B nouvellement infectées peuvent donner naissance à une expansion lymphocytaire. Le projet présenté dans ce mémoire fait partie d’un programme de recherche visant à élucider le rôle de l’infection productive par le VEB dans le développement des SLP. L’objectif précis de ce projet est de développer un anticorps monoclonal chimère contre la glycoprotéine gp350 du VEB dans le but de neutraliser le virus et d’ainsi prévenir son entrée dans les cellules B. Notre laboratoire a construit une version chimère de l’anticorps monoclonal murin 72A1, lequel se lie à la gp350 et bloque l’infection. Les premiers essais ont révélé la présence de chaînes non fonctionnelles (aberrantes) dans l’hybridome produisant l’anticorps 72A1. La construction de la chaîne légère authentique est maintenant complète alors que celle de la chaîne lourde est toujours en cours. Le processus de caractérisation de l’anticorps chimère inclura des essais de cytotoxicité à médiation cellulaire dépendante des anticorps (ADCC). Dans cette optique, une lignée cellulaire exprimant de façon stable la gp350 a été établie. Notre anticorps chimère anti-gp350 pourrait éventuellement être utilisé comme thérapie préventive chez les greffés présentant un risque élevé de SLP en empêchant l’infection des cellules B adjacentes.

Étude de l’infection lytique du Virus Epstein-Barr dans le développement de tumeurs post-greffe

Le virus Epstein-Barr (VEB) est un pathogène opportuniste qui a la capacité d’immortaliser les lymphocytes B et de provoquer une prolifération maligne, appelée syndrome lymphoprolifératif post-transplantation (SLP), chez les individus immunodéprimés. A l’intérieur de ce groupe, les personnes à plus haut risque sont les enfants, puisqu’ils sont à risque de développer une infection primaire par le VEB pendant leur régime d’immunosuppression post-greffe. Dans le but de développer un anticorps préventif, notre laboratoire s’est attardé au rôle du cycle lytique du VEB dans le développement du SLP. À cette fin, le premier objectif du présent projet vise à fournir la preuve expérimentale de l’existence ou non d’une phase réplicative productive pendant l’infection aiguë des lymphocytes B sanguins. Un examen des événements qui se déroulent au tout début de l’infection par le VEB tant au niveau de la réplication virale qu’au niveau de l’expression des gènes lytiques précoces et tardifs a révélé l’existence d’une phase réplicative productive pendant l’infection aiguë. Ceci a permis de justifier l’élaboration, dans notre laboratoire, d’un anticorps chimère (murin-humain) neutralisant, dirigé contre la protéine gp350 située sur l’enveloppe virale. Le deuxième objectif, quant à lui, vise à fournir la preuve expérimentale de la capacité neutralisante de cet anticorps chimère. Des essais de caractérisation in vitro ont démontré une capacité de reconnaissance de la protéine cible, notamment la gp350, et une capacité de neutralisation du virus par l’anticorps chimère. L’anticorps chimère anti-gp350 pourra faire l’objet d’essais précliniques in vivo en vue d’évaluer sa capacité à reconnaître le virus et à prévenir l’apparition de tumeurs de type SLP chez les souris SCID. Il pourrait être éventuellement utilisé, par la suite, comme traitement préemptif contre les tumeurs dans l’espoir de mieux gérer les patients à risque de développer un SLP.

Étude de l'EEG quantifié en éveil et en sommeil chez des adolescents présentant un trouble anxieux

Les troubles anxieux sont parmi les troubles psychiatriques les plus souvent diagnostiqués chez les adolescents. Ces troubles sont souvent accompagnés de nombreuses comorbidités, dont des difficultés de sommeil. L’objectif principal de cette thèse est de caractériser l’activité corticale à l’éveil et pendant le sommeil à l’aide de l’EEG quantifié chez une population d’adolescents présentant un trouble anxieux, et de la comparer à un groupe témoin d’adolescents. Dans un second temps, on cherche à savoir si l’activité EEG des patients anxieux corrèle avec différentes mesures cliniques. Deux études permettent de répondre à ces objectifs, une première portant sur l’activité EEG au cours de l’éveil, et une seconde portant sur l’activité EEG au cours du sommeil (SL et SP). La première étude démontre que l’activité EEG des deux groupes ne présente pas de différence à l’EEG le soir. Par contre, le matin, les patients anxieux présentent une activité significativement supérieure à celle des contrôles aux électrodes centrales (0,75-10 Hz et 13-20 Hz) ainsi qu’aux électrodes occipitales (2,5-7,75 Hz). Dans la seconde étude, nous avons analysé l’activité EEG absolue et relative en SL et en SP. Nous avons trouvé une activité absolue significativement supérieure à l’EEG de la région centrale chez les participants du groupe anxieux : en SLP (stades 3 et 4) sur l’ensemble des bandes de fréquence, en stade 2 sur les bandes de fréquence thêta, alpha et beta seulement. Finalement, en SP, les différences sont trouvées en alpha et beta, et non en thêta et delta. Les résultats obtenus à ces deux études suggèrent la présence de mécanismes de synchronisation et de filtrage inadéquats au niveau de la boucle thalamo-corticale, entraînant une hypervigilance du SNC. Quant aux corrélations entre l’activité EEG et les mesures cliniques, les résultats obtenus dans les deux études révèlent que les fréquences lentes (thêta et delta) de l’activité d’éveil le matin corrèlent à la fois avec l’anxiété de trait et d’état et les fréquences rapides (Alpha et Beta) de l’EEG du sommeil corrèlent sélectivement avec l’anxiété d’état. Il semble donc exister un lien entre les mesures cliniques et l’activité EEG. Une hausse d’activité EEG pourrait être un indicateur de la sévérité accrue des symptômes anxieux.