Notre laboratoire a développé un prototype de vaccin unique contre le VIH-1 / SIDA. C'est un un lentivecteur ADN non-intégratif qui a été testé dans une étude pilote utilisant des modèles animaux. L'étude a montré la protection de tous les macaques (6/6) vaccinés et la réponse était composée de cellules effectrices (EM) et des cellules T mémoire centrale (CM). Plus important encore, elle contenait également des cellules antigène spécifique à haute capacité de prolifération contenant des cellules T mémoire de type cellule souche (TSCM). Durant le travail de cette thèse, le génome vaccinal a été encore amélioré en commutant son enveloppe dotée de tropisme CXCR4 contre des enveloppes à tropisme CCR5 de virus de clade B (WARO) obtenu à partir d'un patient infecté de façon chronique et de trois souches de VIH-1 de Clade C transmetteur foundateur (T/F) de patients Zambiens. Une deuxième amélioration du vaccin a été réalisée en modifiant le génome afin qu’il puisse incorporer des adjuvants moléculaires capables d'améliorer d’avantage son immunogénicité.Etant donné que le lentivirus humain VIH-1 a développé plusieurs stratégies complexes pour persister, l’autre partie de la thèse a été consacrée à développer un outil pour comprendre la latence dans les cellules T CD4 + de la mémoire infectée. Les cellules latentes ont des génomes d'ADN viral intégrés non exprimés. Un des principaux mécanismes de cette latence est l'absence de transactivation du promoteur LTR par Tat. Les développements récents de la thérapie antivirale hautement active (HAART) efficace pour contrôler les cellules infectées circulantes et dans les tissus reste inefficaces contre les cellules du réservoir composé de cellules infectées latentes. Un des obstacles pour ce type d'études est l'absence de prototypes de lentivirus de primates appropriés incapables de d’effectuer la latence pour s’en servir comme modèle d'infection extrême dans l'évaluation. Nous avons émis l'hypothèse qu'un génome SHIV réplicatifdont l’expression est sous le contrôle de LTR du CAEV, Tat-indépendant doté de promoteur constitutif constituera un outil précieux pour de telles études. Nous avons conçu des LTRs chimères de CAEV portant les séquences d'attachement de celles du SIV à leurs extrémités et nous les ont utilisés pour contrôler l’expression du génome complet de SHIV-KU2. La construction résultante est SHIV-YCC qui devrait générer un virus qui ne n’effectue pas de latence en absence de Tat. Nous avons observé que les cellules transfectées avec le génome SHIV-YCC produisent des protéines SHIV qui s’assemblent en particules infectieuses excrétées des cellules. Les virions sont capables d'infecter les lymphocytes T CD4 + cibles tant dans les PBMC primaires que dans les lignées cellulaires. Le passage en série du virus dans les PBMC de macaques augmente la réplication et l'infectiosité du virus. SHIV-YCC est le premier lentivirus chimérique réplicatif de primates qui exprime de manière constitutive toutes les protéines virales. Ce nouveau modèle offre la possibilité d'étudier les événements précoces par lesquels le provirus subit une latence, en particulier lorsque le gène de l'enveloppe sera remplacé par celui du T / F CCR5 tropique VIH-1. / Our lab has previously described the generation of a unique vaccine prototype against HIV-1/AIDS. It is a non-integrative DNA lentivector vaccine tested in pilot studies in animal models of HIV vaccine. The non-human primate study showed protection of all 6/6 macaques and immune response correlates were composed of a variety of effector (EM) and central memory (CM) T cells. More importantly, they also contained high proliferating antigen specific cells containing a type of stem cell-like memory T cells (TSCM). In this thesis the vaccine was enhanced further by switching the CXCR4 envelope of the vaccine to CCR5 tropic envelopes such as the clade B WARO obtained from a chronically infected patient and a series of three transmitted/founder (T/F) HIV Clade C strains from Zambia. To improve further the vaccine we developed new strategies to incorporate molecular adjuvants able to enhance and sustain the newly elicited immune responses.Since the human lentivirus HIV-1 has developed multiple complex strategies to persist, the focus of the next part of my thesis was to develop a tool to ease and better understand the underlying mechanisms of latency in infected memory CD4+ T cells. Latently-infected cells have non-expressed integrated viral DNA genomes. One of the main mechanisms of this latency is absence of Tat transactivation of the LTR promoter. The recent focus post development of efficient highly active antiviral therapy (HAART), is the cure of the reservoir of latently infected cells. One of the obstacles for this type of studies is the lack of proper primate lentivirus prototypes incapable of undergoing latency as extreme infection model in the evaluation. We hypothesized that a replication-competent SHIV genome driven by the Tat-independent constitutive-expression LTRs of CAEV will be a valuable tool for such studies. We designed chimeric CAEV LTRs bearing the attachment sequences of SIV at their extremities and used them to drive the complete genome of SHIV-KU2. The resulting construct is SHIV-YCC which is expected to generate virus that will not undergo latency due to absence of Tat. We found that cells transfected with SHIV-YCC genome produce SHIV proteins that are assembled into infectious particles released out of the cells. Virions are able to infect target CD4+ T cells both in primary PBMCs and cell lines. Passaged virus in macaques PBMCs increased virus replication and infectivity. SHIV-YCC is the first chimeric primate replication-competent lentivirus that constitutively expresses all viral proteins. This new model offers the possibility of studying the early events by which provirus undergoes latency particularly when the envelop gene will be replaced with that of the T/F CCR5 tropic HIV-1.

Standardsystem är ett allt mer förekommande begrepp i dagens moderna verksamheter. Dessa system möjliggör en effektivisering av verksamheten vilket leder till att den blir mer konkurrenskraftig på marknaden. Dock finns det en del problem att lösa vid anpassningen av standardsystem. Eftersom det på marknaden idag finns ett stort utbud av olika standardsystem är det svårt för kunden att välja rätt system för dennes verksamhet. En av riskerna med att anskaffa ett standardsystem är att bunden betalar alldeles för höga summor för onödiga funktioner eller att systemet de anskaffat inte tillfredställer de funktioner företaget eftersträvar. Därför måste grova verksamhetsanalyser utföras innan anskaffningen. Detta för att avgöra om standardsystemet i fråga uppfyller verksamhetskraven samt att det kan anpassas till de delar den inte tillfredställer. Till hjälp finns det kompletta metoder för anskaffningen av standardsystem. En av dessa är SIV-metoden, den är en komplett metod för anskaffning av standardsystem. Efter genomförda val av system är det viktigt att kunna utföra anpassningar av det valda systemet och utifrån den aspekten härstammar idén för denna uppsats. Anpassningen av standardsystem är enligt oss ett outforskat ämne och kräver därför en mer djupgående forskning för att hitta och knyta de delar typiska för denna process. Forskningen i denna uppsats omfattar de delar där anpassningen av standardsystem behandlas. Dessa delar har vi lyft fram från befintliga metoder där anpassnings delen bara är en del av det hela. / A common term in today’s modern businesses is Software packaged system. These system’s are used to make the business operations more efficient thus the enterprises more competitive on the marketplace. But there are still issues regarding the adaptation of these system’s to the specific enterprise that must be resolved. Since the supply of software packages is very high, it is hard for the consumer to find the right kind of system that will fully satisfy company’s requirements. One of the major risks with acquiring these software packaged systems is that the customer pays too much money for a system with functions that are not necessary for the customer business needs. In order avoid this; the customer must make thorough analysis of their business operations before buying and implementing these kinds of systems. There are many obtainable methods that the customer can apply. In this thesis, we have accounted for two of these methods, namely, the SIV-method and the VFS-model. These methods describe different steps that the customer must consider before implementing a software packaged system. One of the significant steps in these methods is the adaptation process of software packaged systems. A matter we believe is unexplored and therefore further research is required on the subject.

BACKGROUND: Natural killer (NK) cells are traditionally considered part of the innate immune system but have recently been shown to possess adaptive qualities similar to T cells in response to an infection with a pathogen. In addition to possessing adaptive features, NK cells have also been found to reside in different organs such as the liver, spleen, and lymph nodes and differ based on phenotypic markers and their responses to different cytokines. Utilizing these findings, several groups have isolated and identified CD49a as a marker for tissue-resident NK cells. In the liver, CD49a has also been shown to be a positive indicator for NK cell memory-like responses in murine models. Building off work that demonstrated antigen-specific responses in rhesus macaques, this project focuses on characterizing the phenotypic markers and functional profile of CD49a+ NK cells in non-human primates. To better understand the role of CD49a in memory-like NK cells outside of the liver, this project utilized spleen samples from rhesus macaques infected with SIV/SHIV. This work aims to help us better understand the dysfunction NK cells experience as a result of HIV-1 infection in humans and also to demonstrate the changes NK cells experience as the disease progresses. A thorough understanding of the adaptive capabilities of NK cells can pave the way for targeted therapies to increase NK cell antiviral activity in HIV and other infections. METHODS: To characterize the functional and phenotypic profiles of CD49a+ NK cells by multiparameter flow cytometry, thirteen samples of spleen from rhesus macaques were thawed and then stained with two different protocols. A phenotyping protocol involved staining with antibodies against surface markers as well as intracellular markers T-Bet and Eomes. For the functional characterization protocol, the same thirteen samples were stained intracellularly after being stimulated with a cocktail of PMA and ionomyocin. The antibodies used in this were for functional markers. Of the thirteen samples used, six were infected with SHIVSF162P3, two were infected with SIVmac239X, and the remaining five were uninfected. After staining, these samples were analyzed on an BD LSRII from BD Biosciences. The data obtained were then analyzed using FlowJo software to study NK cells, which were characterized as CD45+CD14-CD20-CD3-CD159+. RESULTS: The analysis compared NK cells with T cells, B cells, and NKB cells. Some increases were seen among CD49a+ NK cells in the frequency of CD336+ (NCR2/NKp44), CD337+ (NKp30), and CD366+ (Tim-3) after infection. Although there were some mild increases in CD107a and TNF- in infected samples compared to uninfected, a significant increase was observed in the frequency of IFN-ɣ among infected CD49a+ NK cells compared to uninfected. CONCLUSION: When comparing samples that were infected vs uninfected, it appears there were some mild decreases after infection in the ratio of NK cells to other lymphocytes. In addition, there did not appear to be a significant increase in the frequency of CD49a+ among these NK cells as a result of the infection. However, among the CD49a+ subpopulation, there were some observed non-significant decreases in CD56-CD16+ cells. Furthermore, there was found to be an almost significant increase in TNF- (p = 0.06) among CD49a+ cells after infection. These findings demonstrate an increase in cytotoxic activity in splenic NK cells associated with an adaptation to the virus. Although there does not appear to be significant changes in the ratio of NK cell populations in the spleen, the changes observed in phenotypic and functional markers associated with CD49a+ demonstrate an increase in the cytotoxic activity of NK cells as a result of infection with SIV/SHIV. However, it remains to be seen if CD49a is a direct indicator of this type of infection. Future work geared toward memory-like NK cells in non-human primate splenic tissue could look at the contrast in CD49a+ NK cells from different states of infection with HIV-1 and/or SIV (acute vs chronic) to better understand the integrin’s role in adaptation.

Thesis advisor: Tricia H. Burdo / Human immunodeficiency virus-associated peripheral neuropathy (HIVPN) continues to be a prevalent comorbidity of HIV infection, despite virologic control due to effective antiretroviral therapy (ART). Symptoms include bilateral tingling, numbness, and pain in distal extremities. Severity of symptoms is associated with a loss of intraepidermal nerve fiber density (IENFD) in the feet. Damage to the dorsal root ganglia (DRG) has also been observed in postmortem tissue analysis from patients with HIV-PN. Treatment options are limited due to a lack of understanding of the disease pathogenesis. Chronic monocyte activation and accumulation of macrophages in peripheral nervous system (PNS) tissues has been reported but few studies have directly demonstrated the role of monocyte/macrophage activation and traffic in the pathogenesis of HIV-PN. The central hypothesis of this thesis is that monocyte activation and traffic mediates PNS neuronal damage. We addressed this hypothesis in several ways. In chapter 2, we describe pathology seen in a rapid disease progression animal model of HIV-PN. We found that an early loss of IENFD preceded a loss of small diameter DRG neurons. In chapter 3, we associated DRG pathology with an accumulation of inflammatory macrophages surrounding DRG neurons. Increased monocyte traffic to the DRG was associated with severity of DRG pathology and with a loss of IENFD. In chapter 4, we directly tested the impact of monocyte traffic on DRG pathology by blocking leukocyte traffic with an anti-VLA-4 antibody, natalizumab. Blocking cell traffic reduced accumulation of macrophages in the DRG and improved pathology. Next we treated animals with methylglyoxal-bisguanylhydrazone (MGBG) to specifically target myeloid cells and reduce their activation. MGBG treatment improved DRG pathology and reduced accumulation of macrophages in tissues. Having demonstrated the role of monocyte traffic and activation, we aimed to identify signaling proteins and inflammatory proteins associated with PNS pathology. We found elevated monocyte chemoattractants in DRG tissue and elevated markers of monocyte activation in plasma that were associated with a loss of IENFD. Together, these studies demonstrate that systemic monocyte activation, macrophage accumulation in DRG tissue, and monocyte traffic plays a major role in SIV-PN pathogenesis. These studies provide novel insight into immune mechanisms that impact neuronal loss during SIV infection. Thus, modulating macrophage activation and reducing monocyte traffic may have therapeutic benefits to patients suffering from or at risk of developing HIV-PN. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21022号 / 医博第4368号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 朝長 啓造, 教授 萩原 正敏, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Current SME performance models suffer from a number of disadvantages. The models use intensively a business ratio approach, they look at SMEs as a homogenous group, they consider firms to be closed systems, they do not directly incorporate the impact of an enterprise’s innovation activities, and finally they are complex and rely on sophisticated statistical refining methods making them unpractical to use by SME managers. There are four major challenges when one tries to build SME performance models that lack these deficiencies. The first challenge is that the desired performance evaluation model must optimally incorporate both quantitative and qualitative input. The second challenge is that the model must incorporate non-financial input parameters, such as firm size and age (among others), in the performance evaluation models. The third is that the model must consider the variety of SMEs as concerns their business sectors, nationalities, sizes, and ages. The final challenge is that the model must be able to utilize existing limited information available from the SMEs bookkeeping practices in an optimal way. The thesis addresses three questions related to constructing a better SMEperformance model, namely (1) What are the advantages and disadvantages of the existing models used in evaluating SME performance? (2) What characterizes a comprehensive model for measuring SME performance with acknowledgement of the firm’s innovation activities? (3) How can a firm’s innovation activities be enhanced in relation to the firm’s external environment? To construct a model that copes with these challenges, I used a literature-based selection of parameters as well as a theory-based selection. I used both a conceptual approach and an empirical approach to discuss and propose a model, the Survival Index Value (or SIV) model, as an alternative to the existing performance models for SMEs. The major contributions of this thesis to the field of SME performance can be summarized in three outcomes: the SIV model as a new model of SME performance evaluation, the ASPEM as a new tool for strategic utilization of SME performance models, and a new approach to account for innovation in relation to the external environment of the firm using the IBAM tool. The work adds to the theory of the firm, as it presents a new way of evaluating firm performance. It also contributes to bridging the theory of the firm to organizational theory, by elevating the significance of networking and its impact on SME efficiency.

<p>Det här examensarbetet handlar om skrivundervisning. Undersökningen är huvudsakligen en fallstudie. Syftet är att få ökade kunskaper om en gymnasielärares arbetssätt för att utveckla sina elevers skrivande. Den huvudsakliga forskningsfrågan i detta arbete är: Hur beskriver en erfaren gymnasielärare sin skrivundervisning för att öka sina elevers skrivförmåga inom svenskundervisningen i gymnasiet?</p><p>Examensarbetet börjar i en teoretisk utgångspunkt med tre olika teorier om skrivande och skrivundervisning. Sedan fortsätter det med den empiriska delen som är en djupintervju med en lärare vid namn Emmy (fiktivt namn) på en gymnasieskola i Småland. I resultaten, analysen och diskussionen i examensarbetet visas det hur en lärare har försökt att svara på frågeställningen ovan.</p><p>Den huvudsakliga slutsatsen är att läraren är flexibel och att hon har ett antal olika metoder att arbeta med när hon försöker öka sina elevers skrivförmåga i sin dagliga undervisning. Den här fallstudien visar också något av hennes syn på skrivundervisning inom svenskämnet på gymnasiet. Emmys teoretiska grundsyn ligger nära skrivprocessteorin som representeras av den svenska forskaren Siv Strömquist.</p><p>Det är omöjligt att dra några generella slutsatser om alla lärare bara på basis av en enda fallstudie. En försiktig tanke som dock uppstår i diskussionen är att det kan vara svårt att vara lärare, om man tittar på och lever sig in i Emmys bild av sin skrivundervisning. Några av hennes ledstjärnor är värdegrunden, den lokala kursplanen, skrivprocessteorin, en bra lärare–elev-relation, mycket läsning, textanalys, bra skrivuppgifter, tydliga uppgiftsinstruktioner, mönsterexempel, mycket skrivträning, nyttig textrespons, infärgning, goda råd till eleverna samt sist men inte minst humor och lust i skrivundervisningen. Allt detta ska leda henne framåt för att öka skrivförmågan hos eleverna.</p>

Atomic defects in solids offer access to atom-like quantum properties without complex trapping methods while displaying a rich physics due to interactions with their solid-state environment. Such properties have made them an advantageous building block for quantum information processing, in particular to construct a quantum network, where information would be encoded in spins and transferred between nodes through photons. Among defects in solids, the negatively charged silicon-vacancy centre in diamond (SiV$^{−}$) has attracted attention for its very promising optical properties for such a network. In this thesis, we investigate the spin properties of the silicon-vacancy centre as a potential spin-photon interface. First, we use resonant excitation of an SiV$^{−}$ centre in an external magnetic field to selectively address different electronic states and analyse the resulting fluorescence. We find evidence of selection rules in the optical transitions revealing that the centre possesses an electronic spin S = 1/2. Making use of the dependence of such selection rules on the applied magnetic field orientation, we resonantly drive two optical transitions forming a $\Lambda$-scheme. In the double resonance condition, we achieve coherent population trapping, whereby the SiV$^{−}$ is pumped into a dark state corresponding to a superposition of the two addressed ground states of opposite spin. This technique allows us to evaluate the coherence time of the dark state and hence of the spin, while demonstrating the possibility of all-optical control of the spin when a $\Lambda$-scheme is available. We then use resonant optical pulses to initialise and read out the spin state of a single SiV$^{−}$. By tuning a microwave pulse into resonance between two ground states of opposite spin, we demonstrate optically detected magnetic resonance. Subsequently, by varying the duration of a resonant microwave pulse, we achieve coherent control of a single SiV$^{−}$ electronic spin. Through Ramsey interferometry, we measure a spin dephasing time of 115 $\pm$ 9 ns. We then investigate interactions of the SiV$^{−}$ with its environment. We analyse the hyperfine interaction of the SiV$^{−}$ spin with the nuclear spin of $^{29}$Si, with a view to taking advantage of the long-lived nuclear spin in the future. We show that single-phonon-mediated excitations between electronic states of the SiV$^{−}$ are the dominant spin dephasing and population decay mechanism and evaluate how external strain alters optical selection rules and can be used to improve the coherence time of the spin.