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O desenvolvimento de novas ferramentas para manipulação genética é necessário para um melhor entendimento da biologia de protozoários que causam doenças sérias e negligenciadas. Neste contexto, apresentamos uma nova aplicação do sistema Cre recombinase em Leishmania major, utilizado para a expressão condicional de genes de interesse. Como prova de conceito demonstramos por ensaios de PCR, western blotting e imunofluorescência que o gene da Proteína Fluorescente Verde (Green Fluorescent Protein, GFP) é condicionalmente expresso em função do tempo e dose de rapamicina necessários para a ativação da recombinase. A aplicação do sistema diCre em L. major é feita com o estudo da proteína Rad9, que participa na sinalização e resposta a danos ao DNA. A Rad9 de L. major possui um domínio C-terminal desestruturado, que no homólogo humano não é essencial para a formação do complexo 911 (Rad9-Hus1-Rad1), mas possui sítios de fosforilação importantes para a cascata de sinalização que mantém a integridade do DNA. Usando predições da estrutura de Rad9, a proteína foi dividida em domínios e foram geradas linhagens que expressam, condicionalmente, versões truncadas de Rad9. Por análises de PCR, western blotting, citometria de fluxo e imunofluorescência, acessamos alguns dos papeis que o domínio C-terminal de Rad9 pode desempenhar em L. major. / The development of new tools for genetic manipulation is necessary for a better understanding of the biology of protozoa that cause severe and neglected diseases. In this context, we present a new application of the Cre recombinase system in Leishmania major, using it for the conditional expression of genes of interest. As proof of concept we show by PCR, western blotting and immunofluorescence assays that the Green Fluorescent Protein (GFP) gene can be conditionally expressed depending on the time and dose of the rapamycin treatment required for the recombinase activation. The application of the diCre system in L. major was tested with the study of the Rad9 protein, which participates in the parasite\'s DNA damage response. Rad9 from L. major presents an unstructured C-terminal domain, which in the human homolog is not essential for the 911 (Rad9-Hus1-Rad1) complex formation, but has phosphorylation sites that are important in the signaling cascade that maintains the DNA integrity. Using structure predictions of Rad9, the protein was divided into specific domains and cell lines were generated conditionally expressing truncated versions of Rad9. By PCR, western blotting, flow cytometry and immunofluorescence assays, we assessed some of the roles that the C-terminal domain of Rad9 can perform in L. major.

O desenvolvimento de novas ferramentas para manipulação genética é necessário para um melhor entendimento da biologia de protozoários que causam doenças sérias e negligenciadas. Neste contexto, apresentamos uma nova aplicação do sistema Cre recombinase em Leishmania major, utilizado para a expressão condicional de genes de interesse. Como prova de conceito demonstramos por ensaios de PCR, western blotting e imunofluorescência que o gene da Proteína Fluorescente Verde (Green Fluorescent Protein, GFP) é condicionalmente expresso em função do tempo e dose de rapamicina necessários para a ativação da recombinase. A aplicação do sistema diCre em L. major é feita com o estudo da proteína Rad9, que participa na sinalização e resposta a danos ao DNA. A Rad9 de L. major possui um domínio C-terminal desestruturado, que no homólogo humano não é essencial para a formação do complexo 911 (Rad9-Hus1-Rad1), mas possui sítios de fosforilação importantes para a cascata de sinalização que mantém a integridade do DNA. Usando predições da estrutura de Rad9, a proteína foi dividida em domínios e foram geradas linhagens que expressam, condicionalmente, versões truncadas de Rad9. Por análises de PCR, western blotting, citometria de fluxo e imunofluorescência, acessamos alguns dos papeis que o domínio C-terminal de Rad9 pode desempenhar em L. major. / The development of new tools for genetic manipulation is necessary for a better understanding of the biology of protozoa that cause severe and neglected diseases. In this context, we present a new application of the Cre recombinase system in Leishmania major, using it for the conditional expression of genes of interest. As proof of concept we show by PCR, western blotting and immunofluorescence assays that the Green Fluorescent Protein (GFP) gene can be conditionally expressed depending on the time and dose of the rapamycin treatment required for the recombinase activation. The application of the diCre system in L. major was tested with the study of the Rad9 protein, which participates in the parasite\'s DNA damage response. Rad9 from L. major presents an unstructured C-terminal domain, which in the human homolog is not essential for the 911 (Rad9-Hus1-Rad1) complex formation, but has phosphorylation sites that are important in the signaling cascade that maintains the DNA integrity. Using structure predictions of Rad9, the protein was divided into specific domains and cell lines were generated conditionally expressing truncated versions of Rad9. By PCR, western blotting, flow cytometry and immunofluorescence assays, we assessed some of the roles that the C-terminal domain of Rad9 can perform in L. major.

Ulm, Univ., Diss., 2007.

No description available.

Transmissible spongiform encephalopathies (TSEs), such as scrapie and variant Creutzfeldt-Jakob disease are infectious, fatal, neurodegenerative diseases. Following peripheral infection TSE agents usually accumulate in lymhoid tissues before spreading to the central nervous system. In mice, follicular dendritic cells (FDCs) expressing the host prion protein (PrPC) are essential for scrapie agent accumulation in lymphoid tissues. The accumulation of the scrapie agent on FDCs is critical for the efficient spread of infection to the brain. However, it is unknown whether FDCs themselves actively replicate the scrapie agent, or simply accumulate it following production by other cells types such as neurones, lymphocytes or other stromal cell populations. To definitively address this issue a transgenic mouse model was created in which PrPC is switched on or off exclusively on FDCs. Expression of cre-recombinase (Cre) under the action of cell-specific gene promoters can be used to induce or delete the expression of a target gene in specific cell populations. In this model, Cre expression is driven by the complement receptor type 2 gene (Cr2/CD21) which is expressed by FDCs and mature B lymphocytes. Characterisation of the CD21-cre mouse line was achieved by crossing with a ROSA26 reporter strain. The CD21-cre mouse line was subsequently crossed with floxed-PrP mouse lines to produce compound transgenic mouse lines in which PrPC expression was switched on or off, only in FDCs. Cre expression by B lymphocytes was eliminated by γ-irradiation and grafting recipient mice with Cre-deficient bone marrow. Immunohistochemical analysis confirmed the expression PrPC had been switched on or off exclusively on FDCs. Subsequently, the mice were challenged with scrapie by intra-peritoneal injection to determine the precise role of FDCs in the accumulation of scrapie in lymphoid tissues. Switching off PrPC expression exclusively on FDCs prevented the accumulation of TSE agent specific disease-associated PrPSc in the spleen after i.p inoculation. Conversely, in mice in which PrPC was expressed only on FDC, successful replication of the agent occurred on the FDC network in the spleen. Taken together, these data show PrPC-expressing FDCs alone are sufficient to support the accumulation of the scrapie agent within lymphoid tissues. Furthermore, these data suggest FDC replicate the TSE agent and do not simply accumulate it following synthesis by other cell types.

Dans le système nerveux central, les monoamines modulent de nombreuses fonctions essentielles comme la locomotion, la motivation, la cognition, l’humeur et le sommeil. Le niveau extracellulaire de ces neurotransmetteurs est régulé par des transporteurs à haute affinité,cependant d’autres transporteurs, à faible affinité, peuvent contribuer à la recapture des monoamines, comme les transporteurs de cations organiques (OCT) et le transporteur plasmique des monoamines (PMAT). Récemment, l’implication des OCT dans différentes fonctions centrales, notamment le contrôle de l’humeur, la réponse au stress et aux antidépresseurs a été mise en évidence. Le rôle de PMAT dans le cerveau reste quant à lui encore peu caractérisé. Il transporte in vitro les monoamines, avec une préférence pour la dopamine et la sérotonine, avec des affinités submillimolaires. Ce transporteur est exprimé dans de nombreuses régions du cerveau humain et murin et dans différents types neuronaux. Par hybridation in situ fluorescente nous avons déterminé sa distribution cellulaire précise, dans des régions à fort niveau d’expression comme le complexe du cerveau antérieur basal (BFC) et des régions appartenant aux ganglions de la base comme le globus pallidus et la substance noire réticulée (SNr). Nous avons montré qu’il est fortement exprimé dans les neurones GABAergiques exprimant la parvalbumine, dans tous les interneurones cholinergiques dustriatum ainsi qu’une petite fraction des neurones cholinergiques du BFC. Il est également retrouvé dans certains noyaux monoaminergiques comme le locus coeruleus et les noyaux duraphé mais est absent des noyaux dopaminergiques, la substance noire compacte et l’aire tegmentale ventrale.Afin d’étudier sa fonction, nous avons exploité le système Cre-lox, approche couramment utilisée en biologie, en injectant un virus adéno-associé exprimant la recombinase Cre (AAVCre)dans la substance noire (SN) de souris comportant des allèles de PMAT floxés. Cette étude ne nous a pas permis de conclure quant à la fonction de PMAT dans la SN, mais nous a conduit à mettre en évidence une toxicité majeure de cet outil. Nous avons montré que l’injection d’AAV-Cre dans la SN entraine une perturbation anatomique et fonctionnelle des systèmes dopaminergiques et de la SNr, noyau de sortie des ganglions de la base, induisant des altérations comportementales importantes, avec une hyperlocomotion basale robuste et une insensibilité à la cocaïne, potentiellement par une action génotoxique.Nous avons également généré des souris invalidées constitutivement pour PMAT (PMAT-/-). Les tests comportementaux que nous avons commencés récemment nous ont révélé des altérations comportementales significatives chez ces souris de l’activité locomotrice dans un nouvel environnement ainsi que du niveau d’anxiété. Ces altérations pourraient résulter d'une perturbation des voies aminergiques en l’absence de PMAT. Nous poursuivrons cette étude par l'exploration d'autres aspects comportementaux ainsi que par l’évaluation des modifications neurochimiques engendrées par l'invalidation. Ces approches devraient fournir des pistes afin d’identifier les conséquences de l'absence de PMAT sur la signalisation aminergique, que l'on pourra explorer plus précisément par la suite sur le plan fonctionnel / High-affinity reuptake transporters exert a crucial role in the control of synaptic transmissionby ensuring the recycling of the released transmitters into the presynaptic terminals. Other typesof transporters such as Organic Cation Transporters (OCTs) and the Plasma MembranemonoAmine Transporter (PMAT), have been shown to transport, with low-affinity but highcapacity, aminergic neurotransmitters. While the role of OCTs in central nervous system hasbeen partially unraveled, the function of PMAT remains poorly characterized. In vitro, PMATtransports preferentially dopamine and serotonin and its expression is widespread in the brain,encompassing monoamine nuclei but also projection regions. In this study, we determined theprecise neuronal specificity of PMAT in several highly-expressing regions. We show that it isfound mostly in PV+ GABAergic neurons of basal forebrain and basal ganglia, in allcholinergic interneurons of the striatum and in some cholinergic neurons of basal forebraincomplex. These systems, highly regulated by monoamines, are important for locomotion,motivation, learning and wakefulness. Our result show that PMAT is located at a strategicposition to control the aminergic modulation of these integrated functions.To investigate the implication of PMAT in these regions, we used the Cre-lox technology, avalued and widely used approach for the study of gene function in vivo, injecting an adenoassociatedvirus expressing Cre recombinase in substantia nigra (SN) of mice in which PMATgene was floxed. In this study, we could not assess PMAT function in this SN but found thatAAV-CRE expression in this region produces major toxic effects. We showed that AAV-Creinjection in this region engenders a massive decrease of neuronal populations in both parscompacta and reticulata, leading to DA depletion in the nigrostriatal pathway. This wasassociated with a drastic behavioral phenotype with increased basal locomotor activity and lossof locomotor response to cocaine. Several hallmarks of Cre toxicity were found in SN of AAVCreinjected mice, including an increase of the DNA break markers. These observationsunderscore the need for careful control of Cre toxicity in the brain and reassessment of previous studies.To study the role of PMAT, we also generated PMAT knock out mice (PMAT-/-). Behavioralstudies that we just started have revealed significant impairments of locomotor activity in a newenvironment and anxiety level, supporting a possible disruption of monoaminergic systems inthese mice. On-going studies aim to explore other behaviors and search for eventualneurochemical changes provoked by PMAT invalidation. These experiments should providesome cues to understand which monoamines and circuits may be affected, that can beinvestigated functionnally and more specifically in a second step

Uppsatsens titel: Kommer den kommersiella fastighetsvärderingsprocessen automatiseras? En studie i uppfattningarna hos verksamma aktörer inom den svenska kommersiella fastighetsbranschen. Författare: Victoria Fristedt Handledare: Peter Palm Problemformulering: Digitaliseringen av den kommersiella fastighetvärderingsprocessen är på framfart. Genom allt snabbare utvecklad teknologi uppstår nya möjligheter att implementera effektiviserande tekniska lösningar. Det verkar kunna bli tekniskt möjligt att gå från en manuell till en automatiserad värderingsprocess för kommersiella fastigheter. Ingen tidigare forskning har genomförts med syfte att undersöka vad verksamma inom den svenska värderingsbranschen förutspår för framtiden. Således förefaller det relevant att undersöka vilka uppfattningar angående automatisering som finns hos personer som är verksamma inom kommersiell fastighetsvärdering. Dessutom är det intressant att undersöka på vilket sätt teknologi kan effektivisera värderingsprocessen. Syfte: Studiens syfte är att undersöka vilka uppfattningar verksamma aktörer inom den kommersiella fastighetsvärderingsbranschen har rörande automatisering samt framväxande teknologi. I tillägg syftar studien till att undersöka vilka fördelar respektive nackdelar det kan medföra att gå från en manuell till en mer automatiserad värderingsprocess. Frågeställningar: - Vilka uppfattningar finns kring en automatiserad värderingsprocess hos verksamma aktörer inom den kommersiella fastighetsvärderingsbranschen? - Skulle värderingsprocessen kunna effektiviseras med hjälp av teknologi, och om så är fallet, hur? Metod: Studien har en kvalitativ ansats. Semistrukturerade intervjuer har genomförts med professionella som är verksamma inom olika delar av den kommersiella fastighetsvärderingsbranschen. Syftet med detta var att få ett så nyanserat resultat som möjligt. Respondenternas svar har blivit analyserade med avstamp i tidigare forskning för att nå rimliga slutsatser. Slutsatser: Fastighetsbranschens inställning till en automatiserad värderingsprocess är vad som kommer avgöra om processen blir automatiserad eller ej. Det verkar troligt att teknologin kommer att vara tillgänglig innan branschen är övertygad om att den är pålitlig nog. Nyckelord: Kommersiella fastigheter, Fastighetsvärdering, Automatisering, Digitalisering / Title: Will the Appraisal Process for Commercial Real Estate be Automated? A Study on the Perceptions Amongst Professionals Operating Within the Swedish CRE Industry Author: Victoria Fristedt Supervisor: Peter Palm Problem: The digitalization of the appraisal process for commercial real estate is emerging. With rapid development of technologies, the opportunities to implement new efficient solutions arises. There seems to be a possibility that the process of appraising CRE can transfer from manual to automated. It has not yet been researched what professionals operating within the CRE appraisal industry in Sweden predict about the future. Thus, a study that aims to research what the perceptions of implementing automated systems are, as well as looking into how the appraisal process can become more efficient with help from technology, can be considered relevant. Aim: The aim of the study is to investigate the perceptions of automation and emerging technologies amongst Swedish appraisers, system developers, banks and CRE owners. Furthermore, the aim is to investigate potential advantages and disadvantages of going from a manual appraisal process to a more automated one. Research questions: - What is the perception of automation amongst people operating in the CRE appraisal industry in Sweden? - Could technology make the appraisal process for CRE more efficient and if so, then how? Method: The study is performed with a qualitative approach. Semi-structured interviews have been performed with professionals operating within different parts of the CRE appraisal industry in order to achieve an as nuanced results as possible. The respondents’ answers have been analyzed along with what previous research has shown in order to reach reasonable conclusions. Conclusion: The mindset of the industry is likely to determine the future of the automated appraisal process for CRE. The technology is most likely to be accessible long before the industry is convinced of its trustworthiness. Keywords: Commercial Real Estate, Automation, Appraisal, Digitalization

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24782号 / 医博第4974号 / 新制||医||1066(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 濵﨑 洋子, 教授 湊谷 謙司, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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