京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21445号 / 医博第4412号 / 新制||医||1032(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 長船 健二, 教授 篠原 隆司, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21647号 / 医博第4453号 / 新制||医||1034(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 高橋 淳, 教授 藤渕 航, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13559号 / 論医博第2288号 / 新制||医||1067(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 髙折 晃史, 教授 西浦 博, 教授 遊佐 宏介 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Sedan 1960-talet har globala navigationssystem (GNSS) framgångsrikt kartlagt vår planet och möjliggjort en global positionering. Mognaden av GNSS tillsammans med utvecklandet av ny teknik och enheter har skapat en ökad efterfrågan av positionering inomhus. GNSS kräver fri sikt för att uppnå en godtycklig positionering, vilket inte är möjligt i inomhusmiljöer på grund av stora avstånd till satelliter samt byggnaders konstruktion som kraftigt försvagar signalerna. Därför behöver lokala system implementeras för att uppnå positionering i inomhusmiljöer.  Till skillnad mot GNSS är inomhuspositionering inte en mogen teknik. Varje implementering bör utvärderas utifrån kostnad, noggrannhet, skalbarhet, täckning och integritet. Tidigare studier om inomhuspositionering har sin tyngdpunkt i precision snarare än användningsområden. Ett användningsområde för inomhuspositioneringssystem är att observera nyttjande av lokalyta. För företag är kostnaden för kontorsyta en av de största utgiftsposterna. Samtidigt är det en växande kulturell skillnad i arbetskultur där anställda och företag använder sig av distansarbete i större utsträckning än tidigare, något som påskyndats av coronapandemin.  Därför är syftet med detta arbete att använda ett inomhuspositioneringssystem för att beräkna användningen av mötesrum samt identifiera lämpliga kriterier för beräkning av nyttjande. Studien har genomförts hos Sweco digital services i Stockholm och använt data från Sonys Nimway system som finns implementerat i byggnaden. På grund av coronapandemi som inneburit restriktioner har data från mars till april 2022 insamlats för studien.  Genom infraröda sensorer i varje rum upptäcks närvaro och används för beräkning av nyttjande utifrån formler från tidigare studier. Lämpliga kriterier för beräknande av nyttjande har identifierats som rummets kapacitet, placering och dess förhållande till solljus. Vidare har veckodag samt tid på arbetsdagen identifierats som lämpliga kriterier.  Resultatet visar en ineffektiv användning av mötesrum om dryga 30% för den totala perioden. På grund av ett begränsat data går det inte att identifiera några skillnader i nyttjandet utifrån de tidigare identifierade kriterierna bortsett en lägre nyttjandegrad under slutet av veckan. För vidare studier bör mer data insamlas över en längre tidsperiod. Vidare bör data insamlas från flera våningar för att ge en mer trovärdig bild av verkligheten. PIR tekniken är tillräcklig för ändamålet men bör kompletteras med möjligheten att beräkna antal personer i ett rum. Information om kapacitet kan användas för djupare analyser utifrån exempelvis ett säkerhetsperspektiv . / Since the 1960s, global navigation systems (GNSS), has successfully discovered our planet and enabled a technique for worldwide positioning. The development of new techniques and devices has shifted the demand to positioning indoors. GNSS need line of sight to achieve an arbitrary position, which is not possible indoor due to week signal strength. Therefore, a positioning system needs to be implemented locally to achieve positioning indoor.  Indoor positioning systems (IPS) can be used for more than navigation. Unlike GNSS, which is a mature technique, IPS need to be implemented based on its purpose, cost, precision, scalability, coverage, and integrity. Earlier studies focuses on precision rather than use cases. One use case for IPS is to compute the use of office space. According to studies, the cost of office space is one of the largest expenses for companies. Simultaneously there’s a cultural revolution where employees and employers are using teleworking in a greater extent. Accelerated by the COVID pandemic.  Therefore, the purpose of this theses is to compute occupancy of office space by using an IPS. Furthermore, identify criteria’s that can be used to compute occupancy. The study was conducted at Sweco Digital Services on floor 8 in Stockholm. Data has been collected during Mars to April by Sony’s IPS Nimway which is implemented in the building.  Infrared sensors in every room have detected presence which has been used to calculate occupancy. Suitable criterions have been identified as room capacity, location and location to sunlight. Also, weekday and time of the day has been identified as suitable criterions.  The results state an inefficient use of office space by 30% during the extent of the study. Except a lower occupancy during the end of the week, there are no criterion that differs from one another. Probably due to a limited set of data. Further studies should collect more data during an extensive amount of time. Data should also be collected on all floors of the building to provide a more credible picture of reality. Infrared is sufficient for the purpose of this study but should be complemented with the ability to count the number of people in each room. Information of capacity would make it possible to conduct more analysis in more areas such as safety.

No description available.

Activities of daily living (ADLs) are everyday routine tasks which provide insight into the physical and cognitive wellbeing of older adults. ADLs are commonly self-reported to clinicians, which can lead to overestimation and underestimation of a patients’ functional abilities. Remote health monitoring is an emerging field aimed at utilizing technology for monitoring ADLs remotely, improving clinical accuracy and enabling older adults to age safely within their homes. In this dissertation, we report a Smart Home platform and two indoor positioning systems (IPSs) – (i) a hybrid Bluetooth Low Energy (BLE) and radar motion sensor system and (ii) a hybrid radio-frequency identification (RFID) and infrared (IR) range-finding system for tracking occupant mobility, the primary predictor of falls among older adults. For the Smart Home platform, the design methodology and technological features were explained. As for the IPSs’, position accuracy of multiple occupants within multiple rooms of a residential apartment was evaluated. The systems were also evaluated for cost, implementation ease, and scalability, which, upon reviewing literature, were identified as key metrics for developing an IPS for enabling aging in place. Both IPSs enforced a decentralized localization architecture and performed well, achieving high localization accuracy for multiple occupants. / Thesis / Master of Applied Science (MASc) / By 2031, the number of people aged 65 and over is expected to nearly double. This population shift is concerning for healthcare providers as limited resources become increasingly constrained. Resultantly, older adults, the largest consumers of healthcare, face longer wait times and reduced quality of care. Remote health monitoring is an emerging field aimed at utilizing technology for monitoring older adults within their homes. In this thesis, we report a Smart Home platform and two indoor positioning systems (IPSs) for tracking resident mobility, the primary predictor of falls among older adults. For the Smart Home platform, the design methodology and technological features were explained. As for the IPSs’, position accuracy of multiple occupants within multiple rooms of a residential apartment was evaluated. Upon reviewing literature system cost, implementation ease, and scalability, were identified as key metrics for developing an IPS for enabling aging in place. Both IPSs performed well, achieving high localization accuracy for multiple occupants.

Les porphyries érythropoïétiques (PE) : Porphyrie Erythropoïétique Congénitale -PEC- et Protoporphyrie Erythropoïétique -PPE- sont caractérisées par le déficit d’une des enzymes de la voie de biosynthèse de l’hème. Le traitement curatif des formes sévères de PE est la transplantation de moelle osseuse allogénique (TMOA). La PPE est parfois compliquée d’une insuffisance hépatique majeure nécessitant une greffe hépatique. Dans un modèle murin de PPE (Fechm1Pas/Fechm1Pas), nous avons démontré l’apparition progressive de lésions hépatiques dès la 2ème semaine de vie. Une TMO précoce (nouveau-né) a permis de prévenir l’apparition de ces lésions hépatiques et de corriger la photosensibilité cutanée démontrant l’efficacité de cette approche thérapeutique pour les formes sévères de PPE. La thérapie génique par greffe de cellules souches hématopoïétiques autologues corrigées représente une alternative à la TMOA en l’absence de donneur HLA-compatible. Nous avons développé des cellules souches pluripotentes induites (iPS) à partir de cellules épidermiques issues de modèles murins de PE et d’un patient PEC. La correction génique a été obtenue par transfert du gène lentiviral (ferrochélatase ou uroporphyrinogène III synthase (UROS). La pluripotence des cellules iPS a été caractérisée in vitro par la formation de corps embryoïdes et in vivo par la formation de tératomes. In vitro, la correction métabolique a été obtenue après différenciation des cellules iPS humaines en progéniteurs hématopoïétiques. Enfin dans une dernière partie, nous nous sommes intéressés à une approche pharmacologique de la PEC. Nous avons montré que les mutations C73R et P248Q entraînaient une instabilité et une dégradation accélérée de l’UROS par la voie du protéasome. Le traitement de souris UrosP248Q par un inhibiteur du protéasome (Velcade®) a permis la correction de la photosensibilité cutanée. Ces travaux ouvrent de nouvelles perspectives pour le traitement des porphyries érythropoïétiques. / Erythropoietic porphyrias (EP) : Congenital Erythropoietic Porphyria -CEP- and Erythropoietic Protoporphyria -EPP-) are characterized by a deficit of one enzyme implicated in heme biosynthetic pathway. The curative therapy for severe cases of EP is an HLA-compatible Bone Marrow Transplantation (BMT). EPP is sometimes complicated by a major hepatic failure requiring hepatic graft. In a murine model of EPP (Fechm1Pas/Fechm1Pas), we have demonstrated that hepatic lesions progressively appear 2 weeks after birth. Early BMT (in neonates) has made it possible to prevent hepatic lesions and correct skin photosensitivity, demonstrating the efficiency of this therapeutic approach in severe cases of EPP. The gene therapy by graft of corrected autologous hematopoietic stem cells represents an alternative to BMT when HLA-compatible donors are lacking. We have developed induced pluripotent stem cells (iPSC) from epidermic cells of murine models of EP and of one PEC patient. The gene correction was obtained by lentiviral gene transfer (ferrochelatase and uroporphyrinogen III synthase -UROS). The pluripotency of iPSC was characterized in vitro by the formation of embryoid bodies and in vivo by the formation of teratomas. In vitro, the metabolic correction was obtained after differentiation of human IPSC into hematopoietic progenitors. In the last part of this thesis, we have focused on a pharmacological approach of CEP. We have shown that C73R and P248Q mutations lead to instability and accelerated degradation of the UROS protein via the proteasome. Treating UrosP248Q mice with a proteasome inhibitor (Velcade®) has allowed the correction of skin photosensitivity. These works offer new prospects for the treatment of erythropoietic porphyrias.

Biotecnologias reprodutivas como a produção in vitro de embriões e a transferência de núcleo apresentam grande potencial de aplicação na medicina veterinária seja para a correção de infertilidades, para o aumento na eficiência da produção animal ou mesmo para um melhor entendimento sobre os mecanismos envolvidos no desenvolvimento embrionário inicial. Porém, manipulações in vitro de gametas ou embriões levam a alterações na regulação epigenética, podendo causar altas taxas de anormalidades no desenvolvimento e no nascimento de indivíduos derivados. A geração de um modelo de indução da pluripotência in vitro, ou seja, a geração de células iPS (do inglês induced pluripotent stem cells) possibilitou estudar o processo de reprogramação in vitro de maneira robusta e precisa. Os genes OCT4 e SOX2 são fundamentais no processo de aquisição e manutenção da pluripotência celular, e recentemente foi reportado que a ação destes dois fatores exerce grande influência sobre a regulação de alguns genes imprinted, em especial, no locus H19/IGF2, sabidamente importantes para o desenvolvimento normal do embrião e de sua placenta. Este estudo propõe a geração de um modelo experimental in vitro onde os fatores em questão sejam estudados, juntos ou em combinação, quanto à sua influência na regulação do imprinting genômico. Para tal, três linhagens de fibroblastos fetais bovinos (bFF1, bFF2 e bFF3) foram transduzidas com vetores lentivirais contendo cDNAs de OCT4 ou SOX2 humanos. Os fibroblastos foram analisados através de citometria e as células positivas foram separadas e recuperadas (sorted). Os fibroblastos expressando OCT4, SOX2, ambos (OCT4 + SOX2), nenhum (controle) juntamente com um controle recuperado (não sorted) não transgênico (total de cinco tratamentos) foram investigados quanto à expressão de genes relacionados à pluripotência e expressão de genes imprinted, bem como a manutenção dos padrões de metilação do DNA no locus H19/IGF2. Além disso, estas células foram submetidas à reprogramação in vitro e produção de células iPS. A indução à pluripotência foi realizada através da transdução dos fibroblastos com o vetor policistrônico contendo o cDNAs murino ou humano dos fatores de transcrição OCT4, SOX2, c-MYC e KLF4 (OSMK, vetor STEMCCA). Os resultados da análise de fluorescência por citometria de fluxo foram, em média, de 40,4% para OCT4, 6,1% para SOX2 e 0,63% para OCT4 + SOX2. A bFF1 foi a única linhagem a apresentar uma recuperação pós-sorting, o que possibilitou sua utilização para a indução da pluripotência. De maneira interessante, as células que não passaram pela citometria geraram colónias de células iPS, enquanto que os demais grupos não. A quantificação de transcritos por qRT-PCR mostrou que a expressão de OCT4 e de SOX2 estava aumentada nos respectivos grupos, a expressão do gene H19 mostrou-se aumentada no grupo controle que passou pelo procedimento de sorting e a expressão do gene imprinted IGF2R não variou entre os grupos. Já a análise preliminar da manutenção do padrão de metilação de DNA na DMR do locus H19/IGF2 mostrou que o grupo controle sorted apresentou uma leve diferença no padrão de metilação quando comparada aos outros grupos. Neste estudo, portanto, o procedimento de separação e recuperação celular por citometria de fluxo celular, aliado ao elevado número de repiques celulares durante o cultivo prolongado pode ter levado a um efeito prejudicial sobre a eficiência de reprogramação in vitro / Reproductive biotechniques such as in vitro embryo production and somatic cell nuclear transfer may greatly contribute for fertility improvements, to enhance animal production or else to contribute to a better understanding of the underlying mechanism involved during initial embryonic development. However, in vitro manipulation of gametes or embryos may lead to possible disruptions on epigenetic regulation, causing high developmental abnormalities and decreased healthy calves born at term. The generation of induced pluripotency models (induced pluripotent stem cells, or iPS) made it possible to study the process of in vitro reprogramming in a more solid and precise manner. OCT4 and SOX2 are fundamental genes for the acquisition and maintenance process of cellular pluripotency. Recently, it has been reported that both factors may have a huge influence on the regulation of some imprinted genes, specially at locus H19/IGF2, known to be important for the normal development of embryo and placenta. Therefore, this study aimed to generate an in vitro experimental model where the above transcription factors will be studied together or separately regarding their influence on genomic imprinting regulation. For that, three bovine fetal fibroblasts cell lines (bFF1, bFF2 and bFF3) were transduced with lentiviral vectors containing human OCT4 or SOX2 cDNAs. The fibroblasts were analyzed trough cell cytometry and positive cells were sorted. Fibroblasts expressing OCT4, SOX2, both (OCT4+SOX2), none (control) together with a non-sorted and non-transgenic control (five treatments) were investigated regarding pluripotency and imprinted gene expression, as well maintenance of DNA methylation patterns at H19/IGF2 locus. Further, these cells were also submitted to in vitro induced reprogramming and production of iPS cell colonies. Induction into pluripotency was realized by transducing fibroblasts with polycistronic excisable vector containing the murine or human cDNA of OCT4, SOX2, c-MYC and KLF4 transcription factors (OSMK, STEMCCA vector). The results of fluorescence analysis by flow cytometry were, on average, 40.4% for OCT4, 6.1% for SOX2 and 0,63% for OCT4+SOX2 groups. bFF1 was the only lineage presenting a post-sorting recovery that enabled its use for pluripotency induction. Interestingly, non-sorted cells generated biPS colonies whereas sorted cells (control non transgenic, OCT4, SOX2 and OCT4+SOX2 expressing cells) did not generate biPS cells. The transcript quantification by qRT-PCR showed that OCT4 and SOX2 expression were increased in the respective groups, the expression of H19 gene was increased in the control sorted group and IGF2R expression was not different between groups. Preliminary results of imprinting pattern methylation at H19/IGF2 locus showed that sorted group was slightly different from others. In this study, therefore, analysis and sorting procedure by flow citometry, together with an extended period in culture may have lead to a detrimental effect on in vitro reprogramming efficiency

Mutationen im Protein LRRK2 wurden im Zusammenhang mit klinischen Symptomen beschrieben, die dem Idiopathischen Parkinsonsyndrom (IPS) nahezu gleichen. So findet sich neben vielen anderen Mutationen die häufigste pathogene Mutation für das IPS im LRRK2-Gen. Die Aufklärung der molekularbiologischen Mechanismen, die zur Pathologie der spezifischen Neurodegeneration in der Substantia nigra Pars Compacta (SNpc) und somit zur Idiopathischen Parkinsonssyndrom führen, ist mit der Hoffnung auf kausale und kurative Therapieansätze verbunden. In dieser medizinischen Doktorarbeit soll daher versucht werden, die biologische Funktion des LRRK2 in einem dopaminergen Mauszellmodell näher zu beschreiben. Hierfür soll die genetische Aktivität des LRRK2 in mesenzephalen, sogenannten MN9D-Zellen reduziert werden, indem der Mechanismus der RNA-Interferenz in vitro durch Transfektion von siRNA angestoßen wird. Durch die Reduktion der LRRK2-Aktivität sollen Veränderungen in den MN9D-Zellen induziert und diese objektiviert werden. Die Darstellung der Beobachtungen konzentriert sich auf die transkriptionelle Expression von Genen des Zellzyklus sowie der neuralen und dopaminergen Differenzierung (Tyrosinhydroxylase, Nestin und β-Tubulin) durch PCR. Die Proliferation der Zellen vor und nach den RNA-Interferenzexperimenten soll global durch MTT- und BrdU-Test gemessen werden.

Le syndrome d’Andersen est une maladie rare et associée à la perte de fonction du canal potassique Kir2.1. Afin d’étudier sa physiopathologie, nous avons généré et caractérisé des cellules souches pluripotentes induites (iPS) contrôle et Andersen. Nous avons ensuite différencié ces cellules iPS en cellules souches mésenchymateuse (MSC). Les cellules MSC de patients présentent une capacité de différenciation en ostéoblastes et en chondrocytes diminuée par rapport aux cellules contrôle. En effet, la production de matrice extracellulaire et l'expression des master gènes des différenciations osseuses et cartilagineuses, est réduite chez les patients. Ces travaux de thèse montrent que le canal Kir2.1 est essentiel au développement osseux. Les défauts de différentiation observés pourraient expliquer les dysmorphies associées avec le syndrome d’Andersen. / Andersen's syndrome is a rare disorder associated with a Kir2.1 potassium channel loss of fuction. To study the pathophysiology, we have generated and characterized induced Pluripotent Stem cells (iPS) from control and patient cells. We have then differentiated those iPS cells into mesenchymal stem cells (MSC). Patient's MSc have a lower osteoblastic and chondrogenic differnciation ability compared to control cells. Indeed, extracellular matrix production and master gene expression of osteoblastic and chondrogenic differenciation are reduced in patient’s cells. Alltogether, these results shown that Kir2.1 channel is required for bone developement. The differenciation defects saw in patient cells could explain the Andersen's syndrome associated dysmorphies.