A identificação de leveduras por métodos moleculares e bioquímicos teve um grande impacto na sistemática e, devido aos resultados recentes este grupo sofreu alterações taxonômicas. A linhagem de levedura IZ-987 tem sido utilizada na fermentação de caldo de cana-de-açúcar para a obtenção de aguardente no Departamento de Agroindústria, Alimentos e Nutrição da ESALQ/USP. Esta linhagem, catalogada como Saccharomyces cerevisiae, não produz H2S durante o processo fermentativo e apresenta a capacidade de flocular em fermentação. A análise anterior de diversidade genética por marcadores de RAPD demonstrou existir um distanciamento elevado entre esta linhagem e S. cerevisiae. O presente trabalho teve então por objetivo avaliar a diversidade genética entre a linhagem IZ-987, e as linhagens PE-2 e IZ-259 utilizadas como leveduras padrões pertencentes à espécie Saccharomyces cerevisiae e a levedura CR-1 como padrão de Saccharomyces bayanus, por meio das características morfológicas, fisiológicas e bioquímicas, e também por meio de marcadores de RAPD, cariotipagem e análise da seqüência das regiões ITS1 e ITS2 (Internal Transcript Subunit). Os métodos mostraram-se eficientes na diferenciação das linhagens, pois as limitações de um método puderam ser complementadas por outro. A análise morfológica mostrou que a linhagem IZ-987 não apresenta similaridade com as linhagens padrões. Os testes fisiológicos e bioquímicos apresentaram maior similaridade entre as linhagens em estudo não sendo suficiente para identificar com segurança o gênero e a espécie. As análises de diversidade genética por marcadores de RAPD e cariotipagem demonstraram que existe um distanciamento elevado entre IZ-987 e os padrões de S. cerevisiae. A análise da seqüência das regiões ITS1 e ITS2 (incluindo a subunidade 5,8 S do RNA ribossomal) da linhagem IZ-987, mostrou similaridade (>96%) com a espécie Pichia anomala. / Identification of yeast by biochemical and molecular method has changed the systematic of this group. The strain IZ-987 has been used in Department of Agroindustry, Food and Nutrition, ESALQ/USP, Piracicaba/SP for alcohol production from sugarcane. This strain, which flocculates and is not able to produce H2S during fermentation, was previously identified by classical methodology as Saccharomyces cerevisiae. In previous work, RAPD analysis showed a low level of similarity between the IZ-987 and other of S. cerevisiae strains. The aim of the present work was to examines the genetic variability among the IZ-987 strain, S. cerevisae (IZ-259 and PE-2 strains) and S. bayanus (strain CR-1) by nutritional requirements, biochemical, physiological and morphological traits as well as RAPD markers. ITS (Internal Transcript Subunit) sequencing was used in further characterization of the IZ-987 strain. The methodologies used in the present analysis were able to distinguish the strains, since that the limitation observed in one was complemented by other technique. The morphological and molecular analysis distinguished the IZ-987 strain from those of Saccharomyces genera. However, physiological and biochemical evaluation show similarity among the evaluated strains. Therefore, the results were not able to define the specie or genera of the IZ-987 strain. The sequencing of ITS-1, ITS-2 and 5.8S rDNA of the IZ-987 strain and analysis by BLASTn (http://www.ncbi.nlm.nih.gov) showed similarity (>96%) of this strain with Pichia anomala.

Human motion analysis is an important tool for assessing movement, rehabilitation progress, fall risk, progression of neurodegenerative diseases, and classifying gait patterns. Advancements in artificial intelligence models and high-performance computing technologies have given rise to marker-less human motion analysis that determine point correspondences between an array of cameras and estimate 3D joint coordinates using triangulation. However, existing methods have not considered the physical setup and design of a marker-less human motion analysis tool that could be deployed in an institutional environment for active use, such as an institutional hallway where individuals pass regularly on a daily basis (i.e., Smart Hallway). In this thesis, camera locations were modelled, four machine vision grade cameras connected to an NVIDIA Jetson AGX were set up in a simulated institutional hallway environment, and custom software was written to capture synchronized 60 frame per second video of a participant walking through the Smart Hallway capture volume. Software was also written to calculate 3D joint coordinates and extract outcome measures for various test conditions. These outcome measures were compared to ground truth body segment length measurements obtained from direct measurement and ground truth foot event timings obtained from direct observation. Body segment length measurements were within 1.56 (SD=2.77) cm of ground truth values. AI-based stride parameters were comparable with ground truth foot event timings and the implemented foot event detection algorithm was within 4 frames (67 ms), with an absolute error of 3 frames (50 ms) on the ground truth foot event labels. The Smart Hallway can be deployed in an unobtrusive manner and be temporally and spatially calibrated with ease. This multi-camera marker-less approach is viable for calculating useful outcome measures for clinical decision making. With these findings, marker-less motion capture is viable for non-invasive human motion analysis and compares well with marker-based systems. With future research and innovations, marker-less motion analysis will be the ideal approach for human motion analysis that requires minimal human resource to collect meaningful information.

Real-time sensitive network applications are emerging and require ultra-low latency to reach the desired QoS. A main issue that contributes to latency is excessive buffering at intermediate switches and routers. Existing queuing strategies that aim to reduce buffering induced latency typically apply a single queue AQM that does not support service differentiation and treats all packets equally. The recently proposed per packet value framework utilizes a packet value marker and a packet value aware AQM to solve this issue by supporting service differentiation in a single queue and introducing more advanced policies for resource sharing. However, the per packet value framework is implemented and tested in a software environment with no possibility to study the performance on hardware equipment.  This thesis utilizes P4 to design and implement a packet value marker on dataplane programmable devices. The marker should be capable of supporting multiple resource sharing policies, following resource sharing policies accurately, and not being the bottleneck in the network. A target-independent packet value marker is designed and modified with target-dependent P4 constructs to fit the implementation requirements of a Tofino switch and a Netronome smart NIC. An accurate Tofino implementation using this approach is difficult to achieve because of a complicated random number generation process and resource limitation. Evaluation using a testbed with a Netronome marker shows that the marker achieves desired functionality with accurate packet value distribution for throughputs larger than 5000 Kbps. However, the challenge of concurrent packet processing combined with a smart NIC that does not have powerful packet processing cores results in the marker having lower throughput and higher latency than expected. The evaluation also shows that resource limitation in terms of available memory and the number of supported policies affects the maximum number of supported users. We also ported a version to a switching ASIC with limited functionality due to the restrictions of the hardware platform. Our evaluation also provides insights into how such a marking scheme performs on different hardware targets and the limitation imposed by such target specific architecture. / Realtids Känsliga nätverksapplikationer utvecklas och kräver ultra-låg latens för att nå önskad QoS. Befintliga lösningar på detta problem tillämpar AQM på en enda kö och stöder inte tjänst differentiering och behandlar alla paket lika. Det nyligen föreslagna ramverket per packet value löser problemet genom att stödja tjänst differentiering på en kö och införa mer avancerade policyer för resursdelning. Ramverket per packet value implementeras och testas i en mjukvaru miljö utan möjlighet att studera prestanda på hårdvaru utrustning. Denna avhandling använder P4 för att designa och implementera en packet value marker på dataplan programmerbara enheter. Markern bör kunna stödja flera resursdelning principer, följa resursdelning principer exakt, och inte vara bottlenecken i nätverket. En hårdvaruoberoende packet value marker är designad och modifierad med hårdvaruberoende P4-konstruktioner för att passa implementerings kraven för en Tofino switch och en Netronome smart NIC. Slumpmässig talgenerering och resursbegränsning resulterar i en misslyckad implementering av en marker på Tofino med detta tillvägagångssätt. Utvärdering med hjälp av en testbädd med en Netronome marker visar att ett enanvändarscenario och en slumptalsgenerator orsakar lägre genomströmning och högre latens jämfört med forwarding. Resultaten visar att denna metod för Markern är felaktig när man tillämpar policyer vid lägre genomströmningar. Utvärderingen visar också att det maximala antalet användare begränsas av minnet och antalet policyer som stöds. Denna utvärdering ger inblick i hur en sådan marking algoritm är designad och svårigheterna med implementering för olika hårdvara.

This thesis deals with the issues of locating and marking both metalic and optical transmission network. The choice of an appropriate program for creation and management of map document is discussed. In the following chapter some necessary adjustments to the ArcGIS ArcView program are made so that it is applicable to the field of telecommunication networks. The last chapter addresses the design of documentation options in optical transmission network. For metalic cables the possibilities of attaching transmitter to a cable and locating the induced magnetic field is covered. Among the methods for finding optical cables we list ways of locating through the use of markers, GPS location and added metalic conductors. Fundamentals of markers and their types are thoroughly analysed. In the chapter on GPS we examine how this system works and evaluate its accuracy. The next chapter enumerates requirements that a program should meet. We analyse properties of considered programs and evaluate them with respect to these requirements. For a chosen program the possibilities of enriching it with selected map document are discussed. For these map document, information about their applicability and source is presented. We also quote the cost for these maps wherever possible. The last chapter treats ways of documenting optical networks.

Image-guided neurosurgery interventions are becoming sur- gical procedure routines. We suggest a novel method for automatic marker localization in X-ray images for Leksell SurgiPlan® which is an image-based neurosergical treat- ment planning software provided by Elekta Instrument AB. We implemented an algorithm for fiducial marker localiza- tion based on feature detection, classification and prior geo- metrical knowledge of the markers. Automatic localization ca help to decrease the human error associated with manual registration of these fiducial markers which is the current applied method for X-ray images in Leksell SurgiPlan®.

C. elegans is a commonly used genetic model organism due to the ease of genetic screens, transgenesis, and microscopy. Here, I describe methods that improve transgenesis in C. elegans and the development of a genetic screen to identify genes involved in the piRNA pathway. Transgenesis is commonly used for most laboratories that utilize C. elegans and improvements are therefore likely to facilitate research across many research areas. In the first chapter, I characterized a pan-muscular promoter that drives fluorophore expression to help identify C. elegans transgenesis. This promoter is an improved co-injection marker as it drives bright fluorescence with low toxicity and high efficiency. In the second chapter, I study piRNAs which are a large class of non-coding RNA that play important roles in protecting the genome from transposable elements in most animals. The study of piRNAs has mostly focused on their function in the germline, but recent evidence suggests functions in somatic cells such as neurons. To identify genes involved in the piRNA pathway in C. elegans, I performed a chemical genetic screen. I identified one mutant with a somatic phenotype and six mutants with a germline phenotype. I have focused on the germline and sequenced two strains and identified candidate genes involved in the piRNA pathway. Future work will focus on validating and identifying the remaining mutants.

La schizophrénie est un trouble mental qui touche environ 1% de la population mondiale et qui représente l’une des principales causes de handicap psychique. Depuis plusieurs années, des nombreuses études ont montré la présence d’anomalies oculomotrices ainsi que de la motricité fine chez des sujets appartenant au spectre la schizophrénie, soulignant l’intérêt d’approfondir la recherche de biomarqueurs dans cette pathologie. Dans cette thèse nous avons réalisé trois études afin d’examiner l’oculomotricité et la présence de signes neurologiques mineurs (SNM, NSS, Neurological Soft Signs) chez des patients avec schizophrénie, des apparentés sains des patients ainsi que des sujets à haut risque de developper une psychose (UHR) en les comparant avec des sujets contrôles. Pour la première fois nous nous avons enregistré les saccades mémorisées chez des sujets UHR en relation aux SNM (etude 1). Les résultats nous ont montré que les sujets avec schizophrénie, les apparentés sains et les sujets UHR rapportaient plus d’erreurs aux saccades mémorisées par rapport aux sujets contrôles, plus précisément les UHR ayant un nombre plus importants des SNM. L’etude 2 nous a permis d’élargir les investigations en étudiant l’instabilité de la fixation visuelle ainsi que la poursuite. Les résultats nous ont monté une instabilité de la fixation ainsi qu’un default du contrôle saccadique à la poursuite surtout chez les patients avec une présence plus important des SNM. Enfin, l’étude 3, en utilisant la méthode d’apprentissage automatique nous avons pu développer un modèle de machine à vecteurs de support avec l’objectif de pouvoir évaluer les valeurs prédictives différentielles pour mieux discriminer nos quatre groupes de sujets. Cette analyse nous a permis de souligner l’importance de la prise en compte des paramètres oculomoteurs ainsi que des Signes Neurologiques Mineurs dans l’étude sur la détection précoce de la schizophrénie. / Schizophrenia is a mental disorder that affects about 1% of the world's population and it represent one of the main causes of psychological disability. During several years, numerous studies have shown the presence of oculomotor as well as fine motor skills abnormalities in subjects belonging the spectrum of schizophrenia, highlighting the interest to deepen the search for biomarkers in this pathology.During my PhD’s thesis we conducted three studies to examine the oculomotor capability and the presence of Neurological Soft Signs (NSS) in patients with schizophrenia, full siblings of patients and subjects at Ultra High Risk for developing Psychosis (UHR) compared to health volunteers. For the first time we recorded memory guided saccades in UHR subjects in relation to the NSS (study 1). The results showed that subjects with schizophrenia, full siblings and UHR made more errors in this task when they were compared to control, particularly for UHR’s group with a higher score of NSS. The second study (study 2) allowed us to enlarged the investigations of visual fixation as well as of the pursuit eye movements. The results showed an instability of the fixation as well as a lack of saccadic control during pursuit especially for patients with a higher score of NSS. Finally, in the study 3, we used the machine learning method on the data to evaluate predictive values to better discriminate the four groups of subjects. This analysis allowed us to underline the importance of taking into account oculomotor parameters as well as the Neurological Soft Signs for the early detection of schizophrenia.

Indiana University-Purdue University Indianapolis (IUPUI) / The main focus of this study is to determine the impact of Set-Binding Factor 2 (SBF2) on human-derived neurons in the context of taxane-induced peripheral neuropathy. Taxane-induced peripheral neuropathy (TIPN) is a devastating survivorship issue for many cancer patients; SBF2 has been previously identified as a potential germline predictor that has been found to be significantly associated with severe TIPN in African American (AA) patients. The work described here provides ex vivo support for the use of SBF2 as a genotypic biomarker to identify a priori which patients are at a higher risk of manifesting severe TIPN. This study demonstrates that diminished expression of SBF2 exacerbated the effect of paclitaxel on viability and morphology and altered the functional response of a neuronal model exposed to paclitaxel treatment. Furthermore, transcriptomic work showed that reduced expression of SBF2 in a neuronal model treated with paclitaxel impacted the expression of genes that modulate stress-induced cell death and pain threshold. Altogether, these findings suggest that SBF2 plays a role in the development of TIPN. This work sheds light on the pathways potentially involving SBF2 that can be studied to further evaluate the function of this gene in neurons and its contribution to severe TIPN. Further functional approaches investigating these pathways will be pivotal in elucidating the underlying biological mechanism for this toxicity and identifying novel targeted therapeutic strategies to prevent or treat TIPN. / 2021-05-17

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18452号 / 医博第3907号 / 新制||医||1004(附属図書館) / 31330 / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊達 洋至, 教授 武田 俊一, 教授 富樫 かおり / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Single-cell RNA-sequencing (scRNA-seq) has enabled researchers to study interindividual cellular heterogeneity, to explore disease impact on cellular composition of tissue, and to identify novel cell subtypes. However, a major challenge in scRNA-seq analysis is to identify the cell type of individual cells. Accurate cell type identification is crucial for any scRNA-seq analysis to be valid as incorrect cell type assignment will reduce statistical robustness and may lead to incorrect biological conclusions. Therefore, accurate and comprehensive cell type assignment is necessary for reliable biological insights into scRNA-seq datasets. With over 200 distinct cell types in humans alone, the concept of cell identity is large. Even within the same cell type there exists heterogeneity due to cell cycle phase, cell state, cell subtypes, cell health and the tissue microenvironment. This makes cell type classification a complicated biological problem requiring bioinformatics. One approach to classify cell type identity is using marker genes. Marker genes are genes specific for one or a few cell types. When coupled with bioinformatic methods, marker genes show promise of improving cell type classification. However, current scRNA-seq classification methods and databases use marker genes that are non-specific across sources, samples, and/or species leading to bias and errors. Furthermore, many existing tools require manual intervention by the user to provide training datasets or the expected number and name of cell types, which can introduce selection bias. The selection bias negatively impacts the accuracy of cell type classification methods as the model cannot extrapolate outside of the user inputs even when it is biologically meaningful to do so. In this dissertation I developed CellTypeR, a suite of tools to explore the biology governing cell identity in a “normal” state for humans and mice. The work presented here accomplishes three aims: 1. Develop an ontology standardized database of published marker gene literature; 2. Develop and apply a marker gene classification algorithm; and 3. Create user interface and input data structure for scRNA-seq cell type prediction.