No description available.

Thesis advisor: Eranthie Weerapana / Reactive oxygen species (ROS) modulate protein function through cysteine oxidation. Identifying protein targets of ROS can provide insight into uncharacterized ROS-regulated pathways especially within ROS generating organelles such as the mitochondria. There are several known examples of mitochondrial cysteine targets that alter protein and pathway activity resulting in pathological effects. Several chemoproteomic workflows, including ABPP and OxICAT, can be used to identify sites of cysteine oxidation. However, determining ROS targets localized within subcellular regions and ROS hotspots remains challenging with existing workflows. Here, we present combined cysteine- monitoring chemoproteomic platforms (isoTOP-ABPP and OxICAT) with mitochondrial enrichment (organelle isolation and proximity labeling) to monitor cysteine oxidation events within the mitochondria. First, we profile redox- sensitive cysteines under exogenous and endogenous peroxide in isolated mitochondria using isoTOP-ABPP and OxICAT. Next, we introduce PL-OxICAT which combines enzymatic proximity labeling (PL) (TurboID/APEX) and OxICAT to monitor localized cysteine oxidation events within subcellular compartments such as the mitochondrial matrix and intermembrane space as well as ROS hotspots. Together, these platforms further hone our ability to monitor cysteine oxidation events within specific subcellular locations and ROS hotspots and provide a deeper understanding of the protein targets of endogenous and exogenous ROS. / Thesis (PhD) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

No description available.

Hypertension and diabetes have long been closely linked. As such, the major player in the renin, angiotensin system, angiotensin II, has recently been investigated for its effects on metabolism and diabetes. Since skeletal muscle is one of the most metabolically active tissues, this study investigates the effects of angiotensin II specifically on skeletal muscle. In this study, L6 skeletal muscle cells were treated with angiotensin II for either 3 or 24 hours and a number of effects were investigated. Fatty acid oxidation and lipid synthesis was measured using [1-14C]-palmitate, glucose oxidation and glycogen synthesis were measured using 14C-glucose. In addition,mitochondrial oxidative capacity was measured using an XF 24 Flux Analyzer (Seahorse Bioscience) and reactive oxygen species measured using confocal microscopy. The clinical study involving the drug Benicar ® investigated the metabolic effects of blocking angiotensin II on skeletal muscle fatty acid oxidation, glucose oxidation, and oxidative and glycolytic enzyme activity. In L6 cells, angiotensin II significantly reduced fatty acid oxidation after 24 hours (p<0.01) and 3 hours (p<0.001) if angiotensin II was present during oxidation experiments. It also significantly reduced mitochondrial oxidative capacity (p<0.05) after 24 hours and significantly increased reactive oxygen species production (p<0.05) over 3 hours. The clinical study showed no significant effects of Benicar® on fatty acid or glucose oxidation or any enzyme activities. / Master of Science

O projeto de mestrado, denominado de forma abreviada como GPUServices, se insere no contexto da pesquisa e do desenvolvimento de métodos de processamento de dados de sensores tridimensionais aplicados a robótica móvel. Tais métodos serão chamados de serviços neste projeto e incluem algoritmos de pré-processamento de nuvens de pontos 3D com segmentação dos dados, a separação e identificação de zonas planares (chão, vias), e detecção de elementos de interesse (bordas, obstáculos). Devido à grande quantidade de dados a serem tratados em um curto espaço de tempo, esses serviços utilizam processamento paralelo por GPU para realizar o processamento parcial ou completo destes dados. A área de aplicação em foco neste projeto visa prover serviços para um sistema ADAS: veículos autônomos e inteligentes, forçando-os a se aproximarem de um sistema de processamento em tempo real devido ao contexto de direção autônoma. Os serviços são divididos em etapas de acordo com a metodologia do projeto, mas sempre buscando a aceleração com o uso de paralelismo inerente: O pré-projeto consiste de organizar um ambiente que seja capaz de coordenar todas as tecnologias utilizadas e que explore o paralelismo; O primeiro serviço tem a responsabilidade de extrair inteligentemente os dados do sensor que foi usado pelo projeto (Sensor laser Velodyne de múltiplos feixes), que se mostra necessário devido à diversos erros de leitura e ao formato de recebimento, fornecendo os dados em uma estrutura matricial; O segundo serviço em cooperação com o anterior corrige a desestabilidade espacial do sensor devido à base de fixação não estar perfeitamente paralela ao chão e devido aos amortecimentos do veículo; O terceiro serviço separa as zonas semânticas do ambiente, como plano do chão, regiões abaixo e acima do chão; O quarto serviço, similar ao anterior, realiza uma pré-segmentação das guias da rua; O quinto serviço realiza uma segmentação de objetos do ambiente, separando-os em blobs; E o sexto serviço utiliza de todos os anteriores para a detecção e segmentação das guias da rua. Os dados recebidos pelo sensor são na forma de uma nuvem de pontos 3D com grande potencial de exploração do paralelismo baseado na localidade das informações. Porém, sua grande dificuldade é a grande taxa de dados recebidos do sensor (em torno de 700.000 pontos/seg.), sendo esta a motivação deste projeto: usar todo o potencial do sensor de forma eficiente ao usar o paralelismo de programação GPU, disponibilizando assim ao usuário serviços de tratamento destes dados. / The master\'s project, abbreviated hence forth as GPUServices, fits in the context of research and development of three-dimensional sensor data processing methods applied to mobile robotics. Such methods will be called services in this project, which include a 3D point cloud preprocessing algorithms with data segmentation, separation and identification of planar areas (ground track), and also detecting elements of interest (borders, barriers). Due to the large amount of data to be processed in a short time, these services should use parallel processing, using the GPU to perform partial or complete processing of these data. The application area in focus in this project aims to provide services for an ADAS system: autonomous and intelligent vehicles, forcing them to get close to a real-time processing system due to the autonomous direction of context.The services are divided into stages according to the project methodology, but always striving for acceleration using inherent parallelism: The pre-project consists of organizing an environment for development that is able to coordinate all used technologies, to exploit parallelism and to be integrated to the system already used by the autonomous car; The first service has a responsibility to intelligently extract sensor data that will be used by the project (Laser sensor Velodyne multi-beam), it appears necessary because of the many reading errors and the receiving data format, hence providing data in a matrix structure; The second service, in cooperation with the above, corrects the spatial destabilization due to the sensor fixing base not perfectly parallel to the ground and due to the damping of the vehicle; The third service separates the environment into semantics areas such as ground plane and regions below and above the ground; The fourth service, similar to the above, performs a pre-segmentation of street cruds; The fifth service performs an environmental objects segmentation, separating them into blobs; The sixth service uses all prior to detection and segmentation of street guides.The received sensor data is structured in the form of a cloud of points. They allow processing with great potential for exploitation of parallelism based on the location of the information. However, its major difficulty is the high rate of data received from the sensor (around 700,000 points/sec), and this gives the motivation of this project: to use the full potential of sensor to efficiently use the parallelism of GPU programming, therefore providing data processing services to the user, providing services that helps and make the implementation of ADAS systems easier and/or faster.

The daVinci Research Kit (dVRK) is a research platform that consists of the clinical daVinci surgical robot, provided by Intuitive Surgical to Academic Institutions. It provides an open source software and hardware platform for researchers to study and analyze the current architecture and expand the capabilities of the existing technology. The line between general purpose robotics and medical robotics has segregated the two fields. A significant part of the segregation lies at the software end, where new tools and methods developed in general purpose robotics cannot make it to medical robotics in a short amount of time. This research focuses on the integration of a widely used software architecture for general purpose robotics with the dVRK with the hope of utilizing the research and development from one field to the other. As a first step towards this bridging, a motion planning framework and a dynamic simulator has been developed for the dVRK using ROS. The motion planning framework is aimed to assist the surgeon in performing task with additional safety and machine intelligence. A few use cases have been proposed as well. Lastly, a Matlab Interface has been developed that is standalone in terms of usage and provides capabilities to interact with dVRK.

Les plantes prélèvent l’azote nécessaire à leur croissance essentiellement sous forme de nitrate. Pour faire face aux fluctuations spatio-temporelles de la disponibilité de cet ion dans les sols, ces organismes ont développé des mécanismes d’adaptation spécifiques à chaque situation. La réponse de la plante à l’azote met en jeu plusieurs voies de signalisations qui dépendent des scénarios de variations en azote du milieu. Deux grandes voies de signalisation sont étudiées en particulier dans cette thèse. La Primary Nitrate Response (ou PNR) qui correspond aux réponses rapides (minutes) et nitrate-spécifiques de la plante lors de la fourniture de Nitrate. La Nitrogen Starvation response (ou NSR) qui correspond à la réponse plus lente (jours) qui permet de pallier au manque d’azote dans le milieu. Bien que certains acteur moléculaires soient connus dans chacune des voies (PNR et NSR); i) la NSR est largement moins bien documentée que la PNR, ii) rien n’est connu concernant la coordination des 2 voies de signalisations. Au cours de ma thèse j’ai pu démontrer qu’un sous groupe de la famille GARP induit lors de la PNR est directement impliqué dans la régulation de la NSR (répression des gènes de transport à haute affinité de nitrate). Ceci fournit à la fois des nouveaux régulateurs de la NSR et un mécanisme de coordination entre les 2 voies de signalisation. Les phénotypes des plantes altérées dans l’expression des gènes de cette famille de facteurs de transcription ouvrent des perspectives d’améliorations biotechnologiques des plantes car ces dernières présentent des capacités de transport du nitrate bien supérieures aux plantes sauvages.Des résultats quant à la double localisation sub-cellulaire d’HRS1 et du rôle d’HRS1 dans le contrôle du statut redox des plantes sont présentés et discutés dans le contexte du modèle d’interaction entre PNR et NSR proposé précédemment. / Plants take up the nitrogen necessary for their growth mainly in the form of nitrate. To cope with spatio-temporal fluctuations in NO3- availability in soils, these organisms have developed adaptation mechanisms specific to each situation. Plant N response involves several signaling pathways that depend on the N variation scenarios of the medium. Two major signaling pathways are studied in this thesis. The Primary Nitrate Response (or PNR) which corresponds to the rapid (within minutes) and nitrate-specific responses of the plant when provided with Nitrate. The Nitrogen Starvation response (or NSR) which corresponds to the slower response (within days) which makes it possible to overcome the lack of N in the medium. Although some molecular actors are known in each of the pathways (PNR and NSR); i) the NSR is significantly less well documented than the PNR, ii) nothing is known about the coordination of the 2 signaling pathways. During my thesis I was able to demonstrate that a subgroup of the GARP transcription factor family induced during PNR is directly involved in the regulation of NSR (repression of transport genes with a very high nitrate affinity). This provides both new NSR regulators and a coordination mechanism between the 2 signaling pathways. The phenotypes recorded for plants altered in this transcription factors family open up perspectives for crop biotechnological improvements because they have nitrate transport capacities far superior to wild plants.Results regarding the subcellular dual localization of HRS1 and the role of HRS1 in controlling the redox status of plants are presented and discussed in the context of the previously proposed PNR-NSR interaction model.

Le fer est un élément essentiel pour les cellules car il est le cofacteur de nombreuses protéines impliquées dans de multiples processus biologiques comme la photosynthèse et la respiration. Cependant, l'excès de fer peut être délétère pour la cellule, car il peut réagir avec l'oxygène pour former des espèces réactives de l'oxygène (ROS). Les ferritines sont des protéines chloroplastiques codées par le génome nucléaire permettant de stocker le fer en excès sous forme non toxique. Chez les végétaux, la synthèse des ferritines est majoritairement régulée au niveau transcriptionnel en réponse au fer contrairement aux animaux où elle est majoritairement régulée au niveau post-transcriptionnel. Toutefois, une régulation post-transcriptionnelle a été mise en évidence pour le gène de ferritine AtFer1. L'ARNm d'AtFer1 est déstabilisé en réponse à un stress oxydatif généré par un excès de fer. Cette régulation fait intervenir un élément cis nommé DST (DownSTream) localisé dans la région 3' transcrite non traduite de ce transcrit (3'UTR). Chez deux mutants précédemment identifiés comme agissant en trans (dst1 et dst2), cette régulation est affectée. Une caractérisation physiologique de ces mutants a permis de montrer que cette voie de dégradation est un mécanisme essentiel contrôlant la physiologie et la croissance de la plante en réponse à un stress oxydatif. D'autre part, l'expression d'AtFer1 ainsi que d'autres gènes codant des protéines chloroplastiques est régulée par un acteur de la machinerie de dégradation des ARNm, l'exoribonucléase XRN4. Ces ARNm codant des protéines chloroplastiques seraient localisés à la surface des chloroplastes. Cette localisation ferait intervenir des acteurs de la machinerie de dégradation des ARNm. La localisation subcellulaire du transcrit AtFer1 a été estimée par deux approches. L'ARNm d'AtFer1 a été visualisé par une technique d'imagerie, l'hybridation in situ révélé par fluorescence (FISH) (i). L'accumulation d'ARNm codant des protéines chloroplastiques a été évaluée dans deux fractions (chloroplastes isolés et feuilles entière) afin de savoir si certain ARNm se retrouvent enrichis dans la fraction chloroplastique (ii). Les résultats obtenus suggèrent que l'ARNm d'AtFer1 serait localisé autour des chloroplastes, cependant cette localisation ne semble pas être affectée chez le mutant xrn4. Enfin, ce travail a permis de caractériser la régulation et la fonction des ferritines dans les racines d'Arabidopsis. Le fer en excès induit la synthèse de ferritines dans les racines, AtFer1 puis AtFer3 sont les gènes de ferritines les plus exprimés dans cet organe. Les racines de plantes cultivées en excès de fer présentent des spots de fer dans les cellules de l'endoderme et du péricycle, là où l'expression des gènes AtFer1 et AtFer3 est retrouvée. Ces spots sont absents dans un triple mutant fer1-3-4. L'excès de fer diminue la longueur de la racine primaire de manière indépendante des ferritines. Par contre, l'excès de fer modifie la densité et l'élongation des racines latérales, ces deux modifications requièrent la présence des ferritines. Lors d'un excès de fer, les ferritines participent à la mise en place du gradient de H2O2 et de O2.- entre les zones de prolifération et de différentiations. Ce gradient est impliqué dans le contrôle la croissance racinaire. / Iron is essential for cells because it is the cofactor of many proteins involved in many biological processes such as photosynthesis and respiration. However, iron in excess can be deleterious to the cell due to its capacity to react with oxygen to form reactive oxygen species (ROS). Ferritins are plastidial proteins encoded by nuclear genes in order to store iron in a safe form. In plants, ferritin synthesis is mainly regulated at the transcriptional level in response to iron in contrast to animals, where it is mainly regulated at the post-transcriptional level.However, post-transcriptional regulation has been shown for the ferritin gene AtFer1. The AtFer1 mRNA is destabilized in response to oxidative stress generated by an excess of iron. This regulation involves a cis element called DST (DownSTream) located in the 3' untranslated region (3'-UTR) of this transcript. In two mutants previously identified as trans-acting (dst1 and dst2), this regulation is affected. Physiological characterizations of these mutants have shown this pathway is an important mechanism to control physiology and plant growth in response to oxidative stress.On the other hand, AtFer1 expression and expression of other genes encoding chloroplast proteins are regulated by a component of the mRNA decay machinery, the exoribonuclease XRN4. These mRNAs encoding chloroplast proteins would be localized on the surface of chloroplasts. This location would involve component of the mRNA decay machinery. The subcellular localization of AtFer1 mRNA was estimated by two approaches. AtFer1 mRNA was visualized by an imaging technique, fluorescent in situ hybridization revealed by (FISH) (i). Accumulation of mRNA encoding chloroplast proteins was evaluated in two fractions (purified chloroplasts and total leaves) to determine if some mRNAs are found enriched in the chloroplast fraction (ii) . Our results suggest that the AtFer1 mRNA is localized around chloroplasts, however, this location does not seem to be affected in the xrn4 mutant. Finally, this work has shown the regulation and function of ferritins in the roots of Arabidopsis. Iron in excess induces ferritin synthesis in roots, and AtFer1 then AtFer3 are the most expressed ferritin genes in this organ. Roots grown in iron excess present spots of iron in the cellular layers of the endoderm and pericycle, where AtFer1 and AtFer3 ferritin genes are expressed. This staining disappears in a triple fer1-3-4 ferritin mutant. Fe in excess decreases primary root length independently of the ferritins. In contrast, Fe excess mediated alteration of lateral root density and mean length requires ferritins, in particular at the highest Fe concentration tested. During an iron excess, ferritin are involved in the establishment of the H2O2 and O2.- gradient between proliferation and differentiation zones. This gradient is known to control of root growth.

Hibridiniai baltymai, koduojami sulietų lamB ir lacZ genų susiriša su išvirkščiomis E.coli ląstelių plazminės membranos pūslelėmis. Sąveika su plazmine membrane priklauso nuo geno secA produkto buvimo. Hibridinių baltymų susirišimas su membrana priklauso nuo išorinės membranos baltymo LamB polipeptidinės grandinės ilgio. Aktyviosios deguonies formos, susidariusios fotosensibilizuojant eukariotų ląsteles lipofiliniu sensibilizatoriumi sukelia membranų pažaidas, padidina lipidų peoksidacijos lygį ir laktato dehidrogenazės išėjimą iš ląstelių. Vidinių ląstelių pažaidos sumažina ląstelių oksidoreduktazių aktyvumą ir ATP kiekį. Aktyviųjų deguonies formų susidarymas mitochondrijų vidinėje ertmėje buvo indukuotas rodaminu arba safraninu. Oksidacinio streso, sukelto mitochondrijų viduje, poveikis skyrėsi nuo įvairių ląstelės membranų fotopažaidų. Ląstelės atsakas į oksidacinį stresą mitochondrijų viduje priklausė nuo poveikio dozės. Pažaidos iki CD50 nesukėlė žymaus ląstelės gyvybingumo sumažėjimo. Didesnės fotopažaidos virš CD70 indukavo apoptozę, aktyvino kaspazę-3 ir iniciavo citochromo išėjimą iš mitochondrijų. / Hybrid proteins encoded by the fused lamB and lacZ genes were able to reconstitute to the membrane of the inside-out vesicles. The reconstitution of the cytoplasmic membrane vesicles was shown to be dependent on the gene sec A product. The interaction of the hybrid proteins with the membranes depended on the length of the LamB polypeptide chain. Production of ROS after photosensibilization with the lipophylic photosensitizer induced the damage to the cell membranes, enhanced lipid peroxidation and resulted in the leakage of lactate dehydrogenase from the cells. The damage of the inner cell membranes induced the decrease of the activity of cell oxidoreductases and the amount of ATP. For the stimulated production of ROS in the inner space of mitochondria, rhodamine123- and safranin-mediated photodynamic treatment was employed. Cell response to the oxidative insult in the mitochondrial interior was different from the response to the photodamage produced in other cellular membranes. Cell response to the oxidative stress in the mitochondrial interior was dose dependent. Damage up to CD50 did not reveal hallmarks of dead cells. Severe damage (beyond CD70) induced apoptosis following release of cytochrome c and caspase activation.

Cox11 (cytochrome c oxidase 11) is an ancient and conserved protein family present in most respiring organisms. Studies of several family members, mainly in yeast and bacteria, have revealed that these proteins are in charge of Cu+ delivery to the respiratory complex IV (COX). Absence of Cox11 leads to a non-functional COX complex and a complete respiratory deficiency. Although it is assumed that homologues in other species perform the same function, experimental data supporting this notion are lacking. The aim of this work was to characterize the putative Arabidopsis homologue AtCox11 (encoded by locus At1g02410) and to determine its functions. Comparison of AtCox11 with the well-studied ScCox11 in yeast revealed that the two proteins share high similarity in their sequences (32% amino acid identity) and in the predicted secondary structures. Surprisingly, despite this high similarity AtCox11 proved not to be able to functionally replace the yeast protein in ΔSccox11 yeast deletion strains. As presumed, AtCox11 is localized to mitochondria, probably tethered to the inner mitochondrial membrane with its C-terminus facing the intermembrane space. The subsequent experimental work addressed the functions of AtCox11. To this end AtCOX11 knock-down (KD) and overexpression lines (OE) were generated and their impact on plant phenotype was investigated. KD lines that were obtained by artificial micro RNA technology, possess approximately 30% of the WT AtCOX11 mRNA levels. Overexpression resulting in 4-6 fold higher AtCOX11 mRNA levels, was achieved by placing AtCOX11 under the control of the 35S promoter. Remarkably, both KD and OE plants had reduced levels of COX complex activity (~45% and ~80%, respectively) indicating that AtCox11 is, as expected, involved in COX complex assembly. The KD and OE plants exhibited reduced root lengths and pollen germination rates (compared to WT). As both processes are dependent on respiratory energy, these phenotypic changes seemingly result from the reduced COX activity. Interestingly, the short-root phenotype in OE plants was rescued by a surplus of copper in the media, whereas copper deficiency intensified the phenotype. By contrast, KD plants did not respond to changes of the copper concentration. This difference in the copper response between KD and OE plants hints at a different cause for the reduced COX activity. It is proposed that the concentration of AtCox11 in KD plants limits the efficient insertion of Cu+ into COX, independent of the available copper concentration. In OE plants, binding of the limited copper by the high AtCox11 level may lead to a copper deficiency for the copper chaperone AtHcc1 that is required to load copper to subunit AtCoxII. Indeed, addition of copper to the media was able to rescue the phenotype. In line with these data, the analysis of the expression pattern of AtCOX11 revealed that it is expressed in tissues which require substantial mitochondrial and COX biogenesis to sustain their high metabolic and/or cell division rates. Furthermore AtCOX11 was shown to be up-regulated as part of the plant’s response to increased oxidative stress induced by the addition to the plant media of peroxides or inhibitors of respiratory complexes. The up-regulation of AtCOX11 in response to oxidative stress was corroborated with publicly available RNA microarray data and analysis of the AtCOX11 promoter, which revealed the presence of a number of potential oxidative stress responsive elements. Taken together, the experimental results presented in this thesis support the conclusion that AtCox11 is a member of the conserved Cox11 protein family. Most probably, this mitochondrial protein participates in the assembly of the COX complex by inserting Cu+ into the CuB center of the AtCoxI subunit. In addition to this expected role, the data indicate that AtCox11 might participate in cellular oxidative stress response and defense via a yet unknown mechanism.