Investigating the Roles of Master Cell Cycle Regulators during Cytokinesis and Embryonic Development in \u3ci\u3eCaenorhabditis elegans\u3c/i\u3e

Faithful cell division is required to maintain ploidy and generate daughter cells with necessary genetic components for life. During mitosis, dividing cells face the challenge of coordinating multiple processes to ensure that nascent daughter cells inherit an exact copy of the parent cell’s genetic identity to maintain viability. To ensure the proper execution of cell division, multiple core cell cycle proteins, such as Aurora B kinase and separase, are involved in regulating chromosome segregation, cytokinesis and abscission. Interestingly, fundamental roles for these core cell cycle proteins are being characterized in this coordination. Separase regulates chromosome segregation and vesicle trafficking during meiotic and mitotic divisions. Aurora B kinase is well characterized to eliminate incorrect attachments of kinetochore with centromere through its phosphorylation. These faultless attachments initiate a series of signaling pathways to activate separase and promote chromosome segregation. Additionally, Aurora B kinase also phosphorylates centralspindlin to complete cytokinesis and midbody formation. The collection of work presented here addresses the role of these two master cell cycle regulators in cytokinesis, abscission, and cellular events during later morphogenesis. Chapter I outlines the contribution of separase to cytokinesis, highlight how the protease activity of separase regulates exocytosis in anaphase, and suggesting that an unknown substrate is involved in separase’s regulation of exocytosis. Chapter II elucidates how programmed cytokinesis in different tissues contributes to later cellular events during morphogenesis and uncovers the novel migration pattern of midbody to apical surface. Finally, in Chapter III, we present several live imaging methods for observing C. elegans embryogenesis which were applied for this study. Collectively, the work presented here addresses the roles of these master cell cycle regulators in exocytosis, cytokinesis, abscission, and later developmental events, which is critical to understand how failure of cell division promote tumorigenesis and aneuploidy. Finally, our study may provide insightful ideas to generate clinical technologies to cure human infertility, cancer and other genetic diseases

Effet de taille et du dopage sur la structure, les transitions et les propriétés optiques de particules du multiferroïque BiFeO₃ pour des applications photocatalytiques

This experimental PhD work has been dedicated to the synthesis, by wet chemistry methods, and characterization of nanoparticles based on multiferroic BiFeO3, with the aim of using them for photocatalytic applications. This material presents a bandgap of 2.6eV, which allows the charge carrier photoexcitation in the visible range, making BiFeO3 a very interesting system for photoinduced processes. This thesis has been particularly focused on characterizing the properties of BiFeO3 nanoparticles in view of understanding the relationship of their properties on their potential use for photocatalytic applications. First of all, the topic of the size effect on the structural properties, phase transitions, and physics and chemistry of the particles has been developed, keeping as first aim to separate the properties related to the surface from those arising from the bulk/core of the particle. To do so, the mastering and optimization of the synthesis processes of BiFeO3 particles at the nano and microscale were needed, to finally obtain different size compounds with high crystalline quality. Despite the size reduction of the particles, we notice that, thanks to the control of the synthesis process, our BiFeO3 nanoparticles present properties very close to those of the bulk BiFeO3 material, keeping the rhombohedral structure R3c with weak strain effects. In order to indirectly tune the optical properties exploiting the doping, we have succeeded in realizing a homogenous La3+ doping, and a partial Ca2+ doping, on the Bi3+ site. The optical properties of the nanoparticles and their use on the first photocatalytic experiments for degrading rhodamine B dye have shown the complexity of the physics and chemistry phenomena at their surface and of the light-particle processes. After analyzing optical absorbance data as a function of the particle size, we observe that the deduced bandgap for different particles is not the main parameter directing the photocatalytic performances. Other factors have been identified to be at the origin of the localization of the photoexcited charges, as the surface states linked to the skin layer of the nanoparticles, depicting structural defects, a reduction of the oxidation state of Fe3+ towards Fe2+ and the stabilization of other adsorbates, such as FeOOH; all these parameters may contribute to the change on the photocatalytic performances. The photocatalytic results are very encouraging, motivating to continue the study of BiFeO3 based nanoparticles, though depicting a 50% rhodamine B degradation after 4h of photocatalytic reaction using some of the present nanoparticles.Ce travail de thèse expérimentale a été consacré à la synthèse par des méthodes de chimie par voie humide de nanoparticules à base du multiferroïque BiFeO3 et à leur caractérisation, avec comme objectif finale des applications photocatalytiques. Ce matériau présente une bande interdite, avec un gap de 2.6eV, qui permet la photo-génération de porteurs de charges dans le visible faisant ainsi de BiFeO3 un système intéressant pour des processus photo-induits. Ce travail s’est en particulier focalisé à caractériser les propriétés de nanoparticules à base de BiFeO3 en vue de comprendre l’effet de ses propriétés sur leur potentiel dans des applications liées à la photocatalyse. Tout d’abord, l’étude des effets de taille sur les propriétés structurales, de transitions de phase, et physico-chimiques des particules a été réalisée, en gardant comme principal objectif de découpler les propriétés liées à la surface de celles du massif/cœur de la particule. Pour cela, une maîtrise et une optimisation des procédés de synthèse de particules aux échelles nano- et micro-micrométriques de BiFeO3 a été nécessaire pour obtenir des composés de taille variable et de très bonne qualité cristalline. Malgré la diminution de la taille des particules, on constate que, grâce au contrôle de paramètres de synthèse, nos nanoparticules présentent des propriétés très proches à celles du massif de BiFeO3, gardant la structure rhomboédrique R3c avec des faibles effets de contrainte. Afin de contrôler indirectement par le dopage les propriétés optiques des composés à base de BiFeO3, on a réussi à réaliser un dopage très homogène en La3+, et un dopage partiel en Ca2+, sur le site de Bi3+. Les propriétés optiques des nanoparticules et leurs applications dans les premières expériences photocatalytiques sur la dégradation du colorant rhodamine B ont montré la complexité de la physico-chimie de leur surface et du processus d’interaction lumière-particule. Après analyse des données d’absorbance optique en fonction de la taille de nanoparticules, on observe que la bande interdite déduite pour ces différentes particules n’est pas le facteur prédominant sur les performances photocatalytiques. D’autres facteurs ont pu être identifiés comme étant à l’origine de la localisation de charges photo-générées, tels que des états de surface liés à une fine couche de peau ou skin layer sur les nanoparticules, présentant des défauts structuraux, une réduction de l’état d’oxydation du Fe3+ vers le Fe2+ et la stabilisation d’autres adsorbats, tels que FeOOH ; tous ces facteurs peuvent contribuer au changement dans les performances photocatalytiques. Les résultats photocatalytiques restent très encourageants pour poursuivre les études de nanoparticules à base de BiFeO3, montrant une dégradation de la rhodamine B à 50% au bout de 4h de réaction photocatalytique pour certaines des nanoparticules étudiées

Building Marginal Pattern Library with Unbiased Training Dataset for Enhancing Model-Free Load-ED Mapping

Input-output mapping for a given power system problem, such as loads versus economic dispatch (ED) results, has been demonstrated to be learnable through artificial intelligence (AI) techniques, including neural networks. However, the process of identifying and constructing a comprehensive dataset for the training of such input-output mapping remains a challenge to be solved. Conventionally, load samples are generated by a pre-defined distribution, and then ED is solved based on those load samples to form training datasets, but this paper demonstrates that such dataset generation is biased regarding load-ED mapping. The marginal unit and line congestion (i.e., marginal pattern) exhibit a unique characteristic called step change in which the marginal pattern changes when the load goes from one critical loading level (CLL) to another, and there is no change of marginal units within the interval of the two adjacent CLLs. Those loading intervals differ significantly in size. The randomly generated training dataset overfills intervals with large sizes and underfits intervals with small sizes, so it is biased. In this paper, three algorithms are proposed to construct a marginal pattern library to examine this bias according to different computational needs, and an enhancement algorithm is proposed to eliminate the bias for the load-ED dataset generation. Three illustrative test cases demonstrate the proposed algorithms, and comparative studies are constructed to show the superiority of the enhanced, unbiased dataset

Otitis Media in Sperm-Associated Antigen 6 (Spag6)-Deficient Mice

Mammalian SPAG6 protein is localized to the axoneme central apparatus, and it is required for normal flagella and cilia motility. Recent studies demonstrated that the protein also regulates ciliogenesis and cilia polarity in the epithelial cells of brain ventricles and trachea. Motile cilia are also present in the epithelial cells of the middle ear and Eustachian tubes, where the ciliary system participates in the movement of serous fluid and mucus in the middle ear. Cilia defects are associated with otitis media (OM), presumably due to an inability to efficiently transport fluid, mucus and particles including microorganisms. We investigated the potential role of SPAG6 in the middle ear and Eustachian tubes by studying mice with a targeted mutation in theSpag6 gene. SPAG6 is expressed in the ciliated cells of middle ear epithelial cells. The orientation of the ciliary basal feet was random in the middle ear epithelial cells of Spag6-deficient mice, and there was an associated disrupted localization of the planar cell polarity (PCP) protein, FZD6. These features are associated with disordered cilia orientation, confirmed by scanning electron microscopy, which leads to uncoordinated cilia beating. The Spag6 mutant mice were also prone to develop OM. However, there were no significant differences in bacterial populations, epithelial goblet cell density, mucin expression and Eustachian tube angle between the mutant and wild-type mice, suggesting that OM was due to accumulation of fluid and mucus secondary to the ciliary dysfunction. Our studies demonstrate a role for Spag6 in the pathogenesis of OM in mice, possibly through its role in the regulation of cilia/basal body polarity through the PCP-dependent mechanisms in the middle ear and Eustachian tubes

Genetic Identification of Separase Regulators in Caenorhabditis elegans

Separase is a highly conserved protease required for chromosome segregation. Although observations that separase also regulates membrane trafficking events have been made, it is still not clear how separase achieves this function. Here, we present an extensive ENU mutagenesis suppressor screen aimed at identifying suppressors of sep-1(e2406), a temperature-sensitive maternal effect embryonic lethal separase mutant that primarily attenuates membrane trafficking rather than chromosome segregation. We screened nearly a million haploid genomes and isolated 68 suppressed lines. We identified 14 independent intragenic sep-1(e2406) suppressed lines. These intragenic alleles map to seven SEP-1 residues within the N-terminus, compensating for the original mutation within the poorly conserved N-terminal domain. Interestingly, 47 of the suppressed lines have novel mutations throughout the entire coding region of the pph-5 phosphatase, indicating that this is an important regulator of separase. We also found that a mutation near the MEEVD motif of HSP-90, which binds and activates PPH-5, also rescues sep-1(e2406) mutants. Finally, we identified six potentially novel suppressor lines that fall into five complementation groups. These new alleles provide the opportunity to more exhaustively investigate the regulation and function of separase

Muscovy duck reovirus p10.8 protein localizes to the nucleus via a nonconventional nuclear localization signal

BACKGROUND: It was previously report that the first open reading frame of Muscovy duck reocvirus S4 gene encodes a 95-amino-acid protein, designed p10.8, which has no sequence similarity to other known proteins. Its amino acid sequence offers no clues about its function. RESULTS: Subcellular localization and nuclear import signal of p10.8 were characterized. We found that p10.8 protein localizes to the nucleus of infected and transfected cells, suggesting that p10.8 nuclear localization is not facilitated by viral infection or any other viral protein. A functional non-canonical nuclear localization signal (NLS) for p10.8 was identified and mapped to N-terminus residues 1–40. The NLS has the ability to retarget a large cytoplasmic protein to the nucleus. CONCLUSIONS: p10.8 imported into the nucleus might via a nonconventional signal nuclear signal

Understanding Large Language Model Based Fuzz Driver Generation

Fuzz drivers are a necessary component of API fuzzing. However, automatically generating correct and robust fuzz drivers is a difficult task. Compared to existing approaches, LLM-based (Large Language Model) generation is a promising direction due to its ability to operate with low requirements on consumer programs, leverage multiple dimensions of API usage information, and generate human-friendly output code. Nonetheless, the challenges and effectiveness of LLM-based fuzz driver generation remain unclear. To address this, we conducted a study on the effects, challenges, and techniques of LLM-based fuzz driver generation. Our study involved building a quiz with 86 fuzz driver generation questions from 30 popular C projects, constructing precise effectiveness validation criteria for each question, and developing a framework for semi-automated evaluation. We designed five query strategies, evaluated 36,506 generated fuzz drivers. Furthermore, the drivers were compared with manually written ones to obtain practical insights. Our evaluation revealed that: while the overall performance was promising (passing 91% of questions), there were still practical challenges in filtering out the ineffective fuzz drivers for large scale application; basic strategies achieved a decent correctness rate (53%), but struggled with complex API-specific usage questions. In such cases, example code snippets and iterative queries proved helpful; while LLM-generated drivers showed competent fuzzing outcomes compared to manually written ones, there was still significant room for improvement, such as incorporating semantic oracles for logical bugs detection.Comment: 17 pages, 14 figure