Aneuploidy and cell cycle control in the mouse preimplantation embryo

Durant la division cellulaire, la ségrégation des chromosomes et le partage du cytoplasme sont essentiels pour maintenir l'intégrité génomique. Cependant, les erreurs de ségrégation sont fréquentes chez l'embryon préimplantatoire de mammifère et entraînent un gain ou une perte de chromosomes, appelé aneuploïdie. L'aneuploïdie est préjudiciable au développement et est la principale cause de pertes de grossesse. La mitose est coordonnée par cycle cellulaire, notamment la Cycline-B. Comprendre comment la destruction de la Cycline-B contrôle la sortie de la mitose des embryons pourrait expliquer pourquoi l'aneuploïdie est courante en clinique de fertilité. Nous avons étudié la destruction de la Cycline-B en fonction du stade de développement et de l'aneuploïdie. La littérature suggère que l’aneuploïdie perturbe le cycle cellulaire conduisant les cliniques de fertilité à utiliser la durée du cycle cellulaire et la morphologie (morphocinétique) pour prédire la santé de l'embryon. Cependant, la prédiction de la ploïdie par morphocinétique reste à démontrer. Notre objectif était de savoir comment l'aneuploïdie affecte le cycle cellulaire et le développement de l'embryon. Après une micro-injection de CyclineB1:GFP (Cycline-B) et H2B:RFP (chromosomes), les embryons de souris furent imagés par microscopie confocale. Des cellules aneuploïdes furent générées chimiquement pour évaluer leurs morphocinétiques. Curieusement, l'apparition de la Cycline-B après nuclear envelope breakdown a été devancée avec la progression du développement indépendamment de la taille des cellules. De plus, les erreurs de ségrégation ont peu impacté le développement et la destruction de la Cycline-B. Nous concluons que la morphocinétique est un outil prédictif peu fiable pour identifier les embryons aneuploïdes.During cell division, it is essential that chromosome segregation during mitosis, and the partitioning of the cytoplasm at cytokinesis occur in successive timing to maintain genomic integrity. However, segregation errors are frequently observed in the early mammalian embryo, causing daughter cells to inherit whole chromosome gains and losses, termed aneuploidy. Aneuploidy is detrimental to development, being the leading cause of pregnancy loss and developmental disorders. The timing of mitosis is coordinated by the cell cycle component, Cyclin B. Understanding how Cyclin B destruction temporally controls mitotic exit in embryos could help elucidate why aneuploidy is common in IVF clinics. We investigate how Cyclin B destruction changes in different developmental stages and the presence of aneuploidy. Literature suggests aneuploidy disrupts the cell cycle, leading IVF clinics to use cell cycle timings and morphology (morphokinetics) to predict embryo health. However, whether morphokinetics predicts embryo ploidy is uncertain. We seek to investigate how aneuploidy affects the cell cycle and embryo development. We used live-cell confocal imaging and microinjection of CyclinB1:GFP and H2B:RFP mRNA to visualise Cyclin B and chromosomes during mitosis in the 2-, 4- and 8-cell stage mouse embryo. Secondly, we pharmacologically-induced aneuploidy to assess aneuploid morphokinetics. Interestingly, we observe a developmental trend, independent of cell size, where Cyclin B onset begins progressively sooner after NEBD at the 2-, 4- and 8-cell stage. Additionally, chromosome segregation errors had little impact on Cyclin B destruction and development. Finally, we find morphokinetics to be a poor predictive tool in identifying aneuploid embryos

The influence of host and dietary factors on mutagen metabolism in mice

The effects of quantitative and/or qualitative changes to dietary fat and protein on the genotoxicity of dietary carcinogens were evaluated in female BALB/c mice using in vivo and in vitro bacterial mutation assays. An increase in dietary fat content (from 1% to 20%) increased the in vitro activation of MelQ (2-amino-3,4-dimethylimidazo[4,5-f]quinoline) and Trp-P-2 (3-amino-l-methyl-5H-pyrido[4,3b]indole) with the extent of the increase being dependent on the type of fat used although the activation of aflatoxin Bi was unaltered. Changes in in vivo genotoxicity were also seen but these did not always reflect the in vitro results. As the protein content of the diet increased from 5% to 20% there was a decline in the in vivo mutagenicity of aflatoxin Bi (but not MelQ and Trp-P-2). Diet related changes to hepatic enzymes involved in the activation and detoxification of aflatoxin Bi do not explain the reduction in genotoxicity observed in mice fed a high protein diet. Distribution studies indicate that dietary fat and protein may influence the uptake and turnover of mutagens in the liver although fat appeared not to influence the uptake of compounds from the gastro-intestinal tract. The dietary fibre pectin significantly increased caecal nitrate reductase activity but had no apparent effect on the in vivo nitrosation of aminopyrine to the mutagen nitrosodimethylamine. Extreme changes to the gut flora (germ-free mice compared to conventional) however, did result in changes to hepatic metabolism of mutagens in vivo. As the age of mice used in genotoxicity assays increased from 2 - 24 weeks, the mutagenicity in vivo and in vitro of MelQ and Trp-P-2 decreased. Similarly, aflatoxin Bi genotoxicity was reduced in older animals in vivo but not in vitro. Changes in hepatic activation and detoxification capacity of the liver appear to explain these age dependent changes. However age had no consistent effect on gut flora metabolism. To improve the sensitivity of the host-mediated assay, various modifications to the assay were investigated and analysis of this bacterial data was assisted by electron microscopy showing bacterial distribution within the liver

The investigation of a method to generate conformal lattice structures for additive manufacturing

Additive manufacturing (AM) allows a geometric complexity in products not seen in conventional manufacturing. This geometric freedom facilitates the design and fabrication of conformal hierarchical structures. Entire parts or regions of a part can be populated with lattice structure, designed to exhibit properties that differ from the solid material used in fabrication. Current computer aided design (CAD) software used to design products is not suitable for the generation of lattice structure models. Although conceptually simple, the memory requirements to store a virtual CAD model of a lattice structure are prohibitively high. Conventional CAD software defines geometry through boundary representation (B-rep); shapes are described by the connectivity of faces, edges and vertices. While useful for representing accurate models of complex shape, the sheer quantity of individual surfaces required to represent each of the relatively simple individual struts that comprise a lattice structure ensure that memory limitations are soon reached. Additionally, the conventional data flow from CAD to manufactured part is arduous, involving several conversions between file formats. As well as a lengthy process, each conversion risks the generation of geometric errors that must be fixed before manufacture. A method was developed to specifically generate large arrays of lattice structures, based on a general voxel modelling method identified in the literature review. The method is much less sensitive to geometric complexity than conventional methods and thus facilitates the design of considerably more complex structures. The ability to grade structure designs across regions of a part (termed functional grading ) was also investigated, as well as a method to retain connectivity between boundary struts of a conformal structure. In addition, the method streamlines the data flow from design to manufacture: earlier steps of the data conversion process are bypassed entirely. The effect of the modelling method on surface roughness of parts produced was investigated, as voxel models define boundaries with discrete, stepped blocks. It was concluded that the effect of this stepping on surface roughness was minimal. This thesis concludes with suggestions for further work to improve the efficiency, capability and usability of the conformal structure method developed in this work

Impact Absorbent Rapid Manufactured Structures (IARMS)

Rapid Manufacturing (RM) is increasingly becoming a viable manufacturing process due to dramatic advantages that it facilitates in the area of design complexity. Through the exploration of the design freedom afforded by RM, this paper introduces the concept and initial research surrounding Impact Absorbent Rapid Manufactured Structures (IARMS), with an application in sports personal protective equipment (PPE). Designs are based on the cellular structure of foams; the inherent advantages of the cellular structure are used as a basis to create IARMS that have the potential to be optimised for a specific impact absorbent response. The paper provides some initial results from compression testingMechanical Engineerin

A primary effect of palmitic acid on mouse oocytes is the disruption of the structure of the endoplasmic reticulum

Exposure of mouse oocytes to saturated fatty acids (FAs) such as palmitic acid (PA) has been shown to increase lipid content and cause an endoplasmic reticulum (ER) stress response and changes in the mitochondrial redox state. PA can also disrupt Ca2+ stores in other cell types. The links between these intracellular changes, or whether they are prevented by mono-unsaturated FAs such as oleic acid (OA), is unclear. Here, we have investigated the effects of FAs on mouse oocytes, that are maturated in vitro, using coherent anti-Stokes Raman scattering and two-photon fluorescence microscopy. When oocytes were matured in the presence of PA, there were changes in the aggregation pattern and size of lipid droplets that were mitigated by co-incubation in OA. Maturation in PA alone also caused a distinctive disruption of the ER structure. This effect was prevented by incubation of OA with PA. In contrast, maturation of mouse oocytes in medium containing PA was not associated with any significant change in the redox state of mitochondria or the Ca2+ content of intracellular stores. These data suggest that a primary effect of saturated FAs such as PA on oocytes is to disrupt the structure of the ER and this is not due to an effect on the mitochondria or Ca2+ stores

Compressive failure modes and energy absorption in additively manufactured double gyroid lattices

Lattice structures are excellent candidates for lightweight, energy absorbing applications such as personal protective equipment. In this paper we explore several important aspects of lattice design and production by metal additive manufacturing, including the choice of cell size and the application of a post-manufacture heat treatment. Key results include the characterisation of several failure modes in double gyroid lattices made of Al-Si10-Mg, the elimination of brittle fracture and low strain failure by the application of a heat treatment, and the calculation of specific energy absorption under compressive deformation (16x106 J m-3 up to 50% strain). These results demonstrate the suitability of double gyroid lattices for energy absorbing applications, and will enable the design and manufacture of more efficient lightweight parts in the future

An error diffusion based method to generate functionally graded cellular structures

The spatial variation of cell size in a functionally graded cellular structure is achieved using error diffusion to convert a continuous tone image into binary form. Effects of two control parameters, greyscale value and resolution on the resulting cell size measures were investigated. Variation in cell edge length was greatest for the Voronoi connection scheme, particularly at certain parameter combinations. Relationships between these parameters and cell size were identified and applied to an example, where the target was to control the minimum and maximum cell size. In both cases there was an 8% underestimation of cell area for target regions

Strategies for functionally graded lattice structures derived using topology optimisation for additive manufacturing

A number of strategies that enable lattice structures to be derived from Topology Optimisation (TO) results suitable for Additive Manufacturing (AM) are presented. The proposed strategies are evaluated for mechanical performance and assessed for AM specific design related manufacturing considerations. From a manufacturing stand-point, support structure requirement decreases with increased extent of latticing, whereas the design-to-manufacture discrepancies and the processing efforts, both in terms of memory requirements and time, increase. Results from Finite Element (FE) analysis for the two loading scenarios considered: intended loading, and variability in loading, provide insight into the solution optimality and robustness of the design strategies. Lattice strategies that capitalised on TO results were found to be considerably (∼40-50%) superior in terms of specific stiffness when compared to the structures where this was not the case. The Graded strategy was found to be the most desirable from both the design and manufacturing perspective. The presented pros-and-cons for the various proposed design strategies aim to provide insight into their suitability in meeting the challenges faced by the AM design community

The microstructure and mechanical properties of selectively laser melted AlSi10Mg: The effect of a conventional T6-like heat treatment

Selective laser melting (SLM) of aluminium is of research interest because of its potential benefits to high value manufacturing applications in the aerospace and automotive industries. In order to demonstrate the credibility of SLM Al parts, their mechanical properties need to be studied. In this paper, the nano-, micro-, and macro-scale mechanical properties of SLM AlSi10Mg were examined. In addition, the effect of a conventional T6-like heat treatment was investigated and correlated to the generated microstructure. Nanoindentation showed uniform hardness within the SLM material. Significant spatial variation was observed after heat treatment due to phase transformation. It was found that the SLM material's micro-hardness exceeded its die-cast counterpart. Heat treatment softened the material, reducing micro-hardness from 125±1 HV to 100±1 HV. An ultimate tensile strength (333 MPa), surpassing that of the die cast counterpart was achieved, which was slightly reduced by heat treatment (12%) alongside a significant gain in strain-to-failure (~threefold). Significantly high compressive yield strength was recorded for the as-built material with the ability to withstand high compressive strains. The SLM characteristic microstructure yielded enhanced strength under loading, outperforming cast material. The use of a T6-like heat treatment procedure also modified the properties of the material to yield a potentially attractive compromise between the material's strength and ductility making it more suitable for a wider range of applications and opening up further opportunities for the additive manufacturing process and alloy combination