Learning cell representations in temporal and 3D contexts

Cell morphology and its changes under different circumstances is one of the primary ways by which we can understand biology. Computational tools for characterization and analysis, therefore, play a critical role in advancing studies involving cell morphology. In this thesis, I explored the use of representation learning and self-supervised methods to analyze nuclear texture in fluorescence imaging across different contexts and scales. To analyze the cell cycle using 2D temporal imaging data, as well as DNA damage in 3D imaging data, I employed a simple model based on the VAE-GAN architecture. Through the VAE-GAN model, I constructed manifolds in which the latent representations of the data can be grouped and clustered based on textural similarities without the need for exhaustive training annotations. I used these representations, as well as manually engineered features, to perform various analyses both at the single cell and tissue levels. The application on the cell cycle data revealed that common tasks such as cell cycle staging and cell cycle time estimation can be done even with minimal fluorescence information and user annotation. On the other hand, the texture classes derived to characterize DNA damage in 3D histology images unveiled differences between control and treated tissue regions. Lastly, by aggregating cell-level information to characterize local cell neighborhoods, interactions between DNA-damaged cells and immune cells can be quantified and some tissue microstructures can be identified. The results presented in this thesis demonstrated the utility of the representations learned through my approach in supporting biological inquiries involving temporal and 3D spatial data. The quantitative measurements computed using the presented methods have the potential to aid not only similar experiments on the cell cycle and DNA damage but also in exploratory studies in 3D histology

[¹⁸F]fluorination of biorelevant arylboronic acid pinacol ester scaffolds synthesized by convergence techniques

Aim: The development of small molecules through convergent multicomponent reactions (MCR) has been boosted during the last decade due to the ability to synthesize, virtually without any side-products, numerous small drug-like molecules with several degrees of structural diversity.(1) The association of positron emission tomography (PET) labeling techniques in line with the “one-pot” development of biologically active compounds has the potential to become relevant not only for the evaluation and characterization of those MCR products through molecular imaging, but also to increase the library of radiotracers available. Therefore, since the [18F]fluorination of arylboronic acid pinacol ester derivatives tolerates electron-poor and electro-rich arenes and various functional groups,(2) the main goal of this research work was to achieve the 18F-radiolabeling of several different molecules synthesized through MCR. Materials and Methods: [18F]Fluorination of boronic acid pinacol esters was first extensively optimized using a benzaldehyde derivative in relation to the ideal amount of Cu(II) catalyst and precursor to be used, as well as the reaction solvent. Radiochemical conversion (RCC) yields were assessed by TLC-SG. The optimized radiolabeling conditions were subsequently applied to several structurally different MCR scaffolds comprising biologically relevant pharmacophores (e.g. β-lactam, morpholine, tetrazole, oxazole) that were synthesized to specifically contain a boronic acid pinacol ester group. Results: Radiolabeling with fluorine-18 was achieved with volumes (800 μl) and activities (≤ 2 GBq) compatible with most radiochemistry techniques and modules. In summary, an increase in the quantities of precursor or Cu(II) catalyst lead to higher conversion yields. An optimal amount of precursor (0.06 mmol) and Cu(OTf)2(py)4 (0.04 mmol) was defined for further reactions, with DMA being a preferential solvent over DMF. RCC yields from 15% to 76%, depending on the scaffold, were reproducibly achieved. Interestingly, it was noticed that the structure of the scaffolds, beyond the arylboronic acid, exerts some influence in the final RCC, with electron-withdrawing groups in the para position apparently enhancing the radiolabeling yield. Conclusion: The developed method with high RCC and reproducibility has the potential to be applied in line with MCR and also has a possibility to be incorporated in a later stage of this convergent “one-pot” synthesis strategy. Further studies are currently ongoing to apply this radiolabeling concept to fluorine-containing approved drugs whose boronic acid pinacol ester precursors can be synthesized through MCR (e.g. atorvastatin)