Miracle-dz/UniFAR-FTUs huggingface dataset:
Application, Optimisation and Evaluation of Deep Learning for Biomedical Imaging

Wieslander, H. 2022. Application, Optimisation and Evaluation of Deep Learning for Biomedical Imaging. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 2144. 58 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-1489-1. Microscopy imaging is a powerful technique when studying biology at a cellular and sub-cellular level. When combined with digital image analysis it creates an invaluable tool for investigating complex biological processes and phenomena. However, imaging at the cell and sub-cellular level tends to generate large amounts of data which can be difficult to analyse, navigate and store. Despite these difficulties, large data volumes mean more information content which is beneficial for computational methods like machine learning, especially deep learning. The union of microscopy imaging and deep learning thus provides numerous opportunities for advancing our scientific understanding and uncovering interesting and useful biological insights. The work in this thesis explores various means for optimising information extraction from microscopy data utilising image analysis with deep learning. The focus is on three different imaging modalities: bright-field; fluorescence; and transmission electron microscopy. Within these modalities different learning-based image analysis and processing techniques are explored, ranging from image classification and detection to image restoration and translation. The main contributions are: (i) a computational method for diagnosing oral and cervical cancer based on smear samples and bright-field microscopy; (ii) a hierarchical analysis of whole-slide tissue images from fluorescence microscopy and introducing a confidence based measure for pixel classifications; (iii) an image restoration model for motion-degraded images from transmission electron microscopy with an evaluation of model overfitting on underlying textures; and (iv) an image-to-image translation (virtual staining) of cell images from brightfield to fluorescence microscopy, optimised for biological feature relevance. A common theme underlying all the investigations in this thesis is that the evaluation of the methods used is in relation to the biological question at hand.
Single- and duplex TaqMan-quantitative PCR for determining the copy numbers of integrated selection markers during site-specific mutagenesis in Toxoplasma gondii by CRISPR-Cas9

Herein, we developed a single and a duplex TaqMan quantitative qPCR for absolute quantification of copy numbers of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker for pyrimethamine resistance in Toxoplasma gondii knockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the inserted mdhfr-ts and of the wild-type (WT) dhfr-ts (wt-dhfr-ts) which is present as single copy gene in Toxoplasma and encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of the dhfr-ts fragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number of dhfr-ts copies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bp dhfr-ts fragment and the T. gondii 529-bp repeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given by dhfr-ts amplification is equal to the number of tachyzoites determined by amplification of the Toxoplasma 529-bp, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration of mdhfr-ts, the calculated ratio is 2. We then applied both approaches to test T. gondii RH mutants in which the major surface antigen (SAG1) was disrupted through insertion of mdhfr-ts using CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integrated mdhfr-ts. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic of T. gondii KOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions of mdhfr-ts. The principle of the duplex TaqMan qPCR is applicable for other selectable markers in Toxoplasma. TaqMan qPCR tools may contribute to more frequent use of WT Toxoplasma strains during functional genomics.
Predictive model of transcriptional elongation control identifies trans regulatory factors from chromatin signatures

Promoter-proximal Polymerase II (Pol II) pausing is a key rate limiting step for gene expression. DNA and RNA-binding trans-acting factors regulating the extent of pausing have been identified. However, we lack a quantitative model of how interactions of these factors determine pausing, therefore the relative importance of implicated factors is unknown. Moreover, previously unknown regulators might exist. Here we address this gap with a machine learning model that accurately predicts the extent of promoter proximal Pol II pausing from large scale genome and transcriptome binding maps, as well as gene annotation and sequence composition features. We demonstrate high accuracy and generalizability of the model by validation on an independent cell line which reveals the model’s cell line agnostic character. Model interpretation in light of prior knowledge about molecular functions of regulatory factors confirms the interconnection of pausing with other RNA processing steps. Harnessing underlying feature contributions we assess the relative importance of each factor, quantify their predictive effects and systematically identify previously unknown regulators of pausing. We additionally identify 16 previously unknown 7SK ncRNA interacting RNA-binding proteins predictive of pausing. Our work provides a framework to further our understanding of the regulation of the critical early steps in transcriptional elongation. Key Points: Please provide 3 bullet points summarizing the manuscript’s contribution to the field - ML model that accurately predicts promoter proximal Pol II pausing from ChIP and eClip-seq data - Quantification of the interconnection of pausing and other steps of gene regulation - Identification of novel putative trans regulators of pausing GRAPHICAL ABSTRACT
The Chlamydia trachomatis type III-secreted effector protein CteG induces centrosome amplification through interactions with centrin-2

The centrosome is the main microtubule organizing center of the cell and is crucial for mitotic spindle assembly, chromosome segregation, and cell division. Centrosome duplication is tightly controlled, yet several pathogens, most notably oncogenic viruses, perturb this process leading to increased centrosome numbers. Infection by the obligate intracellular pathogen Chlamydia trachomatis (C.t.) correlates with blocked cytokinesis, supernumerary centrosomes, and multipolar spindles; however, the mechanisms behind how C.t. induces these cellular abnormalities from the confines of its inclusion, remain largely unknown. Here we show that the type III secreted effector protein, CteG, binds to centrin-2 (CETN2), a key structural component of centrosomes and regulator of centriole duplication. This interaction requires a functional calcium binding EF hand 4 of CETN2, which is recognized via the C-terminus of CteG. Significantly, we show that deletion of CteG, or knockdown of CETN2, significantly impairs chlamydia’s ability to induce centrosome amplification. Uniquely, we have identified the first bacterial effector to target centrins, crucial regulators of the eukaryotic cell cycle. These findings have not only allowed us to begin addressing how C.t. induces gross cellular abnormalities during infection, but also indicate that obligate intracellular bacteria may contribute to cellular transformation events that negatively impact host physiology even when the pathogen is long removed. Understanding the consequences of CteG-CETN2 interactions, its impact on centrosome amplification, and the long-term effect this has on host cells could explain why chlamydial infection leads to an increased risk of cervical or ovarian cancer. Significance Statement The presence of more than two centrosomes is a hallmark of many types of cancer, including cervical and ovarian cancers of which Chlamydia trachomatis (C.t.) infection is a significant risk factor. Despite the importance of this problem, how C.t. orchestrates these drastic changes in the host cell remains poorly understood. Here, we describe how C.t. uses a single effector protein, CteG, to drive centrosome amplification via manipulation of a key regulator of centriole duplication, centrin-2. This work begins to define how C.t. induces centrosome amplification to promote its replication while potentially contributing to devastating long-term negative consequences for normal host physiology. Further it may help elucidate why chlamydial infection leads to an increased cancer risk.
Phenotypic and genomic signatures of interspecies cooperation and conflict in naturally occurring isolates of a model plant symbiont

Given the need to predict the outcomes of (co)evolution in host-associated microbiomes, whether microbial and host fitnesses tend to trade off, generating conflict, remains a pressing question. Examining the relationships between host and microbe fitness proxies at both the phenotypic and genomic levels can illuminate the mechanisms underlying interspecies cooperation and conflict. We examined naturally-occurring genetic variation in 191 strains of the model microbial symbiont, Ensifer meliloti, paired with each of two host Medicago truncatula genotypes in single- or multi-strain experiments to determine how multiple proxies of microbial and host fitness were related to one another and test key predictions about mutualism evolution at the genomic scale, while also addressing the challenge of measuring microbial fitness. We found little evidence for interspecies fitness conflict; loci tended to have concordant effects on both microbe and host fitnesses, even in environments with multiple co-occurring strains. Our results emphasize the importance of quantifying microbial relative fitness for understanding microbiome evolution and thus harnessing microbiomes to improve host fitness. Additionally, we find that mutualistic coevolution between hosts and microbes acts to maintain, rather than erode, genetic diversity, potentially explaining why variation in mutualism traits persists in nature.
Genome Sequencing Unravelled C3, C4 and CAM Photosynthetic Pathways 5

34 Peepal / Bodhi tree ( Ficus religiosa L.) is an important long-lived keystone ecological species. 35 This plant has been widely used by communities in Indian subcontinent in traditional medicine, 36 Ayurveda and spirituality. Our study aimed to generate molecular resources from whole- 37 genome and transcriptome sequencing approaches. The whole genome of Peepal tree was 38 sequenced using Illumina and MGISEQ-2000 sequencers. We assembled the draft genome 39 (380-Mb) of Peepal tree and annotated 35,093 protein-coding genes; 53% of its genome 40 consists of repetitive sequences. To understand the photosynthetic pathways in leaf tissues, we 41 analysed photosynthetically distinct conditions: bright sunny days and dark nights. The RNA- 42 seq analysis supported the expression of 26,691 genes. The gene expression analysis of the day 43 and night period revealed the molecular switches between the Calvin-Benson (C3) cycle, C4- 44 Dicarboxylic cycle and Crassulacean acid metabolism (CAM) cycle for carbon fixation in 45 leaves of Peepal tree. 46 47
Molecular mechanism of active Cas7-11 in processing CRISPR RNA and interfering target RNA

Cas7-11 is a Type III-E CRISPR Cas effector that confers programmable RNA cleavage and has potential applications in RNA interference. Cas7-11 encodes a single polypeptide containing four Cas7- and one Cas11-like segments that obscures the distinction between the multi-subunit Class 1 and the single-subunit Class-2 CRISPR-Cas systems. We report a cryo-EM structure of the active Cas7-11 from Desulfonema ishimotonii (DiCas7-11) that reveals the molecular basis for RNA processing and interference activities. DiCas7-11 arranges its Cas7- and Cas11-like domains in an extended form that resembles the backbone made up by four Cas7 and one Cas11 subunits in the multi-subunit enzymes. Unlike the multi-subunit enzymes, however, the backbone of DiCas7-11 contains evolutionarily different Cas7 and Cas11 domains, giving rise to their unique functionality. The first Cas7-like domain nearly engulfs the last 15 direct repeat nucleotides and is responsible for processing and recognition of the CRISPR RNA. Whereas both the second and the third Cas7-like domains mediate target RNA cleavage, they differ in metal requirement for catalysis. The long variable insertion to the fourth Cas7-like domain has little impact to RNA processing or targeting, suggesting the possibility for engineering a compact and programmable RNA interference tool. One Sentence Summary Structures of Cas7-11 reveal the molecular basis for processing CRISPR RNA and for cleaving target RNA.
Common mechanism of activated catalysis in P-loop fold nucleoside triphosphatases - in varietate concordia

Although P-loop fold nucleoside triphosphatases (also known as Walker NTPases) are ubiquitous, their catalytic mechanism remains obscure. Based on a comparative structural analysis of 3136 Mg-NTP-containing catalytic sites, we propose a common scheme of activated catalysis for P-loop NTPases where a hydrogen bond (H-bond) between the strictly conserved, Mg-coordinating Ser/Thr of the Walker A motif ([Ser/Thr]WA) and the conserved aspartate of the Walker B motif (AspWB) plays the key role. We found that this H-bond is very short in the structures with bound transition state (TS) analogs. We suggest that the proton affinities of these two residues reverse in the TS so that the proton relocates from [Ser/Thr]WA to AspWB. The anionic [Ser/Thr]WA withdraws then a proton from the (catalytic) water molecule, and the nascent hydroxyl anion attacks gamma-phosphate. When the gamma-phosphate group breaks away, the trapped proton relays from AspWB, via [Ser/Thr]WA, to beta-phosphate and compensates for its developing negative charge.
Feedforward ribosome control mitigates gene activation burden

Heterologous gene activation causes non-physiological burden on cellular resources that cells are unable to adjust to. Here, we introduce a feedforward controller that increases ribosome level upon activation of a gene of interest (GOI) to compensate for such a burden. The controller achieves this by activating a modified SpoT enzyme with sole hydrolysis activity, which lowers ppGpp level and thus de-represses ribosomes. Without the controller, activation of the GOI decreased growth rate by more than 50%. With the controller, we could activate the GOI to the same level without a growth rate decrease. A cell strain armed with the controller in co-culture enabled persistent population-level activation of a GOI, which could not be achieved by a strain devoid of the controller. The feedforward controller is a tunable, modular, and portable tool that for the first time allows dynamic gene activation without growth rate defects for bacterial synthetic biology applications.
The longitudinal cascade development of cosmic-ray showers from observations of atmospheric Cerenkov radiation

This thesis is concerned with the measurement of the longitudinal cascade development of large cosmic-ray showers from observations of atmospheric Cerenkov radiation. The purpose of the measurements is to obtain information on the mass composition of the primary cosmic rays and on the gross features of the high-energy hadronic interactions. The characteristics of the Cerenkov light signal which are strongly coupled to the longitudinal shower development are described with reference to computer simulation results. An experiment designed to measure these characteristics was deployed in Dugway, Utah, U.S.A. between October 1977 and March 1980. Measurements were made in showers of primary energy 10(^15) – 10(^18) eV. Existing data analysis techniques have been refined and new procedures developed in order to optimise the reduction of the digital data. A detailed analysis is given of two aspects of the time structure of the Cerenkov light signal in showers of mean primary energy ~ 2 x 10(^17) eV. The measurements are interpreted in terms of the mean depth of electron cascade maximum and the fluctuations occurring between showers. The many results on the cascade development currently available from the Dugway data are summarised. Interpretation of the results is found to be possible in the framework of scaling-based models of the high energy hadronic interactions which incorporate an enhancement of the central-region multiplicity. This allows certain inferences to be made concerning the primary mass composition. Specifically, primaries of energy ~ 10(^16) eV appear to be predominantly heavy nuclei, and the mass composition becomes lighter with increasing primary energy until at least ~ 2 x 10(^17) eV. While no other single experiment has yet been in a position to corroborate these results, broad consistency is found between the Dugway results and the combined results from a number of other experiments.
In Vitro Evaluation and Mitigation of Niclosamide’s Liabilities as a COVID-19 Treatment

Niclosamide, an FDA-approved oral anthelmintic drug, has broad biological activity including anticancer, antibacterial, and antiviral properties. Niclosamide has also been identified as a potent inhibitor of SARS-CoV-2 infection in vitro, generating interest in its use for the treatment or prevention of COVID-19. Unfortunately, there are several potential issues with using niclosamide for COVID-19, including low bioavailability, significant polypharmacology, high cellular toxicity, and unknown efficacy against emerging SARS-CoV-2 variants of concern. In this study, we used high-content imaging-based immunofluorescence assays in two different cell models to assess these limitations and evaluate the potential for using niclosamide as a COVID-19 antiviral. We show that despite promising preliminary reports, the antiviral efficacy of niclosamide overlaps with its cytotoxicity giving it a poor in vitro selectivity index for anti-SARS-CoV-2 inhibition. We also show that niclosamide has significantly variable potency against the different SARS-CoV-2 variants of concern and is most potent against variants with enhanced cell-to-cell spread including B.1. 1.7. Finally, we report the activity of 33 niclosamide analogs, several of which have reduced cytotoxicity and increased potency relative to niclosamide. A preliminary structure-activity relationship analysis reveals dependence on a protonophore for antiviral efficacy, which implicates nonspecific endolysosomal neutralization as a dominant mechanism of action. Further single-cell morphological profiling suggests niclosamide also inhibits viral entry and cell-to-cell spread by syncytia. Altogether, our results suggest that niclosamide is not an ideal candidate for the treatment of COVID-19, but that there is potential for developing improved analogs with higher clinical translational potential in the future. Importance There is still an urgent need for effective anti-SARS-CoV-2 therapeutics due to waning vaccine efficacy, the emergence of variants of concern, and limited efficacy of existing antivirals. One potential therapeutic option is niclosamide, an FDA approved anthelmintic compound that has shown promising anti-SARS-CoV-2 activity in cell-based assays. Unfortunately, there are significant barriers for the clinical utility of niclosamide as a COVID-19 therapeutic. Our work emphasizes these limitations by showing that niclosamide has high cytotoxicity at antiviral concentrations, variable potency against variants of concern, and significant polypharmacology as a result of its activity as a nonspecific protonophore. Some of these clinical limitations can be mitigated, however, through structural modifications to the niclosamide scaffold, which we demonstrate through a preliminary structure activity relationship analysis. Overall, we show that niclosamide is not a suitable candidate for the treatment of COVID-19, but that structural analogs with improved drug properties may have higher clinical-translational potential.
Optimization of heterojunction c-Si solar cells with front junction architecture

Wafer-based crystalline silicon (c-Si) solar cells currently dominate the photovoltaic (PV) market with high-thermal budget (T > 700 ∘C) architectures (e.g. i-PERC and PERT). However, also low-thermal (T < 250 ∘C) budget heterojunction architecture holds the potential to become mainstream owing to the achievable high efficiency and the relatively simple lithography-free process. A typical heterojunction c-Si solar cell is indeed based on textured n-type and high bulk lifetime wafer. Its front and rear sides are passivated with less than 10-nm thick intrinsic (i) hydrogenated amorphous silicon (a-Si:H) and front and rear side coated with less than 10-nm thick doped a-Si:H layers. Finally, transparent conductive oxide (TCO) and metal at both front and rear side terminate the device. Of course, the front side metal is merely a grid, allowing light to impinge on the c-Si wafer. In this project, an HIT heterojunction architecture is investigated, it is used a double intrinsic passivating layer at the front and an highly hydrogenated (p) a-Si:H forming the emitter; i/n passivating stack at the rear in the role of back surface field; a TCO formed by IO:H and ITO and finally copper as front metal contact.
Cochlear Implantation in Young Mandarin-Speaking Children: One Year After First Fitting

Introduction Bilateral cochlear implantation has been shown to be beneficial in terms of sound localization and speech recognition in children with congenital deafness. However, little is known about the benefits of bilateral cochlear implantationin children who communicate in a tonal language such as Mandarin. This study aims to investigate the auditory perception and speech intelligibility of Mandarin-speaking children 1 year after first fitting. Materials and Methods Twenty-nine children aged between 11.5 and 17.9 months with severe-to-profound bilateral sensorineural hearing loss were recruited; 10 were unilaterally implanted and 19 were bilaterally implanted. A test battery was used to monitor improvements during the first year of cochlear implant use. Results Bilaterally implanted children scored better in the spatial domain of the Speech, Spatial and Qualities of hearing scale for Parents (SSQ-P) in comparison to unilaterally implanted children. Significant improvements were observed in auditory performance and speech intelligibility at 6 and 12 months after first fitting for both groups of children. Discussion Young children that speak a tonal language, such as Mandarin, can obtain significant improvements in hearing and speech abilities within the first year of cochlear implant use. Furthermore, bilateral implantation provides users with better spatial hearing in comparison to unilateral implantation.