Colorectal cancer is one of the most prevalent malignancies globally and recently introduced digital pathology enables the use of machine learning as an aid for fast diagnostics. This project aimed to develop a deep neural network model to specifically identify and differentiate dysplasia in the epithelium of colorectal polyps and was posed as a binary classification problem. The available dataset consisted of 80 whole slide images of different H&E-stained polyp sections, which were parted info smaller patches, annotated by a pathologist. The best performing model was a pre-trained ResNet-18 utilising a weighted sampler, weight decay and augmentation during fine tuning. Reaching an area under precision-recall curve of 0.9989 and 97.41% accuracy on previously unseen data, the model’s performance was determined to underperform compared to the task’s intra-observer variability and be in alignment with the inter-observer variability. Final model made publicly available at https://github.com/stinabr/classification-of-colorectal-polyps.

With the evolution of GPU performance, the interest of using AI for all kinds of purposes has risen. Companies today put a great amount of resources to find new ways of using AI to increase the value of their products or automating processes. An area in the wood industry where AI is widely used and studied is in defect detection. In this thesis, the combination of using AI and hyperspectral images is studied and evaluated in the case of segmenting defects in hardwood with a U- Net network structure. The performance is compared to another known method usually used when dealing with high-dimensional data: PLS-DA. This thesis also compares the use of RGB image data in combination with AI, to further analyze the usefulness that the hyperspectral data provide. The results showed signs of improvement when using hyperspectral images com- pared to RGB images when detecting blue stain and red heartwood defects. De- tection of the defects rot and knots did however show no sign of improvements. Due to the annotations being more accurate in the RGB data, the results from the hyperspectral data-fed networks would suggest that blue stain and red heartwood could be of interest regarding further investigation. Computational performance is shown to vary across the different reduction meth- ods, and the results from this thesis provides some insight that might aid in the reasoning regarding how to choose an appropriate reduction method.

Als Kapazitation bezeichnet man den im weiblichen Reproduktionstrakt stattfindenden Reifungsschritt, der Spermien das volle Fertilisierungspotential verleiht. Die molekularbiologischen Grundlagen dieses für eine erfolgreiche natürliche oder auch artifizielle Befruchtung essenziellen Prozesses sind bis heute nur unvollständig verstanden. Im Rahmen der vorliegenden Dissertation wurden die mit der Kapazitation einhergehenden funktionellen und strukturellen spermalen Veränderungen untersucht. Die kapazitative Stimulation führte zu einer gesteigerten Motilität bis hin zur Hyperaktivierung, zu einer vermehrt induzierten Akrosomenreaktion und zu einer deutlich reduzierten Apoptoseaktivität. Anhand von Inhibitionsexperimenten wurde die Rolle der potentiellen Signaltransduktoren Caspase-1, Calpain und Calmodulin analysiert. Dabei wies die Calmodulinantagonisierung auf eine ausgeprägte Calciumabhängigkeit aller untersuchten kapazitationsassoziierten Prozesse hin. Die Hemmung von Caspase-1 und Calpain führte zu einer Beeinträchtigung der Motilität und der Akrosomenreaktion ohne das Ausmaß der Apoptoseinduktion zu beeinflussen. Die vorstehend genannten Erkenntnisse wurden zur Evaluation verschiedener Ejakulataufbereitungsprotokolle genutzt. Dabei konnte gezeigt werden, dass die Kombination des modernen Verfahrens der immunomagnetische Zellseparation mit der etablierten Methode der Dichtegradientenzentrifugation dem einfachen Standard in Bezug auf die Anreicherung hochmotiler Spermien mit minimaler Apoptoseaktivität aus frischen wie auch aus kryokonservierten Ejakulaten deutlich überlegen war. Bedeutsam im Hinblick auf eine mögliche pratische Anwendung der immunomagnetischen Zellseparation erscheint der Befund, dass die durch das kombinierte Anreicherungsverfahren erhaltene Spermatozoensubpopulation im Hamsteroozytenpenetrationstest ein signifikant höheres Fertilisierungspotential zeigte.

Abstract The function of the transmembrane protein FasL is to complex with the Fas receptor in a target cell and induce target cell apoptosis. Fas/FasL-mediated apoptosis plays important role in immunoregulation. FasL expression is mostly seen in activated lymphocytes. We have characterized endothelial FasL expression in different functional compartments of lymph nodes and gut-associated lymphoid tissue. Furthermore, we have explored the functional role of endothelial FasL expression by analyzing correlation with apoptosis of lymphocyte subpopulations in lymph nodes and by assessing endothelial expression under different conditions by activation of immune functions in gastrointestinal mucosa. Immunohistochemical stainings (Fas, FasL, CD3, CD20, CD19, CD23, CD56, FVIII) were performed on 20 reactive lymph node tissues (I and II), 60 pediatric endoscopy biopsy samples (III) or 60 samples from gut resections (IV). A double-staining method combining apoptosis detection with the TUNEL-method and lymphocyte classification with FasL, Fas and cell lineage markers was optimized. Patient groups included non-pathological lymph nodes, pediatric cow’s milk-sensitive enteropathy, pediatric celiac disease, appendicitis, ulcerative colitis and Crohn’s disease. Control groups included normal biopsy samples from pediatric patients and non-pathological resecate samples from the appendix, colon or ileum to correspond to patient groups. Quantitative analysis (positive vessels or cells per mm2) was performed thoroughly for each anatomical region. In a subset of patients, soluble FasL in the serum was quantified with standard enzyme-linked immunosorbent assay. In reactive lymph nodes FasL expression was predominantly present in high endothelial venules located in the paracortical area, where apoptotic T and B lymphocytes, some expressing Fas, were subsequently found. In the gut wall vascular FasL expression was seen in high endothelial vessels near lymphoid follicles. Serum FasL was elevated in children with an abundance of mucosal lymphoid follicles. In IBD, vascular FasL was upregulated in ulcers and in the submucosa of colons affected by Crohn’s disease. The results indicate that endothelial FasL is characteristically present in high endothelial venules of lymphoid tissues. Detection of apoptotic Fas expressing lymphocytes adjacent to such vessels supports the idea that endothelial FasL functions as a selective gatekeeper by inducing apoptosis of Fas+ lymphocytes entering from the blood stream. / Tiivistelmä Solukalvon läpäisevän proteiinin, FasL:n, tehtävä on sitoutua kohdesolun Fas-reseptoriin ja indusoida kohdesolun apoptoosi. Fas/FasL-välitteinen apoptoosi on merkittävä tekijä immunologisessa säätelyssä. FasL ilmentyy pääsääntöisesti aktivoituneissa lymfosyyteissä. Olemme kuvanneet tutkimuksessamme FasL:n endoteelistä ilmentymistä imukudoksen eri toiminnallisissa alueissa ja suoliston lymfaattisessa kudoksessa. Lisäksi kartoitimme endoteelin FasL:n toiminnallista merkitystä analysoimalla sen yhteyttä lymfosyyttien alaryhmien apoptoosiin imusolmukkeissa ja arvioimalla FasL:n endoteelistä ilmentymistä suoliston limakalvon immunologisesti erilaisissa sairauksissa. Teimme immunohistokemiallisia värjäyksiä (Fas, FasL, CD3, CD20, CD19, CD23, CD56 ja FVIII) 20 reaktiiviselle imusolmukkeelle (I ja II), 60 lapsen endoskooppiselle biopsianäytteelle (III) sekä 60 suoliresekaattinäytteelle (IV). Optimoimme kaksoisvärjäysmenetelmän, missä yhdistettiin apoptoosin havainnointimenetelmä TUNEL ja FasL-, Fas- tai solulinjamarkkeri. Potilasryhmiin kuului potilaita, joilla oli normaalit imusolmukkeet, sekä potilaita, jotka sairastivat lasten viivästynyttä lehmänmaitoallergiaa, lasten keliakiaa, umpilisäketulehdusta, haavaista paksusuolitulehdusta tai Crohnin tautia. Verrokkiryhmiin kuului normaaleja biopsianäytteitä lapsipotilailta sekä terveitä resekaattinäytteitä umpilisäkkeestä sekä paksu- tai sykkyräsuolesta potilasryhmien mukaisesti. Jokaiselle anatomiselle alueelle suoritimme perusteellisen määrällisen analyysin (positiivista suonta tai solua per mm2). Osalle ryhmistä suoritimme seerumin liukoisen FasL:n määrityksen entsyymivälitteisellä immunosorbenttimäärityksellä. Reaktiivisissa imusolmukkeissa FasL:n ilmentyminen näkyi pääsääntöisesti parakortikaalialueen korkeaendoteelisissä venuleissa, missä myös apoptoottiset T- ja B-lymfosyytit (joista osa ilmensi Fasia) sittemmin näkyivät. Suoliston seinämässä havaitsimme verisuoniperäistä FasL:n ilmentymistä korkeaendoteelisissä suonissa lymfaattisten itukeskusten lähettyvillä. Niillä lapsipotilailla, joilla havaitsimme limakalvon lymfaattisten itukeskuksien lisääntymistä, oli myös seerumin FasL-pitoisuus koholla. Tulehduksellisissa suolistosairauksissa verisuoniperäinen FasL oli lisääntynyt limakalvon haavaumissa sekä Crohnin tautia sairastavien potilaiden submukoosassa. Tulokset osoittavat verisuoniperäisen FasL:n tyypillisesti ilmentyvän imukudoksen korkeaendoteelisissa suonissa. Apoptoosin havaitseminen Fasia ilmentävissä lymfosyyteissä näiden suonien läheisyydessä tukee ajatusta siitä, kuinka verisuoniperäinen FasL toimii valikoivana portinvartijana ja aiheuttaa Fas-positiivisten lymfosyyttien apoptoosin estämällä niiden pääsyn verenkierrosta.

Als Kapazitation bezeichnet man den im weiblichen Reproduktionstrakt stattfindenden Reifungsschritt, der Spermien das volle Fertilisierungspotential verleiht. Die molekularbiologischen Grundlagen dieses für eine erfolgreiche natürliche oder auch artifizielle Befruchtung essenziellen Prozesses sind bis heute nur unvollständig verstanden. Im Rahmen der vorliegenden Dissertation wurden die mit der Kapazitation einhergehenden funktionellen und strukturellen spermalen Veränderungen untersucht. Die kapazitative Stimulation führte zu einer gesteigerten Motilität bis hin zur Hyperaktivierung, zu einer vermehrt induzierten Akrosomenreaktion und zu einer deutlich reduzierten Apoptoseaktivität. Anhand von Inhibitionsexperimenten wurde die Rolle der potentiellen Signaltransduktoren Caspase-1, Calpain und Calmodulin analysiert. Dabei wies die Calmodulinantagonisierung auf eine ausgeprägte Calciumabhängigkeit aller untersuchten kapazitationsassoziierten Prozesse hin. Die Hemmung von Caspase-1 und Calpain führte zu einer Beeinträchtigung der Motilität und der Akrosomenreaktion ohne das Ausmaß der Apoptoseinduktion zu beeinflussen. Die vorstehend genannten Erkenntnisse wurden zur Evaluation verschiedener Ejakulataufbereitungsprotokolle genutzt. Dabei konnte gezeigt werden, dass die Kombination des modernen Verfahrens der immunomagnetische Zellseparation mit der etablierten Methode der Dichtegradientenzentrifugation dem einfachen Standard in Bezug auf die Anreicherung hochmotiler Spermien mit minimaler Apoptoseaktivität aus frischen wie auch aus kryokonservierten Ejakulaten deutlich überlegen war. Bedeutsam im Hinblick auf eine mögliche pratische Anwendung der immunomagnetischen Zellseparation erscheint der Befund, dass die durch das kombinierte Anreicherungsverfahren erhaltene Spermatozoensubpopulation im Hamsteroozytenpenetrationstest ein signifikant höheres Fertilisierungspotential zeigte.

Les poly(ADP-ribose) polymérases (PARP) sont des enzymes qui modifient les protéines ou l’ADN par ADP-ribosylation à l’aide du nicotinamide adénine dinucléotide (NAD+). Tankyrase est une PARP qui régule divers processus cellulaires, tels que la maintenance des télomères, la signalisation Wnt et le traitement de l’ARN. Elle est constituée de groupes de répétitions d’ankyrine (ARC) N-terminaux qui se lient aux protéines substrats, d’un domaine de motif α stérile intermédiaire qui médie la polymérisation avec d’autres molécules de tankyrase et d’un domaine catalytique C-terminal. Étant donné que l’activité de la tankyrase favorise la signalisation pro-oncogène Wnt/β-caténine, des inhibiteurs sous forme de petites molécules ont été développés pour cibler son domaine catalytique et se sont révélés prometteurs pour le traitement du cancer colorectal. Certaines protéines se lient à la tankyrase, mais plutôt que d’être ciblées pour l’ADP-ribosylation, elles inhibent son activité catalytique. Ces partenaires de liaison comprennent la GDP-mannose 4,6-déshydratase (GMD) et le gène 4 associé à la prostate (PAGE4). Il est important de noter que la structure de l’enzyme tankyrase complète n’a pas encore été déterminée, et on ignore actuellement pourquoi certaines protéines qui se lient à la tankyrase sont modifiées par l’ADP-ribose, tandis que d’autres restent inchangées et inhibent plutôt son activité catalytique. Comprendre ce mécanisme d’inhibition par analyse structurale pourrait fournir de nouvelles voies thérapeutiques bloquant la signalisation Wnt/β-caténine en ciblant le domaine N-terminal de la tankyrase. Notre objectif était d’utiliser la microscopie à coloration négative et la cryomicroscopie électronique pour déterminer les structures de la tankyrase seule et en complexe avec GMD et PAGE4. Nous avons observé que la tankyrase 1 s’assemble en double hélice lors de la polymérisation, créant des contacts interdomaines susceptibles de faciliter ses rôles catalytiques et d’échafaudage dans la signalisation cellulaire. De plus, GMD compactée et ARC1–5 de la tankyrase 1 se lient dans un rapport molaire 1:1 pour former un complexe bilobé, ce qui aide à expliquer pourquoi la GMD reste inchangée. Ces connaissances permettront le développement d’interventions cliniques plus efficaces ciblant les ARC N-terminaux ainsi que les contacts interdomaines de la tankyrase humaine. / Poly(ADP-ribose) polymerases (PARPs) are enzymes that modify proteins or DNA by ADP-ribosylation using nicotinamide adenine dinucleotide (NAD+). Tankyrase is a PARP that regulates diverse cellular processes, such as telomere maintenance, Wnt signaling, and RNA processing. It is made up of N-terminal ankyrin repeat clusters (ARCs) that bind substrate proteins, a middle sterile α motif domain that mediates polymerization with other tankyrase molecules, and a C-terminal catalytic domain. Due to the fact that tankyrase activity promotes pro-oncogenic Wnt/β-catenin signaling, small molecule inhibitors have been developed that target its catalytic domain and have shown promise for the treatment of colorectal cancer. Certain proteins bind tankyrase, but rather than being targeted for ADP-ribosylation, they inhibit its catalytic activity. These binding partners include GDP-mannose 4,6-dehydratase (GMD) and prostate-associated gene 4 (PAGE4). Importantly, the structure of the full-length tankyrase enzyme has yet to be determined, and it is currently unknown why some proteins that bind to tankyrase are modified with ADP-ribose, while others remain unmodified and instead inhibit its catalytic activity. Understanding this mechanism of inhibition by structural analysis could provide new therapeutic avenues that oppose Wnt/β-catenin signaling by targeting the N-terminal domain of tankyrase. We aimed to use negative stain and cryo-electron microscopy to determine the structures of tankyrase alone and in complex with GMD and PAGE4. We observed that tankyrase 1 assembles as a double helix upon polymerization, creating interdomain contacts that may facilitate its catalytic and scaffolding roles in cellular signaling. Moreover, compacted GMD and ARC1–5 of tankyrase 1 bind in a 1:1 molar ratio to form a bilobal complex, which aids in explaining why GMD remains unmodified. These insights will allow for the development of more effective clinical interventions targeting the N-terminal ARCs as well as interdomain contacts of human tankyrase.

Stains and stained incipient lesions can be challenging to differentiate with established clinical tools. New diagnostic techniques are required for improved distinction to enable early noninvasive treatment. This in vitro study evaluates the performance of artificial intelligence (AI)-based classification of hyperspectral imaging data for early occlusal lesion detection and differentiation from stains. Sixty-five extracted permanent human maxillary and mandibular bicuspids and molars (International Caries Detection and Assessment System [ICDAS] II 0–4) were imaged with a hyperspectral camera (Diaspective Vision TIVITA® Tissue, Diaspective Vision, Pepelow, Germany) at a distance of 350 mm, acquiring spatial and spectral information in the wavelength range 505–1000 nm; 650 fissural spectra were used to train classification algorithms (models) for automated distinction between stained but sound enamel and stained lesions. Stratified 10-fold cross-validation was used. The model with the highest classification performance, a fine k-nearest neighbor classification algorithm, was used to classify five additional tooth fissural areas. Polarization microscopy of ground sections served as reference. Compared to stained lesions, stained intact enamel showed higher reflectance in the wavelength range 525–710 nm but lower reflectance in the wavelength range 710–1000 nm. A fine k-nearest neighbor classification algorithm achieved the highest performance with a Matthews correlation coefficient (MCC) of 0.75, a sensitivity of 0.95 and a specificity of 0.80 when distinguishing between intact stained and stained lesion spectra. The superposition of color-coded classification results on further tooth occlusal projections enabled qualitative assessment of the entire fissure’s enamel health. AI-based evaluation of hyperspectral images is highly promising as a complementary method to visual and radiographic examination for early occlusal lesion detection.

Heteropolymeric O antigen (O-Ag)-capped lipopolysaccharide is the principal constituent of the Gram-negative bacterial cell surface. It is assembled via the integral inner membrane (IM) Wzx/Wzy-dependent pathway. In Pseudomonas aeruginosa, Wzx translocates lipid-linked anionic O-Ag subunits from the cytoplasmic to the periplasmic leaflets of the IM, where Wzy polymerizes the subunits to lengths regulated by Wzz1/2. The Wzx and Wzy IM topologies were mapped using random C-terminal-truncation fusions to PhoALacZα, which displays PhoA/LacZ activity dependent upon its subcellular localization. Twelve transmembrane segments (TMS) containing charged residues were identified for Wzx. Fourteen TMS, two sizeable cytoplasmic loops (CL), and two large periplasmic loops (PL3 and PL5 of comparable size) were characterized for Wzy. Despite Wzy PL3–PL5 sequence homology, these loops were distinguished by respective cationic and anionic charge properties. Site-directed mutagenesis identified functionally-essential Arg residues in both loops. These results led to the proposition of a “catch-and-release” mechanism for Wzy function. The abovementioned Arg residues and intra-Wzy PL3–PL5 sequence homology were conserved among phylogenetically diverse Wzy homologues, indicating widespread potential for the proposed mechanism. Unexpectedly, Wzy CL6 mutations disrupted Wzz1-mediated regulation of shorter O-Ag chains, providing the first evidence for direct Wzy–Wzz interaction. Mutagenesis studies identified functionally-important charged and aromatic TMS residues localized to either the interior vestibule or TMS bundles in a 3D homology model constructed for Wzx. Substrate-binding or energy-coupling roles were proposed for these residues, respectively. The Wzx interior was found to be cationic, consistent with translocation of anionic O-Ag subunits. To test these hypotheses, Wzx was overexpressed, purified, and reconstituted in proteoliposomes loaded with I−. Common transport coupling ions were introduced to “open” the protein and allow detection of I− flux via reconstituted Wzx. Extraliposomal changes in H+ induced I− flux, while Na+ addition had no effect, suggesting H+-dependent Wzx gating. Putative energy-coupling residue mutants demonstrated defective H+-dependent halide flux. Wzx also mediated H+ uptake as detected through fluorescence shifts from proteoliposomes loaded with pH-sensitive dye. Consequently, Wzx was proposed to function via H+-coupled antiport. In summary, this research has contributed structural and functional knowledge leading to novel mechanistic understandings for O-Ag biosynthesis in bacteria. / Bookmarks within the document have been provided for ease of access to a particular section in the body of the thesis. Each entry in the Table of Contents, List of Tables, and List of Figures has been "linked" to its respective position and as such can be clicked for direct access to the entry. Similarly, each in-text Figure or Table reference has been "linked" to its respective figure/table for direct access to the entry. / 1.) Canadian Institutes of Health Research (CIHR) Frederick Banting and Charles Best Canada Graduate Scholarship doctoral award, 2.) CIHR Michael Smith Foreign Study Award, 3.) Cystic Fibrosis Canada (CFC) doctoral studentship, 4.) University of Guelph Dean's Tri-Council Scholarship, 5.) Ontario Graduate Scholarship in Science and Technology, 6.) Operating grants to Dr. Joseph S. Lam from CIHR (MOP-14687) and CFC

This thesis is an exploration of ancestral narratives in the fiction of Thomas Keneally and Christopher Koch. Initially, ancestry in literature creates an historical relationship which articulates the link between the past and the present. In this sense ancestry functions as a type of cultural memory where various issues of inheritance can be negotiated. However, the real value of ancestral narratives lies in their power to aid in the construction of both personal and communal identities. They have the potential to transform these identities, to transgress “natural” boundaries and to reshape conventional identities in the light of historical experience. For Keneally, ancestral narratives depict national forbears who “narrate the nation” into being. His earlier fictions present ancestors of the nation within a mythic and symbolic framework to outline Australian national identity. This identity is static, oppositional, and characterized by the delineation of boundaries which set nations apart from one another. However, Keneally’s more recent work transforms this conventional construction of national identity. It depicts an Irish-Australian diasporic identity which is hyphenated and transgressive: it transcends the conventional notion of nations as separate entities pitted against one another. In this way Keneally’s ancestral narratives enact the potential for transforming identity through ancestral narrative. On the other hand, Koch’s work is primarily concerned with the intergenerational trauma causes by losing or forgetting one’s ancestral narrative. His novels are concerned with male gender identity and the fragmentation which characterizes a self-destructive idea of maleness. While Keneally’s characters recover their lost ancestries in an effort to reshape their idea of what it is to be Australian, Koch’s main protagonist lives in ignorance of his ancestor’s life. He is thus unable to take the opportunity to transform his masculinity due to the pervasive cultural amnesia surrounding his family history and its role in Tasmania’s past. While Keneally and Koch depict different outcomes in their fictional ancestral narratives they are both deeply concerned with the potential to transform national and gender identities through ancestry.

This by-practice project is the first to provide an extensive investigation of the marginalisation of era appropriate science (EAS) and professional investigators by detective and historical detective fiction authors. The purpose of the thesis is to analyse specific detective fiction authors from the earliest formats of the nineteenth century through to the 1990s and contemporary, selected historical detective fiction authors. Its aim is to examine the creation, development and perpetuation of the marginalisation tradition. This generic trend can be read as the authors privileging their detective’s innate skillset, metonymic connectivity and deductive abilities, while underplaying and belittling EAS and professional investigators. Chapter One establishes the project’s critique of the generic trend by considering parental authors, E. T. A Hoffmann, Edgar Allan Poe, Émile Gaboriau and Wilkie Collins. Reading how these authors instigated and purposed the downplaying demonstrates its founding within detective fiction at the earliest point. By comparing how the authors sidelined and omitted specific EAS and professional investigators, alongside science available at the time, this thesis provides a framework for examining how it continued in detective fiction. In following chapters, the framework established in Chapter One and the theoretical views of Charles Rzepka, Lee Horsley, Stephen Knight and Martin Priestman, are used to discuss how minimising EAS and professional investigators developed into a tradition; and became a generic trend in the recognised detective fiction formula that was used by Sir Arthur Conan Doyle, Freeman Wills Crofts, H. C. Bailey, R. Austin Freeman, Agatha Christie, Ruth Rendell and P. D. James. I then examine how the device transferred to historical detective fiction, using the framework to consider Ellis Peters, Umberto Eco and other selected contemporary authors of historical detective fiction. Throughout, the critical aspect considers how the trivialisation developed and perpetuated through a generic trend. The research concludes that there is a trend embedded within detective and historical detective fiction. One that was created, developed and perpetuated by authors to augment their fictional detective’s innate skillset and to help produce narratives using it is a creative process. It further concludes that the trend can be reimagined to plausibly use EAS and professional investigators in detective and historical detective fiction. The aim of the creative aspect of the project is to employ the research and demonstrate how the tradition can be successfully reinterpreted. To do so, the historical detective fiction novel A Hidden Life uses traditional features of the detective fiction formula to support and strengthen plausible EAS and professional investigators within the narrative. The end result is a historical detective fiction novel. One that proves the thesis conclusion and is fundamentally crafted by the critical research.