The notochord is the defining structure of all chordates. A semi-­‐flexible elongated tube of cells, it forms along the central axis of the embryo and provides axial support during development. It also acts as a signalling centre during early embryogenesis, controlling the patterning of a number of tissues and establishing the early body axis of the embryo. In vertebrates, the function of the notochord expands beyond early development. It creates morphogenic gradients for the patterned formation of the vertebral bodies and, in adults, the remnants of the notochord form the nucleus pulposus, a gel-­‐like structure with an integral role in the distribution of vertebral pressure in the intervertebral disc. Little is known about how the notochord copes with damage during embryogenesis, but degeneration of the nucleus pulposus can lead to debilitating spinal disorders. In this thesis, I use a zebrafish model system to present new data that describes the cellular behaviours associated with how the notochord copes with external damage and how this damage can influence the future development of the vertebrae. I have uncovered a novel damage response in the notochord of zebrafish larvae and characterised the morphogenetic changes involved in the process using transgenic fluorescent lines. I have explored the damage in the context of the Wilms’ Tumour 1 (Wt1) gene, a vertebrate-­‐conserved transcription factor, which has recently been associated with several regenerative responses, and discovered that one of its zebrafish orthologues, wt1b, becomes upregulated in the notochord damage response. I have used fluorescent confocal imaging and immunohistochemistry to present new evidence that shows that upon injury, the outer notochord sheath cells upregulate the expression of wt1b. Additionally, I have used time-­‐lapse microscopy to show that damage to the notochord induces novel morphological changes in the injured organ, which include the loss of cellularity of the inner vacuolated cells and the movement of the wt1b-­‐positive outer sheath cells into the injured lumen. Long-­‐term imaging experiments have also demonstrated the capacity of the notochord to heal the damage over time, which ultimately leads to the formation of an extra, smaller vertebra in the wounded area. Skeletal staining of these fish has revealed a previously unknown putative cartilage switch at the site of damage, which leads to the formation of the new vertebral body. This finding has been supported by the microarray analysis of the injured area, which shows the unexpected de-­‐novo expression of cartilage markers at the site of damage The work in this thesis identifies for the first time an endogenous repair mechanism in the notochord of zebrafish larvae and describes the cellular, genetic and molecular processes cotrolling this novel wt1b-­‐associated damage response.

Philosophiae Doctor - PhD / Cancer is a group of diseases that arises from irreversible genomic and epigenomic alterations that result in unrestrained proliferation of abnormal cells. Detailed understanding of the molecular mechanisms underlying a cancer would aid the identification of most, if not all, genes responsible for its progression and the development of molecularly targeted chemotherapy. The challenge of recurrence after treatment shows that our understanding of cancer mechanisms is still poor. As a contribution to overcoming this challenge, we provide an integrative multi-omic analysis on glioblastoma multiforme (GBM) for which large data sets on di erent classes of genomic and epigenomic alterations have been made available in the Cancer Genome Atlas data portal. The rst part of this study involves protein network analysis for the elucidation of GBM tumourigenic molecular mechanisms, identification of driver genes, prioritization of genes in chromosomal regions with copy number alteration, and co-expression and transcriptional analysis. Functional modules were obtained by edge-betweenness clustering of a protein network constructed from genes with predicted functional impact mutations and differentially expressed genes. Pathway enrichment analysis was performed on each module to identify statistical overrepresentation of signaling pathways. Known and novel candidate cancer driver genes were identi ed in the modules, and functionally relevant genes in chromosomal regions altered by homologous deletion or high-level amplication were prioritized with the protein network. Co-expressed modules enriched in cancer biological processes and transcription factor targets were identified using network genes that demonstrated high expression variance. Our findings show that GBM's molecular mechanisms are much more complex than those reported in previous studies. We next identified differentially expressed miRNAs for which target genes associated with the protein network were also differentially expressed. MiRNAs and target genes were prioritized based on the number of targeted genes and targeting miRNAs, respectively. MiRNAs that correlated with time to progression were selected by an elastic net-penalized Cox regression model for survival analysis. These miRNA were combined into a signature that independently predicted adjuvant therapy-linked progression-free survival in GBM and its subtypes and overall survival in GBM. The results show that miRNAs play significant roles in GBM progression and patients' survival finally, a prognostic mRNA signature that independently predicted progression-free and overall survival was identified. Pathway enrichment analysis was carried on genes with high expression variance across a cohort to identify those in chemoradioresistance associated pathways. A support vector machine-based method was then used to identify a set of genes that discriminated between rapidly- and slowly-progressing GBM patients, with minimal 5 % cross-validation error rate. The prognostic value of the gene set was demonstrated by its ability to predict adjuvant therapy-linked progression-free and overall survival in GBM and its subtypes and was validated in an independent data set. We have identified a set of genes involved in tumourigenic mechanisms that could potentially be exploited as targets in drug development for the treatment of primary and recurrent GBM. Furthermore, given their demonstrated accuracy in this study, the identified miRNA and mRNA signatures have strong potential to be combined and developed into a robust clinical test for predicting prognosis and treatment response.

By sensitising the magnetic resonance signal to the diffusion of water molecules in tissue, diffusion-weighted magnetic resonance imaging provides a means of assessing tumour microstructure non-invasively. Such measurements have the potential to provide important information about tumour development and the response of tumours to treatment, but the way in which different tissue properties affect the diffusion-weighted signal remains unclear. Through simulations, in vivo studies and phantom experiments, this thesis investigates the relationship between the diffusion-weighted signal, the pulse sequence parameters used for acquisition, and microstructural properties of tumours. The use of oscillating gradient pulse sequences on a clinical scanner was investigated initially, with theoretical and practical considerations leading subsequent work to focus on pulsed gradient sequences. The forward problem of predicting the diffusion-weighted signal for given combinations of tissue properties and sequence parameters was addressed numerically through Monte Carlo simulations, focussing on how tumour cell size, intracellular volume fraction and membrane permeability affect the signal. These simulations allowed the sensitivity of the signal to changes in these tissue properties to be investigated, revealing how sensitivity depends on sequence parameters as well as the specific microstructural configuration. By repeating the simulations using the specific sequence parameters used in a clinical and preclinical study, the sensitivity of the implemented protocols was assessed, and linked to the experimental findings. The preclinical study illustrated the importance of the diffusion time in determining the sensitivity to treatment-induced changes in tumours, with larger post-treatment signal changes observed at longer diffusion times. These trends were qualitatively reflected in the sensitivity analysis derived from the simulations. Finally, the inverse problem of estimating microstructural properties from the diffusion-weighted signal was addressed using a physical phantom designed as a simple mimic of tumour tissue. By fitting a biophysical model to the diffusion data, the size and volume fraction of the approximately spherical 'cells' were estimated. The radius was slightly underestimated compared with that determined from independent measurements, the fitted volume fraction was plausible, and parameters were found to be estimated with reasonably good precision.

Oncolytic viruses (OVs) have been engineered or selected for cancer cell-specific infection; however, we have found that following intravenous administration of vesicular stomatitis virus (VSV), tumour cell killing rapidly extends far beyond the initial sites of infection. This Bystander Effect is due to the virus’ ability to specifically target tumour vasculature through tumour-specific infection of tumour endothelium and the induction of an inflammatory response resulting in tumour-restricted coagulation, acute vascular disruption, apoptosis and necrosis of the tumour core. VSV-infected tumours, reconstructed in three-dimensions from serial histological sections, revealed that the majority of the tumour mass lacks significant blood flow in contrast to uninfected tumours, which exhibit relatively uniform perfusion. VSV infection rapidly induced intravascular coagulation within 6 hours of intravenous administration. The induction of coagulation was dependent on neutrophils and could be prevented with inhibitors of the coagulation pathway. Normal vasculature was not infected by VSV and no increase in coagulation was observed. Vascular collapse was also observed with the oncolytic poxvirus, JX-594, in patients and preclinical models. Biopsies from patients enrolled in a dose escalation trial for JX-594 were immunoreactive for vaccinia antigens and transgene products in high dose cohorts. Tumour-associated vessels from patients treated with JX-594 were infected with JX-594 and expressed virally encoded transgenes. A decrease in blood flow was also observed 5 days post infection. Several viruses, VSV, JX-594, vvDD, Maraba, and Sindbis, were able to rapidly induce widespread bystander cell death in a subset of mouse models. Tumours responded to OV therapy in three ways, and the type of response was determined by two factors - susceptibility to infection and the heterogeneity of the tumour microenvironment. Heterogeneity correlated with E-cadherin expression. Among tumours that supported viral replication, cancers with low E-cadherin expression were susceptible to vascular collapse. E-cadherin positive tumours were susceptible to infection and direct cell killing but resistant to vascular disruption or bystander cell death. If poorly-differentiated tumours were resistant to infection, no acute cell killing was observed. These histological subtypes provide a potential framework for the rational selection of patients, the integration of combination therapies and the creation of designer viruses to improve the success of OV therapy.

Melanoma is a skin cancer that relies on angiogenesis for growth and progression. Angiogenesis is the growth of new vessels from existing vessels and follows a number of steps that include endothelial cell growth, migration and tubulogenesis. Current anti-angiogenic drugs are not effective in the treatment of melanomas due to serious side effects such as hypertension and the development of resistance. On the other hand, gold nanoparticles (AuNPs) have been reported to be biocompatible in preclinical models. Furthermore, AuNPs were shown to be cytotoxic to prostate cancer cells. In human umbilical vein endothelial cells, AuNPs inhibited the angiogenic protein, vascular endothelial growth factor-A (VEGF-A). Therefore, the study aimed to investigate the possible cytotoxic effects of AuNPs (1.2–3.2 nM) on melanoma cells and angiogenesis parameters (endothelial cell growth and migration) as well as on the levels of angiogenesis promoting proteins, VEGF-A and placental growth factor (PIGF). Melanoma (B16-F10) cells and tumour-derived endothelial (sEnd.2) cells were maintained in an incubator in a humidified atmosphere containing 5% CO2 at a temperature of 37°C. To investigate whether AuNPs were cytotoxic to melanoma cells, the effect of the particles on B16-F10 cell survival was measured using the crystal violet assay. To determine the effects of AuNPs on angiogenesis parameters, endothelial cell (EC) growth and migration were investigated using crystal violet assay and the scratch assay respectively. Also, EC morphology was studied using polarisation-optical interference contrast light microscopy. The enzyme-linked immunosorbent assay (ELISA) was used to determine the effects of AuNPs on the levels of VEGF-A and PIGF. The results showed that AuNPs decreased the viability of melanoma and endothelial cells. The scratch assay showed that more ECs migrated in cultured treated with AuNPs (P < 0.05). The concentration of VEGF-A and PIGF was reduced significantly following treatment with AuNPs, meaning that the particles exhibited anti-angiogenic properties. This outcome provides a basis for further testing of AuNPs as a potential treatment for melanoma. / Dissertation (MSc)--University of Pretoria, 2020. / National Research Foundation / Mintek / Physiology / MSc / Unrestricted

The following master's thesis paper equipped with a short description of CT scans and MR images and the main differences between them, explanation of the structure of convolutional neural networks and how they implemented into biomedical image analysis, besides it was taken a popular modification of U-Net and tested on two loss-functions. As far as segmentation quality plays a highly important role for doctors, in experiment part it was paid significant attention to training quality and prediction results of the model. The experiment has shown the effectiveness of the provided algorithm and performed 100 training cases with the following analysis through the similarity. The proposed outcome gives us certain ideas for future improving the quality of image segmentation via deep learning techniques.

At the Gesellschaft für Schwerionenforschung in Darmstadt a therapy unit for heavy ion cancer treatment has been established in collaboration with the Deutsches Krebsforschungszentrum Heidelberg, the Radiologische Universitätsklinik Heidelberg and the Forschungszentrum Rossendorf. For quality assurance the dual-head positron camera BASTEI (Beta Activity meaSurements at the Therapy with Energetic Ions) has been integrated into this facility. It measures ß+-activity distributions generated via nuclear fragmentation reactions within the target volume. BASTEI has about 4 million coincidence channels. The emission data are acquired in a 3D regime and stored in a list mode data format. Typically counting statstics is two to three orders of magnitude lower than those of typical PET-scans in nuclear medicine. Two iterative 3D reconstruction algorithms based on ISRA (Image Space Reconstruction Algorithm) and MLEM (Maximum Likelihood Expectation Maximization), respectively, have been adapted to this imaging geometry. The major advantage of the developed approaches are run-time Monte-Carlo simulations which are used to calculate the transition matrix. The influences of detector sensitivity variations, randoms, activity from outside of the field of view and attenuation are corrected for the individual coincidence channels. Performance studies show, that the implementation based on MLEM is the algorithm of merit. Since 1997 it has been applied sucessfully to patient data. The localization of distal and lateral gradients of the ß+-activity distribution is guaranteed in the longitudinal sections. Out of the longitudinal sections the lateral gradients of the ß+-activity distribution should be interpreted using a priori knowledge.

Upon responding to cellular stress, p53 protein becomes stabilised and acts as a transcription factor mainly resulting from phosphorylation and acetylation of the protein. Nitration of p53 protein is poorly characterised by comparison with phosphorylation and acetylation. The main aim of this work was to study the effects of nitration on p53 functional activities and on p53-MDM2 protein-protein interactions. Preliminary work was to characterise the nitration of p53 protein over-expressed in E. coli BL21(DE3) which was then purified by a series of column chromatography. GST-MDM2 protein along with control GST protein were also overexpressed in BL21 which were subsequently purified by a single step batch purification before subjected to nitration. Peroxynitrite, a nitrating agent used in this study, was generated in vitro. Preliminary nitration work was carried out using BSA as a model protein as it is easily nitrated owing to its high number of tyrosine residues (19 residues). The present results showed that p53 and GST-MDM2 proteins were hardly nitrated as no strong nitro-tyrosine signals were obtained. This might be due to these proteins, being overexpressed in E. coli, were not properly folded resulting in hidden/cryptic tyrosine residues of which making nitration difficult to achieve. Peroxynitrite was shown to have a degrading property, reducing protein levels of peroxynitrite-treated p53, GST-MDM2 and GST proteins. Immunoprecipitation studies of cancer cell lysates with different p53 status treated with peroxynitrite showed very weak signals of nitro-p53 protein in mutant p53 cells whereby no nitro-p53 protein signal in wild-type p53 MCF7 cells. In addition, NO donor GSNO-treated MCF7 cells showed weak nitro-p53 protein signals. / Ministry of Science, Technology and Innovation (MOSTI) of Malaysia

The integrins play a crucial role in cancer cell proliferation, migration, differentiation, survival and angiogenesis. It has been shown that integrin expression is positively correlated to cancer dissemination, this suggests targeting selected integrins as an anti-metastatic strategy. The aim of this study is to investigate the effect of novel antagonists of α5β1, αvβ3 and αvβ5 integrins on cancer cell migration, a key process in tumour cell dissemination. Immunohistochemistry was used to evaluate the expression of α5, αv, β3 and β5 integrin subunits in prostate cancer tissues. Furthermore the expression of these integrin subunits in tumour and normal human head and neck tissues was compared. The expression profile of these integrin subunits in established human cancer cell lines was subsequently evaluated using immunodetection methods in cells and xenograft tumour samples. The effect of integrin inhibition on cell migration was then assessed using neutralizing antibodies against αvβ3, αvβ5, and α5β1 integrins in the scratch-wound healing assay. This assay was then used to evaluate the potential of novel small molecule integrin antagonists in preventing tumour cell migration. In H & N tissues, αvβ3, αvβ5 and α5β1 integrins are extensively expressed in tumour tissues but weakly expressed in normal tissue from the same patient. Further, prostate cancer tissues expressed variable levels of αvβ3, αvβ5 and α5β1 integrins. αvβ3 and αvβ5 integrins were expressed in variable levels in OSC-19, PC-3, DU145, DLD-1, HT-29, HUVEC, MCF-7, MCF-7ADR and M14 human tumour cell lines and in OSC-19, PC-3, HT-29 and MCF-7 xenografts. α5β1 integrin was expressed in all cell lines and xenografts except in MCF-7 cell line and HT-29 cell line and xenograft. Overall, the expression was elevated in xenografts compared to the corresponding cultured cells. Based on the expression profile and ability of cells to migrate, three cell lines (DLD-1 colon, DU145 prostate and OSC-19 HNSCC) were selected as models to further evaluate the potential of novel small molecule integrin antagonists to inhibit cell migration. The cell lines were characterized by using neutralizing antibodies against αvβ3, αvβ5, and α5β1 integrins to determine which of these three integrins were primarily involved in tumour cell migration. In DLD-1 and DU145, blocking αvβ5 and αvβ3 significantly inhibited migration, whilst the migration of OSC-19 was 50% inhibited by a multi-integrin inhibitor combination. Among the antagonists, ICT9055 and ICT9072 significantly decreased DLD-1 cell migration by 70% and 60% respectively while ICT9023, ICT9024, and ICT9026 significantly decreased DU145 cell migration by 60%, 60% and 50% respectively. The findings suggest that single integrin inhibition is not sufficient to prevent cell migration whereas dual or multiple inhibition is more effective. Two novel anti-migratory agents were identified in colon cancer and three in prostate cancer which would warrant further investigation. / Princess Nora Bint Abdul Rahman University

<p>Loss of chromosome 17p is frequently observed in various cancers. One of the most commonly mutated targets p53 is on chromosome 17p13.1. However, studies have also reported loss of the 17p13.2 region in breast and medulloblastoma, thereby suggesting the residence of potential tumour suppressors in 17p13.2. Cytochrome b5 domain containing 2 (CYB5D2) is located on 17p13.2 implying CYB5D2 being a candidate tumour suppressor. CYB5D2 (neuferricin) belongs to the family of membrane associated progesterone receptors (MAPR). The archetypal member of the family, progesterone receptor membrane component 1 (PGRMC1), has been shown to play a role in domains independently of its function in mediating progesterone signalling. Consistent with this, CYB5D2 was reported to promote neurogenesis and inhibit the proliferation of Neuro2a cells. However, its role in tumorigenesis remains unknown.</p> <p>To investigate the role of CYB5D2 in tumorigenesis, western blot analysis was performed on 20 matched clear cell renal cell carcinomas (ccRCC) and the adjacent non-tumour kidney (ANK) tissues; significant down-regulation of CYB5D2 was demonstrated in ccRCC in comparison to ANK tissues, an observation that was confirmed by immunohistochemistry (IHC) analysis of 9 pairs of ccRCC-ANK tissues. Ectopic expression of CYB5D2 inhibited the proliferation and the invasion of A498 ccRCC along with the inhibition of AKT activation. Collectively, the above results support the possibility of CYB5D2 being a potential tumour suppressor.</p> <p>In support of the results obtained in ccRCC, we were able to show a significant reduction of CYB5D2 in cervical squamous carcinoma compared to normal cervical tissues in our analysis of CYB5D2 expression in 35 cervical squamous tumours. In vitro, overexpression and knockdown of CYB5D2 inhibited and enhanced the invasion of HeLa cells, respectively. As a member of the MAPR family, CYB5D2 contains the signature motif of the family, the cytochrome b5 (cyt-b5) like heme/steroid binding domain. This domain is known for heme binding and research in our laboratory has shown the residue D86 being critical for heme association. Ssubstitution of D86 with G (D86G) abolished not only CYB5D2's ability to bind heme but also its capacity of inhibiting HeLa cell invasion. Taken together, we provide evidence that CYB5D2 possesses activities in suppressing tumorigenesis, at least for the tumorigenesis of ccRCC and cervical squamous carcinoma.</p> / Master of Science (MSc)