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Sequestration of infected erythrocytes during P. falciparum infection can lead to severe malaria. PfEMP1 proteins are central to this process. Multimodular PfEMP1 proteins can bind to a variety of human ligands due to the presence of CIDR and DBL domains. CIDR and DBL domains are classified to three and six families respectively, with each thought to bind to unique human ligands. The interaction between PfEMP1 proteins and human ligands protects the infected erythrocytes against spleen-mediated destruction. Additionally, these PfEMP1 proteins undergo antigenic variation to evade immune surveillance. They also elicit immune suppression by binding to immunoglobulins within the circulatory system. The work described here focuses on two interactions between PfEMP1 on the infected erythrocytes and their human ligands, the first involving CD36 and CIDR1α domains and the second involving human IgM and DBL domains. I have expressed and purified recombinant proteins involved in both interactions. In this thesis, I have confirmed using a variety of biophysical methods that CIDR1α domains are able to bind CD36 in a 1:1 binding stoichiometry, and this accounts for the binding affinity of the full length protein. This interaction was not disrupted after performing deglycosylation procedures on CD36, allowing me to obtain stable complexes of these interacting partners for structural characterisation by crystallography. I have also confirmed the interaction between DBL domains and human IgM Fc region using similar biophysical methods. These experiments are important for paving the way towards further structural characterisation of these interactions that leads to sequestration. .

© 2016 Elsevier Ltd Background Patients with incomplete surgical resection of medulloblastoma are controversially regarded as having a marker of high-risk disease, which leads to patients undergoing aggressive surgical resections, so-called second-look surgeries, and intensified chemoradiotherapy. All previous studies assessing the clinical importance of extent of resection have not accounted for molecular subgroup. We analysed the prognostic value of extent of resection in a subgroup-specific manner. Methods We retrospectively identified patients who had a histological diagnosis of medulloblastoma and complete data about extent of resection and survival from centres participating in the Medulloblastoma Advanced Genomics International Consortium. We collected from resections done between April, 1997, and February, 2013, at 35 international institutions. We established medulloblastoma subgroup affiliation by gene expression profiling on frozen or formalin-fixed paraffin-embedded tissues. We classified extent of resection on the basis of postoperative imaging as gross total resection (no residual tumour), near-total resection (30 Gy vs no craniospinal irradiation). The primary analysis outcome was the effect of extent of resection by molecular subgroup and the effects of other clinical variables on overall and progression-free survival. Findings We included 787 patients with medulloblastoma (86 with WNT tumours, 242 with SHH tumours, 163 with group 3 tumours, and 296 with group 4 tumours) in our multivariable Cox models of progression-free and overall survival. We found that the prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. We identified a progression-free survival benefit for gross total resection over sub-total resection (hazard ratio [HR] 1·45, 95% CI 1·07–1·96, p=0·16) but no overall survival benefit (HR 1·23, 0·87–1·72, p=0·24). We saw no progression-free survival or overall survival benefit for gross total resection compared with near-total resection (HR 1·05, 0·71–1·53, p=0·8158 for progression-free survival and HR 1·14, 0·75–1·72, p=0·55 for overall survival). No significant survival benefit existed for greater extent of resection for patients with WNT, SHH, or group 3 tumours (HR 1·03, 0·67–1·58, p=0·89 for sub-total resection vs gross total resection). For patients with group 4 tumours, gross total resection conferred a benefit to progression-free survival compared with sub-total resection (HR 1·97, 1·22–3·17, p=0·0056), especially for those with metastatic disease (HR 2·22, 1·00–4·93, p=0·050). However, gross total resection had no effect on overall survival compared with sub-total resection in patients with group 4 tumours (HR 1·67, 0·93–2·99, p=0·084). Interpretation The prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. Although maximum safe surgical resection should remain the standard of care, surgical removal of small residual portions of medulloblastoma is not recommended when the likelihood of neurological morbidity is high because there is no definitive benefit to gross total resection compared with near-total resection. Funding Canadian Cancer Society Research Institute, Terry Fox Research Institute, Canadian Institutes of Health Research, National Institutes of Health, Pediatric Brain Tumor Foundation, and the Garron Family Chair in Childhood Cancer Research.

Severe acute respiratory syndrome (SARS) is a new human infectious disease with significant morbidity and mortality. The disease has been shown to be associated with a new coronavirus (SARS‐CoV). The clinical and epidemiological aspects of SARS have been described. Moreover, the viral genome of SARS‐CoV has been fully sequenced. However, much of the biological behaviour of the virus is not known and data on the tissue and cellular tropism of SARS‐CoV are limited. In this study, six fatal cases of SARS were investigated for the tissue and cellular tropism of SARS‐CoV using an in‐situ hybridization (ISH) technique. Among all the tissues studied, positive signals were seen in pneumocytes in the lungs and surface enterocytes in the small bowel. Infected pneumocytes were further confirmed by immunofluorescence–fluorescence in‐situ hybridization (FISH) analysis. These results provide important information concerning the tissue tropism of SARS‐CoV, which is distinct from previously identified human coronaviruses, and suggest the possible involvement of novel receptors in this infection. Whereas the lung pathology was dominated by diffuse alveolar damage, the gut was relatively intact. These findings indicated that tissue responses to SARS‐CoV infection are distinct in different organs. Copyright © 2004 John Wiley & Sons, Ltd.

Acute graft-versus-host disease (GVHD) involves mainly skin, liver and intestines. Other organs such as heart, muscle and central nervous system are seldom affected, although their parenchymal cells also express alloantigens, such as MHC class I antigens. The mechanism of this selective involvement of distinct organs in acute GVHD is not well understood. We postulated that it might be related to the selective migration of activated alloreactive T cells. Indeed, T cell infiltration, revealed by examination of serial samples using flow cytometry and immunohistology, occurred early and continuously in the target organs such as the liver, but not in a non-target organ, the heart, in a murine acute GVHD model. Since T cell migration is largely controlled by the expression of chemokine and chemokine receptors, we investigated the chemokine spectrum in target/non-target organs of mice with acute GVHD. We found that in the spleen and liver MIP-1alpha, MIP-2 and Mig were the predominant chemokines expressed. In another target organ, the skin, MIP-1alpha, MIP-2, MCP-1 and MCP-3 were all highly expressed. In a non-target organ of acute GVHD, the heart, the predominant chemokines expressed were MCP-1 and MCP-3. This distinct pattern of chemokine expression in these organs may contribute to the preferential recruitment of inflammatory cells into the liver and skin, but not into the heart, in acute GVHD.

Protein-protein interactions (PPIs) are increasingly important targets for drug discovery. Efficient fragment-based drug discovery approaches to tackle PPIs are often stymied by difficulties in the production of stable, unliganded target proteins. Here, we report an approach that exploits protein engineering to "humanise" thermophilic archeal surrogate proteins as targets for small-molecule inhibitor discovery and to exemplify this approach in the development of inhibitors against the PPI between the recombinase RAD51 and tumour suppressor BRCA2. As human RAD51 has proved impossible to produce in a form that is compatible with the requirements of fragment-based drug discovery, we have developed a surrogate protein system using RadA from $\textit{Pyrococcus furiosus}$. Using a monomerised RadA as our starting point, we have adopted two parallel and mutually instructive approaches to mimic the human enzyme: firstly by mutating RadA to increase sequence identity with RAD51 in the BRC repeat binding sites, and secondly by generating a chimeric archaeal human protein. Both approaches generate proteins that interact with a fourth BRC repeat with affinity and stoichiometry comparable to human RAD51. Stepwise humanisation has also allowed us to elucidate the determinants of RAD51 binding to BRC repeats and the contributions of key interacting residues to this interaction. These surrogate proteins have enabled the development of biochemical and biophysical assays in our ongoing fragment-based small-molecule inhibitor programme and they have allowed us to determine hundreds of liganded structures in support of our structure-guided design process, demonstrating the feasibility and advantages of using archeal surrogates to overcome difficulties in handling human proteins.

While molecular subgrouping has revolutionized medulloblastoma classification, the extent of heterogeneity within subgroups is unknown. Similarity network fusion (SNF) applied to genome-wide DNA methylation and gene expression data across 763 primary samples identifies very homogeneous clusters of patients, supporting the presence of medulloblastoma subtypes. After integration of somatic copy-number alterations, and clinical features specific to each cluster, we identify 12 different subtypes of medulloblastoma. Integrative analysis using SNF further delineates group 3 from group 4 medulloblastoma, which is not as readily apparent through analyses of individual data types. Two clear subtypes of infants with Sonic Hedgehog medulloblastoma with disparate outcomes and biology are identified. Medulloblastoma subtypes identified through integrative clustering have important implications for stratification of future clinical trials.