An exploration of some aspects of molecular replacement in macromolecular crystallography

This thesis reports work in three areas of X-ray crystallography. An initial chapter describes the structure of a protein, the methods based on the use of X-rays and computer analysis of diffraction patterns to determine crystal structure, and the subsequent derivation of the structure of part or all of a protein molecule. Work to determine the structure of the protein cytokine receptor-like factor 3 (CRLF3) leading to the successful generation of a structural model of a significant part of this molecule is then described in Chapter 2. A variety of techniques had to be deployed to complete this work, and the steps undertaken are described. Analysis was performed principally using phaser, using maximum likelihood methods. Areas for improvement in generating non-crystallographic symmetry (NCS) operators in existing programmes were identified and new and modified algorithms implemented and tested. Searches based on improved single sphere algorithms, and a new two-sphere approach, are reported. These methods showed improvements in many cases and are available for future use. In Chapter 4, work on determining the relative importance of low resolution and high intensity data in molecular replacement solutions is described. This work has shown that high intensity data are more important than the low resolution data, dispelling a common perception and helping in experimental design.Welcome Trust funded PhD with a travel fellowship from Instruc

Somatic TP53 Mutations Are Detectable in Circulating Tumor DNA from Children with Anaplastic Wilms Tumors.

BACKGROUND: Diffuse anaplastic Wilms tumor (DAWT) is a rare, high-risk subtype that is often missed on diagnostic needle biopsy. Somatic mutations in TP53 are associated with the development of anaplasia and with poorer survival, particularly in advanced-stage disease. Early identification of DAWT harboring TP53 abnormalities could improve risk stratification of initial therapy and monitoring for recurrence. METHODS: Droplet digital polymerase chain reaction (ddPCR) was used to evaluate 21 samples from 4 patients with DAWT. For each patient, we assessed TP53 status in frozen tumor, matched germline DNA, and circulating tumor DNA (ctDNA) from plasma, serum, and urine collected throughout treatment. RESULTS: Mutant TP53 was detectable in ctDNA from plasma and serum in all patients. We did not detect variant TP53 in the same volume (200 μl) of urine. One patient displayed heterogeneity of TP53 in the tumor despite both histological sections displaying anaplasia. Concentration of ctDNA from plasma/serum taken prenephrectomy varied significantly between patients, ranging from 0.44 (0.05-0.90) to 125.25 (109.75-140.25) copies/μl. We observed variation in ctDNA throughout treatment, and in all but one patient, ctDNA levels fell significantly following nephrectomy. CONCLUSION: We demonstrate for the first time that ddPCR is an effective method for detection of mutant TP53 in ctDNA from children with DAWT even when there is intratumoral somatic heterogeneity. This should be further explored in a larger cohort of patients, as early detection of circulating variant TP53 may have significant clinical impact on future risk stratification and surveillance

Recurrent mutation of IGF signalling genes and distinct patterns of genomic rearrangement in osteosarcoma

Osteosarcoma is a primary malignancy of bone that affects children and adults. Here, we present the largest sequencing study of osteosarcoma to date, comprising 112 childhood and adult tumours encompassing all major histological subtypes. A key finding of our study is the identification of mutations in insulin-like growth factor (IGF) signalling genes in 8/112 (7%) of cases. We validate this observation using fluorescence in situ hybridization (FISH) in an additional 87 osteosarcomas, with IGF1 receptor (IGF1R) amplification observed in 14% of tumours. These findings may inform patient selection in future trials of IGF1R inhibitors in osteosarcoma. Analysing patterns of mutation, we identify distinct rearrangement profiles including a process characterized by chromothripsis and amplification. This process operates recurrently at discrete genomic regions and generates driver mutations. It may represent an age-independent mutational mechanism that contributes to the development of osteosarcoma in children and adults alike

Recurrent rearrangements of FOS and FOSB define osteoblastoma.

The transcription factor FOS has long been implicated in the pathogenesis of bone tumours, following the discovery that the viral homologue, v-fos, caused osteosarcoma in laboratory mice. However, mutations of FOS have not been found in human bone-forming tumours. Here, we report recurrent rearrangement of FOS and its paralogue, FOSB, in the most common benign tumours of bone, osteoblastoma and osteoid osteoma. Combining whole-genome DNA and RNA sequences, we find rearrangement of FOS in five tumours and of FOSB in one tumour. Extending our findings into a cohort of 55 cases, using FISH and immunohistochemistry, provide evidence of ubiquitous mutation of FOS or FOSB in osteoblastoma and osteoid osteoma. Overall, our findings reveal a human bone tumour defined by mutations of FOS and FOSB

Reduction in podocyte density as a pathologic feature in early diabetic nephropathy in rodents: Prevention by lipoic acid treatment

BACKGROUND: A reduction in the number of podocytes and podocyte density has been documented in the kidneys of patients with diabetes mellitus. Additional studies have shown that podocyte injury and loss occurs in both diabetic animals and humans. However, most studies in animals have examined relatively long-term changes in podocyte number and density and have not examined effects early after initiation of diabetes. We hypothesized that streptozotocin diabetes in rats and mice would result in an early reduction in podocyte density and that this reduction would be prevented by antioxidants. METHODS: The number of podocytes per glomerular section and the podocyte density in glomeruli from rats and mice with streptozotocin (STZ)-diabetes mellitus was determined at several time points based on detection of the glomerular podocyte specific antigens, WT-1 and GLEPP1. The effect of insulin administration or treatment with the antioxidant, α-lipoic acid, on podocyte number was assessed. RESULTS: Experimental diabetes resulted in a rapid decline in apparent podocyte number and podocyte density. A significant reduction in podocytes/glomerular cross-section was found in STZ diabetes in rats at 2 weeks (14%), 6 weeks (18%) and 8 weeks (34%) following STZ injection. Similar declines in apparent podocyte number were found in STZ diabetes in C57BL/6 mice at 2 weeks, but not at 3 days after injection. Treatment with α-lipoic acid substantially prevented podocyte loss in diabetic rats but treatment with insulin had only a modest effect. CONCLUSION: STZ diabetes results in reduction in apparent podocyte number and in podocyte density within 2 weeks after onset of hyperglycemia. Prevention of these effects with antioxidant therapy suggests that this early reduction in podocyte density is due in part to increased levels of reactive oxygen species as well as hyperglycemia

Recurrent intragenic rearrangements of EGFR and BRAF in soft tissue tumors of infants.

Soft tissue tumors of infancy encompass an overlapping spectrum of diseases that pose unique diagnostic and clinical challenges. We studied genomes and transcriptomes of cryptogenic congenital mesoblastic nephroma (CMN), and extended our findings to five anatomically or histologically related soft tissue tumors: infantile fibrosarcoma (IFS), nephroblastomatosis, Wilms tumor, malignant rhabdoid tumor, and clear cell sarcoma of the kidney. A key finding is recurrent mutation of EGFR in CMN by internal tandem duplication of the kinase domain, thus delineating CMN from other childhood renal tumors. Furthermore, we identify BRAF intragenic rearrangements in CMN and IFS. Collectively these findings reveal novel diagnostic markers and therapeutic strategies and highlight a prominent role of isolated intragenic rearrangements as drivers of infant tumors

Figuring Rhetoric: From Antistrophe to Apostrophe through Catastrophe

This essay explores rhetoric tropologically through various strophes: antistrophe, catastrophe, and apostrophe. Our purpose is to delineate problems and possibilities that these tropes pose for rhetoric in an effort to create new rhetorics. We seek to display the antistrophic and catastrophic figurations of rhetoric and then use visual lenses of photography and cinema to disrupt the figurations. Following the disruption, we seek to heighten sensibilities to other figurations, in particular an apostrophic figuration. We cast apostrophe as a figure for change because it marks a deeply felt turn toward difference and otherness. Turned as such, rhetoric becomes erotic

Sunetti ta’ William Shakespeare

Ġabra ta’ poeżiji u proża li tinkludi: Grand Prix ta’ Carmel Azzopardi – Pizza marinara ta’ Carmel Azzopardi – Ħajku ta’ Kit Azzopardi – Ix-xemgħa qiegħda ta’ Charles Bezzina – U taħti ramel, ramel ta’ Charles Bezzina – Vażett ta’ Ġorġ Borg – Bniedem li mhux ta’ Ġorġ Borg – Il-ħajbu ta’ Antoine Cassar – Il-mistoħbija ta’ Manwel Cassar – Għasel ta’ Carmel G. Cauchi – Dgħajsa ta’ Carmel G. Cauchi – Ħitan ta’ Alfred Degabriele – Skeletru silwett...f’realtà moħbija ta’ Stefano Farrugia – Minjatura tal-enimmi ta’ Stefano Farrugia – Mnejn jgħaddi Kristu ta’ Joe Friggieri – Rebbiegħa ta’ Reno Fenech – Blogger ta’ Charles Flores – Veġeterjana ta’ Charles Flores – Mejju ta’ Joe P. Galea – Kien hemm lejla u tmien nisa ta’ Claudia Gauci – Ħobbni ta’ Sergio Grech – Mitlufin ta’ Maria Grech Ganado – Moħħi ta’ Maria Grech Ganado – Viżjoni ta’ Maria Grech Ganado – Inkontinenza ta’ Adrian Grima – Andrew jħebb in-nar ta’ Adrian Grima – It-Tlieta, 20 ta’ Lulju 2004 ta’ Alfred Massa – Fuq l-għolja tal- Verdala ta’ Jane Micallef – Imm’issa ta’ Jane Micallef – Baby blues ta’ Immanuel Mifsud – Ġo dar sawra ta’ Immanuel Mifsud – Lil Dun Karm ta’ Maurice Mifsud Bonnici – Il-fuklar ta’ Achille Mizzi – Ut videam ta’ Achille Mizzi – Karnival solitarju ta’ Patrick Sammut – Mill-baħħ etern ta’ Joe Zammit Tabona – ...fil-ħmieġ ta’ ftit blatiet... ta’ Paul P. Borg – Bħall-qasab ta’ Steve Borg – L-aħħar żjara ta’ Victor Fenech – Ħelwa.morra 18 ta’ Ann Marie Schembri – Jack & Jill ta’ Trevor Żahra – Għadbilura ta’ Russell Davis, traduzzjoni ta’ Toni Aquilina – Sunetti ta’ William Shakespeare, traduzzjoni ta’ Oliver Friggieri.peer-reviewe

Nikteb...

Ġabra ta’ poeżiji u proża li tinkludi: L-iben il-ħali ta’ David Agius Muscat – Kaptan ta’ Kit Azzopardi – Il-lanterna ta’ Charles Bezzina – Li kelli mmur lura ta’ Ġorġ Borg – Firda ta’ Ġorġ Borg – Garden fairy ta’ Charles Briffa – Sejf jinfidlek ruħek ta’ Charles Briffa – Waqt ta’ Joseph Buttigieg – Vjaġġ ta’ John Caruana – Ċaqlembuta ta’ Antoine Cassar – Ħaġa tqila ta’ Carmel G. Cauchi – F’tarf il-blat ta’ Leanne Ellul – Int ta’ Victor Fenech – Pippin u l-bojja ta’ Charles Flores – L-arloġġ ta’ Joe Friggieri – Il-fjur tal-ġakaranda ta’ Joe Friggieri – Fjur tal-kaktus ta’ Joel Galea – Biss is-skiet ta’ Joel Galea – Għalissa ta’ Maria Grech Ganado – Ilsna ta’ Maria Grech Ganado – Is-sried ixoqqna fin fin ta’ Adrian Grima – Ħsieb ħalliel... ta’ Patrick Sammut – Lament lil ommi ta’ Salv Sammut – Hekk kif tinħass ġol-arja x-xitwa ta’ Lillian Sciberras – F’għajnejha, il-ħarsa siekta ta’ Clare Azzopardi – Għad jagħdab l-irdum ta’ Paul P. Borg – Forsi...xi darba ta’ Charles Casha – Faxxa ngħas ta’ Sergio Grech – Il-mejda tal-mogħdija ta’ Pierre J. Mejlak – Min jaf bi Stojan Kurepa? ta’ Immanuel Mifsud – L-eħrex jum tal-gwerra ta’ Maurice Mifsud Bonnici – Il-vażett tal-bewsiet ta’ Rita Saliba – Pjanu ta’ Trevor Żahra – Il-ħalliel ta’ Guy de Maupassant, traduzzjoni ta’ Toni Aquilina – Salvu tal-pasturi ta’ Francis Ebejer, traduzzjoni ta’ Steve Borg – Sunetti ta’ William Shakespeare, traduzzjoni ta’ Oliver Friggieri – Nikteb... ta’ Nizar Qabbani, traduzzjoni ta’ Kevin Saliba.peer-reviewe

Lineage-specific dynamic and pre-established enhancer–promoter contacts cooperate in terminal differentiation

Chromosome conformation is an important feature of metazoan gene regulation; however, enhancer–promoter contact remodeling during cellular differentiation remains poorly understood. To address this, genome-wide promoter capture Hi-C (CHi-C) was performed during epidermal differentiation. Two classes of enhancer–promoter contacts associated with differentiation-induced genes were identified. The first class ('gained') increased in contact strength during differentiation in concert with enhancer acquisition of the H3K27ac activation mark. The second class ('stable') were pre-established in undifferentiated cells, with enhancers constitutively marked by H3K27ac. The stable class was associated with the canonical conformation regulator cohesin, whereas the gained class was not, implying distinct mechanisms of contact formation and regulation. Analysis of stable enhancers identified a new, essential role for a constitutively expressed, lineage-restricted ETS-family transcription factor, EHF, in epidermal differentiation. Furthermore, neither class of contacts was observed in pluripotent cells, suggesting that lineage-specific chromatin structure is established in tissue progenitor cells and is further remodeled in terminal differentiation