Investigation of thermogenic mechanisms in adipose tissue during recovery from sepsis

Background – Sepsis is defined as a dysregulated host response to infection resulting in organ dysfunction and, in some cases, death. Temperature impacts outcomes from sepsis – patients with fever are more likely to survive. However, in the recovery phase, thermogenesis may be detrimental. Survivors frequently develop cachexia and sepsis-induced myopathy which impairs recovery and increases long term mortality. In conditions akin to sepsis, including burn injury and cancer-associated cachexia, this has been attributed to catabolism driven by hypermetabolism due to a process called ‘browning’. Browning describes the switch of energy-storing white adipose tissue to a thermogenic energy-burning brown adipose tissue-like phenotype. Identification and prevention of browning in sepsis may improve outcomes. Hypothesis – In survivors of sepsis, browning of white adipose tissue occurs and drives cachexia and myopathy. Methods – Experimental sepsis was induced in rats using intraperitoneal zymosan. Body mass, muscle mass and myofibre cross sectional area were used to assess cachexia and myopathy. Expression of thermogenic browning mechanisms were studied in epididymal and retroperitoneal adipose tissue (eWAT and rpWAT, respectively) using thermal imaging, respirometry, RNA-sequencing and Western blot. Mitochondrial function and tissue morphology was interrogated by multiphoton imaging in live rpWAT explants. Results – Rats with zymosan peritonitis developed a sepsis-like illness with a 14-day mortality of 17%. In the recovery phase survivors developed hypermetabolism, cachexia and myopathy with reduced muscle mass and myofiber thickness. Oxygen consumption of eWAT and rpWAT per milligram of tissue was elevated at days 3, 7 4 and 14 of sepsis recovery. However, when controlled for protein content, lipid droplet size or mitochondrial or cell number, the increase was abolished. RNA sequencing of rpWAT demonstrated up-regulation of inflammatory genes and down-regulation of genes related to oxidative phosphorylation and thermogenesis during recovery. Notably, SERCA2 mRNA and SERCA2 protein were increased. Multiphoton microscopy showed neither increased mitochondrial density nor lipid multiloculation consistent with browning. The NAD(P)H pool was, however, more oxidised in tissue from animals recovering from sepsis, indicating altered metabolism. Conclusion – Hypermetabolism, cachexia and myopathy in the recovery phase of experimental sepsis are not caused by classical browning. Calcium cycling mechanisms in adipose tissue may be implicated and merit further investigation

Investigating the roles of candidate gap genes from the neuroblast timer series during axial patterning in the beetle Tribolium

The gap genes encode transcription factors that play a central role in the process of segment patterning in Drosophila. Specifically, they interact to form the well-characterised ‘gap gene network’, which directs the formation of segment boundaries (through regulation of pair-rule genes), and the subsequent diversification of segments (through regulation of Hox genes). Although homologues of the gap genes play important roles in segment patterning in many insects, there is as yet no clear understanding of the network as a whole outside of Drosophila. In particular, the existence of a ‘gap gene gap’ (a region of the axis that expresses no gap genes) in embryos of the beetle Tribolium castaneum may indicate the existence of additional, as yet unidentified gap genes in this species. In this work, I investigate the hypothesis that the neuroblast timer genes nubbin (nub) and castor (cas) may act as components of the gap gene network in Tribolium. I first utilise Hybridisation Chain Reaction in situ hybridisation to produce a comprehensive description of the dynamics of gap gene expression in Tribolium, concluding that, although the non-canonical gap genes Tc-mille-pattes and Tc-shavenbaby are expressed in the gap gene gap, their role may be distinct from that of the canonical gap genes, and that there are likely to be other, unknown gap genes expressed alongside them. I show that the four genes of the neuroblast timer series (hunchback, Krüppel, nub and cas) are expressed sequentially in the segment addition zone, with the result that nub and cas are expressed in the gap gene gap. Knocking down the expression of nub, but not cas, using RNAi results in weak homeotic transformations of the first abdominal segment towards a thoracic fate. Finally, I show that this phenotype is dramatically increased in severity and penetrance when Tc-nub is knocked down in addition to the trunk gap genes Tc-gt and/or Tc-kni. In triple knockdowns, all abdominal segments are transformed into thoracic segments due to a posterior expansion of the thoracic gap gene Tc- Kr and subsequent loss of expression of the abdominal Hox genes Tc-abdominal A and Tc- Ultrabithorax. These data indicate that Tc-nub, Tc-gt and Tc-kni act redundantly to repress the expression of Tc-Kr in the abdomen, and that both Tc-nub and Tc-kni should therefore be considered as components of the gap gene network in Tribolium. My work strengthens the hypothesis that the gap gene network may have ancient evolutionary ties to the neuroblast timer network, and promotes a more ‘modular’ view of the gap gene network. I hope that this will inform future studies that aim to unravel the developmental role of the gap genes in sequentially segmenting arthropods, and the evolutionary origins of the gap gene network

Functional Electrical Impedance Tomography of adult and neonatal brain function.

Electrical Impedance Tomography (EIT) is a fast, portable imaging technique that produces tomographic images of the internal impedance of an object from surface electrode measurements. This thesis reports the first use of EIT to image evoked brain activity in adults and neonates and determines whether accurate EIT images could be obtained from the adult and neonatal brain. In addition, a realistic head-tank phantom was developed to test the performance of EIT with known impedance changes placed within a real human skull. Two EIT systems were used. Images were obtained using 31 or 21 Ag/AgCl EEG scalp electrodes in adults and neonates, respectively, with either 256 or 187 individual impedance measurements from different electrode combinations: 2 applied a safe, alternating current and 2 measured the resultant scalp voltage. Imaging was performed using a block design with 6-15 stimulation periods of between 10-75s during either: 1) Visual, 2) Somatosensory or 3) Motor stimuli. Impedance changes were detected in 38/39 adults and 9/9 neonates within 0.6-5.8s after stimulus onset, and returned to baseline 7.6-36s after stimulus cessation. Reconstructed images were noisy: -20-70% images showed correct localisation to the expected area of cortex stimulated by the visual, motor or somatosensory paradigms. As EIT images from the head-tank localised changes within 10% of the impedance perturbation, this indicated that poor localisation in humans was not due to the head-shape or the skull, but may be related to unknown physiological factors. An improved EIT reconstruction algorithm, using a computerised finite-element model of the head, showed improved localisation for the adult images. This is the first demonstration that EIT can detect and image impedance changes in the head, probably due to increased regional cerebral blood volume in the activated cortex. Improvements may enable more accurate neuroimaging of the adult and neonatal brain for use in clinical practice

Cross-Country Differences in Marginal Product of Capital and the Efficient Allocation of the World’s Capital Stock

Since his original publication in 1990, Robert E Lucas Jr’s observation of capital failing to flow between rich and poor countries has stoked debate across international development economics over its theoretical explanation. Since then, economists have sought to rationalize this observation through two explanations: fundamental production structure differences and capital market imperfections. This paper serves to build upon the marginal product of capital (MPK) compositions presented by Caselli and Feyrer (2007) to provide updated and refined data on national accounts. In reassessing cross-country MPK’s, near equalized differentials between rich and poor countries are observed

The synthesis and metal complexes of some unusual phosphines

The high temperature and pressure synthesis of chlorophosphines from an alkene, white phosphorus and phosphorus trichloride yielded both mono- and diphosphine products. Chain and cyclic alkenes, dienes and terpenes were used as substrates forming chain, ring and bicyclic mono- and diphosphines. Many novel, even unique, chlorophosphines were prepared in good yield and characterised using (^31)P and (^13)C NMR, mass spectroscopy and elemental analysis. Simple alkenes readily reacted forming mono- and 1,2-diphosphines. However 1,5-cyclooctadiene formed a 1,4-addition product after double bond conjugation. Butadiene dimerised prior to reaction, generating either a 2,2'-diphospholane or a 1,4-bridged phosphabicyclic alkane in addition to 1,2- and 1,4-diphosphines. Terpenoid dichlorophosphines were prepared but could not be isolated or characterised, because the substrate isomerised under the reaction conditions generating many similar products. Aromatic rings did not react, although the exocyclic double bond of styrene did undergo reaction. Chiral phosphines were readily produced from unsymmetric pro-chiral, alkenes. Chlorophosphines are versatile precursors and, using standard organophosphorus techniques, were readily converted to phosphines, phosphites and other organophosphorus ligands suitable for chelation. Grignard reagents were used to prepare dimethyl derivatives, alcohols reacted with dichlorophosphines producing diethyl-, dimenthyl- or 1,4-butylphosphite derivatives, Piperazine also reacted although the products could not be fully characterised. Electronic properties (^Mn)χ) of the phosphines in manganese carbonyl halide derivatives were measured as a function of the A(_1)carbonyl stretching frequency. Substituent electronegativity was the most important factor in determining the π-acidity. Some dichloropalladium complexes were studied using 3ip NMR, although phosphine impurities complicated spectral interpretation. Iron and molybdenum hydrides reacted with dichlorophosphines to produce some unusual metal-phosphine compounds