Investigation of cilia-associated kinesin families

Kinesins are a protein superfamily that function via microtubules by converting energy released from ATP hydrolysis into mechanical work. Kinesins have been classified into 14 families; the distribution of which can highlight potential roles of the family members. Kinesin-16s are found solely in organisms that build cilia/flagella, whilst Kinesin-13s are only absent in organisms that do not build cilia. The Kinesin-13 family is comprised of three subfamilies. Both 13A and 13B have motors involved in ciliary function, but the roles of 13C are still unknown. The purpose of this research is to identify novel roles of kinesin families and/or subfamilies, with a particular focus on those that may function in the cilia/flagella. Human KIF12 was investigated as a representative of Kinesin-16 whilst the roles of Kinesin-13 subfamilies A and C motors were investigated using the kinesin subfamily members found in Trypanosoma brucei. An inducible system of ciliogenesis in IMCD3 cells was sought to allow investigation into the difference in KIF12 localisation and expression before and after ciliogenesis occurred. Induction of ciliogenesis in IMCD3 cells was unsuccessful with no significant change in the percentage of ciliated cells post-serum starvation and/or contact inhibition. To characterise KIF12-microtubule interactions in vitro, the human RefSeq reference standard KIF12 (rsKif12) sequence was used. After multiple failed attempts to express and purify rsKif12, sequence analysis showed rsKif12 lacks nucleotide-binding motifs conserved across the kinesin superfamily. To determine the KIF12 major splice variant, RNA-sequencing data and sequence alignments of KIF12 homologues were used. kif12ΔPPGGG, harbouring a five amino acid deletion that reduced polycystic kidney disease severity in mice, and KIF12 motor domain only samples were also determined. Having determined the correct major splice variant of KIF12, several expression constructs were created to find a soluble version of KIF12 as well as kif12MD and kif12ΔPPGGG. Expressed KIF12, kif12MD and kif12ΔPPGGG were tagged individually with 6x His or 6x His and GFP at either the N- or C-terminus. Only kif12MD tagged with a C-terminal GFP and 6x His was soluble. T. brucei Kinesin-13s Kin13-1, -3 (both 13A) and -6 (13C) interactions with microtubules were observed in vitro. Kin13-6 does not significantly change microtubule dynamics in vitro. This suggests that Kinesin-13Cs do not function in microtubule regulation. Kin13-3 depolymerises microtubules, consistent with the functions of other Kinesin-13A members. Kin13-1 controls microtubule length in a novel manner by cleaving at the microtubule lattice. Kin13-1 cleaving activity has revealed a possible means of regulating the mitotic spindle, and thus chromosome segregation, which was previously unidentified

Geochemistry of crustally derived leucocratic igneous rocks from the Ulugh Muztagh Area, Northern Tibet and their implications for the formation of the Tibetan Plateau

This is the published version. Copyright 2012 American Geophysical Union. All Rights Reserved.Igneous rocks collected from the Ulugh Muztagh, 200 km south of the northern rim of the Tibetan Plateau (36°28′N, 87°29′E), form intrusive and extrusive bodies whose magmas were produced by partial melting of upper-crustal, primarily pelitic, source rocks. Evidence for source composition includes high initial 87Sr/86Sr ratios (∼0.711 to 0.713), 206Pb/204Pb ratios of 18.72, 207Pb/204Pb of 15.63 and 208Pb/204Pb of 38.73. The degree of melting in the source region was increased by significant heating via in situ decay of radioactive nuclides; a reasonable estimate for the heat production rate in the source is 3.9×10−6 W/m3. The crystallization ages and cooling ages [Burchfiel et al., 1989] of the earliest intrusive rocks within the suite suggest crustal thickening began in the northern Tibetan Plateau before 10.5 Ma, with maximum average unroofing rates in this part of the Tibetan Plateau for the period between 10.5 and 4 Ma at approximately < 2 mm/yr. The Ulugh Muztagh flows are at the northern edge of a widely distributed field of Plio-pliestocene volcanic rocks in the north-central Tibetan Plateau. The crustally derived rocks described here are an endmember component of a wide mixing zone of hybrid magmas; the other end-member forms mantle-derived, potassic basanites and tephrites exposed in the central section of the Plio-Pleistocene field. The compositional trends in these belts strike east-west, at high angle to the N30E strike of the Plateau itself. Consideration of the chemical data and published geophysical data argue that the sub-Plateau mantle is mechanically detached from the overlying continental lithosphère, and that in this section of the plateau the thermal structure of the asthenosphere is not responsible for the formation or maintenance of the plateau's topography

Living standards and plague in London, 1560–1665

This article uses individual records of 930,000 burials and 630,000 baptisms to reconstruct the spatial and temporal patterns of birth and death in London from 1560 to 1665, a period dominated by recurrent plague. The plagues of 1563, 1603, 1625, and 1665 appear of roughly equal magnitude, with deaths running at five to six times their usual rate, but the impact on wealthier central parishes falls markedly through time. Tracking the weekly spread of plague, we find no evidence that plague emerged first in the docks, and in many cases elevated mortality emerges first in the poor northern suburbs. Looking at the seasonal pattern of mortality, we find that the characteristic autumn spike associated with plague continued into the early 1700s. Natural increase improved as smaller crises disappeared after 1590, but fewer than half of those born survived childhood

Max Müller and the Comparative Method

This is an Author’s Accepted Manuscript of an article published by Edinburgh University Press in Comparative Critical Studies. The Version of Record is available online at: https://www.euppublishing.com/doi/10.3366/ccs.2015.016

Investigation of cilia-associated kinesin families

Kinesins are a protein superfamily that function via microtubules by converting energy released from ATP hydrolysis into mechanical work. Kinesins have been classified into 14 families; the distribution of which can highlight potential roles of the family members. Kinesin-16s are found solely in organisms that build cilia/flagella, whilst Kinesin-13s are only absent in organisms that do not build cilia. The Kinesin-13 family is comprised of three subfamilies. Both 13A and 13B have motors involved in ciliary function, but the roles of 13C are still unknown. The purpose of this research is to identify novel roles of kinesin families and/or subfamilies, with a particular focus on those that may function in the cilia/flagella. Human KIF12 was investigated as a representative of Kinesin-16 whilst the roles of Kinesin-13 subfamilies A and C motors were investigated using the kinesin subfamily members found in Trypanosoma brucei. An inducible system of ciliogenesis in IMCD3 cells was sought to allow investigation into the difference in KIF12 localisation and expression before and after ciliogenesis occurred. Induction of ciliogenesis in IMCD3 cells was unsuccessful with no significant change in the percentage of ciliated cells post-serum starvation and/or contact inhibition. To characterise KIF12-microtubule interactions in vitro, the human RefSeq reference standard KIF12 (rsKif12) sequence was used. After multiple failed attempts to express and purify rsKif12, sequence analysis showed rsKif12 lacks nucleotide-binding motifs conserved across the kinesin superfamily. To determine the KIF12 major splice variant, RNA-sequencing data and sequence alignments of KIF12 homologues were used. kif12ΔPPGGG, harbouring a five amino acid deletion that reduced polycystic kidney disease severity in mice, and KIF12 motor domain only samples were also determined. Having determined the correct major splice variant of KIF12, several expression constructs were created to find a soluble version of KIF12 as well as kif12MD and kif12ΔPPGGG. Expressed KIF12, kif12MD and kif12ΔPPGGG were tagged individually with 6x His or 6x His and GFP at either the N- or C-terminus. Only kif12MD tagged with a C-terminal GFP and 6x His was soluble. T. brucei Kinesin-13s Kin13-1, -3 (both 13A) and -6 (13C) interactions with microtubules were observed in vitro. Kin13-6 does not significantly change microtubule dynamics in vitro. This suggests that Kinesin-13Cs do not function in microtubule regulation. Kin13-3 depolymerises microtubules, consistent with the functions of other Kinesin-13A members. Kin13-1 controls microtubule length in a novel manner by cleaving at the microtubule lattice. Kin13-1 cleaving activity has revealed a possible means of regulating the mitotic spindle, and thus chromosome segregation, which was previously unidentified