Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Defining the mechanistic role of hSSB1 : a novel single-stranded DNA binding protein essential for DNA repair

DNA is under constant attack from the external and internal environment. It is imperative to repair and maintain the vital genetic information of DNA. Otherwise it leads to an accumulation of mutations that alters the normal function of DNA and in turn causes a disorder in cellular metabolism. During repair, DNA unwinds into single-stranded DNA (ssDNA) and is even more vulnerable to damage. This is where human single-strand DNA binding protein 1 (hSSB1) binds and protects ssDNA. It is known that hSSB1 exists both as part of the MRN (MRE11, RAD50, NBS1) repair complex and the SOSS1 complex (made up of INTS3 and C9orf80). It is also known to initiate an appropriate repair mechanism by recruiting other proteins to the site of damage. There is an accumulating body of research on hSSB1 function in Double Strand Break Repair (DSBR). It promotes the ataxia telangiectasia mutated (ATM) kinase signalling cascade and also recruits the MRN complex to the site of double strand breaks (DSBs) in order to initiate DSBR via Homologous Recombination (HR). Recently, it was discovered that hSSB1 is capable of forming higher order oligomers and can function in the oxidative DNA damage response. The most common oxidative DNA damage is the 7,8-dihydro-8-oxoguanine (8-oxoG) adduct, which is the direct result of reactive oxygen species (ROS) produced during regular cellular respiration. If this damage goes unrepaired it may result in G:C to T:A transversions. These lesions are normally processed by the Base Excision Repair (BER) pathway, which involves human oxoguanine glycosylase (hOGG1) that cleaves the DNA backbone and removes the offending base. So far, it is understood that hSSB1 levels increase in response to oxidative damage; also, cells depleted of hSSB1 are hypersensitive to oxidative damage and are also unable to efficiently remove 8-oxoG adducts. The recruitment of hOGG1 to chromatin is dependent on hSSB1 and hSSB1 can promote hOGG1 cleavage of 8-oxoG. This thesis examines the mechanism of hSSB1 oligomerisation under oxidative conditions. hSSB1 forms dimers and tetramers and this oligomerisation is likely mediated by inter-domain disulfide bond formation. These oligomers can also be synthetically created through the use of a thiol reactive cross-linker. Oxidised hSSB1 binds to 8-oxoG damaged ssDNA with higher affinity than non-damaged ssDNA, likely indicating a direct role for oxidised hSSB1 in the recognition of 8-oxo-G lesions. Furthermore, hSSB1 and hOGG1 directly interact with a moderate binding affinity in the presence of 8-oxoG damaged ssDNA. Finally, a model of the tetramer is proposed using the recent crystal structure of monomeric hSSB1 as a template. The data presented here along with the proposed structural model allows hSSB1 to be placed in the oxidative DNA damage response pathway and gives crucial insight into the possible role of the oligomer in this process. As heightened levels of oxidative stress are associated with cancer (as well as aging and Alzheimer’s disease), understanding the molecular mechanisms of how cells repair oxidative DNA damage will be essential in the development of novel therapeutic treatments

Mechanical Regulation of Apoptosis and Calcification within Valvular Interstitial Cells

Calcific aortic valvular disease (CAVD) is the most common valvular pathology in the developed world. CAVD results in calcifications forming on the aortic valve leaflets, inhibiting proper closure and causing complications of stenosis and regurgitation. Although, the mechanisms behind the disease initiation are unknown, it is believed to be a cell-mediated phenomenon, and not the result of passive degradation of the valve as once believed due to the increased prevalence with age. Currently, there are no pharmaceutical options for the prevention or reversal of calcifications, the only treatment option is complete valve replacement, an imperfect solution. Hindering the development of potential therapeutics is that currently there are no adequate animal models which replicate the calcification and cell death seen in disease explanted valves. An in vitro model has been develop where valvular interstitial cells (VICs), the main cell type of the valve, are seeded at high density into tissue culture polystyrene dishes and cultured with TGF-β1. This results in VICs activating to the myofibroblast phenotype and forming cell aggregates. Due to currently unknown mechanisms, apoptosis occurs within the center of the aggregates and calcification ensues. Although simplistic, this model has been used to show that rate and frequency of aggregation is affected by cellular tension; conditions of high tension increase aggregation response, while conditions of low tension prevent aggregation and calcification from occurring. It is important to note; however, that despite its wide usage, the current model is limited as the aggregation and subsequent calcification are random occurrences and are not consistent across literature where same conditions for control samples are used. The motivation of the presented work is two-fold. First, high intracellular tension has been suggested as one of the mechanisms leading to disease in the valve. Despite the clear and important role of cell tension, VIC tension has never before been measured in a dynamic environment. The ways in which dynamic stimulation affects individual VIC tension is not known. In aim one, a method is developed to allow for long-term cyclic stretch of VICs with measurement of cell traction force. It was found that cyclic stretch decreased cell tension in cells with high prestress and increased cell tension for conditions of low prestress. Combined, these findings indicate a homeostatic cellular tension which is dependent upon the mechanical environment. In the second aim, a novel method for creating VIC aggregates is validated. Micro-contact printing, essentially “stampingâ€� of a protein in a defined pattern, is used to create circular aggregates on polyacrylamide gels. This method allows for the separation of the aggregation from the subsequent calcification, an improvement over the current in vitro model. The method is then used to explore the role of the distribution of tension in the initiation of diseas

Dichotomic role of NAADP/two-pore channel 2/Ca2+ signaling in regulating neural differentiation of mouse embryonic stem cells

Poster Presentation - Stem Cells and Pluripotency: abstract no. 1866The mobilization of intracellular Ca2+stores is involved in diverse cellular functions, including cell proliferation and differentiation. At least three endogenous Ca2+mobilizing messengers have been identified, including inositol trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and nicotinic adenine acid dinucleotide phosphate (NAADP). Similar to IP3, NAADP can mobilize calcium release in a wide variety of cell types and species, from plants to animals. Moreover, it has been previously shown that NAADP but not IP3-mediated Ca2+increases can potently induce neuronal differentiation in PC12 cells. Recently, two pore channels (TPCs) have been identified as a novel family of NAADP-gated calcium release channels in endolysosome. Therefore, it is of great interest to examine the role of TPC2 in the neural differentiation of mouse ES cells. We found that the expression of TPC2 is markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebound during the late stages of neurogenesis. Correspondingly, perturbing the NAADP signaling by TPC2 knockdown accelerates mouse ES cell differentiation into neural progenitors but inhibits these neural progenitors from committing to the final neural lineage. Interestingly, TPC2 knockdown has no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Overexpression of TPC2, on the other hand, inhibits mouse ES cell from entering the neural lineage. Taken together, our data indicate that the NAADP/TPC2-mediated Ca2+signaling pathway plays a temporal and dichotomic role in modulating the neural lineage entry of ES cells; in that NAADP signaling antagonizes ES cell entry to early neural progenitors, but promotes late neural differentiation.postprin

Progenitor cells in auricular cartilage demonstrate promising cartilage regenerative potential in 3D hydrogel culture

The reconstruction of auricular deformities is a very challenging surgical procedure that could benefit from a tissue engineering approach. Nevertheless, a major obstacle is presented by the acquisition of sufficient amounts of autologous cells to create a cartilage construct the size of the human ear. Extensively expanded chondrocytes are unable to retain their phenotype, while bone marrow-derived mesenchymal stromal cells (MSC) show endochondral terminal differentiation by formation of a calcified matrix. The identification of tissue-specific progenitor cells in auricular cartilage, which can be expanded to high numbers without loss of cartilage phenotype, has great prospects for cartilage regeneration of larger constructs. This study investigates the largely unexplored potential of auricular progenitor cells for cartilage tissue engineering in 3D hydrogels

A complex systems approach to education in Switzerland

The insights gained from the study of complex systems in biological, social, and engineered systems enables us not only to observe and understand, but also to actively design systems which will be capable of successfully coping with complex and dynamically changing situations. The methods and mindset required for this approach have been applied to educational systems with their diverse levels of scale and complexity. Based on the general case made by Yaneer Bar-Yam, this paper applies the complex systems approach to the educational system in Switzerland. It confirms that the complex systems approach is valid. Indeed, many recommendations made for the general case have already been implemented in the Swiss education system. To address existing problems and difficulties, further steps are recommended. This paper contributes to the further establishment complex systems approach by shedding light on an area which concerns us all, which is a frequent topic of discussion and dispute among politicians and the public, where billions of dollars have been spent without achieving the desired results, and where it is difficult to directly derive consequences from actions taken. The analysis of the education system's different levels, their complexity and scale will clarify how such a dynamic system should be approached, and how it can be guided towards the desired performance

Fluorescence lifetime spectroscopy and imaging of FRET probes for the study of cell signalling

This thesis presents the development and application of fluorescence lifetime spectroscopy and imaging to the readout of cellular processes using Förster resonant energy transfer (FRET). For quantitative solution-based studies, a multidimensional fluorometer was refined and applied to characterise two genetically encoded calcium FRET biosensors based on Troponin-C, including a fluorescence lifetime-resolved titration study leading to a quantitative calibration of their calcium response. A study of their time-resolved fluorescence anisotropy was also undertaken to explore the potential to probe molecular conformational changes. For the study of signalling processes in live cells, a novel optically sectioning FLIM microscope was developed to provide multiplexed fluorescence lifetime readouts of different FRET probes in order to facilitate the observation of different components of biological signalling networks by realising FLIM interleaved in different spectral channels. This PhD project was motivated to study the AMP-activated protein kinase (AMPK) cascade, which has a central role in the regulation of the energy level in mammalian cells and is now being studied as a potential drug target for type II diabetes. One pathway leading to activation of AMPK is triggered by an increase in intracellular calcium level leading to activation of calcium-calmodulin dependent protein kinase kinase β (CaMKKβ), an upstream activator of AMPK. This was investigated through a novel inter-molecular FRET system looking at the direct interaction of AMPK and CaMKKβ. The ultimate goal was to multiplex readouts of AMPK activation and intracellular calcium levels, for which new FRET biosensors are required. To this end, work was also undertaken for the design and production of novel FRET sensors in the red part of the spectrum, which are desirable for multiplexing with the common CFP/YFP–based FRET probes and also for use in in vivo imaging applications.Open Acces

Green zone nation : the securitisation and militarisation of the 2010 FIFA World Cup, South Africa

This thesis explores the relationship between the safety and security measures for the 2010 FIFA World Cup and the militarisation of urban space and policing in post-apartheid South Africa. In particular, it focuses upon how the South African state and FIFA, the owners of the World Cup franchise, worked to present the World Cup as an event which required exceptional levels of security – resulting in a historically unprecedented joint police and military operation across host cities. However, in contrast with previous research on these security measures, this thesis aims to interrogate the political and commercial forces which constructed security and positions them against a backdrop of intensified state violence and social exclusion in South Africa. Concurrently, the South African case was indicative of an international militarisation of major events, with policing operations comparable to national states of emergency. This is representative of the ‘new military urbanism’ in which everyday urban life is rendered as a site of ubiquitous risk, leading to the increased diffusion of military tactics and doctrines in policing and policy. While the interpenetration between urbanism and militarism has often been studied against the context of the ‘war on terror’, in the case of South Africa this has primarily been accelerated by a pervasive social fear of violent crime, which has resulted in the securitisation of cities, the remilitarisation of policing and the intensification of a historical legacy of socio-spatial inequalities. The South African government aimed to use the World Cup to ‘rebrand’ the country’s violent international image, while promising that security measures would leave a legacy of safer cities for ordinary South Africans. The concept of legacies was also responsive to the commercial imperatives of FIFA and a range of other security actors, including foreign governments and the private security industry. However these policing measures were primarily cosmetic and designed to allay the fears of foreign tourists and the national middle class. In practice security measures pivoted around the enforcement of social control and urban marginalisation while serving as a training ground for an increasingly repressive state security apparatus. Security was as much a matter of fortifying islands of privilege and aiding a project of financial extraction as protecting the public from harm.Microsoft� Office Word 2007Adobe Acrobat 9.53 Paper Capture Plug-i