Structural and biophysical investigation of +TIPs in yeast and -TIPs in higher eukaryotes

In eukaryotic cells, microtubules represent a highly dynamic protein filament system that is involved in cellular processes as cell division or transport of cargo. Microtubules oscillate between growth and shrinking, and the switch between these states is caused by catastrophe and rescue events. The building block of microtubules is the heterodimer tubulin, which polymerizes into tubular structures and switches from a curved state in the soluble form to a straight state in microtubules. Due to the polarity of tubulin, microtubules feature a plus-end and a minus-end. The highly dynamic plus-end is regulated by the plus-end tracking proteins (+TIPs). Certain +TIPs can function as a microtubule polymerase or rescue shrinking microtubules. Since budding yeast contains only a small number of microtubules, this organism is predestinated to study +TIPs and microtubule dynamics by microscopy on the system level. The exact function and mechanism of yeast +TIPs such as Bik1 remain unresolved. In addition, it is unexplained how kinesins such as Kip2 or Kip3 can act as a microtubule polymerase or rescue factor. In my thesis, the budding yeast +TIPs Bik1, Kip2 and Kip3 were investigated to understand the role of these proteins in the formation of the +TIP network and how these proteins are capable of influencing microtubule dynamics. Recently, it has been discovered that minus-end tracking proteins (-TIPs) recognize the minus-end in cells such as neuronal cells. However, it is enigmatic how -TIPs target the microtubule minus-end. In order to elucidate the mechanism how -TIPs track the minus-end, my work focused on the discovered first -TIP class of CAMSAPs. In all projects, biophysical methods were applied, and besides for Kip2 crystal structures were determined to unravel mechanistic details of the proteins. In budding yeast, Bik1 plays an important role especially in the dynein pathway, which is one of two major pathways for spindle positioning. Bim1 localizes Bik1 to the microtubule plus-end because Bik1 cannot autonomously track the plus-end. Here, we biophysically and structurally describe the interaction of the Bik1 CAP-Gly domain with the C-terminal tail of the +TIP Bim1. The crystal structure of the complex showed that Bik1 CAP-Gly binds specifically to C-terminal phenylalanine residues with a different binding mode compared to CAP-Gly domains of higher eukaryotes. Based on the structure, two different mutants were conceived to perturb the Bik1-Bim1 interaction. Then, the effect of this perturbation on Bik1 localization, microtubule length and Kar9 function was analyzed in yeast cells. Besides, we proved that the coiled-coil of Bik1 interacts with the C-terminal tail of microtubule polymerase Stu2, establishing Bik1 as an adaptor protein between Bim1 and Stu2. Apart from Bim1, the budding yeast kinesin Kip2 also has the ability to transport Bik1 to the plusend. We biophysically characterized the interaction of the Bik1 coiled-coil with the Kip2 coiledcoil. The C-terminal unstructured part of Kip2 turned out to be essential for the Bik1-Kip2 interaction, allowing an elegant way to disrupt this interaction without removing the Kip2 coiledcoil. In addition, Kip2 functions as a microtubule polymerase. By studying the interaction of the Kip2 motor domain with soluble tubulin, we were able to postulate a mechanism how Kip2 can polymerize microtubules. Furthermore, we identified the importance of the Bik1-Kip2 interaction for the polymerase activity. The budding yeast kinesin Kip3 can depolymerize microtubules but exhibits the ability to rescue them as well. The N-terminal motor domain of Kip3 is responsible for the depolymerization activity. We discovered that Kip3 possesses a C-terminal tubulin-binding domain (TBD), followed by a weak microtubule-binding domain. The crystal structure of the Kip3 TBD was solved, and a sophisticated assembly of alpha-helices was revealed. Furthermore, the combination of the Kip3 motor domain together with the Kip3 TBD was identified as the minimal construct that can rescue microtubules. Therefore, we proposed that the Kip3 motor domain can also act as an anchor at the microtubule plus-end so that the Kip3 TBD can fulfill its rescue function by either increasing the tubulin concentration or facilitating the exchange of tubulin. Most microtubules minus-ends are attached to the centrosome. However, some microtubules can occur with free minus-ends because not all microtubules are attached to the centrosome or cells such as neuronal cells entirely lack the centrosome. Thus, -TIPs like CAMSAPs can stabilize these free minus-ends. CAMSAP proteins have a CKK domain that can autonomously track the microtubule minus-end. In this study, we determined the crystal structure of this CKK domain. Our collaborator used this structure for fitting into a cryo-EM map of microtubules decorated by the CKK domain. Combined with other experimental results, we found that the CKK domain recognizes a unique curved state of tubulin that only occurs at the microtubule minus-end. Overall, important insights into the mechanisms of Bik1 Kip2, Kip3 and CAMSAP were obtained. In the +TIP network, the understanding of Bik1 as a critical adaptor protein was considerably increased. Furthermore, we revealed new insights into the function of Kip2 as a microtubule polymerase. For Kip3, a mechanism for its microtubule rescue function was postulated. In the case of CAMSAP, it was discovered how this protein can recognize the microtubule minus-end. This represents the first described mechanism of a -TIP

Transfer of manualized CBT for social phobia into clinical practice (SOPHO-PRAX) : a study protocol for a cluster-randomized controlled trial

Background: Cognitive-behavioral therapy (CBT) is generally known to be efficacious in the treatment of social phobia when applied in RCT's, namely when the treatment manual is based on the Clark-Wells approach. However, little is known about the efficacy of manualized treatments in routine clinical practice (Phase IV of psychotherapy research). The present study (SOPHO-PRAX) is a continuation of a large multi-centre randomized clinical trial (SOPHO-NET) and analyses the extent to which additional training practitioners in manualized procedures enhances treatment effect. Methods: N = 36 private practitioners will be included in three treatment centres and randomly designated to either training in manualized CBT or no specific training. The treatment effects of the therapies conducted by both groups of therapists will be compared. A total of 162 patients (N = 116 completers; N = 58 per condition) will be enrolled. Liebowitz Social Anxiety Scale (LSAS) will serve as primary outcome measure. Remission from social phobia is defined as LSAS total [less than or equal to] 30 points. Data will be collected at treatment begin, after 8, 15, and 25 sessions (50 mins. each), at treatment completion, as well at 6 and 12 months post-treatment. Discussion: The present CBT trial combines elements of randomized-controlled trials and naturalistic studies in an innovative way. It will directly inform about the incremental effects of procedures established in a controlled trial into clinical practice. Study results are relevant to health care decisions and policy. They may serve to improve quality of treatment, and shorten the timeframe between the development and widespread dissemination of effective methods, thereby reducing health cost expenditures. The results of this study will not only inform about the degree to which the new methods lead to an improvement of treatment course and outcome, but also about whether the effects of routine psychotherapeutic treatment are comparable to those of the controlled, strictly manualized treatments of the SOPHO-NET study. Trial Registration: ClinicalTrials.gov identifier: NCT01388231. This study was funded by the German Federal Ministry of Education and Research (SOPHO-NET: BMBF 01GV0607; SOPHO-PRAX: BMBF 01GV1001)

Weighted Fractional and Integral k-Matching in Hypergraphs

We consider the problem of finding polynomial-time approximations of maximal weighted k-matchings in a hypergraph and investigate the relationship between the integral and fractional maxima of the corresponding 0-1 integer linear program and its LP-relaxation. We extend results of Raghavan, who gave a deterministic approximation algorithm for unweighted k-matching, to the weighted case and compare the so obtained lower bound for the ratio of the integer and fractional maximum with a lower bound of Aharoni, Erdös and Linial

On Derandomized Approximation Algorithms

With the design of powerful randomized algorithms the transformation of a randomized algorithm or probabilistic existence result for combinatorial problems into an efficient deterministic algorithm (called derandomization) became an important issue in algorithmic discrete mathematics. In the last years several interesting examples of derandomization have been published, like discrepancy in hypergraph colouring, packing integer programs and an algorithmic version of the Lovász-Local-Lemma. In this paper the derandomization method of conditional probabilities of Raghavan/Spencer is extended using discrete martingales. As a main result pessimistic estimators are constructed for combinatorial approximation problems involving non-linear objective functions with bounded martingale differences. The theory gives polynomial-time algorithms for the linear and quadratic lattice approximation problem and a quadratic variant of the matrix balancing problem extending results of Spencer, Beck/Fiala and Raghavan. Finally a probabilistic existence result of Erdös on the average graph bisection is transformed into a deterministic algorithm

Algorithmic Chernoff-Hoeffding Inequalities in Integer Programming

Proofs of classical Chernoff-Hoeffding bounds have been used to obtain polynomial-time implementations of Spencer's derandomization method of conditional probabilities on usual finite machine models: given m events whose complements are large deviations corresponding to weighted sums of n mutually independent Bernoulli trials, Raghavan's lattice approximation algorithm constructs for 0-1 weights and integer deviation terms in O(mn)-time a point for which all events hold. For rational weighted sums of Bernoulli trials the lattice approximation algorithm or Spencer's hyperbolic cosine algorithm are deterministic procedures, but a polynomial-time implementation was not known. We resolve this problem with an O(mn^2log frac{mn}{epsilon})-time algorithm, whenever the probability that all events hold is at least epsilon > 0. Since such algorithms simulate the proof of the underlying large deviation inequality in a constructive way, we call it the algorithmic version of the inequality. Applications to general packing integer programs and resource constrained scheduling result in tight and polynomial-time approximations algorithms

Impact of tungsten incorporation on the tribomechanical behavior of AlCrWxSiN films at room and elevated temperature

AlCrWxSiN thin films (0 ≤ x ≤ 17.1 at.%) were synthesized by means of a hybrid magnetron sputtering process, merging direct current (DC) as well as tungsten high power impulse magnetron sputtering (HiPIMS) supplies. The influences of increasing the tungsten contents on the structural as well as the friction and wear behavior at room and high temperatures (500 °C) were elaborated. As a reference, a W61.4N38.6 system served to analyze synergetic effects on the oxidation behavior. Increased tungsten contents in AlCrWxSiN resulted in more distinctive (200)-, (202)-, and (311)- crystal orientations. A W/Cr ratio of ~1 could be correlated with a denser film growth, the highest hardness (24.3 ± 0.7 GPa), and a significantly decreased wear coefficient (<0.3 × 10−5 mm3/Nm). Tribological tests performed at room temperature revealed that the coefficient of friction decreased with higher tungsten contents to µ~0.35. In contrast, at elevated temperatures, the coefficient of friction increased with higher W concentrations due to spotty oxidations in the wear track, which resulted in a locally increased surface roughness. Finally, a phase transformation of the WN film to m-WO3 did not contribute to a friction reduction at 500 °C

Sprint interval training on the vertical treadmill improves aerobic and anaerobic running performance

The vertical treadmill (VertiRun) is an unresearched mode of exercise where users engage in a “running-like” action whilst body weight is supported by a recumbent bench and overhanging resistance cables are tethered to the user’s ankles. The purpose of this study was to determine the effects of training on a VertiRun and any cross-training effect on running performance. Thirty active males (age, 22±4 years; stature, 1.79±0.08 m; body mass, 78.5±12.6 kg) volunteered for this study. Participants’ aerobic and anaerobic running performance were determined by incremental maximum rate of oxygen consumption (VO2max) treadmill test and a maximum anaerobic running test (MART), respectively. Participants were matched and then randomly assigned to either a VertiRun group, 20-m shuttle sprint group or control group. The intervention consisted of 4–6, 30-sec all-out efforts with 4-min recovery between bouts, 3 days a week for 6 weeks. The pre- and postintervention VO2max and MART were analysed using a mixed repeated measures analysis of variance. MART increased by 4.5% in the VertiRun group (P=0.006) and 4% in the sprint group (P<0.001). VO2max increased by 6.2% in the VertiRun group (P=0.009) and 5.5% in the sprint group (P=0.020). The MART and VO2max of the control group were unchanged (P=0.910 and P=0.915, respectively). These data suggest that the VertiRun could be an effective cross-training mode for running and could supplement training programmes. Also, as VertiRun is a low-impact exercise it might be useful in the physical preparation of athletes returning to sport following lower limb injury

Atmospheric Thermal Properties of Venus and Mars. Investigation of CO2 Absorption Lines using Ground-Based Mid-Infrared Heterodyne Spectroscopy

Atmospheric thermal properties of different altitude layers of terrestrial planets can be deduced from pressure-broadened molecular transition features. Ground-based heterodyne spectroscopy is used to observe the nightside of Venus by probing single pressure-broadened CO2 absorption lines at around 10 µm. In addition, a dayside spectrum of Mars, also containing a pressure-broadened absorption feature was investigated. Infrared heterodyne spectroscopy is sensitive to those atmospheric layers, which can be identified as the absorption line formation region. These layers correspond to an altitude range in the Venusian mesosphere between ~60 and ~95km. On Mars, the line formation region is located in the troposphere between the surface and an altitude of ~35km. Retrieval of atmospheric parameters is based on a Levenberg-Marquard chi-squared optimization that iteratively compares observed data to telluric transmittance corrected planetary top-of-atmosphere spectra calculated using a radiative transfer algorithm. A sophisticated proof of concept is performed to investigate the influence of the altitude resolution and to demonstrate the reliability of the newly developed retrieval technique. During two observing campaigns in March and May 2012, four different locations on the Venusian nightside hemisphere were investigated. In this thesis, the retrieval of vertical temperature profiles in the nightside atmosphere of Venus using mid-infrared heterodyne spectroscopy is reported for the first time. The retrieval can be deduced with an altitude resolution of ~4.5km. The retrieved profiles are compared to existing space- and ground-based observations as well as to the Venus International Reference Atmosphere. The temperatures found are in good agreement to other retrieval techniques. Emphasis is given to the comparison of the temperatures from one specific location to thermal profiles simultaneously obtained with the Venus Express Radio Science Experiment during a coordinated observing campaign in May 2012. Sub-Doppler resolution infrared heterodyne observations can now provide temperature measurements on the dark side of Venus that complement those techniques. Analysis of a broad CO2 absorption feature obtained at the Martian dayside during an observing campaign in 2010 is performed and a preliminary temperature profile is retrieved. This profile is compared to predictions from the Mars Climate Database and found to be in satisfactory agreement. A further detailed poof of concept is provided, addressing the specific preconditions of the Martian atmosphere and analyzing the contribution of the solar induced non-thermal CO2 emission on the retrieval method. It is found, that the deduction of atmospheric dayside temperatures on Mars is subject to additional restrictions, which are due to the thin atmosphere and the multifarious topography

Application of the eutectic high entropy alloy Nb0.73CoCrFeNi2.1 for high temperature joints

The eutectic high entropy alloy Nb0.73CoCrFeNi2.1 was manufactured by means of arc smelting and the obtained ingots were cut into 300-μm-thick foils, which were used as filler alloys to braze Crofer 22 APU to Hf-metallized yttria-stabilized zirconia (3YSZ). The brazing process was conducted in a vacuum furnace at 1200 °C for 5 min at a vacuum of 4.3·10–4 mbar. In order to minimize the intense diffusion and erosion of the steel substrate, a heating and cooling rate of 50 K/min was applied. Sound joints without any pores or flaws were obtained. The microstructure of the joints consisted of an HfO2 reaction layer at the ceramic interface and the same eutectic structure consisting of a Laves phase and a solid solution that was already detected in the smelted foil. The average hardness of the microstructure in the joint seam amounted to 352 ± 17 HV0.01 and the joints reached strength values up to 97 ± 7 MPa while the fracture area was always located at the ceramic interface in the HfO2 layer. Comparable joints, with AgCuTi3 as filler metal, brazed at 920 °C, only reached a shear strength of ~ 52 ± 2 MPa