Palladin, a novel microfilament protein

Palladin is a novel actin microfilament associated protein, which together with myotilin and myopalladin forms a novel cytoskeletal IgC2 domain protein family. Whereas the expression of myotilin and myopalladin is limited mainly to striated muscle, palladin is widely expressed in both epithelial and mesenchymal tissues, including heart and the nervous system. Palladin has a complex genetic structure and it is expressed as several different sized and structured splice variants, which also display differences in their expression pattern and interactions. In muscle cells, all the family members localize to the sarcomeric Z-disc, and in non-muscle cells palladin also localizes to the stress-fiber-dense regions, lamellipodia, podosomes and focal adhesions. A common feature of this protein family is the binding to α-actinin, but other interactions are mostly unique to each member. Palladin has been shown to interact with several proteins, including VASP, profilin, Eps8, LASP-1 and LPP. Its domain structure, lack of enzymatic activity and multiple interactions define it as a molecular scaffolding protein, which links together proteins with different functional modalities into large complexes. Palladin has an important role in cytoskeletal regulation, particularly in stress fiber formation and stabilization. This assumption is supported by several experimental results. First, over-expression of palladin in non-muscle cells results in rapid reorganization of the actin cytoskeleton and formation of thick actin bundles. Second, the knock-down of palladin with anti-sense and siRNA techniques or knock-out by genetic methods leads to defective stress fiber formation. Furthermore, palladin is usually up-regulated in situations requiring a highly organized cytoskeleton, such as differentiation of dendritic cells, trophoblasts and myofibroblasts, and activation of astrocytes during glial scar formation. The protein family members have also direct disease linkages; myotilin missense mutations are the cause of LGMD1A and myofibrillar myopathy. Palladin mutations and polymorphisms, on the other hand, have been linked to hereditary pancreatic cancer and myocardial infarction, respectively. In this study we set out to characterize human palladin. We identified several palladin isoforms, studied their tissue distribution and sub-cellular localization. Four novel interaction partners were identified; ezrin, ArgBP2, SPIN90 and Src-kinase.The previously identified interaction between palladin and α-actinin was also characterized in detail. All the identified new binding partners are actin cytoskeleton associated proteins; ezrin links the plasma membrane to the cytoskeleton, ArgBP2 and SPIN90 localize, among other structures, to the lamellipodia and in cardiomyocytes to the Z-disc. Src is a transforming tyrosine kinase, which besides its role in oncogenesis has also important cytoskeletal associations. We also studied palladin in myofibroblasts, which are specialized cells involved in diverse physiological and pathological processes, such as wound healing and tissue fibrosis. We demonstrated that palladin is up-regulated during the differentiation of myofibroblasts in an isoform specific manner, and that this up-regulation is induced by TGF-β via activation of both the SMAD and MAPK signalling cascades. In summary, the results presented here describe the initial characterization of human palladin and offer a basis for further studies.

Novel insights on functions of myotilin/palladin family members

Poikkijuovaisen luuranko- ja sydänlihaksen supistumisyksikkö, sarkomeeri, koostuu tarkoin järjestyneistä aktiini- ja myosiinisäikeistä. Rakenne eroaa muista solutyypeistä, joissa aktiinisäikeistö muovautuu jatkuvasti ja sen järjestyminen säätelee solun muotoa, solujakautumista, soluliikettä ja solunsisäisten organellien kuljetusta. Myotilin, palladin ja myopalladin kuuluvat proteiiniperheeseen, jonka yhteispiirteenä ovat immunoglobuliinin kaltaiset (Igl) domeenit. Proteiinit liittyvät aktiinitukirankaan ja niiden arvellaan toimivan solutukirangan rakenne-elementteinä ja säätelijöinä. Myotilinia ja myopalladinia ilmennetään poikkijuovaisessa lihaksessa. Sen sijaan palladinin eri silmukointimuotoja tavataan monissa kudostyypeissä kuten hermostossa, ja eri muodoilla saattaa olla solutyypistä riippuvia tehtäviä. Poikkijuovaisessa lihaksessa kaikki perheen jäsenet sijaitsevat aktiinisäikeitä yhdistävässä Z-levyssä ja ne sitovat Z-levyn rakenneproteiinia, -aktiniinia. Myotilingeenin pistemutaatiot johtavat periytyviin lihastauteihin, kun taas palladinin mutaatioiden on kuvattu liittyvän periytyvään haimasyöpään ja lisääntyneeseen sydäninfarktin riskiin. Tässä tutkimuksessa selvitettin myotilinin ja pallainin toimintaa. Kokeissa löydettiin uusia palladinin 90-92kDa alatyyppiin sitoutuvia proteiineja. Yksi niistä on aktiinidynamiikkaa säätelevä profilin. Profilinilla on kahdenlaisia tehtäviä; se edesauttaa aktiinisäikeiden muodostumista, mutta se voi myös eristää yksittäisiä aktiinimolekyylejä ja edistää säikeiden hajoamista. Solutasolla palladinin ja profilinin sijainti on yhtenevä runsaasti aktiinia sisältävillä solujen reuna-alueilla. Palladinin ja profilinin sidos on heikko ja hyvin dynaaminen, joka sopii palladinin tehtävään aktiinisäideiden muodostumisen koordinoijana. Toinen palladinin sitoutumiskumppani on aktiinisäikeitä yhteensitova -aktiniini. -Aktiniini liittää solutukirangan solukalvon proteiineihin ja ankkuroi solunsisäisiä viestintämolekyylejä. Sitoutumista välittävä alue on hyvin samankaltainen palladinissa ja myotilinissa. Luurankolihaksen liiallinen toistuva venytys muuttaa Z-levyjen rakennetta ja muotoa. Prosessin aikana syntyy uusia aktiinifilamenttejä sisältäviä tiivistymiä ja lopulta uusia sarkomeereja. Löydöstemme perusteella myotilinin uudelleenjärjestyminen noudattaa aktiinin muutoksia. Tämä viittaa siihen, että myotilin liittää yhteen uudismuodostuvia aktiinisäikeitä ja vakauttaa niitä. Myotilin saattaa myös ankkuroida viesti- tai rakennemolekyylejä, joiden tehtävänä on edesauttaa Z-levyjen uudismuodostusta. Tulostemme perusteella arvelemme, että myotilin toimii Z-levyjen rakenteen vakaajana ja aktiinisäikeiden säätelijänä. Palladinin puute johtaa sikiöaikaiseen kuolemaan hiirillä, mutta myotilinin puutoksella ei ole samanlaisia vaikutuksia. Tuotettujen myotilin poistogeenisten hiirten todetiin syntyvän ja kehittyvän normaalisti eikä niillä esiintynyt rakenteellisia tai toiminnallisia häiriöitä. Toisaalta aiemmissa kokeissa, joissa hiirille on siirretty ihmisen lihastautia aikaansaava myotilingeeni, nähdään samankaltaisia kuin sairailla ihmisillä. Näin ollen muuntunut myotilin näyttä olevan lihaksen toiminnalle haitallisempi kuin myotilinin puute. Myotilinin ja palladinin yhteisvaikutusta selvittääksemme risteytimme myotilin poistegeenisen hiiren ja hiirilinjan, joka ilmentää puutteellisesti palladinin 200 kDa muotoa. Puutteellisesti 200 kDa palladinia ilmentävien hiirten sydänlihaksessa todettiin vähäisiä hienorakenteen muutoksia, mutta risteytetyillä hiirillä tavattiin rakenteellisia ja toiminnallisia muutoksia myös luurankolihaksessa. Tulosten perusteella voidaan todeta, että palladinin 200 kDa muoto säätelee sydänlihassolujen rakennetta. Luurankolihaksessa sen sijaan myotilinilla ja palladinilla näyttäisi olevan päällekkäisiä tehtäviä.

NMR Spectroscopy of Multi-domain Proteins : Immunoglobulin-like Domains of Human Filamin A

Proteins are complex biomacromolecules playing fundamental roles in the physiological processes of all living organisms. They function as structural units, enzymes, transporters, process regulators, and signal transducers. Defects in protein functions often derive from genetic mutations altering the protein structure, and impairment of essential protein functions manifests itself as pathological conditions. Proteins operate through interactions, and all protein functions depend on protein structure. In order to understand biological mechanisms at the molecular level, one has to know the structures of the proteins involved. This thesis covers structural and functional characterization of human filamins. Filamins are actin-binding and -bundling proteins that have numerous interaction partners. In addition to their actin-organizing functions, filamins are also known to have roles in cell adhesion and locomotion, and to participate in the logistics of cell membrane receptors, and in the coordination of intracellular signaling pathways. Filamin mutations in humans induce severe pathological conditions affecting the brain, bones, limbs, and the cardiovascular system. Filamins are large modular proteins composed of an N-terminal actin-binding domain and 24 consecutive immunoglobulin-like domains (IgFLNs). Nuclear magnetic resonance (NMR) spectroscopy is a versatile method of gaining insight into protein structure, dynamics and interactions. NMR spectroscopy was employed in this thesis to study the atomic structure and interaction mechanisms of C-terminal IgFLNs, which are known to house the majority of the filamin interaction sites. The structures of IgFLN single-domains 17 and 23 and IgFLN domain pairs 16-17 and 18-19 were determined using NMR spectroscopy. The structures of domain pairs 16 17 and 18 19 both revealed novel domain domain interaction modes of IgFLNs. NMR titrations were employed to characterize the interactions of filamins with glycoprotein Ibα, FilGAP, integrin β7 and dopamine receptors. Domain packing of IgFLN domain sextet 16 21 was further characterized using residual dipolar couplings and NMR relaxation analysis. This thesis demonstrates the versatility and potential of NMR spectroscopy in structural and functional studies of multi-domain proteins.

Generalised regression estimation for domain class frequencies

This study examines the properties of Generalised Regression (GREG) estimators for domain class frequencies and proportions. The family of GREG estimators forms the class of design-based model-assisted estimators. All GREG estimators utilise auxiliary information via modelling. The classic GREG estimator with a linear fixed effects assisting model (GREG-lin) is one example. But when estimating class frequencies, the study variable is binary or polytomous. Therefore logistic-type assisting models (e.g. logistic or probit model) should be preferred over the linear one. However, other GREG estimators than GREG-lin are rarely used, and knowledge about their properties is limited. This study examines the properties of L-GREG estimators, which are GREG estimators with fixed-effects logistic-type models. Three research questions are addressed. First, I study whether and when L-GREG estimators are more accurate than GREG-lin. Theoretical results and Monte Carlo experiments which cover both equal and unequal probability sampling designs and a wide variety of model formulations show that in standard situations, the difference between L-GREG and GREG-lin is small. But in the case of a strong assisting model, two interesting situations arise: if the domain sample size is reasonably large, L-GREG is more accurate than GREG-lin, and if the domain sample size is very small, estimation of assisting model parameters may be inaccurate, resulting in bias for L-GREG. Second, I study variance estimation for the L-GREG estimators. The standard variance estimator (S) for all GREG estimators resembles the Sen-Yates-Grundy variance estimator, but it is a double sum of prediction errors, not of the observed values of the study variable. Monte Carlo experiments show that S underestimates the variance of L-GREG especially if the domain sample size is minor, or if the assisting model is strong. Third, since the standard variance estimator S often fails for the L-GREG estimators, I propose a new augmented variance estimator (A). The difference between S and the new estimator A is that the latter takes into account the difference between the sample fit model and the census fit model. In Monte Carlo experiments, the new estimator A outperformed the standard estimator S in terms of bias, root mean square error and coverage rate. Thus the new estimator provides a good alternative to the standard estimator.

Measurement of $d\sigma/dy$ of Drell-Yan $e^+e^-$ pairs in the $Z$ Mass Region from $p\bar{p}$ Collisions at $\sqrt{s}=1.96$ TeV

We report on a CDF measurement of the total cross section and rapidity distribution, $d\sigma/dy$, for $q\bar{q}\to \gamma^{*}/Z\to e^{+}e^{-}$ events in the $Z$ boson mass region ($66M_{ee}

Search for exclusive Z boson production and observation of high mass ppbar->gammagamma->p+ll+pbar events in ppbar collisions at sqrt(s) = 1.96 TeV

We present a search for exclusive Z boson production in proton-antiproton collisions at sqrt(s) = 1.96 TeV, using the CDF II detector at Fermilab. We observe no exclusive Z->ll candidates and place the first upper limit on the exclusive Z cross section in hadron collisions, sigma(exclu) gammagamma->p+ll+pbar, and measure the cross section for M(ll) > 40 GeV/c2 and |eta(l)|

Search for the Higgs boson produced in association with Z→ℓ+ℓ- using the matrix element method at CDF II

We present a search for associated production of the standard model (SM) Higgs boson and a $Z$ boson where the $Z$ boson decays to two leptons and the Higgs decays to a pair of $b$ quarks in $p\bar{p}$ collisions at the Fermilab Tevatron. We use event probabilities based on SM matrix elements to construct a likelihood function of the Higgs content of the data sample. In a CDF data sample corresponding to an integrated luminosity of 2.7 fb$^{-1}$ we see no evidence of a Higgs boson with a mass between 100 GeV$/c^2$ and 150 GeV$/c^2$. We set 95% confidence level (C.L.) upper limits on the cross-section for $ZH$ production as a function of the Higgs boson mass $m_H$; the limit is 8.2 times the SM prediction at $m_H = 115$ GeV$/c^2$.

Search for the Higgs Boson Produced in Association with Z→ℓ+ℓ- in pp̅ Collisions at √s=1.96 TeV

We present a search for the Higgs boson in the process $q\bar{q} \to ZH \to \ell^+\ell^- b\bar{b}$. The analysis uses an integrated luminosity of 1 fb$^{-1}$ of $p\bar{p}$ collisions produced at $\sqrt{s} =$ 1.96 TeV and accumulated by the upgraded Collider Detector at Fermilab (CDF II). We employ artificial neural networks both to correct jets mismeasured in the calorimeter, and to distinguish the signal kinematic distributions from those of the background. We see no evidence for Higgs boson production, and set 95% CL upper limits on $\sigma_{ZH} \cdot {\cal B}(H \to b\bar{b}$), ranging from 1.5 pb to 1.2 pb for a Higgs boson mass ($m_H$) of 110 to 150 GeV/$c^2$.

Measurement of the Ratio σtt̅ /σZ/γ*→ll and Precise Extraction of the tt̅ Cross Section

We report a measurement of the ratio of the tt̅ to Z/γ* production cross sections in √s=1.96  TeV pp̅ collisions using data corresponding to an integrated luminosity of up to 4.6  fb-1, collected by the CDF II detector. The tt̅ cross section ratio is measured using two complementary methods, a b-jet tagging measurement and a topological approach. By multiplying the ratios by the well-known theoretical Z/γ*→ll cross section predicted by the standard model, the extracted tt̅ cross sections are effectively insensitive to the uncertainty on luminosity. A best linear unbiased estimate is used to combine both measurements with the result σtt̅ =7.70±0.52  pb, for a top-quark mass of 172.5  GeV/c2.

Measurement of the Ratio σtt̅ /σZ/γ*→ll and Precise Extraction of the tt̅ Cross Section

We report a measurement of the ratio of the tt̅ to Z/γ* production cross sections in √s=1.96  TeV pp̅ collisions using data corresponding to an integrated luminosity of up to 4.6  fb-1, collected by the CDF II detector. The tt̅ cross section ratio is measured using two complementary methods, a b-jet tagging measurement and a topological approach. By multiplying the ratios by the well-known theoretical Z/γ*→ll cross section predicted by the standard model, the extracted tt̅ cross sections are effectively insensitive to the uncertainty on luminosity. A best linear unbiased estimate is used to combine both measurements with the result σtt̅ =7.70±0.52  pb, for a top-quark mass of 172.5  GeV/c2.

Measurement of Zγ production in pp̅ collisions at √s=1.96 TeV

The production rate and kinematics of photons produced in association with Z bosons are studied using 2/fb of p\bar{p} collision data collected at the Collider Detector at Fermilab. The cross section for p\bar{p} -> l^+ l^- gamma + X (where the leptons l are either muons or electrons with dilepton mass M_{ll} > 40 GeV/c^2, and where the photon has transverse energy Et_{gamma} > 7 GeV and is well separated from the leptons) is 4.6 +/- 0.2 (stat) +/- 0.3 (syst) +/- 0.3 (lum) pb, which is consistent with standard model expectations. We use the photon Et distribution from Z-gamma events where the Z has decayed to mu^+ mu^-, e^+ e^-, or nu\bar{nu} to set limits on anomalous (non-standard-model) trilinear couplings between photons and Z bosons.

Diffractive W and Z production at the Fermilab Tevatron

We report on a measurement of the fraction of events with a W or Z boson produced diffractively in antiproton-proton collisions at a center of mass energy of 1.96 TeV, using data from 0.6 inverse femtobarns of integrated luminosity collected with the CDF-II detector equipped with a Roman-pot spectrometer that detects the antiproton (pbar) from pbar+p --> pbar+[X+W/Z]. We find that (0.97 +/- 0.11)% of Ws and (0.85 +/- 0.22)% of Zs are produced diffractively in a region of (anti)proton fractional momentum loss (\xi) of 0.03-1t p+[X+W/Z]+pbar, and on exclusive Z production, pbar+p-->pbar+Z+p. No signal is seen above background for these processes, and comparisons are made with expectations.

Measurement of the Ratio σtt̅ /σZ/γ*→ll and Precise Extraction of the tt̅ Cross Section

We report a measurement of the ratio of the top-antitop to Z/gamma* production cross sections in sqrt(s) = 1.96 TeV proton-antiproton collisions using data corresponding to an integrated luminosity of up to 4.6 fb-1, collected by the CDF II detector. The top-antitop cross section ratio is measured using two complementary methods, a b-jet tagging measurement and a topological approach. By multiplying the ratios by the well-known theoretical Z/gamma*->ll cross section, the extracted top-antitop cross sections are effectively insensitive to the uncertainty on luminosity. A best linear unbiased estimate is used to combine both measurements with the result sigma_(top-antitop) = 7.70 +/- 0.52 pb, for a top-quark mass of 172.5 GeV/c^2.