U12-type Spliceosome: Localization and Effects of Splicing Efficiency on Gene Expression

The removal of non-coding sequences, introns, is an essential part of messenger RNA processing. In most metazoan organisms, the U12-type spliceosome processes a subset of introns containing highly conserved recognition sequences. U12-type introns constitute less than 0,5% of all introns and reside preferentially in genes related to information processing functions, as opposed to genes encoding for metabolic enzymes. It has previously been shown that the excision of U12-type introns is inefficient compared to that of U2-type introns, supporting the model that these introns could provide a rate-limiting control for gene expression. The low efficiency of U12-type splicing is believed to have important consequences to gene expression by limiting the production of mature mRNAs from genes containing U12-type introns. The inefficiency of U12-type splicing has been attributed to the low abundance of the components of the U12-type spliceosome in cells, but this hypothesis has not been proven. The aim of the first part of this work was to study the effect of the abundance of the spliceosomal snRNA components on splicing. Cells with a low abundance of the U12-type spliceosome were found to inefficiently process U12-type introns encoded by a transfected construct, but the expression levels of endogenous genes were not found to be affected by the abundance of the U12-type spliceosome. However, significant levels of endogenous unspliced U12-type intron-containing pre-mRNAs were detected in cells. Together these results support the idea that U12-type splicing may limit gene expression in some situations. The inefficiency of U12-type splicing has also promoted the idea that the U12-type spliceosome may control gene expression, limiting the mRNA levels of some U12-type intron-containing genes. While the identities of the primary target genes that contain U12-type introns are relatively well known, little has previously been known about the downstream genes and pathways potentially affected by the efficiency of U12-type intron processing. Here, the effects of U12-type splicing efficiency on a whole organism were studied in a Drosophila line with a mutation in an essential U12-type spliceosome component. Genes containing U12-type introns showed variable gene-specific responses to the splicing defect, which points to variation in the susceptibility of different genes to changes in splicing efficiency. Surprisingly, microarray screening revealed that metabolic genes were enriched among downstream effects, and that the phenotype could largely be attributed to one U12-type intron-containing mitochondrial gene. Gene expression control by the U12-type spliceosome could thus have widespread effects on metabolic functions in the organism. The subcellular localization of the U12-type spliceosome components was studied as a response to a recent dispute on the localization of the U12-type spliceosome. All components studied were found to be nuclear indicating that the processing of U12-type introns occurs within the nucleus, thus clarifying a question central to the field. The results suggest that the U12-type spliceosome can limit the expression of genes that contain U12-type introns in a gene-specific manner. Through its limiting role in pre-mRNA processing, the U12-type splicing activity can affect specific genetic pathways, which in the case of Drosophila are involved in metabolic functions.

Expression, Enzymatic Activities and Subcellular Localization of Hepatitis E Virus and Semliki Forest Virus Replicase Proteins

Viruses are submicroscopic, infectious agents that are obligate intracellular parasites. They adopt various types of strategies for their parasitic replication and proliferation in infected cells. The nucleic acid genome of a virus contains information that redirects molecular machinery of the cell to the replication and production of new virions. Viruses that replicate in the cytoplasm and are unable to use the nuclear transcription machinery of the host cell have developed their own transcription and capping systems. This thesis describes replication strategies of two distantly related viruses, hepatitis E virus (HEV) and Semliki Forest virus (SFV), which belong to the alphavirus-like superfamily of positive-strand RNA viruses. We have demonstrated that HEV and SFV share a unique cap formation pathway specific for alphavirus-like superfamily. The capping enzyme first acts as a methyltransferase, catalyzing the transfer of a methyl group from S-adenosylmethionine to GTP to yield m7GTP. It then transfers the methylated guanosine to the end of viral mRNA. Both reactions are virus-specific and differ from those described for the host cell. Therefore, these capping reactions offer attractive targets for the development of antiviral drugs. Additionally, it has been shown that replication of SFV and HEV takes place in association with cellular membranes. The origin of these membranes and the intracellular localization of the components of the replication complex were studied by modern microscopy techniques. It was demonstrated that SFV replicates in cytoplasmic membranes that are derived from endosomes and lysosomes. According to our studies, site for HEV replication seems to be the intermediate compartment which mediates the traffic between endoplasmic reticulum and the Golgi complex. As a result of this work, a unique mechanism of cap formation for hepatitis E virus replicase has been characterized. It represents a novel target for the development of specific inhibitors against viral replication.

Synthesis of Thioesters by Simultaneous Activation of Carboxylic Acids and Alcohols Using PPh3/NBS with Benzyltriethylammonium Tetrathiomolybdate as the Sulfur Transfer Reagent

A new and simple route for the synthesis of thioesters starting from carboxylic acids and alcohols is reported by using tetrathiomolybdate as the key sulfur transfer reagent. Triphenylphosphane and N-bromosuccinimide were used for the activation of the carboxylic acid and alcohol in the same pot followed by the transfer of sulfur from tetrathiomolybdate. Thioesters were obtained in good to moderate yields. Primary alcohols show excellent reactivity and gave good yields of the corresponding thioesters, whereas secondary alcohols gave moderate yields and tertiary alcohols were very less reactive and gave poor yields of the corresponding thioesters.

Localization of handwritten text in documents using moment invariants and Delaunay triangulation

This paper describes an approach based on Zernike moments and Delaunay triangulation for localization of hand-written text in machine printed text documents. The Zernike moments of the image are first evaluated and we classify the text as hand-written using the nearest neighbor classifier. These features are independent of size, slant, orientation, translation and other variations in handwritten text. We then use Delaunay triangulation to reclassify the misclassified text regions. When imposing Delaunay triangulation on the centroid points of the connected components, we extract features based on the triangles and reclassify the text. We remove the noise components in the document as part of the preprocessing step so this method works well on noisy documents. The success rate of the method is found to be 86%. Also for specific hand-written elements such as signatures or similar text the accuracy is found to be even higher at 93%.

Simultaneous material selection and geometry design of statically determinate trusses using continuous optimization

In this work, we explore simultaneous geometry design and material selection for statically determinate trusses by posing it as a continuous optimization problem. The underlying principles of our approach are structural optimization and Ashby’s procedure for material selection from a database. For simplicity and ease of initial implementation, only static loads are considered in this work with the intent of maximum stiffness, minimum weight/cost, and safety against failure. Safety of tensile and compression members in the truss is treated differently to prevent yield and buckling failures, respectively. Geometry variables such as lengths and orientations of members are taken to be the design variables in an assumed layout. Areas of cross-section of the members are determined to satisfy the failure constraints in each member. Along the lines of Ashby’s material indices, a new design index is derived for trusses. The design index helps in choosing the most suitable material for any geometry of the truss. Using the design index, both the design space and the material database are searched simultaneously using gradient-based optimization algorithms. The important feature of our approach is that the formulated optimization problem is continuous, although the material selection from a database is an inherently discrete problem. A few illustrative examples are included. It is observed that the method is capable of determining the optimal topology in addition to optimal geometry when the assumed layout contains more links than are necessary for optimality.

Simultaneous observation of positive and negative nuclear Overhauser effects in oligopeptides due to segmental motio

Nuclear Overhauser effect (NOE) studies of the symmetrical cystine peptides (Formula: see text) (n = 1-3) in dimethylsulfoxide, have resulted in the simultaneous observation of both positive and negative NOEs. Positive NOEs are observed on the Trp C2H and C4H protons of the indole ring upon irradiation of Trp C alpha H and C beta H2 resonances in the peptides where n = 1 and 2. Negative NOEs are observed between backbone NH and C alpha H protons. The magnitudes of the observed NOEs are sensitive to changes in molecular size and solvent viscosity. The results demonstrate that NOEs may be a useful probe of sidechain segmental motion in oligopeptides.

Binaural Localization and Masking

Binaural experiments are described which indicate that the ability of the brain to localize a desired sound and to suppress undesired sounds coming from other directions can be traced in part to the different times of arrival of a sound at the two ears. It is suggested that the brain inserts a time delay in one of the two nerve paths associated with the ears so as to be able to compare, and thus concentrate on, those sounds arriving at the ears with this particular time of arrival distance.The ability to perceive weak sounds binaurally in the presence of noise is shown to be a simple function of the direction of the desired sound and noise. An explanation is given for the effect reported by Koenig that front and rear confusion is avoided by head movements.

Oncolytic Adenoviruses with Radiation Therapy for Treatment of Prostate Cancer

Prostate cancer is the most common cancer in males. Although many patients with localized disease can be cured with surgery and radiotherapy, advanced disease and especially castration resistant metastatic disease remains incurable, with a median life expectancy of less than 18 months. Oncolytic adenoviruses (Ads) are a new promising treatment against cancer due to their innate capacity to kill cancer cells. Viral replication in tumor cells leads to oncolysis and production of a multiplicity of new virions that are capable of further destroying cancerous tissue. Oncolytic Ads can be modified for tumor targeted infection and replication and be armed with therapeutic transgenes to maximize the oncolytic effect. Worldwide, clinical trials with oncolytic Ads have demonstrated good safety while the antitumor efficacy remains to be improved. Importantly, the best responses have been reported when oncolytic adenoviruses have been combined with standard cancer treatments, such as chemotherapy and radiation. Further, a challenge in many virotherapy approaches has been the monitoring of virus replication in vivo. Reporter genes have been extensively used as transgenes to evaluate the biodistribution of the virus and activity of specific promoters. However, these techniques are often limited to preclinical evaluation and not amenable to human use. The aim of the thesis was to find and develop new oncolytic Ads with maximum efficacy against metastatic, castration resistant prostate cancer and study them in vitro and in vivo combined to different forms of radiation therapy. Using combination therapy, we were aiming for better antitumor efficacy with reduced side effects. Capsid modified Ads for enhanced transduction were studied. Serotype 3 targeted chimera, Ad5/3, was found to have enhanced infectivity for prostate cancer and was used for developing new viruses for the study. Correlation between Ad-encoded marker peptide secretion and simultaneous viral replication was evaluated and the effects of radiotherapy on viral replication were studied in detail. We found that the repair of double strand breaks caused by ionizing radiation was inhibited by adenoviral proteins and led to autophagic cell death. Both subcutaneous models and intrapulmonary tumor models mimicking metastatic, aggressive disease were used in vivo. Virus efficacy was evaluated by intratumoral injections. Also, intravenous administration was evaluated to study the effectiveness in metastatic disease. Oncolytic adenovirus treatment led to significant tumor growth control and increased the survival rate of the mice. These results were further improved when oncolytic Ads were combined with radiation therapy. Oncolytic Ads expressing human sodium/iodide transporter (hNIS) as a transgene were evaluated for their oncolytic potency and for the functionality of hNIS in vitro and in vivo. Monitoring of viral replication was also assessed using different imaging modalities relative to clinical use. SPECT imaging of tumor-bearing mice was evaluated and combined with simultaneous CT-scanning to obtain important anatomical information on biodistribution, also in a three-dimensional form. It was shown that hNIS-expressing adenoviruses could harbour a bi-functional transgene allowing for localization and imaging of viral replication. Targeted radiotherapy was applied by systemic radioiodide administration and resulted in iodide accumulation into Ad-infected tumor. The combination treatment showed significantly enhanced antitumor efficacy in mice bearing prostate cancer tumors. In summary, the results presented above aim to provide new treatment modalities for castration resistant prostate cancer. Molecular insights were provided for better understanding of the benefits of combined radiation therapy and oncolytic adenoviruses, which will hopefully facilitate the translation of the approach into clinical use for humans.

First simultaneous measurement of the top quark mass in the lepton+jets and dilepton channels at CDF

We present a measurement of the mass of the top quark using data corresponding to an integrated luminosity of 1.9fb^-1 of ppbar collisions collected at sqrt{s}=1.96 TeV with the CDF II detector at Fermilab's Tevatron. This is the first measurement of the top quark mass using top-antitop pair candidate events in the lepton + jets and dilepton decay channels simultaneously. We reconstruct two observables in each channel and use a non-parametric kernel density estimation technique to derive two-dimensional probability density functions from simulated signal and background samples. The observables are the top quark mass and the invariant mass of two jets from the W decay in the lepton + jets channel, and the top quark mass and the scalar sum of transverse energy of the event in the dilepton channel. We perform a simultaneous fit for the top quark mass and the jet energy scale, which is constrained in situ by the hadronic W boson mass. Using 332 lepton + jets candidate events and 144 dilepton candidate events, we measure the top quark mass to be mtop=171.9 +/- 1.7 (stat. + JES) +/- 1.1 (syst.) GeV/c^2 = 171.9 +/- 2.0 GeV/c^2.

Intrinsic Electron localization in manganites

We mention here an unusual disorder effect in manganites, namely the ubiquitous hopping behavior for electron transport observed in them over a wide range of doping. We argue that the implied Anderson localization is intrinsic to manganites, because of the existence of polarons in them which are spatially localized, generally at random sites (unless there is polaron ordering). We have developed a microscopic two fluid lb model for manganites, where l denotes lattice site localized l polarons, and b denotes band electrons. Using this, and the self-consistent theory of localization, we show that the occupied b states are Anderson localized in a large range of doping due to the scattering of b electrons from l polarons. Numerical simulations which further include the effect of long range Coulomb interactions support this, as well the existence of a novel polaronic Coulomb glass. A consequence is the inevitable hopping behaviour for electron transport observed in doped insulating manganites.

Simultaneous heat and mass transfer in unsteady free convection from two-dimensional and axisymmetric bodies

The unsteady laminar free convection boundary layer flows around two-dimensional and axisymmetric bodies placed in an ambient fluid of infinite extent have been studied when the flow is driven by thermal buoyancy forces and buoyancy forces from species diffusion. The unsteadiness in the flow field is caused by both temperature and concentration at the wall which vary arbitrarily with time. The coupled nonlinear partial differential equations with three independent variables governing the flow have been solved numerically using an implicit finite-difference scheme in combination with the quasilinearization technique. Computations have been performed for a circular cylinder and a sphere. The skin friction, heat transfer and mass transfer are strongly dependent on the variation of the wall temperature and concentration with time. Also the skin friction and heat transfer increase or decrease as the buoyancy forces from species diffusion assist and oppose, respectively, the thermal buoyancy force, whereas the mass transfer rate is higher for small values of the ratio of the buoyancy parameters than for large values. The local heat and mass transfer rates are maximum at the stagnation point and they decrease progressively with increase of the angular position from the stagnation point.

Text localization and extraction from complex gray images

We propose two texture-based approaches, one involving Gabor filters and the other employing log-polar wavelets, for separating text from non-text elements in a document image. Both the proposed algorithms compute local energy at some information-rich points, which are marked by Harris' corner detector. The advantage of this approach is that the algorithm calculates the local energy at selected points and not throughout the image, thus saving a lot of computational time. The algorithm has been tested on a large set of scanned text pages and the results have been seen to be better than the results from the existing algorithms. Among the proposed schemes, the Gabor filter based scheme marginally outperforms the wavelet based scheme.

Binarization and localization of text images captured on a mobile phone camera

This paper proposes and compares four methods of binarzing text images captured using a camera mounted on a cell phone. The advantages and disadvantages(image clarity and computational complexity) of each method over the others are demonstrated through binarized results. The images are of VGA or lower resolution.