A inserção de DNA exógeno no genoma hospedeiro é conseguida principalmente através da utilização de vias de reparo como a junção de pontas não homólogas, que possui caráter aleatório, e a recombinação homóloga, que possibilita o gene targeting. Algumas ferramentas como as TAL Effector Nucleases (TALENs) e o RNA interferência (RNAi) podem ser utilizadas para aumentar a taxa de integração específica e assim melhorar a eficiência e o direcionamento da edição gênica. Nesse trabalho utilizamos o silenciamento gênico mediano por short interference RNA (siRNA) para inibição temporária dos genes ATF7IP uma metiltrasferase, EP300 uma acetiltransferase e KU70 (NHEJ) e um par de TALENs complementares a uma região do gene da distrofina canina. Células Caninas MDCK I foram transfectadas por lipofectamina 2000 (Invitrogen) com 320pmol de siRNAs para ATF7IP e Ep300; e 64 pmol do SiRNA para KU70 em diferentes grupos, 40 horas depois as células foram transfectadas com 15 μg vetor molde derivado do pEGFP-N1 (Clonatech) e com 10 μg dos RNAm das TALENs. A seleção se deu em meio DMEM high com 600μg/ mL de G418 (Lonza) por 14-16 dias. As colônias coletadas através de biópsias foram analisadas por Polimerase Chain Reaction e sequenciamento gênico. Três pares de primers foram utilizados; um controle endógeno (GAPDH), um controle interno do inserto (Neo qPCR) e um para confirmação da recombinação homóloga (DMD3). Os grupos apresentaram grande variação na taxa de mortalidade celular e consequentemente no número de colônias: Com o grupo ATF7IP+Vetor (648c) apresentando maior número de colônias e o grupo EP300+Ku70+Vetor+TALENs o menor (1c). A maior taxa de recombinação ocorreu nos grupos no grupo ATF7IP +Ku70+Vetor+TALENs com 40% das células positivas para neomicina apresentado o evento gene targeting, um aumento considerável na taxa de recombinação quando comparada a porcentagem de 3,1% do controle transfectado somente com o vetor molde. Mostrando que o uso conjunto das TALENs com siRNAs foi um sucesso para o aumento de eventos de edição gênica direcionada. / The insertion of exogenous DNA into a host genome is achieved primarily through the use of DNA repair pathways such as Non-Homologous End Joining (NHEJ) and the Homologous Recombination (HR). The integration by NHEJ has a random feature and is much more common than HR insertions, which are more likely to produce gene targeting events . TAL effector nucleases (TALENs) and RNA interference (RNAi) can be used to increase the rate of specific integration and thus improving the efficiency of gene editing. In this work, we used short interference RNA (siRNA)-mediated gene silencing for transient inhibition of genes ATF7IP (implicated in histone methylation), EP300 (acetyltransferase) and Ku70 (essential to NHEJ) and a pair of TALENs RNAm complementary to canine muscle dystrophin (DMD) gene. MDCK I Canine Cells were transfected by lipofectamine 2000 (Invitrogen) with 320 pmol of siRNAs for ATF7IP and EP300; and 64 pmol of siRNA for Ku70 in different groups. After 40 hours cells were transfected with 15 μg of a vector derived from pEGFP- N1 (Clontech) containing two regions homologous to the canine DMD gene (left arm length: 873 bp and right arm length: 1370 bp) and 10 μg of TALEN mRNA. The cell selection was achieved with DMEM high glucose with 600μg/ml G418 for 14-16 days. The colonies collected through biopsies were analyzed by polymerase chain reaction and gene sequencing. Three pairs of primers were used; an endogenous control (GAPDH) , an internal control of the insert (Neo qPCR) and a primer set to confirm the occurrence of homologous recombination events (DMD3). .Groups showed great variation in cell death rate and consequently in the number of colonies: ATF7IP+Vector had highest number of colonies (648c) and the group EP300+Ku70+Vetor+TALENs the lowest one (1c) The highest rate of homologous recombination was in ATF7IP +Ku70+Vetor+TALENs group that had 40% of the neomycin positives cells confirmed as gene targeting events, a considerable increase in the recombination rate compared to the 3.1% in the control group transfected only with the template vector. That shows that the combined use of siRNAs and TALENs was a success for increasing directed gene editing events.

Advertising in mobile apps are increasing and so is the need to show the right ad to the right user. This study was conducted in cooperation with Seekly AB, a company whose app displays a feed with upcoming events in a users immediate area. In this app, every event is associated with an interest and users can choose interests to follow in a list. Seekly wanted to use so called behavioral targeting to show ads in their feed. The solution that was developed is useful for all Seekly-like systems and consists of a content based recommender system that chooses ads based on the interests a user has selected and events and ads that the user has shown an interest in. Apps that in some way or another resembles the Seekly app are not uncommon and recommender systems for behavioral targeting suited for this kind of system are to the best of our knowledge not described in the literature. The resulting recommender system has been implemented and shown to be able to recommend ads that has been associated with interests that match the interests selected by the user and/or amplified by his or her behavior. There are also indications that the system would be able to increase the number of ad clicks compared to randomly selected ads, but no statistically significant proof was found.

The theme of this thesis is the marketing strategy of Expensa company. Expensa joint-stock company provides a unique managing and controlling system for company cash expenditures. This system includes Expensa company payment cards.The aim of this thesis is to (based on current situation) raise potential clients awareness of Expensa company by proposing a partial improvement of marketing strategy. Proposal of new segment which will help Expensa to increase higher market share and competitive ability is the specific output. Theoretical section describes marketing strategies, elements of the marketing mix, forms of situation analysis and three stages of STP strategy. The practical section reflects the theoretical composition.The presented thesis also contains a survey focused on current clients and their perception of marketing communication. The outcome emerging from the analysis is a good source for making a marketing strategy improvement in the future.

Because of shared physiological, anatomical and metabolical features with humans, mice have served for a long time as mammalian disease models. In particular, these last ten years have been the golden age for this favoured model animal. Human and mouse genome projects show that there is 95% genome homology. Spurred by this fact, research attention has shifted from reading these sequences to deciphering the functions of these genes. The 1980s saw the remarkable achievement of homologous recombination in mammalian cell culture systems. Later in the 1990s, innovative gene trapping strategies were developed to enabled random mutagenesis. Today, the goal is to generate more versatile tools to avoid limitations posed by these earlier mutagenesis strategies. Many public and private research centers have united with the aim of mutating all mouse genes. In order to achieve this mutagenesis, the first requirement is a set of practical and efficient viral or plasmid based vectors that can be used globally in the genome. This will be aided by advances in understanding of biological events such as gene transcription, recombination, and embryonic stem cell cycle. In addition, technical improvements such as vector development, precise cell culture assay, and recombinant DNA delivery will also be important. The vector design work in this PhD thesis encompasses 0.00001 % ofthese efforts but may to out to be highly relevant...

Designer nucleases have greatly simplified small genome modifications in many genomes. They can precisely target a specific DNA sequence within a genome and make a double stranded break (DSB). DNA repair mechanisms of the DSB lead to gene mutations or gene modification by homologous directed repair (HDR) if a repair template is exogenously supplied. Thus, small, site directed mutations are easily and quickly achieved. However, strategies that utilize designer nucleases for more complex tasks are emerging and require optimization. To optimize CRISPR/Cas9 assisted targeting, an HPRT rescue assay was utilized to measure the relationship between targeting frequency and homology arm length in targeting constructs in mouse embryonic stem cells. The results show that different gene engineering exercises had different homology requirements.

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Early stage disease diagnosis still remains a challenge despite much efforts to develop novel imaging and diagnostic techniques. Nanoparticles used as molecular imaging contrast agents with multifunctionality and flexibility provide a platform for targeting the specific disease biomarkers and integration of imaging modalities. In this work, we developed a simplified method for synthesis of radiolabeled targeted super paramagnetic iron oxide nanoparticles (SPIOs). This method takes advantage of the chelator BAT that is conjugated to the PEG before the coating process begins. The effect of nanoparticle size and PEG density was investigated in a series of in vivo experiments. The 64Cu-VINP-SPIOs were used in the PET imaging of inflammation and 64Cu-CD105-SPIOs were used in imaging of 4T1 murine tumor model. In summary, we investigated the potential of the radiolabeled, targeted SPIOs in imaging atherosclerotic plaque and tumor in vivo using magnetic resonance imaging (MRI) and Positron emission tomography (PET). Our results show that dual modality SPIOs with active targeting mediated by affinity ligands can be a great tool in molecular imaging and diagnosis of early stage plaque and tumor.

DISC1, a strong genetic candidate for psychiatric illness, is a molecular scaffold residing in multiple subcellular compartments, where it regulates the function of interacting proteins with key roles in neurodevelopment and plasticity. Both common and rare DISC1 missense variants are associated with risk of mental illness and/or brain abnormalities in healthy carriers, but the underlying mechanisms are unclear. In this thesis, I initially examine the effect of a panel of common and rare single amino acid substitutions on DISC1 subcellular targeting, establishing that the rare mutation R37W and the common variant L607F disrupt DISC1 nuclear targeting in a dominant-negative fashion. This finding predicts that DISC1 nuclear expression is severely impaired in 37W and 607F carriers. In addition, I show that the L607F substitution results in aberrant cytoplasmic and cytoskeletal distribution of DISC1. In the nucleus, DISC1 interacts with the transcription factor ATF4, which is involved in the regulation of cellular stress responses and memory consolidation. Here I show that at basal cAMP levels, wild-type DISC1 strongly inhibits the transcriptional activity of ATF4, and this effect is ablated by 37W and 607F, most likely as a consequence of their defective nuclear targeting. 607F additionally reduces DISC1/ATF4 interaction, which likely contributes to its weakened inhibitory effect. I also demonstrate that DISC1 modulates transcriptional responses to endoplasmic reticulum stress, and that this modulatory effect is also ablated by 37W and 607F. By providing evidence that single amino acid substitutions of DISC1 associated with psychiatric illness impair its regulatory function on ATF4-dependent transcription, I highlight a potential mechanism by which these protein variants may impact on molecular pathways underlying cognition and stress responses, two processes of direct relevance to psychiatric disease.

The purpose of this professional report is to more precisely define social media tools and consumer-generated media and consider their effects on advertising campaigns in the digital age. By examining the rather controversial “Motrin Mom” campaign, certain insights arose, including the necessity to embrace the concepts within the micro-targeting of demographics and the testing and measuring of consumer-generated media. / text

<p>The optimal way to detect and treat cancer is to target cancer cells exclusively without affecting the surrounding tissue. One promising approach is to use radiolabelled molecules to target receptors that are overexpressed in cancer cells. Since the epidermal growth factor receptor (EGFR) family is overexpressed in many types of cancer, it is an attractive target for both diagnostic and therapeutic applications.</p><p>This thesis can be divided into two parts. In part one (paper I), studies were conducted to modulate radionuclide uptake in tumour cells. The results showed that it was possible to modulate the cellular uptake of ¹²⁵I delivered by trastuzumab (targeting HER2) by adding EGF (targeting EGFR).</p><p>In part two (papers II-V) a high affinity EGFR-targeting affibody molecule (Z_EGFR:955)₂ was selected and analysed both <i>in vitro</i> and <i>in vivo</i>. In papers II, III and V, the results obtained when using (Z_EGFR:955)₂ were compared with those obtained with the two EGFR-binding molecules, EGF and cetuximab. These studies demonstrated that the affibody molecule bound specifically to EGFR (probably to subdomain III) with high affinity (~50 nM in biosensor analysis and ~1 nM in cellular studies) and produced intracellular signalling changes similar to those with cetuximab. In paper IV, <i>in vivo</i> studies were made, demonstrating that [¹¹¹In](Z_EGFR:955)₂ gave a tumour-specific ¹¹¹In uptake of 3.8±1.4% of injected dose per gram tumour tissue, 4 h post-injection. The tumours could be easily visualized with a gamma camera at this time-point. </p><p>The results of these studies indicated that the affibody molecule (Z_EGFR:955)₂ is a possible candidate for radionuclide-based imaging of EGFR-expressing tumours. The biological effects of (Z_EGFR:955)₂ might be of interest for therapy applications.</p>