Systemic gene therapy: biodistribution and long-term expression of a transgene in mice.

We have investigated the in vivo efficacy of a systemic gene transfer method, which combines a liposomal delivery system (DLS liposomes) with episomally replicative DNA plasmids to effect long-term expression of a transgene in cells. A single i.v. injection of a plasmid DNA vector containing the luciferase gene as a marker was administered with the DLS liposomes in BALB/c mice. The luciferase gene and its product were found in all mouse tissues tested as determined by PCR analysis and immunohistochemistry. Luciferase activity was also detected in all tissues tested and was present in lung, liver, spleen, and heart up to 3 months postinjection. In contrast to the nonepisomal vectors tested (pRSV-luc and pCMVintlux), human papovavirus (BKV)-derived episomal vectors showed long-term transgene expression. We found that these episomal vectors replicated extrachromosomally in lung 2 weeks postinjection. Results indicated that transgene expression in specific tissues depended on the promoter element used, DNA/liposome formulation, dose of DNA per injection, and route of administration.

Gene therapy for long-term expression of erythropoietin in rats.

The injection of recombinant erythropoietin (Epo) is now widely used for long-term treatment of anemia associated with chronic renal failure, cancer, and human immunodeficiency virus infections. The ability to deliver this hormone by gene therapy rather than by repeated injections could provide substantial clinical and economic benefits. As a preliminary approach, we investigated in rats the expression and biological effects of transplanting autologous vascular smooth muscle cells transduced with a retroviral vector encoding rat Epo cDNA. Vector-derived Epo secretion caused increases in reticulocytes, with peak levels of 7.8-9.6% around day 10 after implantation. The initial elevation in reticulocytes was followed by clinically significant increases in hematocrit and hemoglobin for up to 11 weeks. Ten control and treated animals showed mean hematocrits of 44.9 +/- 0.4% and 58.7 +/- 3.1%, respectively (P < 0.001), and hemoglobin values of 15.6 +/- 0.1 g/dl and 19.8 +/- 0.9 g/dl, respectively (P < 0.001). There were no significant differences between control and treated animals in the number of white blood cells and platelets. Kidney and to a lesser extent liver are specific organs that synthesize Epo in response to tissue oxygenation. In the treated animals, endogenous Epo mRNA was largely down regulated in kidney and absent from liver. These results indicate that vascular smooth muscle cells can be genetically modified to provide treatment of anemias due to Epo deficiency and suggest that this cell type may be targeted in the treatment of other diseases requiring systemic therapeutic protein delivery.

Preparation and properties of nido-carborane-specific monoclonal antibodies for potential use in boron neutron capture therapy for cancer.

As the first step of a research program aimed at developing a bispecific monoclonal antibody system for the delivery of boron-rich molecules to tumor cells for boron neutron capture therapy, monoclonal antibodies (mAbs) were produced against an anionic nido-carborane derivative, 4-[7,8-dicarbadodecahydroundecaborat(-1)-7-yl]butanoic acid. Two IgG subclass mAbs, designated HAW101 and HAW102, were identified that specifically bound the anionic nido-carborane hapten, as well as a variety of other anionic nido-carborane cage derivatives. By using surface plasmon resonance technology, the affinity constants of HAW101 and HAW102 were determined to be 1.9 x 10(9) and 6.8 x 10(8) M-1, respectively. A diverse array of 7-substituted and 7,8-disubstituted anionic nido-carborane derivatives reacted with the mAb HAW101 in competition ELISA, whereas anionic closo-polyhedral boranes showed negligible binding, suggesting a role for the open nido-carborane cage structure. These results suggest that mAbs such as HAW101, which bind anionic nido-carboranes, are useful in the development of bispecific mAbs for specific targeting and enhanced boron delivery to tumor sites.

A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo.

The application of DNA technology to regulate the transcription of disease-related genes in vivo has important therapeutic potentials. The transcription factor E2F plays a pivotal role in the coordinated transactivation of cell cycle-regulatory genes such as c-myc, cdc2, and the gene encoding proliferating-cell nuclear antigen (PCNA) that are involved in lesion formation after vascular injury. We hypothesized that double-stranded DNA with high affinity for E2F may be introduced in vivo as a decoy to bind E2F and block the activation of genes mediating cell cycle progression and intimal hyperplasia after vascular injury. Gel mobility-shift assays showed complete competition for E2F binding protein by the E2F decoy. Transfection with E2F decoy inhibited expression of c-myc, cdc2, and the PCNA gene as well as vascular smooth muscle cell proliferation both in vitro and in the in vivo model of rat carotid injury. Furthermore, 2 weeks after in vivo transfection, neointimal formation was significantly prevented by the E2F decoy, and this inhibition continued up to 8 weeks after a single transfection in a dose-dependent manner. Transfer of an E2F decoy can therefore modulate gene expression and inhibit smooth muscle proliferation and vascular lesion formation in vivo.

Nanopartículas lipídicas sólidas e vesículas cataniônicas contendo ftalocianina de cloro alumínio aplicadas nos processos fotodinâmicos

O trabalho apresentado foi realizado em duas etapas independentes e baseou-se no estudo de diferentes sistemas nanométricos para viabilizar a aplicação da ftalocianina de cloro alumínio (ClAlPc) na terapia fotodinâmica (TFD) para o tratamento do câncer de pele do tipo melanoma. O fármaco fotossensibilizante (FS) utilizado apresenta propriedades físico-químicas que lhe permitem exercer sua atividade fotodinâmica com excelência, sem a interferência do cromóforo endógeno melanina existente nas células melanocíticas. Para driblar sua elevada hidrofobicidade, ClAlPc foi encapsulada em sistemas nanométricos para administração em meio fisiológico. Inicialmente nanopartículas lipídicas sólidas (NLS) foram desenvolvidas por emulsificação direta, após um estudo de elaboração do diagrama de fases. O compritol foi o lipídio sólido escolhido para compor as NLS, com diferentes concentrações de ClAlPc. Todas as formulações desenvolvidas foram devidamente caracterizadas, com tamanho médio entre 100 e 200 nm, baixa polidispersão, potencial zeta adequadamente negativo (~|30| mV), drug loading de ClAlPc entre 76-94% (com pequena redução após 24 meses) e alta eficiência de encapsulação (E.E.). A morfologia arredondada das nanopartículas foi confirmada por microscopia eletrônica de transmissão e de força atômica. A estabilidade das NLS foi de 24 meses. A avaliação da cristalinidade do lipídio revelou a integração da ClAlPc à matriz lipídica da NLS, presença de estruturas polimórficas e grau de cristalinidade adequado, sem alterações após 24 meses. Nos estudos de difusão in vitro, observou-se que ftalocianina encapsulada nas NLS acumulam-se preferencialmente na epiderme e derme do que no estrato córneo, sem traços de permeação do ativo. Foi confirmado o caráter biocompatível das NLS sobre fibroblastos NIH-3T3. A ftalocianina encapsulada nas NLS não foi tóxica na linhagem de melanoma B16-F10 na ausência de luz, porém, apresentou excelente efeito fototóxico (0,75 ?g mL-1 de ClAlPc nanoencapsulada e irradiação entre 0,5 e 2,0 J cm-2), com redução da viabilidade celular de 87%. O segundo sistema de veiculação estudado foram as vesículas cataniônicas (VesCat), que se formam espontaneamente em água com o tensoativo TriCat 12. A obtenção das vesículas contendo ClAlPc envolve uma etapa adicional, para remoção de solvente orgânico, que foi aprimorada, reduzindo o tempo de produção em 55%. As VesCat/ClAlPc obtidas mantiveram suas propriedades físico-químicas e morfologia arredondada (confirmada por microscopia eletrônica de varredura), drug loading de 47% e alta E.E. Os resultados comprovaram que a aplicação desses dois sistemas nanométricos é altamente eficiente para aplicação da TFD no tratamento do câncer de pele do tipo melanoma ou outras doenças cutâneas, apresentando características favoráveis para avanços nos estudos de fase clínica e pré-clínica.

Anatomofisiologia da sucção e deglutição do bebê em computação gráfica 3D como instrumento educacional

A eficiência da amamentação exige uma complexa coordenação entre sucção, deglutição e respiração, sendo que a tecnologia tem possibilitado importantes avanços na compreensão desse processo. Porém, não foram encontrados vídeos disponíveis na internet que demonstrassem a anatomia e fisiologia da amamentação, de modo didático e fidedigno à ciência atual. Este trabalho teve por objetivo descrever o desenvolvimento de uma sequência em computação gráfica sobre a sucção e a deglutição, resultante da produção digital do Bebê Virtual, bem como validar tal produção quanto ao conteúdo e prover adequações necessárias ao material educacional. Para a produção das iconografias em 3D da sucção e deglutição no Bebê Virtual, inicialmente foi elaborado um mapa conceitual e uma matriz de conteúdos, objetivos e competências voltadas ao material educacional. Posteriormente foi elaborado um roteiro científico que abordou a anatomia do crânio, face, cavidade oral, faringe, laringe e esôfago do recém-nascido, bem como, a descrição dos mecanismos fisiológicos relacionados à sucção e às fases oral e faríngea da deglutição no bebê. Para isso foram utilizadas 14 publicações do período de 1998 a 2008, que continham informações relevantes para demonstrar a amamentação. Os conteúdos teóricos foram organizados em cenas principais, possibilitando a criação de previews das sequências dinâmicas, as quais foram avaliadas por profissionais de anatomia, fonoaudiologia e medicina, possibilitando os ajustes necessários e a construção das imagens em computação gráfica 3D. Para análise da validade de conteúdo dessas imagens foi verificada a representatividade dos itens que o compõe, por meio de consulta à literatura. Foram incluídos estudos que utilizaram auxílio tecnológico e abordaram o tema proposto em bebês a termo e saudáveis, sem alterações neurológicas ou anomalias craniofaciais. Foram excluídas as publicações realizadas com bebês pré-termo, sindrômicos, com anomalias, doenças neurológicas ou qualquer alteração que pudesse interferir na amamentação, revisões de literatura e relatos de caso. Os artigos selecionados foram analisados e classificados quanto ao nível de evidência científica, predominando o nível três de evidência. A análise de conteúdo demonstrou a necessidade de adequações nas iconografias 3D, para que o processo de sucção e deglutição demonstrado no bebê virtual pudesse corresponder ao conhecimento científico atual. Tais adequações foram propostas a partir dos achados de 9 estudos, publicados entre 2008 e 2014, que utilizaram ultrassonografia para demonstrar como ocorre o processo de amamentação. Desta forma, foram modificados os aspectos da pega, da movimentação de língua, mandíbula, palato mole e laringe, além da sincronização da sucção/deglutição/respiração e deslocamento do mamilo, num processo desenvolvido em cinco etapas. Assim, o presente estudo descreveu o processo de desenvolvimento das iconografias em 3D sobre a anatomia e fisiologia da sucção e deglutição no recém-nascido a termo, sendo que a validade de conteúdo permitiu atualizar vários aspectos da amamentação do Bebê Virtual, quebrando velhos paradigmas e possibilitando ilustrar didaticamente as evidências científicas relacionadas.

The Perks of Playing Dress-Up: Psychological Impressions of Fashion and Feminism.

Research on the psychological impact of women's fashion has focused on fashion's negative influence over how women think and feel about themselves. Several studies have examined the relationship between fashion and women's self-appraisals (Martin & Gentry, 1997; Pinhas, Toner, Ali, Garfinkel, & Stuckless, 1999; Tiggemann, Polivy, & Hargreaves, 2009), although few investigations have explored the range of viewpoints that arise when women interact with their own personal style or with other forms of fashion media. This paper presents a narrative review of what has been written about fashion in clinical research. I briefly discuss why this is an important topic and why fashion has psychological meaning. Cognitive behavioral therapy (CBT) is considered in the exploration of fashion's impact on conjuring unproductive and productive schemas (Beck, 1976; Wright, Basco, & Thase, 2006). This discussion includes a presentation of interviews with female consultants, hypothetical examples, my own accounts, and feminist perspectives. While emphasizing the potential biases of women's interactions with fashion, I discuss matters of gender performance and reflections on clinical work. The purpose of this article is to present a pro-social defense of fashion. I do this by acquiring personal chronicles, applying those findings to the current body of research, and adding to the continued investigation of why women's fashion is still important in a postfeminist world.

O efeito da estimulação top-down e bottom-up no potencial evocado auditivo de tronco encefálico com estímulo complexo

Introdução: Crianças com transtorno fonológico (TF) apresentam dificuldade na percepção de fala, em processar estímulos acústicos quando apresentados de forma rápida e em sequência. A percepção dos sons complexos da fala, dependem da integridade no processo de codificação analisado pelo Sistema Nervoso Auditivo. Por meio do Potencial Evocado Auditivo de Tronco Encefálico com estímulo complexo (PEATEc) é possível investigar a representação neural dos sons em níveis corticais e obter informações diretas sobre como a estrutura do som da sílaba falada é codificada no sistema auditivo. Porém, acredita-se que esse potencial sofre interferências tanto de processos bottom-up quanto top-down, o que não se sabe é quanto e como cada um desses processos modifica as respostas do PEATEc. Uma das formas de investigar a real influência dos aspectos top-down e bottom-up nos resultados do PEATEc é estimulando separadamente esses dois processos por meio do treinamento auditivo e da terapia fonoaudiológica. Objetivo: Verificar o impacto da estimulação sensorial (processamento bottom-up) e cognitiva (processamento top-down), separadamente, nos diferentes domínios da resposta eletrofisiológica do PEATEc. Método: Participaram deste estudo 11 crianças diagnosticadas com TF, com idades entre 7 e 10:11, submetidas a avaliação comportamental e eletrofisiológica e então dividas nos grupos Bottom-up (B-U) (N=6) e Top-down T-D (N=5). A estimulação bottom-up foi voltada ao treinamento das habilidades sensoriais, através de softwares de computador. A estimulação top-down foi realizada por meio de tarefas para estimular as habilidades cognitiva por meio do Programa de Estimulação Fonoaudiológica (PEF). Ambas as estimulações foram aplicadas uma vez por semana, num período de aproximadamente 45 minutos por 12 semanas. Resultados: O grupo B-U apresentou melhoras em relação aos domínios onset e harmônicos e no valor da pontuação do escore após ser submetido à estimulação bottom-up. Por sua vez, após serem submetidos à estimulação top-down, o grupo T-D apresentou melhoras em relação aos domínios onset, espectro-temporal, fronteiras do envelope e harmônicos e para os valores da pontuação do escore. Conclusão: Diante dos resultados obtidos neste estudo, foi possível observar que a estimulação sensorial (processamento bottom-up) e a estimulação cognitiva (processamento top-down) mostraram impactar de forma diferente a resposta eletrofisiológica do PEATEc

The Impact of Prefrontal Cortex "Warm Up" on Immediate Cognitive Reappraisal Ability in Older Adolescents with Elevated Symptoms of Depression

Cognitive Reappraisal (CR) is a central component of Cognitive Behavioral Therapy for adolescent depression. Yet, previous research indicates that a brain region highly associated with successful CR in adults, the Prefrontal Cortex (PFC), is not fully developed until early adulthood. Thus, there is growing concern that CBT interventions directed at building CR abilities in depressed teens might be constrained by PFC immaturity. However, CR is an effective strategy for regulating affect. The current study evaluated an intervention aimed at enhancing CR performance through PFC “warm up” with a working memory task. Additionally, the study examined moderators of intervention response, as well as cognitive correlates of self-reported CR use. Participants included 48 older adolescents (mean age=19.1, 89% female) with elevated symptoms of depression who were randomly assigned to a lab-based WM or control activity followed by a CR task. Overall, results failed to support the effectiveness of “warm up” to augment CR performance. However, current level of depression predicted negative bias and sadness ratings after CR instructions, and this effect was qualified by an interaction with condition. The moderator analysis showed that depressive symptoms interacted with condition such that in the control condition, participants with higher depressive symptoms had significantly lower negative bias scores than individuals with lower depressive symptoms, but this pattern was not found in the experimental condition. Contrary to hypotheses, history of depression did not moderate treatment response. Additional analyses explored alternative explanations for the lack of intervention effects. There was some evidence to suggest that the WM task was frustrating and cognitively taxing. However, irritation scores and overall WM task accuracy did not predict subsequent CR performance. Lastly, multiple cognitive variables emerged as correlates of self-reported CR use, with cognitive flexibility contributing unique variance to self-reported CR use. Results pointed to new directions for improving CR performance among youth with elevated symptoms of depression.

Hashimoto’s thyroiditis follow up along 10 years

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

The Single Supervisory Mechanism: A Sound First Step in Europe’s Banking Union? Egmont European Affairs Paper, March 2013

The financial and economic crisis has hit Europe in its core. While the crisis may not have originated in the European Union, it has laid bare structural weaknesses in the EU’s policy framework. Both public finances and the banking sector have been heavily affected. For a long time, the EU failed to take into account sufficiently the perverse link that existed between the two. Negative evolutions in one field of the crisis often dragged along the other in its downward spiral. In June 2012, in the early hours of a yet another EU Summit, the leaders of the eurozone finally decided to address the link between the banking and sovereign debt crises. Faced with soaring public borrowing costs in Spain and Italy, they decided to allow for the direct European recapitalisation of banks when the Member State itself would no longer be in a position to do so. In exchange, supervision of the banking sector would be lifted to the European level by means of a Single Supervisory Mechanism. The Single Supervisory Mechanism, or SSM in the EU jargon, is a first step in the broader revision of policies towards banks in Europe. The eventual goal is the creation of a Banking Union, which is to carry out effective surveillance and – if needed – crisis management of the banking sector. The SSM is to rely on national supervisors and the ECB, with the ECB having final authority on the matter. The involvement of the latter made it clear that the SSM would be centred on the eurozone – while it is to remain open to other Member States willing to join. Due to the ongoing problems and the link between the creation of the SSM and the recapitalisation of banks, the SSM became one of the key legislative priorities of the EU. In December 2012, Member States reached an agreement on the design of the SSM. After discussions with the European Parliament (which were still ongoing at the time of writing), the process towards making the SSM operational can be initiated. The goal is to have the SSM fully up and running in the first half of 2014. The decisions that were taken in June 2012 are likely to have had a bigger impact than the eurozone’s Heads of State and Government could have realised at the time for two important reasons. On the one hand, creating the SSM necessitates a full Banking Union and therefore shared risk. On the other hand, the decisions improved the ECB’s perception of the willingness of governments to take far-reaching measures. This undoubtedly played a significant role in the creation of the Outright Monetary Transactions programme by the ECB, which has led to a substantial easing of the crisis in the short-term. 1 These short-term gains should now be matched with a stable long-term framework for bank supervision and crisis management. The agreement on the SSM should be the first step in the direction of this goal. This paper provides an analysis of the SSM and its role in the creation of a Banking Union. The paper starts with a reminder of why the EU decided to put in place the SSM (§1) and the state of play of the ongoing negotiations on the SSM (§2). Subsequently, the supervisory responsibilities of the SSM are detailed, including its scope and the division of labour between the national supervisors and the ECB (§3). The internal functioning of the SSM (§4) and its relation to the other supervisors are discussed afterwards (§5). As mentioned earlier, the SSM is part of a wider move towards a Banking Union. Therefore, this paper sheds light on the other building blocks of this ambitious project (§6). The transition towards the Banking Union is important and will prove to be a bumpy ride. Before formulating a number of conclusions, this Working Paper therefore provides an overview of the planned road ahead (§7).

Was the ECB’s Comprehensive Assessment up to standard? CEPS Policy Brief No. 325, 10 November 2014

The Comprehensive Assessment conducted by the European Central Bank (ECB) represents a considerable step forward in enhancing transparency in euro-area banks’ balance sheets. The most notable progress since the previous European stress test has been the harmonisation of the definition of non-performing loans and other concepts as well as uncovering hidden losses, which resulted in a €34 billion aggregate capital-charge net of taxes. Despite this tightening, most banks were able to meet the 5.5% common equity tier 1 (CET1) threshold applied in the test, which suggests that the large majority of the euro-area banks have improved their financial position sufficiently that they should no longer be constrained in financing the economy. As shown in this CEPS Policy Brief by Willem Pieter de Groen, however, the detailed results provide a more nuanced picture: there remain a large number of the banks in the euro area that are still highly leveraged and in many cases unable to meet the regulatory capital requirements that will be introduced in the coming years under the adverse stress test scenario.

Neospora caninum calcium-dependent protein kinase 1 is an effective drug target for neosporosis therapy.

Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis.

Non-Viral Gene Therapy to Cells of the bovine Intervertebral Disc

Background. Low back pain is an increasing global health problem, which is associated with intervertebral disc (IVD) damage and degeneration. Major changes occur in the nucleus pulposus (NP), with the degradation of the extracellular matrix (ECM).1 Further studies showed that growth factors from transforming growth factor β (TGFβ) and bone morphogenic proteins (BMP) family may induce chondrogenic differentiation of mesenchymal stem cells (MSC).2 Focusing on non-viral gene therapies and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition. Methods. Bovine nucleus pulposus (bNPC) and annulus fibrosus cells (bAFC) were harvested from bovine coccygeal IVD. Primary cells were then electroporized with plasmid GDF6 (Origene, vector RG211366) by optimizing parameters using the Neon Transfection system (Life Technologies, Basel). After transfection, cells were cultured in 2D monolayer or 3D alginate beads for 7, 14 or 21 days. Transfection efficiency of pGDF6 was analyzed by immunohistochemistry and fluorescent microscopy. Cell phenotype was quantified by real-time RT-PCR. To test a non-viral gene therapy applied directly to 3D whole organ culture, coccygeal bovine IVDs were harvested as previously described. Bovine IVDs were transfected by injection of plasmid GDF6 into the center. Electroporation was performed with ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) using 2-needle array electrode or tweezertrodes. 72 h after tranfection discs were fixed and cryosectioned and analyzed by immunofluorescence against GDF6. Results. RT-PCR and immunohistochemistry confirmed up-regulation of GFP and GDF6 in the primary bNPC/bAFC culture. The GFP-tagged GDF6 protein, however, was not visible, possibly due to failure of dimer formation as a result of fusion structure. Organ IVD culture transfection revealed GDF6 positive staining in the center of the disc using 2-needle array electrode. Results from tweezertrodes did not show any GDF6 positive cells. Conclusion. Non-viral transfection is an appealing approach for gene therapy as it fulfills the translational safety aspects of transiency and lacks the toxic effects of viral transduction. We identified novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD explants electroporation parameters need to be further optimized. Acknowledgements. This project was funded by the Lindenhof Foundation (Funds “Research & Teaching”) Project no. 13-02-F. The imaging part of this study was performed with the facility of the Microscopy Imaging Center (MIC), University of Bern. References. Roughly PJ (2004): Spine (Phila), 29:2691-2699 Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014), Arthritis Research & Therapy, 16:R67

Potted fiction : being a series of extracts from the world's best sellers, put up in thin slices for hurried consumers /

Verso of t.p.: Published July, 1908.