Orientador: Andrigo Barboza De Nardi / Resumo: A medicina comparativa torna-se cada vez mais importante para obtenção de estudos aprofundados em diversas áreas incluindo a oncologia, possibilitando melhor conhecimento da genética do câncer e desenvolvimentos de novas terapias. A expressão do IGF2BP-1 tem sido correlacionada com lesões pré-neoplásicas e neoplasias de maior agressividade, além de estar correlacionada com mau prognóstico em pacientes oncológicos com melanomas, sarcomas e carcinomas. A proposta geral desta pesquisa é identificar a expressão do IGF2BP-1 em linhagens celulares de osteossarcoma (OSA) canino e murino, bem como correlacionar sua expressão com o comportamento celular e avaliar a intensidade de marcação desta proteína através de imuno-histoquímica em amostras de OSA canino. Os resultados deste ensaio demonstraram que o IGF2BP-1 reduziu o potencial de formação de colônias, aumentou o potencial de invasão e migração celular e gerou aumento da expressão dos genes ABCG2, AXIN2, BTrCP, VEGF, CD133 e C-MYC em células de OSA murinho. A expressão deste gene em células de cão aumentou a formação de colônias, reduziu o potencial de invasão e migração celular e o silenciamento parcial do IGF2BP-1 gerou redução da expressão dos genes CCND1, CD133, CMYC e FZD6 em células de OSA canino. O silenciamento parcial do IGF2BP-1 gerou aumento dos genes AXIN-2 e VEGF em células de OSA canino. A imuno-histoquímica é um método eficaz para detecção do IGF2BP-1 em amostras de OSA canino evidenciando marcação citoplasmática f... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

This thesis explored the processes underlying interprofessional working relationships between midwives and health visitors in UK maternity services; using a multi method approach consisting of a systematic review, interviews, and focus groups. The systematic review synthesised the literature on midwife-health visitor collaboration, identifying barriers and enablers that are influential to successful interprofessional collaboration. Thus, the subsequent empirical studies attempted to explore these barriers and enablers in greater depth, from the perspectives of midwives and health visitors. Two studies utilised the Theoretical Domains Framework to explore the barriers and enablers to midwife-health visitor collaboration (Chapters 3 6). These are the first studies to examine midwives’ and health visitors’ perceived barriers and enablers to interprofessional collaboration using a psychologically grounded theoretical framework. Midwives and health visitors identified barriers and enablers to interprofessional collaboration across each of the 12 theoretical domains, such as ‘Knowledge’ (e.g. awareness of processes involved in contacting midwives) and ‘Memory, attention, and decision processes’ (e.g. contacting health visitors when there is a concern). Chapter 6 compared midwives’ and health visitors’ perceived barriers and enablers to interprofessional collaboration, and discussed its research and practice implications, including approaches to intervention development for improving interprofessional collaboration. For example, various behaviour change techniques can be integrated as part of interventions aiming to enhance interprofessional collaboration. The final empirical study attempted to address the gap in the interprofessional literature by involving service users’ views. Focus groups with recent mothers were conducted to gain explore their perspectives of interprofessional collaboration in maternity services. Findings suggest that women observe fragmentation between midwifery and health visiting. Participants recommended service changes including group based antenatal classes jointly provided by midwives and health visitors. In summary, the findings indicate that midwife-health visitor interprofessional collaboration is important to professionals and women, but will require health professional behaviour change along with service changes.

Introduction: Difficulties persist in conceptualising spiritual needs and understanding their relationship to religious needs and wellbeing in healthcare and particularly in nursing education. This research was undertaken to explore approaches and challenges associated with this area in undergraduate nursing education in England. Methods/Methodology: Using a systematic approach, a literature review covering the period 1993-2017 was undertaken to explore potential issues and challenges reported. Applying case-study methodology, data were collected from three university nursing schools from different parts of England. Sources for data triangulation within schools included, curriculum review of undergraduate nursing courses, exploring the views of nursing educators through semi structured interviews and focus group studies with nursing students. Template analysis was used to identify themes in the data. Findings: Due to the module based curriculum, the integration of spirituality in nursing education appeared to be treated as a matter of personal choice and convenience rather than as an essential domain of teaching and learning practice in England. Owing to conceptual complexity, addressing religious needs was often considered to be synonymous with addressing spiritual care need. Factors were identified contributing to the difficulties in the issue of educating nurses in this area. These were: lack of clarity in curriculum documents; uncertainty as to how far nurses should address these issues and how far this was a specialist chaplaincy function; fear of being judged or rejected in a multicultural environment; and the dominance of disease-centred care. The participants voiced a desire for developing a shared understanding through developing a more explicit representation of spirituality in nursing education and the recognition of appropriate educational approaches in this area. Based on the findings of this study a learning framework is proposed; SOPHIE (Self-exploration through Ontological, Phenomenological, Humanistic, Ideological, and Existential expressions), to encourage self-awareness and reflexivity among nursing educators and students. SOPHIE aims to bring ontological authenticity and congruency to the forefront of nursing knowledge and practice. Conclusion: Constructing knowledge through ontological learning engagements among educators and students is essential to develop role clarity, authenticity and empowerment in understanding and addressing spiritual care needs. A multidisciplinary teaching approach integrating medical anthropology, humanistic psychology and existential phenomenology should be explored as a basis for an integrated nursing curriculum that could explore spirituality in its widest sense.

Oligodendroglial tumours originate from oligodendrocytes usually arising in the white matter and could be classified into grade-II oligodendrogliomas (OD) and anaplastic oligodendrogliomas (AOD, grade-III) according to the 2016 World Health Organization (WHO) grading scheme. ODs1 could be diagnosed by pathological and immunohistochemical analyses, however recent evidence suggests that they could be better diagnosed on the basis of defined genetic entities, such as the combined loss of chromosome 1p and 19q arms and IDH mutation. 1p/19q co-deletion is molecular hallmark of ODs and is clinically associated with better prognosis, response to chemo/radio-therapy and overall survival. Typical oligodendroglial histological features are strongly associated with 1p/19q loss and IDH mutation, which is critically important as diagnostic point of view. The examining of exclusive molecular signatures and transcriptome expression profiles added to histological class could compliment the classification of OD subtypes. In this regard, microRNAs (miRNAs, miRs) profiles could serve classifier signatures for tumour subsets. MiRNAs are 22nt short non-coding RNAs which are expressed endogenously and regulate diverse cellular process through negative control on gene expression at the posttranscriptional level by direct or imperfect interaction with their target mRNAs. MiRNAs are involved in regulating human tumorigenesis acting as either tumour suppressors or oncogenes. During the passage of tumorigenesis miRNA expression level is significantly increased or decreased compared to corresponding normal tissue. The same is observed with their mRNAs. Therefore, transcriptome profiling of human tumours could identify signatures associated with progression, diagnosis, prognosis and response to therapy. However, until recently the information regarding the expression of miRNAs and mRNA in oligodendroglial tumours is scarce. In this study we performed miRNA and mRNA differential expression profiling between grade II and grade III ODs using microarray based expression profiling platforms (723 transcripts and 41,000 genes, respectively). 7 cases for OD grade-II, and 7 for AOD grade-III, and 15 non neoplastic white matter (nnWM) samples were used after microdissection with no previous history of treatment. We performed a systematic evaluation of miRNAs and mRNAs expressions and determined miRNAs and putative target genes that are differentially expressed in grade III AOD, but not in grade II OD and in non-neoplastic white matter (nnWM). 1 ODs when used with ,,s\" will represent both OD and AOD. 50 miRNAs were overexpressed and 43 were down regulated in AOD-III, whereas 7 miRNAs showed significant reduction in expressions in OD-II group. 3 miRNAs were commonly down regulated in comparisons of both groups. The hsa-miR-23a was strongly upregulated and hsamiR-27a was strongly downregulated in AOD-III. The functions of hsa-miR-23a and hsa-miR- 27a were tested in human adult fibroblasts for cell proliferation assay and apoptosis detection. Cells treated with pre-miR-23a and pre-miR-27a showed 20% reduction in cell proliferation as compared with controls. Further, the functional relevance of miRNAs to their target mRNAs was validated for each group, using real time qPCR. 10 key-miRNAs from AOD were subjected to validation by qPCR. We were able to confirm 7 miRNAs (p? 0.05). Among these, 5 miRs (miR- 193a-3p, miR-24, miR-27a, miR-30a-5p and miR-30c) showed reduced expression whose target genes (CCND1, HDAC2, PDGFA and RAB-26) were upregulated. Whereas, 2 miRNAs likewise miR-301b and miR-378 were overexpressed whose target genes BCL2, FGF2, CD44 and PPP4R4 confirmed by qPCR (p? 0.05). Bioinformatics based gene ontology (GO), and networking analysis revealed that differential expression and targets are attributed to differentiation of embryonic stem cells, cell adhesion, angiogenesis and neurogenesis, resistance to apoptosis, protein-protein interactions and cell proliferation. It was possible to identify and validate miRNAs and their mRNA-targets potentially involved in the progression of oligodendrogliomas particularly in grade III-AOD. Collectively, this analysis provides new insights to malignant progression of oligodendroglial tumours and could compliment WHO-2016 diagnosis scheme and may provide predictive outcome in patients as well as decision to therapy. / Oligodendrogliomas originários de oligodendrócitos que geralmente surgem na substância branca podendo ser classificados em grau oligodendroglioma (II-OD), e anaplastic oligodendrogliomas (grau III-AOD). Os ODs2 podem ser diagnosticados por análises patológicas e imuno-histoquímicas, porém evidências recentes sugerem que poderiam ser melhor diagnosticados com base em assinaturas moleculares, como a deleção combinada dos cromossomas 1p e 19q - marcadores moleculares dos OD associados clinicamente a um melhor prognóstico, resposta à terapia e melhor sobrevida. As características histológicas típicas dos oligodendrogliomas também estão fortemente associadas à deleção de 1p/19q, que é criticamente importante como ponto de vista diagnóstico. Assim, os subtipos de gliomas podem ser fortemente diferenciados não somente em relação ao seu perfil histológico mas também com base em seu perfil de expressão genica e suas assinaturas moleculares exclusivas. Os microRNAs (miRNAs, miRs) emergiram como assinaturas moleculares para os diferentes graus. Os miRNAs são RNAs não codificantes, contendo em torno de 22 nucleótidos. São expressos endogenamente e regulam diversos processos celulares através do controle negativo da expressão gênica em nivel pós-transcricional e por interacção directa ou imperfeita com o RNAm-alvo. Os miRNAs estão envolvidos na regulação da tumorigenese humana atuando como supressores de tumour ou oncogenes. Durante o processo da tumorigenese o nível de expressão dos miRNAs é aumentado ou diminuído significativamente em comparação com tecido normal correspondente. O perfil de expressão de miRNA de tumores humanos poderia identificar assinaturas associadas com progressão, diagnóstico, prognóstico e resposta à terapia. Contudo, até recentemente a informação sobre a expressão de miRNAs em oligodendrogliomas é escassa. Neste estudo, avaliamos o perfil de expressão diferencial de miRNA e RNAm em ODs graus II e III usando plataformas de perfis de expressão baseadas em microarray (723 transcritos e 41.000 genes, respectivamente). Foram utilizados 14 casos de ODs microdissecados, sendo 7 OD grau II, e 7 AOD grau III (anaplasicos) sem histórico prévio de tratamento, além de 15 amostras de substancia branca não neoplásica (nnSB). Por meio de avaliações sistemáticas foram determinados miRNAs e mRNAs expressos em AOD grau III, mas não em OD grau II e em substancias brancas não neoplásicas (nnSB). 2 ODs when used with ,,s\" will represent both OD and AOD. Assim, foram encontrados 50 miRNAs com alta expressão e 43 miRNAs com baixa expressão em AOD-III, enquanto que 7 miRNAs apresentaram expressões reduzidas no grupo OD-II. Na comparação entre os dois grupos, 3 miRNAs apresentaram baixa expressão. A hsa-miR-23a mostrou alta expressão e a hsa-miR-27a apresentou uma diminuição de expressão importante em AOD III. A atividade dos hsa-miR-23a e hsa-miR-27a foram testadas em células de fibroblastos adultos humanos usando ensaios de proliferação celular e detecção de apoptose. As células tratadas com pre-miR-23a e pre-miR-27a mostraram 20% redução de proliferação celular em comparação com os controles. Para cada grupo, a relevância funcional dos miRNAs e seus mRNAs alvos foi validada utilizando qPCR. Dos 10 miRNAs submetidos a validação em grau III, foi possivel confirmar 7 miRNA(p<0,05). Entre esses, 5 miRs (miR-193a-3p, miR-24, miR- 27a, miR-30a-5p e miR-30c) mostraram expressão reduzida, cujos genes alvos (CCND1, HDAC2, PDGFA e RAB-26) apresentavam alta expressão. Enquanto que, 2 miRNAs como miR-301b e miR-378 apresentaram alta expressão cujos genes alvo BCL2, FGF2, CD44 e PPP4R4 foram confirmados por qPCR (p<0,05). Ferramentas de bioinformática (Gene Ontology) e a análises em rede revelaram que a expressão diferencial e os alvos são atribuídos à diferenciação de células-tronco embrionárias, adesão de celular, angiogênese e neurogênese, resistência à apoptose, interações proteína-proteína e proliferação celular. Foi possível identificar e validar miRNAs e RNAm-alvos potencialmente envolvidos na progressão de oligodendrogliomas. Coletivamente, esta análise fornece novos achados relacionados a progressão maligna de tumores oligodendrogliais e poderia facilitar o diagnóstico preciso e mais restritivo, o desfecho preditivo em pacientes, bem como auxiliar na decisão da terapia.

This research is an exploration of nurse education leadership, as identified and experienced by nurse teachers working in a Higher Education (HE) environment. In total 18 nurse teachers were interviewed who formed a whole population sample. The author is identified as a coworker and part of the community being researched. Semi-structured interviews and field notes were used to triangulate the data and thematic analysis was used to analyse the data. From the findings, the interviewees can be seen to have a strong professional focus and look to their own health and nursing structures to provide leadership. The acceptance of the authority of these institutions and people can be broadly located in a bureaucratic model. Their expectations of people they identified as leaders conforms to a transformational model of leadership, as they identified that leaders were people who had vision, values and influence and of particular importance was the identification of how these individuals made them feel valued. They identified themselves as problematic within the HE environment in which they work and identify being excluded from the collegiate structures and working practices of the university. The findings illuminate problems in the identification and role of nurse education leadership and their position within the university, which this author suggests are areas that need further exploration and have policy implications.

Ascochyta blight, caused by Ascochyta rabiei (Pass.) Labrousse, is one of the most destructive diseases of chickpea (Cicer arietinum L.) worldwide. Despite the existence of highly resistant uncultivated genotypes, attempts to develop cultivars with a high level of durable resistance have been unsuccessful. This study investigated the chickpea defence response to A. rabiei using a functional genomics approach, which has the capacity to improve the overall understanding of the coordinated defence response at a molecular level. An existing cDNA library was used to generate a resource of Expressed Sequence Tags (ESTs) that, after clustering, comprised 516 unigenes. The unigenes were functionally annotated resulting in the identification of 20 specific defence-related unigenes, as well as numerous transcripts with possible involvement in the coordination of defence responses. To explore the expression patterns of the defence-related unigenes in an A. rabiei resistant and susceptible genotype, the unigenes were employed as probes in microarrays. Resulting expression data was analysed to identify differentially expressed unigenes over a time-course after infection. Comparison of the expression profiles from the resistant and susceptible genotype identified three putative genes that were exclusively up-regulated in the resistant genotype, thus may be involved in an effective defence response. Considering that a defence response can involve hundreds of genes, the entire set of chickpea unigenes were used to construct large-scale microarrays. To supplement the chickpea probes, 156 putative defence-related grasspea (Lathyrus sativus L.) ESTs and 41 lentil (Lens culinaris Med.) Resistance Gene Analogs (RGAs) were also included. Expression profiles for three chickpeas and one wild relative were generated over a time course. 97 differentially expressed ESTs were identified using a robust experimental system that included confirmation by quantitative RT-PCR. The results indicated that genes involved in the active defence response were similar to those governed by R-gene mediated resistance, including the production of reactive oxygen species and the hypersensitive response, down-regulation of 'housekeeping' gene expression, and expression of pathogenesis-related proteins. The comparison between resistant and susceptible genotypes identified certain gene expression 'signatures' that may be predictiv e of resistance. To further characterise the regulation of potential defence-related genes, the microarray was used to study expression profiles of the three chickpea genotypes (excluding the wild relative) after treatment with the defence signalling compounds, ethylene (E), salicylic acid (SA), and jasmonate (JA). 425 ESTs were differentially expressed, and comparison between genotypes revealed the presence of a wider range of inducible defence responses in resistant genotypes. Linking the results with the previous microarray results indicated the presence of other pathogen-specific signalling mechanisms in addition to E, SA and JA. The lower arsenal of defence-related gene expression observed in the susceptible genotype may be a result of 'breaks' in the pathways of defence-related gene activation. To draw together the findings of all experiments, a model was constructed for a hypothetical mechanism of chickpea resistance to A. rabiei. The model was synthesised based on the evidence gathered in this study and previously documented defence mechanisms in chickpea, and identified signal transduction as a key to resistance.

Home networking is becoming an attractive application not only for the Internet access but also for home automation. Being a high-speed and dominant standard presently, HomePlug has an important role in home LAN connecting to the Internet. For industrial applications, the Power Line Communication also has significant advances. However, the PHY/MAC technology provided by HomePlug still cannot be employed with some critical features such as real time performance, implications in the event of link and node loss. In this paper, the characteristics of HomePlug PHY/MAC, the property of power line channel, as well as the noise features of power line are analyzed. Based on HomePlug, a model of high level real-time protocol applied to industrial environment is proposed. The protocol simultaneously belongs to layer two and three, and can support real-time implementation with no loss and small delay according to the requirement in PLC networks, for targeting to develop a real time network with high speed power line media and advanced modulation. / Singapore-MIT Alliance (SMA)

Replication of HIV-1 is inhibited by azidothymidine (AZT), which leads to chain termination and inhibition of DNA synthesis. Resistance to AZT is frequently the result of mutations that increase the ability of RT to remove the chain-terminating nucleotides after they have been incorporated. It has been proposed that RNase H cleavage of the RNA template can occur when RT is stalled near the site of chain termination and contributes to the inhibition by causing the dissociation of the primer-template before the chain terminator can be excised. Mutations in the connection and RNase H domains of RT have been shown to increase excision. It has long been known that resistance to thymidine analogs is conferred by the mutations M41L, D67N, K70R, L210W, T215F/Y and T219Q/E in RT and that this resistance is suppressed by the additional presence of the M184V mutation. Changes in excision activity on DNA templates have been observed with these mutant RTs, but effects on RNase H cleavage resulting in indirect effects on excision activity is also possible with RNA templates. We used a 5'-labeled -3'-chain-terminated DNA primer annealed to either a DNA or RNA template to evaluate primer rescue activities, a 5'-labeled RNA template to evaluate RNA cleavage activity and a biotin-tagged chain-terminated oligodeoxynucleotide to monitor primer-template dissociation. We first investigated differences between RNA and DNA templates when the primers were chain terminated and observed a correlation between RNase H activities and template/primer (T/P) dissociation. An inverse correlation was observed between excision rescue rates and RNase H cleavages leading to T/P dissociation. We observed that the chain terminator (i.e. AZTMP or ddAMP) affected RNase H cleavages and excision rates with RNA template and dNTPs. When we investigated mutations in the N-terminal domain of RT associated with nucleoside reverse transcriptase inhibitor (NRTI) resistance we found that primer rescue was decreased when M184V was present in combination with thymidine analog mutations (TAMs) and the template was RNA with either ATP or PPi as excision substrate. RNase H cleavage at secondary cleavage sites (-7, -8) was substantially reduced with M41L/T215Y RT in comparison with wild type RT, and primer-template dissociation was decreased. In contrast, when M184V was present, RNase H cleavage at the secondary cleavage sites and dissociation of the primer-template occurred at higher levels and excision rescue was decreased. The ability of RT to rescue an AZT terminated primer in the presence of the 184V mutation was restored when the RNase H activity was inactivated by the RNase H negative mutation E478Q. Electromobility shift assay (EMSA) analysis of AZT-resistant mutant RT with M184V showed an increased Kd for formation of the ternary complex. These results suggest that RNase H-mediated RNA-DNA template-primer dissociation is influenced by mutations associated with thymidine analog resistance, and that suppression of resistance to nucleoside RT inhibitors by M184V may be partly explained by effects on RNase H cleavage that decrease the time available for excision to occur. This is the first time that mutations in the polymerase domain are shown to affect excision rescue through an RNase H-dependent mechanism.

Reduction of cardiac myocyte loss and repair of the vasculature post myocardial infarction are important therapeutic goals because the potential for intrinsic repair is limited. Preclinical and limited clinical data support the possibility that bone marrow-derived mesenchymal stem cells may be a suitable cell type for cellular therapy. The goal of this research was to determine the effectiveness of using MSCs from aged mice in cellular therapy for the treatment of AMI. The central hypothesis for this research was that therapeutic potential of mesenchymal stem cells decreases with age. This research utilized global gene expression analysis to investigate molecular differences in MSCs harvested from three different age groups of mice. Microarray analysis was performed to investigate changes in gene expression with respect to aging. Furthermore, both in vitro and in vivo experiments were completed to analyze the functional and molecular characteristics of the MSCs. The data identified age-related defects in mouse MSCs as well as determined the molecular basis for these deficiencies. This study indicates that MSCs from 26m mice are severely deficient in the induction of angiogenesis and cardiac repair due to defective paracrine factor secretion caused by decreased expression of growth factor/cytokine genes. Hypoxia attenuates the deficiency in the aged mice, whereas in young mice low oxygen promotes secretion of paracrine growth factors. It was determined a dysfunction in HIF-1 alpha signaling was present in MSCs from 26m mice and is regulated by the PI3K/Akt signaling in MSCs. Furthermore, two novel and important and novel aspects of this study were the discovery that cell cycle regulation gene expression decreases with age and MSCs have increased insulin resistance with age. Increased insulin resistance in this cell type with aging is likely to have profound effects on the clinical outcomes of using these cells therapeutically. Likewise, loss of cell cycle regulation during proliferation could also lead to undesirable clinical effects. Gaining insight to the repair potential of these cells with respect to age will help to better define future trials of autologous stem cells not only for heart disease but for all of the many applications proposed for these cells.

Drought is a major constraint for attaining economic yield in tree crops. As an initial step to understand molecular response to water-deficit-stress in trees, gene expression in response to water stress was quantified using real-time RT-PCR. The specific objectives established for this to were I. to identify and characterize the genes induced by drought stress in Aleppo pine (Pinus halepensis) and II to identify and quantify the differentially expressed genes in different populations of Loblolly pine (Pinus taeda.L) due to water deficit (chapter III). Results of these studies may be used to identify candidate genes for future breeding programs against water-deficit-stress.