Cellular reprogramming strategies for degenerative disorders involving the retinal pigment epithelium

RESUMO: A reprogramação celular permite que uma célula somática seja reprogramada para outra célula diferente através da expressão forçada de factores de transcrição (FTs) específicos de determinada linhagem celular, e constitui uma área de investigação emergente nos últimos anos. As células somáticas podem ser experimentalmente manipuladas de modo a obter células estaminais pluripotentes induzidas (CEPi), ou convertidas directamente noutro tipo de célula somática. Estas descobertas inovadoras oferecem oportunidades promissoras para o desenvolvimento de novas terapias de substituição celular e modelos de doença, funcionando também como ferramentas valiosas para o estudo dos mecanismos moleculares que estabelecem a identidade celular e regulam os processos de desenvolvimento. Existem várias doenças degenerativas hereditárias e adquiridas da retina que causam deficiência visual devido a uma disfunção no tecido de suporte da retina, o epitélio pigmentar da retina (EPR). Uma destas doenças é a Coroideremia (CHM), uma doença hereditária monogénica ligada ao cromossoma X causada por mutações que implicam a perda de função duma proteína com funções importantes na regulação do tráfico intracelular. A CHM é caracterizada pela degenerescência progressiva do EPR, assim como dos foto-receptores e da coróide. Resultados experimentais sugerem que o EPR desempenha um papel importante na patogénese da CHM, o que parece indicar uma possível vantagem terapêutica na substituição do EPR nos doentes com CHM. Por outro lado, existe uma lacuna em termos de modelos in vitro de EPR para estudar a CHM, o que pode explicar o ainda desconhecimento dos mecanismos moleculares que explicam a patogénese desta doença. Assim, este trabalho focou-se principalmente na exploração das potencialidades das técnicas de reprogramação celular no contexto das doenças de degenerescência da retina, em particular no caso da CHM. Células de murganho de estirpe selvagem, bem como células derivadas de um ratinho modelo de knockout condicional de Chm, foram convertidos com sucesso em CEPi recorrendo a um sistema lentiviral induzido que permite a expressão forçada dos 4 factores clássicos de reprogramação, a saber Oct4, Sox2, Klf4 e c-Myc. Estas células mostraram ter equivalência morfológica, molecular e funcional a células estaminais embrionárias (CES). As CEPi obtidas foram seguidamente submetidas a protocolos de diferenciação com o objectivo final de obter células do EPR. Os resultados promissores obtidos revelam a possibilidade de gerar um valioso modelo de EPR-CHM para estudos in vitro. Em alternativa, a conversão directa de linhagens partindo de fibroblastos para obter células do EPR foi também abordada. Uma vasta gama de ferramentas moleculares foi gerada de modo a implementar uma estratégia mediada por FTs-chave, seleccionados devido ao seu papel fundamental no desenvolvimento embrionário e especificação do EPR. Conjuntos de 10 ou menos FTs foram usados para transduzir fibroblastos, que adquiriram morfologia pigmentada e expressão de alguns marcadores específicos do EPR. Adicionalmente, observou-se a activação de regiões promotoras de genes específicos de EPR, indicando que a identidade transcricional das células foi alterada no sentido pretendido. Em conclusão, avanços significativos foram atingidos no sentido da implementação de tecnologias de reprogramação celular já estabelecidas, bem como na concepção de novas estratégias inovadoras. Metodologias de reprogramação, quer para pluripotência, quer via conversão directa, foram aplicadas com o objectivo final de gerar células do EPR. O trabalho aqui descrito abre novos caminhos para o estabelecimento de terapias de substituição celular e, de uma maneira mais directa, levanta a possibilidade de modelar doenças degenerativas da retina com disfunção do EPR numa placa de petri, em particular no caso da CHM.---------------ABSTRACT: Cellular reprogramming is an emerging research field in which a somatic cell is reprogrammed into a different cell type by forcing the expression of lineage-specific transcription factors (TFs). Cellular identities can be manipulated using experimental techniques with the attainment of pluripotency properties and the generation of induced Pluripotent Stem (iPS) cells, or the direct conversion of one somatic cell into another somatic cell type. These pioneering discoveries offer new unprecedented opportunities for the establishment of novel cell-based therapies and disease models, as well as serving as valuable tools for the study of molecular mechanisms governing cell fate establishment and developmental processes. Several retinal degenerative disorders, inherited and acquired, lead to visual impairment due to an underlying dysfunction of the support cells of the retina, the retinal pigment epithelium (RPE). Choroideremia (CHM), an X-linked monogenic disease caused by a loss of function mutation in a key regulator of intracellular trafficking, is characterized by a progressive degeneration of the RPE and other components of the retina, such as the photoreceptors and the choroid. Evidence suggest that RPE plays an important role in CHM pathogenesis, thus implying that regenerative approaches aiming at rescuing RPE function may be of great benefit for CHM patients. Additionally, lack of appropriate in vitro models has contributed to the still poorly-characterized molecular events in the base of CHM degenerative process. Therefore, the main focus of this work was to explore the potential applications of cellular reprogramming technology in the context of RPE-related retinal degenerations. The generation of mouse iPS cells was established and optimized using an inducible lentiviral system to force the expression of the classic set of TFs, namely Oct4, Sox2, Klf4 and c-Myc. Wild-type cells, as well as cells derived from a conditional knockout (KO) mouse model of Chm, were successfully converted into a pluripotent state, that displayed morphology, molecular and functional equivalence to Embryonic Stem (ES) cells. Generated iPS cells were then subjected to differentiation protocols towards the attainment of a RPE cell fate, with promising results highlighting the possibility of generating a valuable Chm-RPE in vitro model. In alternative, direct lineage conversion of fibroblasts into RPE-like cells was also tackled. A TF-mediated approach was implemented after the generation of a panoply of molecular tools needed for such studies. After transduction with pools of 10 or less TFs, selected for their key role on RPE developmental process and specification, fibroblasts acquired a pigmented morphology and expression of some RPE-specific markers. Additionally, promoter regions of RPE-specific genes were activated indicating that the transcriptional identity of the cells was being altered into the pursued cell fate. In conclusion, highly significant progress was made towards the implementation of already established cellular reprogramming technologies, as well as the designing of new innovative ones. Reprogramming into pluripotency and lineage conversion methodologies were applied to ultimately generate RPE cells. These studies open new avenues for the establishment of cell replacement therapies and, more straightforwardly,raise the possibility of modelling retinal degenerations with underlying RPE defects in apetri dish, particularly CHM.

Recurrent herpes simplex infections: laser therapy as a potential tool for long-term successful treatment

Herpes simplex virus types 1 and 2 are the main infectious agents associated with oral and genital ulcerations. These infections are now widely recognized as sexually transmitted diseases. Among treatment options, low-level laser therapy (LLLT) has shown promising clinical results as a longer-lasting suppression therapy. Two clinical cases are described with recurrent labial herpes for which LLLT was used. Following treatment, both patients remained symptom free during the 17-month clinical follow-up period.

Differentiation between Candida albicans and Candida dubliniensis using hypertonic Sabouraud broth and tobacco agar

INTRODUCTION: Opportunistic fungal infections in immunocompromised hosts are caused by Candida species, and the majority of such infections are due to Candida albicans. However, the emerging pathogen Candida dubliniensis demonstrates several phenotypic characteristics in common with C. albicans, such as production of germ tubes and chlamydospores, calling attention to the development of stable resistance to fluconazole in vitro. The aim of this study was to evaluate the performance of biochemistry identification in the differentiating between C. albicans and C. dubliniensis, by phenotyping of yeast identified as C. albicans. METHODS: Seventy-nine isolates identified as C. albicans by the API system ID 32C were grown on Sabouraud dextrose agar at 30°C for 24-48h and then inoculated on hypertonic Sabouraud broth and tobacco agar. RESULTS: Our results showed that 17 (21.5%) isolates were growth-inhibited on hypertonic Sabouraud broth, a phenotypic trait inconsistent with C. albicans in this medium. However, the results observed on tobacco agar showed that only 9 (11.4%) of the growth-inhibited isolates produced characteristic colonies of C. dubliniensis (rough colonies, yellowish-brown with abundant fragments of hyphae and chlamydospores). CONCLUSIONS: The results suggest that this method is a simple tool for screening C. albicans and non-albicans yeast and for verification of automated identification.

A model to screen and improve business potential of micro and small enterprises in developing countries: Evidence from the Island of Mozambique

Programmes supporting micro and small enterprises in developing countries have been showing that capital is not enough to allow business success: survival and growth. Literature does not provide comprehensive and practical tool to support business development in this context, but allowed the collection of forty-nine success variables that were studied in a sample of successful and unsuccessful businesses in the Island of Mozambique to discover what were the key factors affecting those businesses’ performance. Empirical data gave the insights for the development of a model to screen and improve business potential of micro and small enterprises in this context.

Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) in the region of Saquarema: potential area of visceral leishmaniasis transmission in the state of Rio de Janeiro, Brazil

INTRODUCTION: Lutzomyia longipalpis is the main vector of Leishmania infantum chagasi in the Americas. METHODS: Phlebotomine captures were conducted during 2008 and 2009 in a rural area endemic for cutaneous leishmaniasis located in the municipality of Saquarema, Rio de Janeiro. RESULTS: Among other species captured, we observed the presence of Lutzomyia longipalpis. CONCLUSIONS: This is the first report of the occurrence of Lutzomyia longipalpis in this region, demonstrating the potential risk of visceral leishmaniasis transmission in the coastal area of the State of Rio de Janeiro. Therefore, active vigilance by all municipalities in the area is necessary.

Potential M&A between TIM Brazil and Oi S.A: The irreversible force of Telecommunications convergence

The intrinsic forces of market aiming for telecom industry convergence has arrived to Brazil. This case presents real characters, a sequence of events and other public information that has been impacting two corporations studied in this case. TIM Brazil and Oi S.A, two top players in the Brazilian telecom industry mobile and fixed segment respectively. While a merge between the two of them looks perfect and simple in an operational perspective due to its vertical complementarity, bring to them opportunities to win over a bundle offer (multi service package) that will consolidate their market predominance. Macroeconomic and internal corporate contrasts between these companies’ environment might signal that an impulsive could have a high price to pay in the future.

The financial system in Mozambique: Opportunities, barriers and market potential

This research is empirical and exploratory intending to analyse the attractiveness of banking in Mozambique, considering its positive outlook. To identify the opportunities and barriers, the methods adopted were elite interviews with banking executives, complemented by secondary data. The opportunities for new entrants seem to include bankarization and the emergence of micro and smallmedium enterprises; other avenues seem to include investment banking, support of mega-projects (e.g. energy, infrastructures) through syndicates and cooperation with multilaterals, and the participation in developing capital markets. Conversely, the main barriers include shortage of talent, inadequate infrastructures, poverty, unsophisticated entrepreneurial culture (e.g. informal economy, inadequate financial reporting), burdensome bureaucracy (e.g. visas), foreign exchange regulation, as well as low liquidity and high funding costs for banks. The key conclusions suggest a window of opportunity for niche markets, and new products and services in retail and investment banking.

Evaluating the potential of yeast strains to produce added value products for the food and/or pharmaceutical industries

This study focus in the valorization of the apple pomace with the main goal of obtaining added value products. For that, hot compressed water technology was used for the extraction of phenolic compounds and hydrolysis of polysaccharides presents in the lignocellulosic structure of apple pomace to obtain simple sugars. The sugars have been utilized as alternative carbon source for growth, lipid accumulation and carotenoids production by five different yeast Yarrowia lipolytica, Rhodotorula mucilaginosa, Rhodotorula glutinis, Rhodosporidium babjevae and Rhodosporidium toruloides. Hydrolysis experiments were carried out with constant pressure of 100 bar, flow rate of 2mL/min and temperatures between 50°C and 250°C. The amount of total sugars present in apple pomace hydrolysates showed maximum values for the hydrolysis temperatures of 110°C and 190°C. In fact, these temperatures revealed the best results regarding the monosaccharides quantities. The amount of 5-HMF and furfural in each hydrolysate varied through the different temperatures. Maximum values for 5-HMF were obtained with 170°C, while furfural showed to be maximum at 210°C. Extraction of phenolic compounds were performed in simultaneously with hydrolysis reactions. Total phenolic compounds (TPC) increased along the temperature, however with small variations between 170°C and 250°C. Hydrolysates were then used as alternative carbon source to yeast growth. R. mucilaginosa shows the highest optical density, with the hydrolysate obtained at 130°C. Carotenoids produced by these yeast scored a total of 7.02μg carotenoids/g cell dry weight, while for the control assay, the same yeast scored 9.31μg caratonoides/g cell dry weight. β-carotene was quantified by HPLC, were 33% of the carotenoid production by R. mucilaginosa with hydrolysate as carbon source, corresponded to β-caroteno.

How to take advantage of wine tourism: Creation of a new concept for central Alentejo based on its potential and worldwide success factors

Wine Tourism is gaining importance in today’s world and more destinations and establishments have been arising. After understanding the importance of this economic activity and the factors it must have to succeed, a new project was conceived for Central Alentejo taking into account its potential. This project is an example of how to take advantage of Wine Tourism in wine regions that are underexplored, such as Aldeias de Montoito, the village near Redondo to which a Business Plan will be created, explaining the strategies to pursue in order to have a successful Wine Tourism destination.

Rapid detection and differentiation of mycobacterial species using a multiplex PCR system

Introduction The early diagnosis of mycobacterial infections is a critical step for initiating treatment and curing the patient. Molecular analytical methods have led to considerable improvements in the speed and accuracy of mycobacteria detection. Methods The purpose of this study was to evaluate a multiplex polymerase chain reaction system using mycobacterial strains as an auxiliary tool in the differential diagnosis of tuberculosis and diseases caused by nontuberculous mycobacteria (NTM) Results Forty mycobacterial strains isolated from pulmonary and extrapulmonary origin specimens from 37 patients diagnosed with tuberculosis were processed. Using phenotypic and biochemical characteristics of the 40 mycobacteria isolated in LJ medium, 57.5% (n=23) were characterized as the Mycobacterium tuberculosis complex (MTBC) and 20% (n=8) as nontuberculous mycobacteria (NTM), with 22.5% (n=9) of the results being inconclusive. When the results of the phenotypic and biochemical tests in 30 strains of mycobacteria were compared with the results of the multiplex PCR, there was 100% concordance in the identification of the MTBC and NTM species, respectively. A total of 32.5% (n=13) of the samples in multiplex PCR exhibited a molecular pattern consistent with NTM, thus disagreeing with the final diagnosis from the attending physician. Conclusions Multiplex PCR can be used as a differential method for determining TB infections caused by NTM a valuable tool in reducing the time necessary to make clinical diagnoses and begin treatment. It is also useful for identifying species that were previously not identifiable using conventional biochemical and phenotypic techniques.

The Pampulha reservoir remains a potential urban focus of schistosomiasis mansoni in Brazil: changes in the occurrence patterns of Biomphalaria species and a new record of the parasite

Introduction The Pampulha reservoir has long been a focus of schistosomiasis transmission in Belo Horizonte, State of Minas Gerais, Brazil. The last malacological study conducted in this urban reservoir was more than two decades ago, and thus, an update on the distribution of the species of Biomphalaria as well as new data on the presence of Schistosoma mansoni in this water body are required. Methods The current distribution of Biomphalaria spp. in the Pampulha reservoir and their infection with S. mansoni was evaluated during 55 malacological surveys conducted between 2009 and 2012. Results Biomphalaria straminea displayed a high population density and distribution, and 13.7% (2,233/16,235) of the specimens collected were infected with larval trematodes other than Schistosoma mansoni. Biomphalaria tenagophila and Biomphalaria glabrata, species currently presenting a restricted distribution and small populations, displayed trematode infection rates of 15.2% (98/644) and 13% (83/640), respectively. Thirteen (2%) specimens of B. glabrata were found to be infected with S. mansoni. In addition, a historical review based on previous and new data on the occurrence patterns of Biomphalaria species in this reservoir is presented. Conclusions The results indicate that the Pampulha reservoir remains a potential focus of urban schistosomiasis in Brazil, and significant changes in the occurrence patterns of Biomphalaria species were verified.

Ecological aspects of mosquitoes (Diptera: Culicidae) in the gallery forest of Brasilia National Park, Brazil, with an emphasis on potential vectors of yellow fever

Introduction We analyzed the vertical and monthly distributions of culicid species in the gallery forest of Brasília National Park, with an emphasis on the potential vectors of yellow fever (YF). Methods Between September 2010 and August 2011, mosquitoes were captured on the ground and in the canopy of the forest for five consecutive days per month, from nine to 15 hours. The mosquitoes were examined to verify natural infection with flaviviruses by isolation in Aedes albopictus Skuse, 1864 cells followed by indirect immunofluorescence. Results We identified 2,677 culicids distributed in 29 species. Most of the mosquitoes were captured at ground level (69%) during the rainy season (86%). The most abundant species were Sabethes (Sabethes) albiprivus Theobald, 1903; Limatus durhamii Theobald, 1901; Haemagogus (Conopostegus) leucocelaenus Dyar & Shannon, 1924; Haemagogus (Haemagogus) janthinomys Dyar, 1921; Aedes (Ochlerotatus) scapularis Rondani, 1848; Psorophora (Janthinosoma) ferox Von Humboldt, 1819; and Aedes (Ochlerotatus) serratus Theobald, 1901. Limatus durhamii, Limatus durhamii, Psorophora ferox, Aedes scapularis and Aedes serratus showed significant differences (p<0.05) in their habitat use. Limatus durhamii was found more often in the canopy, unlike the other species. During the rainy season, the most abundant species were Sa. albiprivus, Haemagogus leucocelaenus and Limatus durhamii. During the dry season, the potential YF vectors exhibited a very low frequency and abundance, except Aedes scapularis and Aedes serratus. No flavivirus was detected in the 2,677 examined mosquitoes. Conclusions We recommend continued and systematic entomological monitoring in areas vulnerable to the transmission of YF in the Federal District of Brazil.

Ants in a hospital environment and their potential as mechanical bacterial vectors

Introduction We studied the richness and abundance of ant species, their bacteria and the bacteria isolated from patient clinical samples. Methods Ants were collected with baited traps at 64 sites in a public hospital in São Luis, State of Maranhão, Brazil. Results In total, 1,659 ants from 14 species were captured. The most frequent species were Crematogaster victima, Solenopsis saevissima, Tapinoma melanocephalum, Camponotus vittatus and Paratrechina fulva. Forty-one species of bacteria were isolated from the ants and 18 from patients. Conclusions Ants are potential vehicles for pathogenic and opportunistic bacteria, and they can represent a risk factor in nosocomial infections.