Integrin genetic variants and stage-specific tumor recurrence in patients with stage II and III colon cancer

Integrins (ITGs) are key elements in cancer biology, regulating tumor growth, angiogenesis and lymphangiogenesis through interactions of the tumor cells with the microenvironment. Moving from the hypothesis that ITGs could have different effects in stage II and III colon cancer, we tested whether a comprehensive panel of germline single-nucleotide polymorphisms (SNPs) in ITG genes could predict stage-specific time to tumor recurrence (TTR). A total of 234 patients treated with 5-fluorouracil-based chemotherapy at the University of Southern California were included in this study. Whole-blood samples were analyzed for germline SNPs in ITG genes using PCR-restriction fragment length polymorphism or direct DNA sequencing. In the multivariable analysis, stage II colon cancer patients with at least one G allele for ITGB3 rs4642 had higher risk of recurrence (hazard ratio (HR)=4.027, 95% confidence interval (95% CI) 1.556-10.421, P=0.004). This association was also significant in the combined stage II-III cohort (HR=1.975, 95% CI 1.194-3.269, P=0.008). The predominant role of ITGB3 rs4642 in stage II diseases was confirmed using recursive partitioning, showing that ITGB3 rs4642 was the most important factor in stage II diseases. In contrast, in stage III diseases the combined analysis of ITGB1 rs2298141 and ITGA4 rs7562325 allowed to identify three distinct prognostic subgroups (P=0.009). The interaction between stage and the combined ITGB1 rs2298141 and ITGA4 rs7562325 on TTR was significant (P=0.025). This study identifies germline polymorphisms in ITG genes as independent stage-specific prognostic markers for stage II and III colon cancer. These data may help to select subgroups of patients who may benefit from ITG-targeted treatments.

Tumor suppressor Ras-association domain family 1 isoform A is a novel regulator of cardiac hypertrophy

BACKGROUND: Ras signaling regulates a number of important processes in the heart, including cell growth and hypertrophy. Although it is known that defective Ras signaling is associated with Noonan, Costello, and other syndromes that are characterized by tumor formation and cardiac hypertrophy, little is known about factors that may control it. Here we investigate the role of Ras effector Ras-association domain family 1 isoform A (RASSF1A) in regulating myocardial hypertrophy.

METHODS AND RESULTS: A significant downregulation of RASSF1A expression was observed in hypertrophic mouse hearts, as well as in failing human hearts. To further investigate the role of RASSF1A in cardiac (patho)physiology, we used RASSF1A knock-out (RASSF1A(-)(/)(-)) mice and neonatal rat cardiomyocytes with adenoviral overexpression of RASSF1A. Ablation of RASSF1A in mice significantly enhanced the hypertrophic response to transverse aortic constriction (64.2% increase in heart weight/body weight ratio in RASSF1A(-)(/)(-) mice compared with 32.4% in wild type). Consistent with the in vivo data, overexpression of RASSF1A in cardiomyocytes markedly reduced the cellular hypertrophic response to phenylephrine stimulation. Analysis of molecular signaling events in isolated cardiomyocytes indicated that RASSF1A inhibited extracellular regulated kinase 1/2 activation, likely by blocking the binding of Raf1 to active Ras.

CONCLUSIONS: Our data establish RASSF1A as a novel inhibitor of cardiac hypertrophy by modulating the extracellular regulated kinase 1/2 pathway.

IGF-Binding Protein 2 - Oncogene or Tumor Suppressor?

The role of insulin-like growth factor binding protein 2 (IGFBP2) in cancer is unclear. In general, IGFBP2 is considered to be oncogenic and its expression is often observed to be elevated in cancer. However, there are a number of conflicting reports in vitro and in vivo where IGFBP2 acts in a tumor suppressor manner. In this mini-review, we discuss the factors influencing the variation in IGFBP2 expression in cancer and our interpretation of these findings.

Low-grade malignant Triton tumor of the oral cavity:a case report

Malignant Triton tumor (MTT) is a malignant peripheral nerve sheath tumor showing rhabdomyoblastic differentiation. It is considered a high-grade neoplasm with poor outcome. This report describes an MTT appearing in the oral cavity. On histologic examination the encapsulated lesion was composed of interlacing fascicles of spindle cells and scattered, large, strap-like pleomorphic cells with abundant eosinophilic cytoplasm. No cross striations were seen. Examination of levels through the tissue showed a total of only 4 normal mitoses and no necrosis. Immunohistochemistry demonstrated diffuse S100 positivity in the spindle cells. The large pleomorphic cells were weakly positive for alpha-sarcomeric actin and myoglobin, although variably but strongly positive for desmin. Management involved a small en bloc resection of the maxilla. After 33 months there was no sign of recurrence or distant metastasis. It was concluded that low-grade variants of MTT occur that do not have an aggressive clinical course.

Immunocytochemical Localization of Substance P Receptors (NK-1 Receptors) in Human Gingival Tissue

Substance P (SP) is a member of the structurally related family of neuropeptides known as the tachykinins. In addition to neurotransmitter roles, the tachykinins are also known to modulate local inflammation which depends on signalling between the neuropeptide molecules and target cells and tissues. SP mediates its effects through a specific receptor, known as the substance P receptor or the neurokinin 1 (NK-1) receptor. The NK-1 receptor is a G-protein associated integral membrane protein and although it has been studied in a wide range of tissues, to date there has been no published data on the localisation of the NK-1 receptor in human gingival tissue. Objective: The aim of this study was to examine the distribution of the NK-1 receptor in human gingival tissue using immunocytochemistry. Method: Gingival tissue was obtained from patients undergoing periodontal surgery. Tissue was fixed in paraformaldehyde and embedded in wax for sectioning. Sections were dewaxed in xylene and then rehydrated in alcohols and phosphate buffered saline. Rehydrated sections were probed with rabbit polyclonal antibody to human NK-1 receptor which was subsequently detected using anti-rabbit horseradish peroxidase conjugate and diaminobenzidine as substrate. Results: Immunocytochemistry revealed that the NK-1 receptor was distributed along nerve fibres and blood vessel endothelial cells, suggesting these areas are main targets for the actions of SP via the NK-1 receptor. Conclusion: This is the first immunocytochemical report of NK-1 receptors in human gingival tissue and provides evidence for possible NK-1 mediated biological effects of SP in human gingival tissue from periodontitis patients.

Novos derivados porfirínicos: síntese e potenciais aplicações em PDT

O trabalho apresentado nesta dissertação descreve estudos desenvolvidos segundo três metodologias para a funcionalização de meso-tetra-arilporfirinas; uma delas baseada na funcionalização com diazo compostos, outra na reacção de Suzuki-Miyaura e a terceira na reacção de metátese. O presente documento é composto por quatro partes distintas. Na primeira parte são feitas considerações gerais sobre propriedades e aplicações de macrociclos porfirínicos. Na segunda parte é apresentado o trabalho realizado na funcionalização de porfirinas com diazo compostos através de reacções de inserção com um catalisador de ródio(II). Este estudo envolveu a preparação de duas porfirinas com grupos O-H e N-H nas posições para dum substituinte meso-fenilo. Os estudos iniciaram-se com a reacção de 5-(4-hidroxifenil)-10,15,20-trifenilporfirinatozinco(II) com diazoacetato de etilo na presença de Rh2(OAc)4. Obteve-se o produto esperado de inserção com bons rendimentos. A reacção de 5-(4-aminofenil)- 10,15,20-trifenilporfirinatozinco(II) com esse diazo composto na presença de Rh2(OAc)4 mostrou um perfil diferente do da reacção com o outro porfirinato. Foram obtidos três produtos, a saber: produto de bis-inserção e dois outros contendo o grupo funcional amida. Este capítulo descreve ainda a síntese de glicoporfirinas e de glicoclorinas através de reacções de inserção e de ciclopropanação envolvendo, respectivamente, os complexos 5-(4-hidroxifenil)- 10,15,20-trifenilporfirinatozinco(II) e 5,10,15,20- tetraquis(pentafluorofenil)porfirinatozinco(II) com α-diazoacetatos com substituintes sacarídicos. Foram usados, como catalisadores, Rh2(OAc)4 e CuCl, respectivamente, na reacção de inserção e na de ciclopropanação. Tendo em vista a sua aplicação em PDT, os novos produtos foram descomplexados e a unidade glicosídica foi desprotegida. Estudos de geração de oxigénio singuleto mostraram que os compostos obtidos são bons geradores dessa espécie citotóxica. Foram também realizados estudos de avaliação da actividade fotodinâmica em células humanas tumorais HeLa e em células humanas saudáveis HaCaT. Estudos de viabilidade celular, após tratamento fotodinâmico, mostraram que as glicoclorinas são mais eficazes na fotoinactivação das células tumorais. Apenas o derivado da clorina com a unidade de galactose mostrou selectividade para a linha celular tumoral, inibindo o crescimento desta e não causando efeito significativo na linha celular saudável. Este fotossensibilizador não foi tóxico na ausência de luz e depois do tratamento fotodinâmico e em condições sub-letais, provocou vacuolização citoplasmática e retracção pronunciada nas células tumorais, enquanto que nas células saudáveis os danos sofridos foram escassos. Nos estudos de localização celular este fotossensibilizador localizou-se na membrana citoplasmática e nos lisossomas tanto nas células HeLa como nas HaCaT. Na terceira parte deste trabalho são descritos os resultados obtidos nos estudos de funcionalização de porfirinas através da reacção de Suzuki-Miyaura. O complexo 2-(4,4,5,5,-tetrametil-1,3,2-dioxaborolan-2-il)-5,10,15,20- tetrafenilporfirinatozinco(II) foi o composto escolhido como reagente de partida. Usando a reacção de Suzuki foi possível sintetizar complexos β-bromoarilporfirínicos em bons rendimentos. Estes complexos foram sujeitos à reacção de Suzuki com pinacolborano, tendo sido possível obter os produtos de acoplamento esperados e ainda derivados diméricos de complexos porfirínicos ligados por unidades fenileno. Esta metodologia reacional, catalisada por paládio, permitiu ainda sintetizar novos derivados quinolona-porfirina através da reacção do complexo porfirínico β-borilado anteriormente referido com bromo-quinolonas Nsubstiuídas com grupos alquílicos e glicosídicos. Deste modo foram obtidos derivados quinolona-porfirina em bons rendimentos. Com vista a realizar estudos de potencial aplicação em PACT, foram hidrolisados os grupos éster da unidade de quinolona e clivados os grupos protectores das unidades glicosídicas desses derivados. No estudo por MS Tandem (MS/MS) foi possível verificar que os isómeros dos derivados quinolona-porfirina sofrem as mesmas vias de fragmentação, confirmando as suas estruturas análogas. Foi possível ainda distinguir os derivados em que a unidade de porfirina está ligada à quinolona através da posição C-6 da quinolona daqueles em que a unidade de porfirina está ligada à quinolona através da sua posição C-7. Os estudos de geração de oxigénio singuleto mostraram que as porfirinas β- substituídas com unidades de quinolona são melhores geradoras desta espécie citotóxica do que a tetrafenilporfirina e que a eficiência que têm em gerar essa espécie é afectada pela posição da ligação entre a porfirina e a quinolona, assim como com o tipo do N-substituinte da quinolona. As bases livres dos conjugados quinolona-porfirinatos foram testadas como fotossensibilizadores em PACT em células do parasita Leishmania Braziliensis, tendo-se observado que estes compostos têm capacidade para fotoinactivar este parasita. Na quarta parte deste trabalho são descritos os resultados dos estudos de funcionalização de porfirinas através da reacção de metátese com catalisador de Grubbs, visando a obtenção de novos macrociclos com grupos triazolilo. Verificouse que a eficiência da reacção de metátese cruzada entre 2-vinil-5,10,15,20- tetrafenilporfirinatozinco(II) e 4-vinil-1,2,3-triazóis N-substituídos dependia das condições reaccionais e do triazol usado em cada caso. Foi possível ainda concluir que o impedimento estéreo das espécies envolvidas é um dos responsáveis pelo ciclo catalítico prosseguir por uma via secundária, originando uma espécie de ruténio pouco eficiente como catalisador. Este estudo foi estendido à reacção dos 4-vinil-1,2,3-triazois seleccionados com 2-butadienil-5,10,15,20- tetrafenilporfirinatoniquel(II); em cada caso foi possível obter uma mistura de isómeros dos derivados triazol-porfirina, geralmente em maiores rendimentos globais do que na reacção com 2-vinil-5,10,15,20-tetrafenilporfirinatozinco(II). Este facto será indicativo de que a reacção de metátase com esse derivado porfirínico segue, predominantemente, a via mais eficiente.

Sectorial antennas for WSN localization system

This work investigates low cost localization systems (LS) based on received signal strength (RSS) and integrated with different types of antennas with main emphasis on sectorial antennas. The last few years have witnessed an outstanding growth in wireless sensor networks (WSN). Among its various possible applications, the localization field became a major area of research. The localization techniques based on RSS are characterized by simplicity and low cost of integration. The integration of LS based on RSS and sectorial antennas (SA) was proven to provide an effective solution for reducing the number of required nodes of the networks and allows the combination of several techniques, such as RSS and angle of arrival (AoA). This PhD thesis focuses on studying techniques, antennas and protocols that best meet the needs of each LS with main focus on low cost systems based on RSS and AoA. Firstly there are studied localization techniques and system that best suit the requirements of the user and the antennas that are most appropriate according to the nature of the signal. In this step it is intended to provide a fundamental understanding of the undertaken work. Then the developed antennas are presented according to the following categories: sectorial and microstrip antennas. Two sectorial antennas are presented: a narrowband antenna operating at 2.4 to 2.5 GHz and a broadband antenna operating at 800MHz-2.4GHz. The low cost printed antennas were designed to operate at 5 GHz, which may be used for vehicular communication. After presenting the various antennas, several prototypes of indoor/outdoor LS are implemented and analyzed. Localization protocols are also proposed, one based on simplicity and low power, and the other on interoperability with different types of antennas and system requirements.

Concentration of tumor biomarkers using aqueous biphasic systems

According to the World Health Organization, around 8.2 million people die each year with cancer. Most patients do not perform routine diagnoses and the symptoms, in most situations, occur when the patient is already at an advanced stage of the disease, consequently resulting in a high cancer mortality. Currently, prostate cancer is the second leading cause of death among males worldwide. In Portugal, this is the most diagnosed type of cancer and the third that causes more deaths. Taking into account that there is no cure for advanced stages of prostate cancer, the main strategy comprises an early diagnosis to increase the successful rate of the treatment. The prostate specific antigen (PSA) is an important biomarker of prostate cancer that can be detected in biological fluids, including blood, urine and semen. However, the commercial kits available are addressed for blood samples and the commonly used analytical methods for their detection and quantification requires specialized staff, specific equipment and extensive sample processing, resulting in an expensive process. Thus, the aim of this MSc thesis consisted on the development of a simple, efficient and less expensive method for the extraction and concentration of PSA from urine samples using aqueous biphasic systems (ABS) composed of ionic liquids. Initially, the phase diagrams of a set of aqueous biphasic systems composed of an organic salt and ionic liquids were determined. Then, their ability to extract PSA was ascertained. The obtained results reveal that in the tested systems the prostate specific antigen is completely extracted to the ionic-liquid-rich phase in a single step. Subsequently, the applicability of the investigated ABS for the concentration of PSA was addressed, either from aqueous solutions or urine samples. The low concentration of this biomarker in urine (clinically significant below 150 ng/mL) usually hinders its detection by conventional analytical techniques. The obtained results showed that it is possible to extract and concentrate PSA, up to 250 times in a single-step, so that it can be identified and quantified using less expensive techniques.

Concentration of tumor biomarkers using aqueous biphasic systems

According to the World Health Organization, around 8.2 million people die each year with cancer. Most patients do not perform routine diagnoses and the symptoms, in most situations, occur when the patient is already at an advanced stage of the disease, consequently resulting in a high cancer mortality. Currently, prostate cancer is the second leading cause of death among males worldwide. In Portugal, this is the most diagnosed type of cancer and the third that causes more deaths. Taking into account that there is no cure for advanced stages of prostate cancer, the main strategy comprises an early diagnosis to increase the successful rate of the treatment. The prostate specific antigen (PSA) is an important biomarker of prostate cancer that can be detected in biological fluids, including blood, urine and semen. However, the commercial kits available are addressed for blood samples and the commonly used analytical methods for their detection and quantification requires specialized staff, specific equipment and extensive sample processing, resulting in an expensive process. Thus, the aim of this MSc thesis consisted on the development of a simple, efficient and less expensive method for the extraction and concentration of PSA from urine samples using aqueous biphasic systems (ABS) composed of ionic liquids. Initially, the phase diagrams of a set of aqueous biphasic systems composed of an organic salt and ionic liquids were determined. Then, their ability to extract PSA was ascertained. The obtained results reveal that in the tested systems the prostate specific antigen is completely extracted to the ionic-liquid-rich phase in a single step. Subsequently, the applicability of the investigated ABS for the concentration of PSA was addressed, either from aqueous solutions or urine samples. The low concentration of this biomarker in urine (clinically significant below 150 ng/mL) usually hinders its detection by conventional analytical techniques. The obtained results showed that it is possible to extract and concentrate PSA, up to 250 times in a single-step, so that it can be identified and quantified using less expensive techniques.

Source localization with vector sensor array during the makai experiment

Vector sensors measure both the acoustic pressure and the three components of particle velocity. Because of this, a vector sensor array (VSA) has the advantage of being able to provide substantially higher directivity with a much smaller aperture than an array of traditional scalar (pressure only) hydrophones. Although several, most of them theoretic, works were published from early nineties, only in the last years due to improvements and availability of vector sensor technology, the interest on field experiments with VSA increased in the scientific community. During the Makai Experiment, that took place off the coast of Kauai I., Hawaii, in September 2005, real data were collected with a 4 element vertical VSA. These data will be discussed in the present paper. The acoustic signals were emitted from a near source (low frequency ship noise) and two high frequency controlled acoustic sources located within a range of 2km from the VSA. The advantages of the VSA over traditional scalar hydrophone arrays in source localization will be addressed using conventional beamforming.

Co-localization of GnRH ligands and their receptors in the European sea bass, Dicentrarchus labrax

The migration of the hypophysiotropic GnRH (GnRH-I) neurons during early development is a crucial step in establishing a normally functioning reproductive system in all vertebrates. These neurons derive from progenitor cells in the olfactory placode and subsequently migrate to their final position in the ventral forebrain, where they mediate hypophysiotropic control over Lh. We use zebrafish as a model to investigate the path and the factors that mediate the migration of the GnRH-I neurons during early development. A transgenic line of zebrafish, in which GnRH- I neurons specifically express a reporter gene (GFP) has been developed in our lab. This was achieved by integrating a GnRH-I promoter/GFP reporter transgene into the zebrafish genome. The resulting transgenic line allows us to track the route of the GnRH-I neuronal migration in real time and in vivo. We have used this line to conduct time lapse imaging to ascertain the exact migrational path and the final position in the ventral forebrain of the GnRH-I neurons.

Searching biocompounds in marine natural extracts with potential use in tumor prevention and treatment

Cancer is a multistage process characterized by three stages: initiation, promotion and progression; and is one of the major killers worldwide. Oxidative stress acts as initiator in tumorigenesis; chronic inflammation promotes cancer; and apoptosis inactivation is an issue in cancer progression. In this study, it was investigated the antioxidant, antiinflammatory and antitumor properties of hexane, ether, chloroform, methanol and water extracts of five species of halophytes: A. macrostachyum, P. coronopus, J. acutus, C. edulis and A. halimus. Antioxidant activity was assessed by DPPH• and ABTS•+ methods, and the total phenolics content (TPC) was evaluated by the Folin-Ciocalteau method. The anti-inflammatory activity of the extracts was determined by the Griess method, and by evaluating the inhibition of NO production in LPS-stimulated RAW- 264.7 macrophages. The cytotoxic activity of the extracts against HepG2 and THP1 cell lines was estimated by the MTT assay, and the results obtained were further compared with the S17 non-tumor cell line. The induction of apoptosis of J. acutus ether extract was assessed by DAPI staining. The highest antioxidant activities was observed in C. edulis methanol and the J. acutus ether extracts against the DPPH• radical; and J. acutus ether and A. halimus ether extracts against the ABTS•+ radical. The methanol extracts of C. edulis and P. coronopus, and the ether extract of J. acutus revealed a high TPC. Generally the antioxidant activity had no correlation with the TPC. The A. halimus chloroform and P. coronopus hexane extracts demonstrated ability to reduce NO production in macrophages (> 50%), revealing their anti-inflammatory capacity. The ether extract of J. acutus showed high cytotoxicity against HepG2 cancer cells, with reduced cellular viability even at the lowest concentrations. This outcome was significantly lower than the obtained with the non-tumor cells (S17). This result was complemented by the induction of apoptosis.

Localization and regulation by GABA of anion transporter present in pollen grain protoplasts and tube of Arabidopsis thaliana

Tese de doutoramento, Biologia (Biologia do Desenvolvimento), Universidade de Lisboa, Faculdade de Ciências, 2015

Distinct H3F3A and H3F3B driver mutations define chondroblastoma and giant cell tumor of bone

It is recognized that some mutated cancer genes contribute to the development of many cancer types, whereas others are cancer type specific. For genes that are mutated in multiple cancer classes, mutations are usually similar in the different affected cancer types. Here, however, we report exquisite tumor type specificity for different histone H3.3 driver alterations. In 73 of 77 cases of chondroblastoma (95%), we found p.Lys36Met alterations predominantly encoded in H3F3B, which is one of two genes for histone H3.3. In contrast, in 92% (49/53) of giant cell tumors of bone, we found histone H3.3 alterations exclusively in H3F3A, leading to p.Gly34Trp or, in one case, p.Gly34Leu alterations. The mutations were restricted to the stromal cell population and were not detected in osteoclasts or their precursors. In the context of previously reported H3F3A mutations encoding p.Lys27Met and p.Gly34Arg or p.Gly34Val alterations in childhood brain tumors, a remarkable picture of tumor type specificity for histone H3.3 driver alterations emerges, indicating that histone H3.3 residues, mutations and genes have distinct functions.

Performance of microstrip antennas in the presence of EBG in an indoor localization system

Indoor localization systems in nowadays is a huge area of interest not only at academic but also at industry and commercial level. The correct location in these systems is strongly influenced by antennas performance which can provide several gains, bandwidths, polarizations and radiation patterns, due to large variety of antennas types and formats. This paper presents the design, manufacture and measurement of a compact microstrip antenna, for a 2.4 GHZ frequency band, enhanced with the use of Electromagnetic Band-Gap (EBG) structures, which improve the electromagnetic behavior of the conventional antennas. The microstrip antenna with an EBG structure integrated allows an improvement of the location system performance in about 25% to 30% relatively to a conventional microstrip antenna.