Determination of sialic acids in the serum of cancer patients by capillary electrophoresis

A novel method for the determination of N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) was developed by using high-performance capillary electrophoresis (HPCE) with UV detection at 195 nm. NANA and NGNA were separated directly and analyzed without pre- or postcolumn derivation. The detection limit of NANA is 9.6 x 10(-6) mol L-1 and for mass 3.879 x 10(-14) mol (39 fmol). This method was applied for the determination of NANA in 30 normal human and 72 cancer patients. The results demonstrated that NANA in the sera of cancer patients increased significantly as compared with the normal human (P < 0.001). The new method is simple and sensitive, and is suitable for basic research and clinical application to malignant tumors.

Characteristic infrared spectroscopic patterns in the protein bands of human breast cancer tissue

Infrared (IR) spectra of normal, hyperplasia, fibroadenoma and carcinoma tissues of human breast obtained from 96 patients have been determined and analyzed statistically. Several spectral differences were detected in the frequency regions of N-H stretching, amide I, II and III bands: (1) the bands in the region 3000-3600cm-1 shifted to lower frequencies for the carcinomatous tissue; (2) the A(3300)/A(3075) absorbance ratio was significantly higher for the fibroadenoma than for the other types of tissues; (3) the frequency of the a-helix amide I band decreased for the malignant tissue, while the corresponding beta -sheet amide I band frequency increased; (4) the A(1657)/A(1635) and A(1553)/A(1540) absorbance ratios were the highest for fibroadenoma and carcinoma tissues; (5) the A(1680)/A(1657) absorbance ratio decreased significantly in the order of normal > hyperplasia > fibroadenoma > carcinoma; (6) the A(1651)/A(1545) absorbance ratio increased slightly for the fibroadenoma and the carcinoma tissues; (7) the bands at 1204 and 1278 cm(-1), assigned to the vibrational modes of the collagen, did not appear in the original spectra as resolved peaks and were distinctly stronger in the deconvoluted spectra of the carcinoma tissue and (8) the A(1657)/A(1204) and A(1657)/A(1278) absorbance ratios, both yielding information on the relative content of collagen, increased in the order of normal < hyperplasia < carcinoma < fibroadenoma. The said differences imply that the information is useful for the diagnosis of breast cancer and malignant breast abnormalities, and may serve as a basis for further studies on conformational changes in tissue proteins during carcinogenesis. (C) 2001 Elsevier Science B.V. All rights reserved.

Age-dependence of urinary normal and modified nucleosides in childhood as determined by reversed-phase high-performance liquid chromatography

Modified nucleosides have been characterized as tumor markers for a number of malignant diseases. In order to use these markers in children, the age-dependence of the nucleoside levels in healthy children has to be established and taken into account in diagnostic decisions. In this study, the levels of 12 normal and modified nucleosides in urine of 166 healthy children and adolescents with an age between 1 day and 19 years are determined by reversed-phase HPLC, and age-dependent reference ranges are defined. The urinary nucleoside concentrations are related to the creatinine concentrations, which allows the use of randomly collected urine samples. All nucleoside levels in urine of children decrease with age, most pronounced during the first 4 years of life, and the age-dependence of the reference values of the individual nucleosides can be approximated by a mathematical function y = b(0) + b(1) (1/x) with the regression coefficients b(0) and b(1), the nucleoside levels y and the age x between 1 year and 19 years. In the very young children, the shifts in the nucleoside concentrations are more differentiated. Starting with low levels on the first day of life, the concentrations of all studied nucleosides rise up to an age of 1-2 months, when they reach their absolute maximum for all age periods, and then decrease. (C) 2004 Elsevier B.V. All rights reserved.

Caratterizzazione proteomica di fluidi e tessuti in diverse condizioni fisio-patologiche

This thesis has been focused on the proteomic characterization of human saliva from donors of different ages, starting from birth up to adult age, and pediatric brain tumor tissues. The first study has been performed in order to compare the acid-insoluble fraction of saliva from preterm with at-term newborns and adults and establish if differences exist. In the second study medulloblastoma and pilocytic astrocytoma pediatric brain tumor extracts have been compared. In both studies 2- DE analysis was coupled with high resolution tandem mass spectrometry (MS/MS). The proteomic characterization of the acid-insoluble fractions of saliva from preterm newborns allowed to integrate data previously obtained on the acid-soluble fraction by HPLC-electrospray ionization (ESI)-mass spectrometry (MS), and to evidence several differences between preterm newborns, at-term newborns and adults. Spots differentially expressed between the three groups, according to image analysis of the gels, were submitted to in-gel tryptic digestion and the peptide mixture analyzed by high performance HPLC-ESI-MS/MS for their characterization. By this strategy, we identified three over-expressed proteins in atterm newborns with respect to preterm newborns and adults (BPI fold-containing family A member 1, two proteoforms of annexin A1, and keratin type 1 cytoskeletal 13), and several over-expressed proteins in adults (fatty acid-binding protein, S100A6, S100A7, two proteoforms of S100A9, several proteoforms of prolactin-inducible protein, Ig kappa chain, two proteoforms of cystatin SN, one proteoform of cystatin S and several proteoforms of α-amylase 1). Moreover, for the first time, it was possible to assign by MS/MS four spots of human saliva 2-DE, already detected by other authors, to different proteoforms of S100A9. The strategy applied used a sequential staining protocol to the 2-DE gels, first with Pro-Q Diamond, that allows specific detection of phosphoproteins, and successively with total protein SYPRO Ruby stain. In the second study, proteomic analysis of two pediatric brain tumor tissues pointed out differences between medulloblastoma, the prevalent malignant tumor in childhood, and pilocytic astrocytoma, the most common, that only rarely shows a malignant progression. Due to the limited availability of bioptic tissue, the study was performed on pooled tumor tissues, and was focused on acid-insoluble fraction to integrate the characterization performed by a group of colleagues in Rome on the acid-soluble fraction by high performance HPLC-ESI-MS/MS. The results indicated that the two tumors exhibit different proteomic profiles and evidenced interesting differential expression of several proteins. Among them, peroxiredoxin- 1, peptidyl-prolyl cis–trans isomerase A, heterogeneous nuclear ribonucleoproteins A2/B1, mitochondrial isoform of malate dehydrogenase, nucleoside diphosphate kinase A, glutathione S-transferase P and fructose bisphosphate aldolase A resulted significantly over-expressed in medulloblastoma while glial fibrillary acidic protein, serotransferrin, α crystallin B chain, ferritin light chain, annexin A5, fatty acid-binding protein (brain), sorcin and apolipoprotein A-I resulted significantly over-expressed in pilocytic astrocytoma. In conclusion, the work done allowed to evidence the usefulness of using an integrated bottom-up/top-down approach, based on 2-DE-MS analysis and high performance MS in order to obtain a complete characterization of the proteome under investigation, revealing and identifying, not only peptides and small proteins, but also proteins with higher MW, that often it is not possible to identify by using exclusively a top-down ESI-MS approach.

Sinusite odontogénica

Monografia apresentada à Universidade Fernando Pessoa para obtenção do grau de Licenciada em Medicina Dentária.

Via de sinalização mTOR e efeito dos seus inibidores no tratamento do cancro

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Cancro oral

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária

Avaliação do perfil de referenciação dos doentes com neoplasia oral para o Instituto Português de Oncologia do Porto no ano de 2013

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária

Frutas e produtos hortícolas na prevenção do cancro

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

EVALUATION OF HARMONIC MOTION ELASTOGRAPHY AND ACOUSTO-­OPTIC IMAGING FOR MONITORING LESION FORMATION BY HIGH INTENSITY FOCUSED ULTRASOUND

Malignant or benign tumors may be ablated with high‐intensity focused ultrasound (HIFU). This technique, known as focused ultrasound surgery (FUS), has been actively investigated for decades, but slow to be implemented and difficult to control due to lack of real‐time feedback during ablation. Two methods of imaging and monitoring HIFU lesions during formation were implemented simultaneously, in order to investigate the efficacy of each and to increase confidence in the detection of the lesion. The first, Acousto‐Optic Imaging (AOI) detects the increasing optical absorption and scattering in the lesion. The intensity of a diffuse optical field in illuminated tissue is mapped at the spatial resolution of an ultrasound focal spot, using the acousto‐optic effect. The second, Harmonic Motion Imaging (HMI), detects the changing stiffness in the lesion. The HIFU beam is modulated to force oscillatory motion in the tissue, and the amplitude of this motion, measured by ultrasound pulse‐echo techniques, is influenced by the stiffness. Experiments were performed on store‐bought chicken breast and freshly slaughtered bovine liver. The AOI results correlated with the onset and relative size of forming lesions much better than prior knowledge of the HIFU power and duration. For HMI, a significant artifact was discovered due to acoustic nonlinearity. The artifact was mitigated by adjusting the phase of the HIFU and imaging pulses. A more detailed model of the HMI process than previously published was made using finite element analysis. The model showed that the amplitude of harmonic motion was primarily affected by increases in acoustic attenuation and stiffness as the lesion formed and the interaction of these effects was complex and often counteracted each other. Further biological variability in tissue properties meant that changes in motion were masked by sample‐to‐sample variation. The HMI experiments predicted lesion formation in only about a quarter of the lesions made. In simultaneous AOI/HMI experiments it appeared that AOI was a more robust method for lesion detection.

Evaluation of a WHO-Validated Serotype-Specific Serological Assay for the Diagnosis of Pneumococcal Etiology in Children with Community-Acquired Pneumonia.

BACKGROUND: The etiologic diagnosis of community-acquired pneumonia (CAP) remains challenging in children because blood cultures have low sensitivity. Novel approaches are needed to confirm the role of Streptococcus pneumoniae. METHODS: In this study, pneumococcal aetiology was determined by serology using a subset of blood samples collected during a prospective multicentre observational study of children <15 years of age hospitalised in Belgium with X-ray-confirmed CAP. Blood samples were collected at admission and 3-4 weeks later. Pneumococcal (P)-CAP was defined in the presence of a positive blood or pleural fluid culture. Serotyping of Streptococcus pneumoniae isolates was done with the Quellung reaction. Serological diagnosis was assessed for nine serotypes using World Health Organization validated IgG and IgA serotype-specific enzyme-linked immunosorbent assays (ELISAs). RESULTS: Paired admission/convalescent sera from 163 children were evaluated by ELISA (35 with proven P-CAP and 128 with non proven P-CAP). ELISA detected pneumococci in 82.8% of patients with proven P-CAP. The serotypes identified were the same as with the Quellung reaction in 82% and 59% of cases by IgG ELISA and IgA ELISA, respectively. Overall, ELISA identified a pneumococcal aetiology in 55% of patients with non-proven P-CAP. Serotypes 1 (51.6%), 7F (19%), and 5 (15.7%) were the most frequent according to IgG ELISA. CONCLUSIONS: In conclusion, the serological assay allows recognition of pneumococcal origin in 55% of CAP patients with negative culture. This assay should improve the diagnosis of P-CAP in children and could be a useful tool for future epidemiological studies on childhood CAP etiology.

Hand-held spectroscopic device for in vivo and intraoperative tumor detection: contrast enhancement, detection sensitivity, and tissue penetration.

Surgery is one of the most effective and widely used procedures in treating human cancers, but a major problem is that the surgeon often fails to remove the entire tumor, leaving behind tumor-positive margins, metastatic lymph nodes, and/or satellite tumor nodules. Here we report the use of a hand-held spectroscopic pen device (termed SpectroPen) and near-infrared contrast agents for intraoperative detection of malignant tumors, based on wavelength-resolved measurements of fluorescence and surface-enhanced Raman scattering (SERS) signals. The SpectroPen utilizes a near-infrared diode laser (emitting at 785 nm) coupled to a compact head unit for light excitation and collection. This pen-shaped device effectively removes silica Raman peaks from the fiber optics and attenuates the reflected excitation light, allowing sensitive analysis of both fluorescence and Raman signals. Its overall performance has been evaluated by using a fluorescent contrast agent (indocyanine green, or ICG) as well as a surface-enhanced Raman scattering (SERS) contrast agent (pegylated colloidal gold). Under in vitro conditions, the detection limits are approximately 2-5 × 10(-11) M for the indocyanine dye and 0.5-1 × 10(-13) M for the SERS contrast agent. Ex vivo tissue penetration data show attenuated but resolvable fluorescence and Raman signals when the contrast agents are buried 5-10 mm deep in fresh animal tissues. In vivo studies using mice bearing bioluminescent 4T1 breast tumors further demonstrate that the tumor borders can be precisely detected preoperatively and intraoperatively, and that the contrast signals are strongly correlated with tumor bioluminescence. After surgery, the SpectroPen device permits further evaluation of both positive and negative tumor margins around the surgical cavity, raising new possibilities for real-time tumor detection and image-guided surgery.

Airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling.

The small airways of the human lung undergo pathological changes in pulmonary disorders, such as chronic obstructive pulmonary disease (COPD), asthma, bronchiolitis obliterans and cystic fibrosis. These clinical problems impose huge personal and societal healthcare burdens. The changes, termed 'pathological airway remodeling', affect the epithelium, the underlying mesenchyme and the reciprocal trophic interactions that occur between these tissues. Most of the normal human airway is lined by a pseudostratified epithelium of ciliated cells, secretory cells and 6-30% basal cells, the proportion of which varies along the proximal-distal axis. Epithelial abnormalities range from hypoplasia (failure to differentiate) to basal- and goblet-cell hyperplasia, squamous- and goblet-cell metaplasia, dysplasia and malignant transformation. Mesenchymal alterations include thickening of the basal lamina, smooth muscle hyperplasia, fibrosis and inflammatory cell accumulation. Paradoxically, given the prevalence and importance of airway remodeling in lung disease, its etiology is poorly understood. This is due, in part, to a lack of basic knowledge of the mechanisms that regulate the differentiation, maintenance and repair of the airway epithelium. Specifically, little is known about the proliferation and differentiation of basal cells, a multipotent stem cell population of the pseudostratified airway epithelium. This Perspective summarizes what we know, and what we need to know, about airway basal cells to evaluate their contributions to normal and abnormal airway remodeling. We contend that exploiting well-described model systems using both human airway epithelial cells and the pseudostratified epithelium of the genetically tractable mouse trachea will enable crucial discoveries regarding the pathogenesis of airway disease.

MRP3: a molecular target for human glioblastoma multiforme immunotherapy.

BACKGROUND: Glioblastoma multiforme (GBM) is refractory to conventional therapies. To overcome the problem of heterogeneity, more brain tumor markers are required for prognosis and targeted therapy. We have identified and validated a promising molecular therapeutic target that is expressed by GBM: human multidrug-resistance protein 3 (MRP3). METHODS: We investigated MRP3 by genetic and immunohistochemical (IHC) analysis of human gliomas to determine the incidence, distribution, and localization of MRP3 antigens in GBM and their potential correlation with survival. To determine MRP3 mRNA transcript and protein expression levels, we performed quantitative RT-PCR, raising MRP3-specific antibodies, and IHC analysis with biopsies of newly diagnosed GBM patients. We used univariate and multivariate analyses to assess the correlation of RNA expression and IHC of MRP3 with patient survival, with and without adjustment for age, extent of resection, and KPS. RESULTS: Real-time PCR results from 67 GBM biopsies indicated that 59/67 (88%) samples highly expressed MRP3 mRNA transcripts, in contrast with minimal expression in normal brain samples. Rabbit polyvalent and murine monoclonal antibodies generated against an extracellular span of MRP3 protein demonstrated reactivity with defined MRP3-expressing cell lines and GBM patient biopsies by Western blotting and FACS analyses, the latter establishing cell surface MRP3 protein expression. IHC evaluation of 46 GBM biopsy samples with anti-MRP3 IgG revealed MRP3 in a primarily membranous and cytoplasmic pattern in 42 (91%) of the 46 samples. Relative RNA expression was a strong predictor of survival for newly diagnosed GBM patients. Hazard of death for GBM patients with high levels of MRP3 RNA expression was 2.71 (95% CI: 1.54-4.80) times that of patients with low/moderate levels (p = 0.002). CONCLUSIONS: Human GBMs overexpress MRP3 at both mRNA and protein levels, and elevated MRP3 mRNA levels in GBM biopsy samples correlated with a higher risk of death. These data suggest that the tumor-associated antigen MRP3 has potential use for prognosis and as a target for malignant glioma immunotherapy.

c-Myc is required for maintenance of glioma cancer stem cells.

BACKGROUND: Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. METHODOLOGY/PRINCIPAL FINDINGS: Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G(0)/G(1) phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice. CONCLUSIONS/SIGNIFICANCE: These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.