Detection of Bartonella henselae DNA in clinical samples including peripheral blood of immune competent and immune compromised patients by three nested amplifications

Bacteria of the genus Bartonella are emerging pathogens detected in lymph node biopsies and aspirates probably caused by increased concentration of bacteria. Twenty-three samples of 18 patients with clinical, laboratory and/or epidemiological data suggesting bartonellosis were subjected to three nested amplifications targeting a fragment of the 60-kDa heat shock protein (HSP), the internal transcribed spacer 16S-23S rRNA (ITS) and the cell division (FtsZ) of Bartonella henselae, in order to improve detection in clinical samples. In the first amplification 01, 04 and 05 samples, were positive by HSP (4.3%), FtsZ (17.4%) and ITS (21.7%), respectively. After the second round six positive samples were identified by nested-HSP (26%), eight by nested-ITS (34.8%) and 18 by nested-FtsZ (78.2%), corresponding to 10 peripheral blood samples, five lymph node biopsies, two skin biopsies and one lymph node aspirate. The nested-FtsZ was more sensitive than nested-HSP and nested-ITS (p < 0.0001), enabling the detection of Bartonella henselae DNA in 15 of 18 patients (83.3%). In this study, three nested-PCR that should be specific for Bartonella henselae amplification were developed, but only the nested-FtsZ did not amplify DNA from Bartonella quintana. We conclude that nested amplifications increased detection of B. henselae DNA, and that the nested-FtsZ was the most sensitive and the only specific to B. henselae in different biological samples. As all samples detected by nested-HSP and nested-ITS, were also by nested-FtsZ, we infer that in our series infections were caused by Bartonella henselae. The high number of positive blood samples draws attention to the use of this biological material in the investigation of bartonellosis, regardless of the immune status of patients. This fact is important in the case of critically ill patients and young children to avoid more invasive procedures such as lymph nodes biopsies and aspirates.

A go-to-market strategy for green extration and impregnation

Dissertação para obtenção do Grau de Mestre em Biotecnologia

The Role of the Humoral Immune Response in the Molecular Evolution of the Envelope C2, V3 and C3 Regions in Chronically HIV-2 Infected Patients

BACKGROUND: This study was designed to investigate, for the first time, the short-term molecular evolution of the HIV-2 C2, V3 and C3 envelope regions and its association with the immune response. Clonal sequences of the env C2V3C3 region were obtained from a cohort of eighteen HIV-2 chronically infected patients followed prospectively during 2-4 years. Genetic diversity, divergence, positive selection and glycosylation in the C2V3C3 region were analysed as a function of the number of CD4+ T cells and the anti-C2V3C3 IgG and IgA antibody reactivity RESULTS: The mean intra-host nucleotide diversity was 2.1% (SD, 1.1%), increasing along the course of infection in most patients. Diversity at the amino acid level was significantly lower for the V3 region and higher for the C2 region. The average divergence rate was 0.014 substitutions/site/year, which is similar to that reported in chronic HIV-1 infection. The number and position of positively selected sites was highly variable, except for codons 267 and 270 in C2 that were under strong and persistent positive selection in most patients. N-glycosylation sites located in C2 and V3 were conserved in all patients along the course of infection. Intra-host variation of C2V3C3-specific IgG response over time was inversely associated with the variation in nucleotide and amino acid diversity of the C2V3C3 region. Variation of the C2V3C3-specific IgA response was inversely associated with variation in the number of N-glycosylation sites. CONCLUSION: The evolutionary dynamics of HIV-2 envelope during chronic aviremic infection is similar to HIV-1 implying that the virus should be actively replicating in cellular compartments. Convergent evolution of N-glycosylation in C2 and V3, and the limited diversification of V3, indicates that there are important functional constraints to the potential diversity of the HIV-2 envelope. C2V3C3-specific IgG antibodies are effective at reducing viral population size limiting the number of virus escape mutants. The C3 region seems to be a target for IgA antibodies and increasing N-linked glycosylation may prevent HIV-2 envelope recognition by these antibodies. Our results provide new insights into the biology of HIV-2 and its relation with the human host and may have important implications for vaccine design.

IPTV data reduction strategy to measure real users’ behaviours

Dissertação para obtenção do Grau de Mestre em Engenharia Informática

EFFICACY OF NITAZOXANIDE AGAINST Toxocara canis: LARVAL RECOVERY AND HUMORAL IMMUNE RESPONSE IN EXPERIMENTALLY INFECTED MICE

SUMMARY The efficacy of nitazoxanide (NTZ) against toxocariasis was investigated in an experimental murine model and results were compared to those obtained using mebendazole. Sixty male BALB/c mice, aged six to eight weeks-old, were divided into groups of 10 each; fifty were orally infected with 300 larvaed eggs of T. canisand grouped as follows, G I: infected untreated mice; G II: infected mice treated with MBZ (15 mg/kg/day) 10 days postinfection (dpi); G III: infected mice treated with NTZ (20 mg/kg/day) 10 dpi; G IV: infected mice treated with MBZ 60 dpi; G V: infected mice treated with NTZ 60 dpi; GVI: control group comprising uninfected mice. Mice were bled via retro-orbital plexus on four occasions between 30 and 120 dpi. Sera were processed using the ELISA technique to detect IgG anti- Toxocaraantibodies. At 120 dpi, mice were sacrificed for larval recovery in the CNS, liver, lungs, kidneys, eyes and carcass. Results showed similar levels of anti- ToxocaraIgG antibodies among mice infected but not submitted to treatment and groups treated with MBZ or NTZ, 10 and 60 dpi. Larval recovery showed similar values in groups treated with NTZ and MBZ 10 dpi. MBZ showed better efficacy 60 dpi, with a 72.6% reduction in the parasite load compared with NTZ, which showed only 46.5% reduction. We conclude that administration of these anthelmintics did not modify the humoral response in experimental infection by T. canis. No parasitological cure was observed with either drug; however, a greater reduction in parasite load was achieved following treatment with MBZ.

Sialyl Tn-Expressing Bladder Cancer Cells Induce a Tolerogenic Phenotype in Innate and Adaptive Immune Cells

Despite the wide acceptance that glycans are centrally implicated in immunity, exactly how they contribute to the tilt immune response remains poorly defined. In this study, we sought to evaluate the impact of the malignant phenotype-associated glycan, sialyl-Tn (STn) in the function of the key orchestrators of the immune response, the dendritic cells (DCs). In high grade bladder cancer tissue, the STn antigen is significantly overexpressed and correlated with the increased expression of ST6GALNAC1 sialyltransferase. Bladder cancer tissue presenting elevated expression of ST6GALNAC1 showed a correlation with increased expression of CD1a, a marker for bladder immature DCs and showed concomitant low levels of Th1-inducing cytokines IL-12 and TNF-α. In vitro, human DCs co-incubated with STn+ bladder cancer cells, had an immature phenotype (MHC-IIlow, CD80low and CD86low) and were unresponsive to further maturation stimuli. When contacting with STn+ cancer cells, DCs expressed significantly less IL-12 and TNF-α. Consistent with a tolerogenic DC profile, T cells that were primed by DCs pulsed with antigens derived from STn+ cancer cells were not activated and showed a FoxP3high IFN-γlow phenotype. Blockade of STn antigens and of STn+ glycoprotein, CD44 and MUC1, in STn+ cancer cells was able to lower the induction of tolerance and DCs become more mature. Overall, our data suggest that STn-expressing cancer cells impair DC maturation and endow DCs with a tolerogenic function, limiting their capacity to trigger protective anti-tumour T cell responses. STn antigens and, in particular, STn+ glycoproteins are potential targets for circumventing tumour-induced tolerogenic mechanisms.

Personality, Brain Asymmetry, and Neuroendocrine Reactivity in Two Immune-Mediated Disorders: a Preliminary Report

Development of some immune-mediated disorders may depend on dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. To explore neuropsychologic mechanisms in relation to the abnormal endocrine reactivity in patients with systemic lupus erythematosus (SLE) and chronic hepatitis C (CHC) we used the corticotropin releasing hormone (CRH) test, the Minnesota Multiphasic Personality Inventory (MMPI), and the Edinburgh Inventory of Manual Preference Inventory (EIMP). Compared to controls, the adrenocorticotrophic hormone (ACTH) response to CRH was reduced in CHC, while SLE presented reduced baseline dehydroepiandrosterone sulfate levels; higher neurotic scores were found in SLE and higher behavior deviant scores in CHC. Peak ACTH levels were a significant factor for the MMPI profile variability, while the manual preference score was a significant factor for the ACTH response. Personality and manual preference contribute to neuroendocrine abnormalities. Different behavioral and neuroimmunoendocrine models emerge for these disorders.

IWP strategy: TechFrame

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics

Internationalization strategy: HOLOS Mobile - Angola

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics

Internationalization strategy of Shoes Closet to the United Kingdom

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics

Internationalization strategy of ABotoa/Skypro shoes to United States of America

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics

Leber Congenital Amaurosis: Comprehensive Survey of the Genetic Heterogeneity, Refinement of the Clinical Definition, and Genotype-Phenotype Correlations as a Strategy for Molecular Diagnosis

Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies, responsible for congenital blindness. Disease-associated mutations have been hitherto reported in seven genes. These genes are all expressed preferentially in the photoreceptor cells or the retinal pigment epithelium but they are involved in strikingly different physiologic pathways resulting in an unforeseeable physiopathologic variety. This wide genetic and physiologic heterogeneity that could largely increase in the coming years, hinders the molecular diagnosis in LCA patients. The genotyping is, however, required to establish genetically defined subgroups of patients ready for therapy. Here, we report a comprehensive mutational analysis of the all known genes in 179 unrelated LCA patients, including 52 familial and 127 sporadic (27/127 consanguineous) cases. Mutations were identified in 47.5% patients. GUCY2D appeared to account for most LCA cases of our series (21.2%), followed by CRB1 (10%), RPE65 (6.1%), RPGRIP1 (4.5%), AIPL1 (3.4%), TULP1 (1.7%), and CRX (0.6%). The clinical history of all patients with mutations was carefully revisited to search for phenotype variations. Sound genotype-phenotype correlations were found that allowed us to divide patients into two main groups. The first one includes patients whose symptoms fit the traditional definition of LCA, i.e., congenital or very early cone-rod dystrophy, while the second group gathers patients affected with severe yet progressive rod-cone dystrophy. Besides, objective ophthalmologic data allowed us to subdivide each group into two subtypes. Based on these findings, we have drawn decisional flowcharts directing the molecular analysis of LCA genes in a given case. These flowcharts will hopefully lighten the heavy task of genotyping new patients but only if one has access to the most precise clinical history since birth.

A communication strategy for Continente: proposals to promote fresh products of Continente’s own brands

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics

Communication strategy for Continente online store: improving the Ecommerce experience

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics

Internationalization strategy of Aveleda’s Wine in the Chinese market

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics