Characterization of NKR+ T-cell subsets in human bone marrow:implications for immunosurveillance of neoplasia

In addition to hematopoietic progenitors, human bone marrow contains mature T/NK lymphocytes. Valpha24Vbeta11 NKT-cells, a subset of NK receptor+ (NKR+) T-cells in humans, are rare in bone marrow, suggesting the presence of other NKR+ T-cells which may contribute to tumor surveillance. NKR+/- T-cells were examined in blood (PB), and bone marrow from donors (DM) and patients with active hematopoietic malignancy (PM), or in remission (PR). T-cells in PR & PM were enriched for CD56+ and CD57+ subsets, compared to DM. All marrow NKR+/- T-cell subsets were more activated than PB. PM and, surprisingly, PR marrow contained more activated cells than DM. CD8+ cells were significantly increased in all patient marrows and there was evidence of the formation of an effector/memory pool in malignant marrow. These data suggest that NKR+ T-cell enrichment in human bone marrow that has been exposed to neoplastic transformation is compatible with a role in localized tumor surveillance/eradication.

Polymerase chain reaction based methods for assessing chimerism following allogeneic bone marrow transplantation

It is important to be able to assess the contribution of donor cells to the graft followmg bone marrow transplantation (BMT), as complete engraftment of marrow progenitors that can give rise to long term donor derived hemopoiesis may be important in long-term disease-free survival. The contribution of the donor marrow, both in terms of filling the marrow "space" created by the intense conditioning regimen and in its ability to mediate a graft versus leukemia effect may be assessed by studying the kinetics of the engraftment process. As BMT involves repopulation of the host hemopoietic system with donor cells, recipients of allogeneic marrow are referred to as hemopoietic chimeras. A donor chimera is an individual who exhibits complete donor hemopoiesis and we would imagine that donor chimertsm carries the best long-term prognosis. A patient who has both donor and recipient cells coexistmg in a stable fashion post-BMT without hematological evidence of relapse or graft rejection is referred to as a mixed chimera. Mixed chimerism may be a prelude to graft rejection or leukemic relapse; therefore, it is important to be able to monitor the presence of these cells in a precise manner.

5.9-keV Mn K-shell X-ray luminosity from the decay of 55Fe in Type Ia supernova models

We show that the X-ray line flux of the Mn Kα line at 5.9 keV from the decay of 55Fe is a promising diagnostic to distinguish between Type Ia supernova (SN Ia) explosion models. Using radiation transport calculations, we compute the line flux for two three-dimensional explosion models: a near-Chandrasekhar mass delayed detonation and a violent merger of two (1.1 and 0.9 M⊙) white dwarfs. Both models are based on solar metallicity zero-age main-sequence progenitors. Due to explosive nuclear burning at higher density, the delayed-detonation model synthesizes ˜3.5 times more radioactive 55Fe than the merger model. As a result, we find that the peak Mn Kα line flux of the delayed-detonation model exceeds that of the merger model by a factor of ˜4.5. Since in both models the 5.9-keV X-ray flux peaks five to six years after the explosion, a single measurement of the X-ray line emission at this time can place a constraint on the explosion physics that is complementary to those derived from earlier phase optical spectra or light curves. We perform detector simulations of current and future X-ray telescopes to investigate the possibilities of detecting the X-ray line at 5.9 keV. Of the currently existing telescopes, XMM-Newton/pn is the best instrument for close (≲1-2 Mpc), non-background limited SNe Ia because of its large effective area. Due to its low instrumental background, Chandra/ACIS is currently the best choice for SNe Ia at distances above ˜2 Mpc. For the delayed-detonation scenario, a line detection is feasible with Chandra up to ˜3 Mpc for an exposure time of 106 s. We find that it should be possible with currently existing X-ray instruments (with exposure times ≲5 × 105 s) to detect both of our models at sufficiently high S/N to distinguish between them for hypothetical events within the Local Group. The prospects for detection will be better with future missions. For example, the proposed Athena/X-IFU instrument could detect our delayed-detonation model out to a distance of ˜5 Mpc. This would make it possible to study future events occurring during its operational life at distances comparable to those of the recent supernovae SN 2011fe (˜6.4 Mpc) and SN 2014J (˜3.5 Mpc).

Locations of peculiar supernovae as a diagnostic of their origins

We put constraints on the properties of the progenitors of peculiar calcium-rich transients using the distribution of locations within their host galaxies. We confirm that this class of transients do not follow the galaxy stellar mass profile and are more likely to be found in remote locations of their apparent hosts. We test the hypothesis that these transients are from low-metallicity progenitors by comparing their spatial distributions with the predictions of self-consistent cosmological simulations that include star formation and chemical enrichment. We find that while metal-poor stars and our transient sample show a consistent preference for large offsets, metallicity alone cannot explain the extreme cases. Invoking a lower age limit on the progenitor helps to improve the match, indicating these events may result from a very old metal-poor population. We also investigate the radial distribution of globular cluster systems, and show that they too are consistent with the class of calcium-rich transients. Because photometric upper limits exist for globular clusters for some members of the class, a production mechanism related to the dense environment of globular clusters is not favoured for the calcium-rich events. However, the methods developed in this paper may be used in the future to constrain the effects of low metallicity on radially distant core-collapse events or help establish a correlation with globular clusters for other classes of peculiar explosions.

A comparative study of Type II-P and II-L supernova rise times as exemplified by the case of LSQ13cuw

We report on our findings based on the analysis of observations of the Type II-L supernova LSQ13cuw within the framework of currently accepted physical predictions of core-collapse supernova explosions. LSQ13cuw was discovered within a day of explosion, hitherto unprecedented for Type II-L supernovae. This motivated a comparative study of Type II-P and II-L supernovae with relatively well-constrained explosion epochs and rise times to maximum (optical) light. From our sample of twenty such events, we find evidence of a positive correlation between the duration of the rise and the peak brightness. On average, SNe II-L tend to have brighter peak magnitudes and longer rise times than SNe II-P. However, this difference is clearest only at the extreme ends of the rise time versus peak brightness relation. Using two different analytical models, we performed a parameter study to investigate the physical parameters that control the rise time behaviour. In general, the models qualitatively reproduce aspects of the observed trends. We find that the brightness of the optical peak increases for larger progenitor radii and explosion energies, and decreases for larger masses. The dependence of the rise time on mass and explosion energy is smaller than the dependence on the progenitor radius. We find no evidence that the progenitors of SNe II-L have significantly smaller radii than those of SNe II-P.

Beneficial Effects of Berberine on Oxidized LDL-induced Cytotoxicity in Human Retinal Müller Cells

PURPOSE. Limited mechanistic understanding of diabetic retinopathy (DR) has hindered therapeutic advances. Berberine, an isoquinolone alkaloid, has shown favorable effects on glucose and lipid metabolism in animal and human studies, but effects on DR are unknown. We previously demonstrated intraretinal extravasation and modification of LDL in human diabetes, and toxicity of modified LDL to human retinal M¨uller cells. We now explore pathogenic effects of modified LDL on M¨uller cells, and the efficacy of berberine in mitigating this cytotoxicity. METHODS. Confluent human M¨uller cells were exposed to in vitro–modified ‘highly oxidized, glycated (HOG-) LDL versus native-LDL (N-LDL; 200 mg protein/L) for 6 or 24 hours, with/ without pretreatment with berberine (5 lM, 1 hour) and/or the adenosine monophosphate (AMP)-activated protein kinase (AMPK) inhibitor, Compound C (5 lM, 1 hour). Using techniques including Western blots, reactive oxygen species (ROS) detection assay, and quantitative real-time PCR, the following outcomes were assessed: cell viability (CCK-8 assay), autophagy (LC3, Beclin-1, ATG-5), apoptosis (cleaved caspase 3, cleaved poly-ADP ribose polymerase), oxidative stress (ROS, nuclear factor erythroid 2-related factor 2, glutathione peroxidase 1, NADPH oxidase 4), angiogenesis (VEGF, pigment epithelium-derived factor), inflammation (inducible nitric oxide synthase, intercellular adhesion molecule 1, IL-6, IL-8, TNF-a), and glial cell activation (glial fibrillary acidic protein). RESULTS. Native-LDL had no effect on cultured human M¨uller cells, but HOG-LDL exhibited marked toxicity, significantly decreasing viability and inducing autophagy, apoptosis, oxidative stress, expression of angiogenic factors, inflammation, and glial cell activation. Berberine attenuated all the effects of HOG-LDL (all P < 0.05), and its effects were mitigated by AMPK inhibition (P < 0.05). CONCLUSIONS. Berberine inhibits modified LDL-induced M¨uller cell injury by activating the AMPK pathway, and merits further study as an agent for preventing and/or treating DR.

c-Met inhibition in a HOXA9/Meis1 model of CN-AML

BACKGROUND: Hematopoiesis is a paradigm for developmental processes, hierarchically organized, with stem cells at its origin. Hematopoietic stem cells (HSCs) replenish progenitor and precursor cells of multiple lineages, which normally differentiate into short-lived mature circulating cells. Hematopoiesis has provided insight into the molecular basis of tissue homeostasis and malignancy. Malignant hematopoiesis, in particular acute myeloid leukemia (AML), results from impaired development or differentiation of HSCs and progenitors. Co-overexpression of HOX and TALE genes, particularly the HOXA cluster and MEIS1, is associated with AML. Clinically relevant models of AML are required to advance drug development for an aging patient cohort.

RESULTS: Molecular analysis identified altered gene, microRNA, and protein expression in HOXA9/Meis1 leukemic bone marrow compared to normal controls. A candidate drug screen identified the c-Met inhibitor SU11274 for further analysis. Altered cell cycle status, apoptosis, differentiation, and impaired colony formation were shown for SU11274 in AML cell lines and primary leukemic bone marrow.

CONCLUSIONS: The clonal HOXA9/Meis1 AML model is amenable to drug screening analysis. The data presented indicate that human AML cells respond in a similar manner to the HOXA9/Meis1 cells, indicating pre-clinical relevance of the mouse model.

O lugar da memória na obra de jovens autores de expressão alemã

O presente trabalho propõe-se reflectir sobre o efeito transgeracional do Holocausto, responsável pela formação de traumas e sentimentos de culpa, como se testemunham tanto em estudos psicológicos como em textos literários. Trata-se de um factor nuclear da problemática identitária verificada junto da geração de indivíduos, nascidos após o final da ditadura nacionalsocialista. Embora esta geração não tenha vivido durante os anos da ditadura hitleriana, recai sobre ela uma herança histórica que, inevitavelmente, deixa marcas profundas no processo de construção e consolidação da identidade de cada um: se os filhos dos sobreviventes se debatem com o sentimento de "culpa pela sobrevivência" sentido pelos pais, aos filhos dos criminosos é imputada uma culpa moral pela barbárie perpetrada pelos progenitores. Esta reflexão assenta no estudo da obra dos escritores Jan Koneffke e Doron Rabinovici, jovens autores de expressão alemã, que representam os conceitos de trauma, identidade e memória no contexto histórico, político e social da actualidade.

Contribuição parental do linguado do Senegal, Solea senegalensis (KAUP 1858), em aquacultura

Dissertação mest., Biologia Marinha, Universidade do Algarve, 2008

Molecular genetic analysis of a cattle population to reconstitute the extinct Algarvia breed

Background: Decisions to initiate conservation programmes need to account for extant variability, diversity loss and cultural and economic aspects. Molecular markers were used to investigate if putative Algarvia animals could be identified for use as progenitors in a breeding programme to recover this nearly extinct breed. Methods: 46 individuals phenotypically representative of Algarvia cattle were genotyped for 27 microsatellite loci and compared with 11 Portuguese autochthonous and three imported breeds. Genetic distances and factorial correspondence analyses (FCA) were performed to investigate the relationship among Algarvia and related breeds. Assignment tests were done to identify representative individuals of the breed. Y chromosome and mtDNA analyses were used to further characterize Algarvia animals. Gene- and allelic-based conservation analyses were used to determine breed contributions to overall genetic diversity. Results: Genetic distance and FCA results confirmed the close relationship between Algarvia and southern Portuguese breeds. Assignment tests without breed information classified 17 Algarvia animals in this cluster with a high probability (q > 0.95). With breed information, 30 cows and three bulls were identified (q > 0.95) that could be used to reconstitute the Algarvia breed. Molecular and morphological results were concordant. These animals showed intermediate levels of genetic diversity (MNA = 6.0 ± 1.6, Rt = 5.7 ± 1.4, Ho = 0.63 ± 0.19 and He = 0.69 ± 0.10) relative to other Portuguese breeds. Evidence of inbreeding was also detected (Fis = 0.083, P < 0.001). The four Algarvia bulls had Y-haplotypes H6Y2 and H11Y2, common in Portuguese cattle. The mtDNA composition showed prevalence of T3 matrilines and presence of the African-derived T1a haplogroup. This analysis confirmed the genetic proximity of Algarvia and Garvonesa breeds (Fst = 0.028, P > 0.05). Algarvia cattle provide an intermediate contribution (CB = 6.18, CW = -0.06 and D1 = 0.50) to the overall gene diversity of Portuguese cattle. Algarvia and seven other autochthonous breeds made no contribution to the overall allelic diversity. Conclusions: Molecular analyses complemented previous morphological findings to identify 33 animals that can be considered remnants of the Algarvia breed. Results of genetic diversity and conservation analyses provide objective information to establish a management program to reconstitute the Algarvia breed.

Identification and study of novel genes expressed in the heart precursor cells

Understanding heart development on a molecular level is a requirement for uncovering the causes of congenital heart diseases. Several genes have been implicated as critical for heart development. However, the inducers of these genes as well as their targets and pathways, remain largely unknown. We have identified a promoter element of chick cCer able to drive EGFP expression in a population of cells that consistently exit from the anterior primitive streak region, from as early as stage HH3+, and that later will populate the heart. Using this promoter element as a tool allowed us to identify novel genes previously not known to potentially play a role in heart development. In order to identify and study genes expressed and involved in the correct development and differentiation of the vertebrate heart precursor cell (HPC) lineages, a differential screening using Affymetrix GeneChip® system technologies was performed. Remarkably, this screening led to the identification of more than 700 transcripts differentially expressed in the heart forming regions (HFR). Bioinformatic tools allowed us to filter the large amount of data generated from this approach and to select a few transcripts for in vivo validation. Five genes were selected for further characterization by whole mount in situ hybridization leading to the validation of their expression in the HPC. From those, Adtk1 and Ccbe1 were selected for functional analysis. Regarding to ccbe1, a more detailed WISH analysis was performed and showed that Ccbe1 is expressed specifically on the cardiac progenitors regions at HH4, more specifically in primary heart field and at later stages is present in the second heart field. Further functional analyses by knockdown and overexpression revealed an important role for Ccbe1 in early heart tube formation. Moreover, the results presented in this thesis suggested that Ccbe1 is a key gene during heart development and might be limited to multipotent and highly proliferative progenitors and downregulated upon cellular commitment into more specific cardiac phenotypes. Other of the genes identified, Adtk1 was also subjected to further functional studies. Knockdown of Adtk1 using morpholino oligonucleotides suggested that it might be necessary for the migration and fusion of the heart tube as well as for neural tube closure.

From embryonic stem cells to sensory hair cells : a programming approach

Tese de doutoramento, Ciências Biomédicas (Biologia do Desenvolvimento), Universidade de Lisboa, Faculdade de Medicina, 2014

HES1 in immunity and cancer

Hairy and enhancer of split homolog-1 (HES1) is a part of an extensive family of basic helix-loop-helix (bHLH) proteins and plays a crucial role in the control and regulation of cell cycle, proliferation, cell differentiation, survival and apoptosis in neuronal, endocrine, T-lymphocyte progenitors as well as various cancers. HES1 is a transcription factor which is regulated by the NOTCH, Hedgehog and Wnt signalling pathways. Aberrant expression of these pathways is a common feature of cancerous cells. There appears to be a fine and complicated crosstalk at the molecular level between the various signalling pathways and HES1, which contributes to its effects on the immune response and cancers such as leukaemia. Several mechanisms have been proposed, including an enhanced invasiveness and metastasis by inducing epithelial mesenchymal transition (EMT), in addition to its strict requirement for tumour cell survival. In this review, we summarize the current biology and molecular mechanisms as well as its use as a clinical target in cancer therapeutics.

Cellular polarity in aging: role of redox regulation and nutrition

Cellular polarity concerns the spatial asymmetric organization of cellular components and structures. Such organization is important not only for biological behavior at the individual cell level, but also for the 3D organization of tissues and organs in living organisms. Processes like cell migration and motility, asymmetric inheritance, and spatial organization of daughter cells in tissues are all dependent of cell polarity. Many of these processes are compromised during aging and cellular senescence. For example, permeability epithelium barriers are leakier during aging; elderly people have impaired vascular function and increased frequency of cancer, and asymmetrical inheritance is compromised in senescent cells, including stem cells. Here, we review the cellular regulation of polarity, as well as the signaling mechanisms and respective redox regulation of the pathways involved in defining cellular polarity. Emphasis will be put on the role of cytoskeleton and the AMP-activated protein kinase pathway. We also discuss how nutrients can affect polarity-dependent processes, both by direct exposure of the gastrointestinal epithelium to nutrients and by indirect effects elicited by the metabolism of nutrients, such as activation of antioxidant response and phase-II detoxification enzymes through the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2). In summary, cellular polarity emerges as a key process whose redox deregulation is hypothesized to have a central role in aging and cellular senescence.

Extension and patterning of the vertebrate body

Dissertation presented to obrain the Ph.D degree in Biology. Developmental Biology