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BACKGROUND: Neurofibromatosis type 1 (NF1) is a multisystem genetic disorder, predisposing development of benign and malignant tumours. Given the oncogenic potential, long-term surveillance is important in patients with NF1. Proposals for NF1 care and its specific manifestations have been developed, but lack integration within routine care. This guideline aims to assimilate available information on NF1 associated tumours (based on evidence and/or expert opinion) to assist healthcare professionals in undertaking tumour surveillance of NF1 individuals. METHODS: By comprehensive literature review, performed March 18th 2020, guidelines were developed by a NF1 expert group and patient representatives, conversant with clinical care of the wide NF1 disease spectrum. We used a modified Delphi procedure to overcome issues of variability in recommendations for specific (national) health care settings, and to deal with recommendations based on indirect (scarce) evidence. FINDINGS: We defined proposals for personalised and targeted tumour management in NF1, ensuring appropriate care for those in need, whilst reducing unnecessary intervention. We also incorporated the tumour-related psychosocial and quality of life impact of NF1. INTERPRETATION: The guideline reflects the current care for NF1 in Europe. They are not meant to be prescriptive and may be adjusted to local available resources at the treating centre, both within and outside EU countries. FUNDING: This guideline has been supported by the European Reference Network on Genetic Tumour Risk Syndromes (ERN GENTURIS). ERN GENTURIS is funded by the European Union. DGE is supported by the Manchester NIHRBiomedical Research Centre (IS-BRC-1215-20007). ispartof: ECLINICALMEDICINE vol:56 ispartof: location:England status: published

A large odontometric reference database is essential for a clear and reliable differentiation between the teeth of dogs and wolves found in archaeological contexts. However, the data compilation of skeletal material is a slow process because access to dog remains may be difficult. The expansion of a dental database could benefit from computed tomography (CT) scans performed on live dogs during medical appointments and archived in veterinary clinics. To enable it, however, the reliability and accuracy of digital measurements must first be validated. This is the main objective of this paper. We tested the accuracy of dental measurements obtained from CT scans of deceased modern dogs and one Mesolithic dog. CT measurements were obtained using multi-planar reconstruction (MPR) (bi-dimensional images) and three-dimensional volume rendering (3D VR), either using bone or soft tissue windows. Then, measurements were compared with data obtained with a conventional caliper (Fischer Darex®, France). The sample comprised 25 maxillary fourth premolars and 17 maxillary canines. Measurements included the mesiodistal length (MDL) and the vestibular palatine width (VPW). Intra- and inter-observer variations were assessed by calculating the technical error of measurement (TEM). This approach was also used to assess the level of agreement between both techniques, complemented by a Bland-Altman analysis. Intra-observer (TEM = 0.52% to 5.98%) and inter-observer variations (TEM = 3.15% to 7.14%) were relatively small for most standard measurements, windows, and CT approaches. In the case of the MPR approach, the soft tissue window provided less observer variation on the fourth premolar; the same occurred for the bone window regarding the canine. The soft tissue window generally provided less observer variation with the VR approach. CT and caliper agreement ranged from 2.89% to 7.31% (MPR), and from 2.92% to 9.42% (3D VR). At least 91% of the CT measurements were within the Bland-Altman 95% confidence interval. We were able to carry out a thorough odontometric study of the Muge dog skull – a specimen dated to the Mesolithic period, using the MPR approach (with both bone and soft tissue windows). Both the MPR and VR CT scan approaches were validated, thus confirming digital data as an alternative way to generate more comprehensive odontometric references.

Studies undertaken several years ago indicated little change in cattle size in Portugal before the fifteenth century AD, indeed even Roman cattle were not very different from preceding Iron Age ones. The Roman province of Lusitania com- prised the southwestern part of the Iberian Peninsula (present-day central and southern Portugal and Spanish Extremad- ura). Here we present some evidence of cattle size increase in the newly founded Lusitanian Roman cities, that is, those with no previous occupation. These cities include, for example, Emerita Augusta (Mérida, Spain) and ancient Ammaia (near Marvão, Portugal). We compare their measurements with those from smaller Roman towns and sites from other periods. Our hypothesis is that the size increase reflects better nourishment or the presence of different cattle morpho- types (by selection or introduction of new animals). Osteometric analyses suggest that cattle from the main Roman cities were generally bigger in Roman newly founded cities, contrary to sites with pre-Roman occupation. Innovations could have been implemented via the import of new stock and/or new breeding strategies.

Domesticated dogs have been present in the Iberian Peninsula long before other domesticated species, back to the late Palaeolithic period. Their origin is still uncertain, but dogs were already well established during the Chalcolithic period (ca. 5000–4000 BP). This study employed a multidisciplinary approach comprising osteometric, radiographic and palaeogenomic analyses to characterize Chalcolithic Iberian Canis remains. Two Chalcolithic archaeological sites – Leceia, Oeiras, in Portugal, and El Casetón de la Era, Villalba de los Alcores, Valladolid, in Spain – were the main focus of this study. Osteometric and odontometric data from eleven other sites in Iberia were also included. Osteometric results show signs of phenotypic variability, likely the result of human-driven selective pressure. Dental radiographic and dental wear analyses allowed age at death estimation for four individuals (two juvenile and two adults). Three Chalcolithic Iberian dogs had their mitogenomes resequenced and the mitochondrial DNA analysis allowed to assign each individual to two of the major known haplogroups – A and C. Molecular sex infered by the chromosomeX/chromosome1 coverage ratio allowed to identify one female and two males. This study unveils some aspects of the Iberian Chalcolithic dogs: these dogs already exhibited various morphotypes whose profiles might be associated to the performance of certain tasks, as well as mitogenomes of two distinct lineages that help tracking the evolutionary paths of Iberian dogs., The research leading to these results has received funding from FEDER Funds through the Operational Competitiveness Factors Program “COMPETE”, and by National Funds through the Foundation for Science and Technology (FCT Grant POCI-01-0145-FEDER-029545 – PTDC/HAR-ARQ/29545/2017), by the project UIDB/CVT/00772/2020 and associated laboratory AL4AnimalS, as well as from individual grants and contracts: DL57/2016/CP1440/CT0029 (A.E. Pires), DL57/2016/CP1375/CT0003 (I.R. Amorim), 2020.02754.CEECIND (C. Ginja), CEECINS/00127/2018 (J. Requicha), DL 57/2016/CP1443/CT0029 (C. Detry), 2020.06605.BD (A.B. Santos) and 2020.05503.BD (C. Delicado). A considerable amount of the osteometric data included in this research were obtained during the development of the Master thesis and PhD of A. Daza-Perea at Universidad Autónoma de Madrid, thanks to the granting of materials and expertise from C. Liesau, P. Ríos, Argea Consultores S.L., and Trébede, Patrimonio y Cultura S.L. We must thank the support of the FPI PhD BES-2012-056461 grant, which was associated with the HAR2011-28731 Project, both financed by the Spanish Ministry of Economy and Competitiveness.

Inheritable traits of cattle were modified in various ways at different times by diverse cultures. A large collection of seventeenth century cattle remains excavated from 71 silos in the Largo do Coreto in Carnide, Lisbon, included 47 com- plete and 44 distal ends of metacarpals. These provide an opportunity to make a detailed osteometric and archaeogenetics study. While morphological changes such as size increase, as detected in bone measurements, indicate improvement for larger animals, ancient DNA is useful for studying evolutionary trajectories and modes of improvement of domestic animals. High-throughput sequencing can show evolutionary processes at unprecedented resolution. We generated whole-genome shotgun resequencing data for 6 Carnide specimens on the Illumina HiSeq X Ten platform. Mean en- dogenous DNA content for these samples varied between 1% (CAR008) and 4% (CAR014), and their mitogenomes were sequenced to between 3.56x and 33.44x mean depth of coverage, respectively. The Carnide cattle belong to the Europe- an-T3 (CAR002 and CAR021) and African-T1 haplogroups (CAR008, CAR009, CAR014, and CAR016). These haplogroups are also present in extant native breeds from the Iberian Peninsula, corroborating the hypothesis of a genetic continuity of maternal lineages over time. The admixture of African taurine cattle observed in these specimens from the Modern period supports the idea that current genetic diversity of Iberian cattle derives from the dispersal of a heterogeneous population since early times along the Mediterranean basin. In addition, the biological sex of the Carnide specimens was consistently determined by our genomic analysis and osteometric measurements. Further analyses of whole-genome nuclear data are needed to infer specific biological properties, for example, the coat color of these animals, as well as the variability of genes linked to body size, meat, and milk production traits.

The high levels of degradation characteristic of ancient DNA molecules severely hinder the recovery of endogenous DNA fragments and the discovery of genetic variation, limiting downstream population analyses. Optimization of read mapping strategies for ancient DNA is therefore essential to maximize the information we are able to recover from archaeological specimens. In this paper we assess Burrows-Wheeler Aligner (BWA) effectiveness for mapping of ancient DNA sequence data, comparing different sets of parameters and their effect on the number of endogenous sequences mapped and variants called. We also consider different filtering options for SNP calling, which include minimum values for depth of coverage and base quality in addition to mapping quality. Considering our results, as well as those of previous studies, we conclude that BWA-MEM is a good alternative to the current standard BWA-backtrack strategy for ancient DNA studies, especially when the computational resources are limited and time is a constraint., This work received funding from: the project PORBIOTA- Portuguese E-Infrastructure for Information and Research on Biodiversity (POCI-01-0145-FEDER-022127), supported by Operational Thematic Program for Competitiveness and Internationalization (POCI), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER); Fundação Nacional para a Ciência e a Tecnologia (FCT), Portugal, contract grant 2020.02754.CEECIND (C.G.), Norma Transitória contract grant DL 57/2016/CP1440/CT0029 (A.E.P.) and the ARCHAIC Project grant PTDC/CVTLIV/2827/2014 co-funded by COMPETE 2020 POCI-01-0145-FEDER-016647 and LISBOA-01-0145-FEDER-016647 (C.G.). This work was also supported by National Funds through FCT/MCTES under the UIDB/50027/2020 funding.

Highlights • Dental pathology of wild Iberian wolf was studied in 61 complete skulls and 4 mandibles from a museum collection. • Museum collections can provide crucial information about wild elusive species. • The most frequent observed abnormalities were tooth wear, periodontitis and tooth fractures. • Dental radiography is of a great importance for the diagnose and classification of the dental and periodontal conditions. • Dental radiography can be useful for the age estimation of archaeological Canis., For some wild canids, such as the Iberian wolf, there is a lack of in-depth knowledge about dental pathology. We aimed to evaluate it, in a standardized manner, in specimens from a Portuguese museum collection. Sixty-five deceased specimens of wild Iberian wolves, 61 complete skulls and 4 mandibles, collected in Portugal between 1977 and 1995, were analyzed. Sample comprised 18 females, 24 males and 23 individuals of undetermined sex. Teeth were evaluated by visual observation and dental radiography for tooth wear, periodontitis, fractures and other dental lesions. We have found several causes for teeth absence: artefactual, secondary to periodontitis and agenesia. About 30% of the teeth showed signs of wear. Only a small (

Tese de mestrado, Biologia Humana e Ambiente, Universidade de Lisboa, Faculdade de Ciências, 2020 Submitted by Teresa Boa (tdboa@fc.ul.pt) on 2021-05-11T15:09:21Z No. of bitstreams: 1 ulfc126260_tm_Ana_Elisabete_Lima.pdf: 859177 bytes, checksum: be77a62b26fa73dfaf41bbf38fc84c33 (MD5) Made available in DSpace on 2021-05-11T15:09:33Z (GMT). No. of bitstreams: 1 ulfc126260_tm_Ana_Elisabete_Lima.pdf: 859177 bytes, checksum: be77a62b26fa73dfaf41bbf38fc84c33 (MD5) Previous issue date: 2020

We investigated the genetic, composition of six Canis remains from western Iberia, directly radiocarbon dated to 7,903-7,570 years (cal BP). They were identified as dogs via their qrchaeological and depositional context, osteometry, and a high percentage of aquatic diet shared with humans. For comparison, genetic data were obtained from an additional 37 Iberian dog remains from the Neolithic to Late Antiquity, as well as two Palaeolithic anda a Chalcolithic Canis identified as wolves. Previous data indicated that dog mtDNA haplogroup A (HgA) is prevalent in extant European dogs (>50%), in the Near East and Asia, but rare or absent (

BackgroundIberian primitive breeds exhibit a remarkable phenotypic diversity over a very limited geographical space. While genomic data are accumulating for most commercial cattle, it is still lacking for these primitive breeds. Whole genome data is key to understand the consequences of historic breed formation and the putative role of earlier admixture events in the observed diversity patterns.ResultsWe sequenced 48 genomes belonging to eight Iberian native breeds and found that the individual breeds are genetically very distinct with FST values ranging from 4 to 16% and have levels of nucleotide diversity similar or larger than those of their European counterparts, namely Jersey and Holstein. All eight breeds display significant gene flow or admixture from African taurine cattle and include mtDNA and Y‐chromosome haplotypes from multiple origins. Furthermore, we detected a very low differentiation of chromosome X relative to autosomes within all analyzed taurine breeds, potentially reflecting male‐biased gene flow.ConclusionsOur results show that an overall complex history of admixture resulted in unexpectedly high levels of genomic diversity for breeds with seemingly limited geographic ranges that are distantly located from the main domestication center for taurine cattle in the Near East. This is likely to result from a combination of trading traditions and breeding practices in Mediterranean countries. We also found that the levels of differentiation of autosomes vs sex chromosomes across all studied taurine and indicine breeds are likely to have been affected by widespread breeding practices associated with male-biased gene flow.

Multifunctional liposomes containing manganese ferrite/gold core/shell nanoparticles were developed. These magnetic/plasmonic nanoparticles were covered by a lipid bilayer or entrapped in liposomes, which form solid or aqueous magnetoliposomes as nanocarriers for simultaneous chemotherapy and phototherapy. The core/shell nanoparticles were characterized by UV/Visible absorption, X-Ray Diffraction (XRD), Transmission Electron Microscopy (TEM), and Superconducting Quantum Interference Device (SQUID). The magnetoliposomes were characterized by Dynamic Light Scattering (DLS) and TEM. Fluorescence-based techniques (FRET, steady-state emission, and anisotropy) investigated the incorporation of a potential anti-tumor drug (a thienopyridine derivative) in these nanosystems. The core/shell nanoparticles exhibit sizes of 25 &plusmn, 2 nm (from TEM), a plasmonic absorption band (&lambda, max = 550 nm), and keep magnetic character. XRD measurements allowed for the estimation of 13.3 nm diameter for manganese ferrite core and 11.7 nm due to the gold shell. Aqueous magnetoliposomes, with hydrodynamic diameters of 152 &plusmn, 18 nm, interact with model membranes by fusion and are able to transport the anti-tumor compound in the lipid membrane, with a high encapsulation efficiency (EE (%) = 98.4 &plusmn, 0.8). Solid magnetoliposomes exhibit hydrodynamic diameters around 140 nm and also carry successfully the anticancer drug (with EE (%) = 91.2 &plusmn, 5.2), while also being promising as agents for phototherapy. The developed multifunctional liposomes can be promising as therapeutic agents for combined chemo/phototherapy.

This study investigates the gene pool of Portuguese autochthonous dog breeds and their wild counterpart, the Iberian wolf subspecies (Canis lupus signatus), using standard molecular markers. A combination of paternal and maternal molecular markers was used to investigate the genetic composition, genetic differentiation and genetic relationship of native Portuguese dogs and the Iberian wolf. A total of 196 unrelated dogs, including breed and village dogs from Portugal, and other dogs from Spain and North Africa, and 56 Iberian wolves (wild and captive) were analyzed for nuclear markers, namely Y chromosome SNPs, Y chromosome STR loci, autosomal STR loci, and a mitochondrial fragment of the control region I. Our data reveal new variants for the molecular markers and confirm significant genetic differentiation between Iberian wolf and native domestic dogs from Portugal. Based on our sampling, no signs of recent introgression between the two subspecies were detected. Y chromosome data do not reveal genetic differentiation among the analyzed dog breeds, suggesting they share the same patrilineal origin. Moreover, the genetic distinctiveness of the Iberian wolf from other wolf populations is further confirmed with the description of new mtDNA variants for this endemism. Our research also discloses new molecular markers for wolf and dog subspecies assignment, which might become particularly relevant in the case of forensic or noninvasive genetic studies. The Iberian wolf represents a relic of the once widespread wolf population in Europe and our study reveals that it is a reservoir of unique genetic diversity of the grey wolf, Canis lupus. These results stress the need for conservation plans that will guarantee the sustainability of this threatened top predator in Iberia.

In this study, we integrate osteometric and palaeogenetic data to investigate dog variability in the Roman Empire in Iberia and North Africa. Osteometry was used to distinguish the statusddomestic or wild, of approximately 2000 years old Canis remains and to understand to what extent teeth and long bones varied in dogs in the Roman provinces of Mauretania Tingitana, Lusitania and Tarraconensis. Highthroughput 454-DNA sequencing technology was used to obtain mitochondrial DNA (mtDNA) sequences from 15 bone and teeth samples.We identified five dog haplotypes from partial sequences of the hypervariable D-loop region. MtDNA haplotypes were grouped into two of the four major clades found in present-day dogs. We detected three clade A haplotypes in 12 samples from Portugal, Spain and Morocco, and a single clade D haplotype in 3 samples from Spain. So far, this is the oldest evidence for the presence of dog clade D in Iberia. It is dated to the late Roman occupation in the 4th-5th cent. AD (ca. 1,600 years ago). Our results confirm the existence of distinct dog morphotypes in Roman times that also harboured distinct genetic lineages. According to our data, dogs from distinct mtDNA lineages (clades A and D) have been continuously bred in the Iberian Peninsula since at least 1600 years ago. Moreover, the sharing of matrilines between dogs from Spain and North Africa may indicate gene flow. Dogs could have been easily transported between these regions by humans along maritime and terrestrial trade routes. These results provide new insights into pre-Roman and Roman domestication practices, confirming selection practices were extensively applied to dogs during the first centuries of our era in the Iberian Peninsula. We show that the greater size variability of teeth length (and consequently cranium) and long bone breadths (and consequently phenotype) of Roman dogs in the Iberian Peninsula, is concomitant with the detection of diverse and rare maternal lineages. This would reflect an intensification of dog breeding and the use of non-local dogs for breeding., This study was funded by the FCT (Fundação para a Ciência e a Tecnologia) within the research PTDC/HIS-ARQ/100225/2008; and AE Pires grant SFRH/BPD/112653/2015; C Detry grant SFRH/BPD/108326/2015 and C Ginja contract grant IF/00866/2014.

The study of bioarchaeological evidence associated with burials is essential for achieving a global perspective on cremation as a funerary practice, its chronological and geographical distribution, as well as its inner socio-cultural and technological diversity. However, for that purpose, similar and consistent analyses must be adopted by bioarchaeologists to enable intra- and inter-sites comparisons. The 1995–2015 literature encompassing 84 geographically representative articles concerning bioarchaeological studies of burned human skeletal remains is reviewed herein. The objective was to assess methodological variability. Information concerning colour, fragmentation, skeleton completeness, ‘skeletal region’ representation, non-human funerary assemblage, pre-burning condition of the remains, minimum number of individuals, biological profile, trauma and pathologies was considered. The results demonstrate that certain methods were used by almost all researchers. That was the case for colour description (91 %), skeleton completeness (91 %), minimum number of individuals (96 %), age-at-death (100 %) and sex of the individuals (95 %). Researchers are much more divided about the implementation of the remaining methods. Methodological choices also vary. The asymmetries in the selection of the analyses that are undertaken can lead to different interpretations and conclusions of the contexts under study. This may prevent consistent comparisons within the same site and between different sites. We emphasize the need for bioarchaeologists to discuss and standardize analytical procedures for studying cremated remains.

Purpose Intravenous IgG (ivIg) is a therapeutic alternative for lupus erythematosus, the mechanism of which remains to be fully understood. Here we investigated whether ivIg affects two established sub-phenotypes of SLE, namely relative oligoclonality of circulating T-cells and reduced activity of CD4+Foxp3+ regulatory T-cells (Tregs) reflected by lower CD25 surface density. Methods We conducted a longitudinal study of 15 lupus patients (14 with SLE and one with discoid LE) treated with ivIg in cycles of 2–6 consecutive monthly infusions. Among these 15 patients, 10 responded to ivIg therapy with clear clinical improvement. We characterized Tregs and determined TCR spectratypes of four Vβ families with reported oligoclonality. Cell counts, cytometry and TCR spectratypes were obtained from peripheral blood at various time points before, during and after ivIg treatment. T-cell oligoclonality was assessed as Vβ-familywise repertoire perturbation, calculated for each patient in respect to an individual reference profile averaged over all available time points. Results For 11 out of 15 patients, average Vβ1/Vβ2/Vβ11/ Vβ14 repertoires were less perturbed under than outside ivIg therapy. The four exceptions with relatively increased average perturbation during ivIg therapy included three patients who failed to respond clinically to an ivIg therapy cycle. Patients' Treg CD25 surface density (cytometric MFI) was clearly reducedwhencomparedtohealthycontrols,but not obviously influenced by ivIg. However, patients' average Treg CD25 MFI was found negatively correlated with both Vβ11 and Vβ14 perturbations measured under ivIg therapy. Conclusions This indicates a role of active Tregs in the therapeutic effect of ivIg.

[corrected] After autologous stem cell transplantation (ASCT) the immunological B cell compartment recovers slowly. Delays on the recovery of B cell function after autologous stem cell transplantation are due to the low lymphocytes count and to their intrinsic dysfunction.We studied the in vivo B cell reconstitution after ASCT examining the independent effect of polyclonal IgG (PolyIg), Fab or Fc fragments infusions in a murine animal model during a period of 12 weeks. These molecules were used in low doses, mimicking the recommended use of IVIg in the case of hypogammaglobulinemia in humans. Flow cytometry analysis and ELISA tests were conducted to monitor the reconstitution of B cells and serum immunoglobulin production. Panama blot and PCA factor 1 analysis were used to study the kinetics of immunoglobulin repertoires reconstitution. Mechanistic studies were also performed using in vitro cell culture.During follow-up after ASCT, peripheral B cells expand independently of treatment, correcting the immediate increase in sBAFF (soluble B cell activating factor) induced by previous intense myeloablation. Treatments with Fab and Fc fragments infusions promote significant IgM and IgG production comparing to control. Although the complete recovery of antibody repertoire is only achieved at the end of follow-up after ASCT, there is an earlier and significantly stronger recovery in the treated mice, which is evident at 9 weeks after ASCT. At 30 weeks after ASCT, normal values of antibody repertoire were detected in all individuals. Mechanistic studies show that Fab and Fc fragments promote IgG1 production by indirect pathways.The results presented here demonstrate that polyclonal immunoglobulin indirectly improves the function of the reconstituted B cells and their IgG production by means of Fc-mediated effects on bystander cells. These results further stimulate the discussion about the advantages of IVIg therapy during immune reconstitution after human ASCT.

Genetic variability in purebred dogs is known to be highly structured, with differences among breeds accounting for approximately 30% of the genetic variation. However, analysis of the genetic structure in non-cosmopolitan breeds and local populations is still limited. Nine Portuguese native dog breeds, and other peripheral dog populations (five) with regional affinities, were characterized using 16 microsatellites and 225 amplified fragment length polymorphism (AFLP) markers, and the pattern of genetic differentiation was investigated. Although the level of breed differentiation detected is below that of other dog breeds, there is in most cases a correlation between breed affiliation and molecular structure. AFLP markers and Bayesian clustering methods allowed an average of 73.1% of individuals to be correctly assigned to source populations, providing robust genotypic assessment of breed affiliation. A geographical genetic structure was also detected, which suggests a limited influence of African dogs on the Iberian breeds. The sampling effect on the estimation of population structure was evaluated and there was a 2.2% decrease in genetic differentiation among breeds when working animals were included. Genetic diversity of stray dogs was also assessed and there is no evidence that they pose a threat to the preservation of the gene pool of native dog breeds.

In an extensive survey of the genetic diversity in Portuguese dogs, we have examined an 887-bp fragment of the mitochondrial DNA (mtDNA) from 8 Portuguese, 1 Spanish, and 2 North African native dog breeds, including village dogs from Portugal and Tunisia. Forty-nine haplotypes were found in the 164 individuals analyzed, with private haplotypes being found in several breeds. For example, the Castro Laboreiro Watchdog, a rare breed from a small and isolated region in Portugal, was monomorphic for mtDNA and possessed a new haplotype, which may be provisionally considered a breed-specific marker. Phylogenetic analyses recapitulated 4 major clades identified in other studies, but new haplotypes, grouping within a clade that was previously thought as geographically restricted, were detected in Portugal and Morocco. Portuguese village dogs showed no genetic differentiation from nonnative dogs or from local breeds of the areas in which the village dogs were sampled. Although Iberian and North African dog breeds possessed breed-specific mtDNA haplotypes, no significant geographic structure could be detected among them. There is no evidence for introgression of North African haplotypes in Iberian dogs, contrary to previous results for other domestic animals.

The Iberian lynx is the most threatened felidin the world and has suffered a declinethroughout its range. Effective monitoring ofthe species' presence is essential. Fieldwork inpreviously identified areas of lynx occurrencein Portugal has resulted in the collection of104 possible lynx scats. Recently, there hasbeen little or no evidence of lynx presence andscats could be confused with others from moreabundant carnivores such as wildcat, fox anddog. In order to confirm or not exclude thepresence of the species, identification ofscats was performed through the amplificationof lynx-specific mitochondrial DNA sequences.Two samples collected in Malcata NaturalReserve in 1997 were identified as lynx. Thisis the most recent and reliable proof of lynxpresence in Portugal*. Given the territorialbehavior of lynx, stable resident populationswould have produced a higher proportion ofpositively identified scats. Local extinctionsmight have taken place, and this genetic datasupports a suspected national pre-extinctionscenario for the species. Genetic analysisusing a non-invasive approach has proved to bean informative part of the lynx monitoringprogram. Technical problems faced and overcomeare also presented.

In the course of a zooarchaeological survey of Holocene sites in southern Portugal, a substantial size increase of cattle bones was noted following the Christian reconquista of the 11the13th centuries AD. A size increase in the course of time within a lineage of domestic livestock is usually considered to represent animal improvement. However several other factors including sex may influence the average size of a sample of mammal bones e cattle exhibit considerable sexual size dimorphism, with bulls being larger than cows. A histogram of the distal widths of a large (n ¼ 44) sample of cattle metacarpals from 15th century Beja (Alentejo, Portugal), revealed a bimodal distribution. It was assumed that the large measurements belonged to males and the small to females. In order to rule out the possibility of a post- Moslem change in the sex ratio of cattle, a sub-sample of 21 cattle metacarpals from Beja was selected and we used genetic markers to identify the sex of the animals to which these metacarpals belonged. The ancient DNA sex of all specimens agreed with the previously assumed sex as determined osteometrically. We conclude that the two nearly separated peaks for the metacarpal distal width measurements do indeed indicate sex. A similar bimodal distribution was obtained from another large but earlier sample of cattle metacarpals from Moslem Alcáçova de Santarém (9the12th century AD). Although these have not been molecularly sexed and since osteometric sexing has now been validated, we conclude that both small (female) and large (male) peaks are smaller than the 15th century ones and that there was an overall size increase or improvement of cattle in this region. Why the Christians improved cattle is unclear, but a selection for larger beeves for meat is one possibility as is the selection of more robust cattle for power. The spread of the quadrangular or chariot plough in Iberia is known to have occurred at this time. We then use the genetically sexed metacarpals to determine which measurements provide reasonable distinction between the sexes. Both the distal width (BFd; as already noted by Svensson et al., 2008; in Swedish medieval cattle) and the width of the lateral condyle (WCL) offer the best distinction. We also used them as a reference ‘collection’ to sex the medieval and post-medieval cattle metacarpals from Launceston Castle in England. This re-visit of the Launceston data corroborates other evidence indicating increased specialisation (milk and veal) in post-medieval cattle husbandry in England.

Intravenous IgG (ivIg) is a therapeutic alternative for lupus erythematosus. Relative oligoclonality of circulating T-cells in SLE has been reported. Also CD4+Foxp3+ regulatory T-cells (Tregs) have a characteristically reduced activity in SLE, reflected by CD25 surface density. Aiming to study the role of Tregs for ivIg therapy, we characterized Tregs and determined TCR spectratypes of four Vb families with reported oligoclonality, in 15 lupus patients (14 with SLE and one with discoid LE) treated with ivIg in cycles of 2-6 consecutive monthly infusions. Among these 15 patients, 11 responded with clinical improvement. Cell counts, cytometry and TCR spectratypes were obtained from peripheral blood at various time points before, during and after ivIg treatment. T-cell oligoclonality was assessed as Vb-familywise repertoire perturbation, calculated for each patient in respect to an individual reference profile averaged over all available time points. For 11/15 patients, average Vb1/Vb2/Vb11/Vb14 repertoires were less perturbed under ivIg treatment than outside ivIg therapy. The four exceptions with relatively increased average perturbation during ivIg therapy included three patients who failed to respond clinically to an ivIg therapy cycle. Patients' Treg CD25 surface density (cytometric MFI) was, other than Treg/CD4+ frequency, clearly reduced when compared to healthy controls, but not obviously influenced by ivIg. However, patients' average Treg CD25 MFI was found negatively correlated with both Vb11 and Vb14 perturbations measured under ivIg therapy, which indicates a role of active Tregs in the therapeutic effect of ivIg.

A visit to the past through ancient DNA analysis; Uma visita ao passado através da análise de DNA antigo; Ana Elisabete Pires Molecular and serological evaluation of vector-borne diseases infection in dogs with non-Hodgkin lymphoma; Análise molecular e sorológica da infecção por doenças transmitidas por vetores em cães com linfoma não-Hodgkin; Henriques, J.; Almeida B.; Ramos, J.; Pereira V.; Santos, A.S.; Sousa, R.; Heeney, J.; Dobson, J. & Alves, M. Time-lapse imaging in the IVF laboratory: clinical applications through understanding embryo development; Imagem de "time-lapse" no laboratório FIV: aplicações clínicas através do desenvolvimento embrionário; Ana Sousa Lopes Ionic liquids synthesis and their application in drug delivery systems; Síntese de líquidos iónicos e sua aplicação em sistemas de veiculação; Tânia Almeida

Summary: Natural antibodies are unique self molecules endowed with both suppressive and activating functions on various cells of the immune system and are recognized as a fundamental link between the adaptive and innate immune system. Here, we examine the role of natural antibodies, using polyclonal immunoglobulins (Ig), as a promoter of T-cell reconstitution in a context of lymphopenia. We have established a mouse model to mimic immunologic recovery in adult patients with severe hypothymic function subjected to autologous hematopoietic precursor cell transplantation. Thymectomized mice were transplanted and treated with low doses of Ig or its Fab or Fc fragments. The animals displayed, during early phases of Ig treatment, a significant increase of T-cell reconstitution displaying a naive CD4 + phenotype. In addition, the Igtreated animals exhibited an increase dilution of single-joint T-cell receptor excision circles (sjTRECs) in peripheral blood, suggesting an early increase in proliferation of T cells stimulated by the natural antibodies. These results unveil a novel and considerable effect of intravenous Ig treatment in situations of severe lymphopenia as a stimulator of proliferation of peripheral naive T cells, possibly protecting diverse immune repertoires.

O presente artigo consiste numa breve resenha da aplicabilidade dos testes moleculares ao serviço da Clínica Veterinária. Os testes moleculares actualmente disponíveis são muito abrangentes, permitindo por exemplo a identificação de agentes patogénicos causadores de doença ao nível da espécie, o diagnóstico de doenças genéticas e até a identificação molecular do sexo em espécies de aves que não apresentam dimorfismo sexual. O recurso a estes testes durante a prática clínica pode acelerar o diagnóstico, permitir uma prescrição médica mais específica e ser de apoio à criação animal. São, ainda, apresentados os testes moleculares que presentemente estão disponíveis no Laboratório de Análises Clínicas e Histopatologia da Universidade Lusófona de Humanidades e Tecnologias