Vaginal delivery of the recombinant HIV-1 clade-C trimeric gp140 envelope protein CN54gp140 within novel rheologically structured vehicles elicits specific immune responses

Rheologically structured vehicle (RSV) gels were developed as delivery systems for vaginal mucosal vaccination with an HIV-1 envelope glycoprotein (CN54gp140). RSVs comprised a mucoadhesive matrix forming and vaginal fluid absorbing polymer. The mucoadhesive and rheological properties of the RSVs were evaluated in vitro, and the distribution, antigenicity and release of CN54gp140 were analysed by ELISA. CN54gp140 was uniformly distributed within the RSVs and continuously released in vitro in an antigenically intact form over 24 h. Vaginal administration to rabbits induced specific serum IgG, and IgG and IgA in genital tract secretions. The RSVs are a viable delivery modality for vaginal immunization.

Primitivismo y Feminismo en el arte contemporáneo

Para la mentalidad colonialista del siglo XIX la raza negra estaba provista de una extraordinaria sexualidad y lujuria. En la metrópolis el equivalente era la mujer sexuada, la prostituta. En los primeros años del siglo XX este tándem, prostituta-colonizado, sirve a los artistas vanguardistas como elemento de confrontación con la moral imperante. Son las artistas mujeres, con una intención feminista, quienes principalmente utilizan el primitivismo asociado a la mujer en su crítica a los estereotipos. Figura pionera en ello es Hannah Höch, especialmente en su serie De un Museo Etnográfico, en el que asocia figuras femeninas y objetos de arte primitivo. En los años 70 del siglo XX, algunas artistas de la performance, como Carolee Schneemann o Ana Mendieta reivindican el cuerpo femenino con el significado de Origen y Fertilidad que tiene en las culturas primitivas. En este mismo contexto puede inscribirse la escultura Maman de Louise Bourgeois. El elemento de crítica social, que está vigente aún en nuestros días, puede verse en las obras de las Guerrilla Girls.

Intravaginal immunization using the recombinant HIV-1 clade-C trimeric envelope glycoprotein CN54gp140 formulated within lyophilized solid dosage forms

Vaccine-mediated prevention of primary HIV-1 infection at the heterosexual mucosal portal of entry may be facilitated by highly optimised formulations or drug delivery devices for intravaginal (i.vag) immunization. Previously we described hydroxyethylcellulose (HEC)-based rheologically structured gel vehicles (RSVs) for vaginal immunization of an HIV-1 vaccine candidate, a soluble recombinant trimeric HIV-1 clade-C envelope glycoprotein designated CN54gp140. Here we investigated the efficacy of lyophilized solid dosage formulations (LSDFs) for prolonging antigen stability and as i.vag delivery modalities. LSDFs were designed and developed that upon i.vag administration they would reconstitute with the imbibing of vaginal fluid to mucoadhesive, site-retentive semi-solids. Mice were immunized with lyophilized equivalents of (i) RSVs, (ii) modified versions of the RSVs more suited to lyophilization (sodium carboxymethyl cellulose (NaCMC)-based gels) and (iii) Carbopol® gel, all containing CN54gp140. NaCMC-based LSDFs provided significantly enhanced antigen stability compared to aqueous-based RSVs. Rheological analysis indicated the NaCMC-based LSDFs would offer enhanced vaginal retention in woman compared to more conventional vaginal gel formulations. All LSDFs were well tolerated in the mouse model. Following i.vag administration, all LSDFs boosted systemic CN54gp140-specific antibody responses in sub-cutaneously primed mice. Induction of CN54gp140-specific antibody responses in the female genital tract was evident. Of all the LSDFs the fastest releasing which was lyophilized Carbopol® gel elicited immune responses comparable to buffer instillation of antigen suggesting that rather than slower sustained release, initial high burst release from the LSDFs may suffice. The boosting of specific immune responses upon i.vag administration indicates that LSDFs are viable mucosal vaccine delivery modalities promoting antigen stability and facilitating intimate exposure of CN54gp140 to the mucosal-associated lymphoid tissue of the female genital tract.

IMMUNOCYTOCHEMICAL LOCALIZATION OF SEROTONIN (5-HT) IN THE NERVOUS-SYSTEM OF THE HYDATID ORGANISM, ECHINOCOCCUS-GRANULOSUS (CESTODA, CYCLOPHYLLIDEA)

The localization and distribution of the serotoninergic components of the nervous system in the hydatid organism, Echinococcus granulosus, were determined by immunocytochemical techniques in conjunction with confocal scanning laser microscopy (CSLM). The distribution of serotonin immunoreactivity (IR) paralleled that previously described for cholinesterase activity, although it was more widespread. Nerve cell bodies and nerve fibres immunoreactive for 5-HT were present throughout the central nervous system (CNS), occurring in the paired lateral, posterior lateral and rostellar ganglia, their connecting commissures and nerve rings in the scolex and in the ten longitudinal nerve cords that run posteriorly throughout the body of the worm. A large population of nerve cell bodies was associated with the lateral nerve cords. In the peripheral nervous system (PNS), immunoreactive nerve fibres occurred in well-developed nerve plexuses innervating the somatic musculature and the musculature of the rostellum and suckers. The genital atrium and associated reproductive ducts were richly innervated with serotoninergic nerve cell bodies and nerve fibres.

IMMUNOCYTOCHEMICAL AND RADIOIMMUNOMETRICAL DEMONSTRATION OF SEROTONIN-IMMUNOREACTIVITIES AND NEUROPEPTIDE-IMMUNOREACTIVITIES IN THE ADULT-RAT TAPEWORM, HYMENOLEPIS-DIMINUTA (CESTODA, CYCLOPHYLLIDEA)

Standard indirect immunocytochemical techniques have been interfaced with confocal scanning laser microscopy (for whole-mount preparations) and epifluorescence microscopy (for cryosections) to investigate the occurrence and distribution of serotoninergic and peptidergic nerve elements in adult H. diminuta. Serotonin (5-HT)-immunoreactivity (IR) was widespread throughout the worm, occurring in the paired cerebral ganglia, transverse commissure, the 10 longitudinal nerve cords and in a plethora of small nerve fibres of the peripheral nervous system. An abundance of serotoninergic nerve cell bodies was found in association with the lateral nerve cords. The genital atrium and accessory reproductive ducts were richly innervated with serotoninergic nerve fibres. Thirty-five antisera to 20 vertebrate regulatory peptides and 1 invertebrate peptide (FMRFamide) were used to screen the worm for neuropeptide IR. Immunostaining was obtained with antisera raised to pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), substance P (SP), peptide histidine isoleucine (PHI), xenopsin (XP) and FMRFamide. The most extensive pattern of IR occurred with antisera to PP and PYY, IR being evident in the cerebral ganglia, transverse commissure, longitudinal nerve cords and in small nerve fibres that ramified throughout the parenchyma. A series of bipolar nerve cell bodies between the median nerve cords displayed PP/PYY-IR. The distribution of FMRFamide-IR was reminiscent of the PP/PYY pattern but was less extensive. Comparison of the serotoninergic and peptidergic nervous systems has revealed general similarities and some distinct differences, especially with regard to the distribution of immunoreactive nerve cell bodies. Quantitative data are presented on the levels of PP-, SP-, PHI-, and gastrin-releasing peptide (GRP)-immunoreactivities demonstrable in acid-alcohol extracts of whole worms. The highest level of peptide IR determined was recorded for PP.

THE CHOLINERGIC, SEROTONINERGIC AND PEPTIDERGIC COMPONENTS OF THE NERVOUS-SYSTEM OF MONIEZIA-EXPANSA (CESTODA, CYCLOPHYLLIDEA)

The central (CNS) and peripheral (PNS) nervous systems of the cyclophyllidean tapeworm, Moniezia expansa, were examined for the presence of cholinergic, serotoninergic and peptidergic elements using enzyme cytochemical and immunocytochemical techniques in conjunction with light and confocal scanning laser microscopy. Cholinesterase activity and 5-hydroxytryptamine- and regulatory peptide-immunoreactivities (IRs) were localized to the nerve fibres and cell bodies of all of the major neuronal components in the CNS of the worm, including the cerebral ganglia and connecting commissure, the 10 longitudinal nerve cords and associated transverse ring commissures. Although each of the 3 systems appeared well developed and comprised a significant portion of the nervous system, the serotoninergic constituent was the most highly developed, consisting of a vast array of nerve fibres and cell bodies distributed throughout the strobila of the worm. A close association of cholinesterase reactivity and peptide-IRs was evident throughout the CNS, indicating the possible co-localization of acetylcholine and neuropeptides. Within the PNS, cholinergic activity and serotoninergic- and peptidergic-IRs occurred in the subtegumental network of nerve fibres and somatic musculature. Although all 3 neurochemical elements were present in the acetabula, they were found in different nerve fibres; only cholinergic and peptidergic cell bodies were found. The common genital opening, vagina and ootype regions of the reproductive system displayed a rich innervation of all 3 types of neuronal populations. Within the peptidergic system, immunostaining with antisera raised to the C-terminus of the neuropeptide Y superfamily of peptides and the invertebrate peptides, neuropeptide F (M. expansa) and FMRFamide was the most prevalent. Limited positive-IR for substance P and neurokinin A were also recorded in the CNS of the worm.

Clonal complex Pseudomonas aeruginosa in horses

Pseudomonas aeruginosa is associated with infectious endometritis in horses. Although infectious endometritis is often considered a venereal infection, there is relatively limited genotypic-based evidence to support this mode of transmission. The study sought to determine the relatedness between genital P. aeruginosa isolates collected from a limited geographical region using molecular strain typing. Enterobacterial repetitive intergenic consensus PCR typing was performed on 93 isolates collected between 2005 and 2009 from 2058 thoroughbred horses (including 18 stallions) at 66 studs. While P. aeruginosa was not detected in the stallions, 53/93 (57%) mares harbouring P. aeruginosa had clonally related strains, which included a single dominant genotype detected in 42 (45%) mares from 13 different studs. These novel findings suggest that most equine genital P. aeruginosa infections in this region may have been acquired from mechanisms other than direct horse to horse transmission. Instead, other potential acquisition pathways, as well as strain specific adaptation to the equine genital tract, should be investigated.

Computerized key of the genus genus Laimaphelenchus Fuchs, 1937

The Order Aphelenchida contains several genera of economic importance, namely Aphelenchoides and Bursaphelenchus. Nematode species belonging to these 2 genera frequently co-habit with other genera such as Laimaphelenchus. It is therefore important to clearly distinguish them, as well as understand the group´s biodiversity. A computerized, or e-key, for the genus Laimaphelenchus Fuchs has been developed in the BiKey Identification system (Dianov & Lobanov, 1996-2004). The e-key includes 14 species and 34 characters (from 2 to 6 character states each). It also includes the built-in algorithm ranging characters according their diagnostic values to minimize the number of the diagnosis steps (average number of steps is 2.7; values are re-calculated at each step). The most important characters (as calculated by BiKey) are: length of posterior branch of the female genital system; excretory pore position; vulval anterior flap shape; number pairs of mail tale papillae; male bursa shape (ventral view); number of tail tip setae in female; female tail tip stub shape; presence of mucro on tail tip in male. Key is pictorial (image-operating), multientry, as other BiKey products.

Progress in the use of scanning electron microscopy (SEM) applied to the taxonomy of the genus Bursaphelenchus (Nematoda: Parasitaphelenchidae)

The scanning electron microscope (SEM) has been a major tool in detailed morphological observations of plant parasitic nematodes during the last 30 years, efficiently complementing light microscopical (LM) studies. Nematodes are extremely difficult to observe and characterize due to their small size (aprox. 1 mm long) and paucity of morphological characters, so detailed surface observations of several organs and nematode regions are of the highest value. Among plant parasitic nematodes, one of the most devastating species is the “pinewood nematode” (PWN), Bursaphelenchus xylophilus, which has been a major problem for forest species, and in particular pines, in Asia (Japan, China, Korea) and has been recently detected in the European Union (Portugal). B. xylophilus belongs to a closely related, morphologically similar group of species, within the genus Bursaphelenchus, and designated by the “xylophilus group”. SEM has become a crucial tool in observing several genital characters of males and females, such as male genital papillae, male copulatory spicules, female vulval flap and female genital papillae.s In this presentation, we will show how SEM has been utilized to observe and characterize the shape of the vulval flap, the presence/ absence of papillae near the flap, and confirm the presence and the arrangement of the male genital papillae. LM is also used in this work to show its value as a complementary tool to SEM, in both genital characteristics and other, general, characters of the genus Bursaphelenchus, such as the male bursa and cephalic region.

Pesquisa de Campylobacter fetus em amostras prepuciais de touros da região do Alentejo

A bactéria Campylobacter fetus divide-se em duas subespécies C. fetus fetus (Cff) e C. fetus venerealis (Cfv). Cfv é o agente patogénico responsável pela campilobacteriose genital bovina (CGB), uma doença que provoca infertilidade em vacas. Cff pode provocar abortos esporádicos, mas não provoca CGB. Neste trabalho procedeu-se à pesquisa de C. fetus por imunofluorescência direta (IFD) em 117 amostras prepuciais, recolhidas por raspagem, de touros pertencentes a 25 explorações na região do Alentejo. A prevalência real de CGB na amostra, considerando a sensibilidade e especificidade do teste, foi de 52,89% de animais e 80% de explorações. Foi também realizada uma comparação entre dois testes de diagnóstico de CGB, a IFD e o isolamento bacteriológico. O isolamento bacteriológico foi difícil de executar e a presença de bactérias contaminantes foi bastante problemática, levando a resultados falso-negativos. A sua correlação com a IFD foi muito baixa, tendo esta revelado ser muito mais sensível; #### Abstract Search for Campylobacter fetus in preputial samples of bulls from Alentejo region The bacteria Campylobacter fetus comprises two subspecies, C. fetus fetus (Cff) and C. fetus venerealis (Cfv). Cfv is the pathogen responsible for bovine genital campylobacteriosis (BGC), a disease that causes infertility in cows. Cff may lead to sporadic miscarriages, but does not cause BGC. In this work we searched for C. fetus by direct immunofluorescence (DIF) in 117 preputial samples, collected by scraping, from bulls from Alentejo region. The actual prevalence of BGC, considering the sensitivity and specificity of the test was 52.89% of animals and 80 % of farms. It was also performed a comparison between two BGC diagnostic tests, bacterial isolation and DIF. The bacterial isolation was difficult to perform and the presence of bacterial contaminants was very problematic, leading to false-negative results. The presence of bacterial contaminants in bacterial isolation was problematic, leading to false-negative results. Its correlation with DIF was quite low, and DIF proved to be much more sensitive.

Campilobacteriose em bovinos de carne no Alentejo – avaliação preliminar

The aim of this study was to assess the prevalence of infection by Campylobacter fetus in beef cattle reared in extensive systems in Alentejo region by direct fluorescent antibody test (DFAT), a technique widely used for determining the prevalence of this disease.

Morphological and ecological studies on the phylum Cycliophora

Cycliophora é um filo animal descrito recentemente que acomoda, apenas, duas espécies: Symbion pandora Funch e Kristensen, 1995 e S. americanus Obst, Funch e Kristensen, 2006. Este filo é caracterizado por um ciclo de vida assaz complexo, cuja posição filogenética tem sido debatida desde a sua descoberta. Esta dissertação visa aprofundar o conhecimento geral existente acerca destes enigmáticos e pouco explorados metazoários. Assim, vários aspectos da morfologia e ecologia de ciclióforos foram estudados através de observações in vivo, técnicas de microscopia e reconstrução tridimensional. A mioanatomia de várias fases do ciclo de vida é descrita para S. pandora e S. americanus. Os nossos resultados revelam uma similaridade contundente entre a musculatura das duas espécies. A mioanatomia geral de Symbion é, ainda, comparada à de outros metazoários. A expressão de algumas substâncias imunorreactivas, como são exemplo a serotonina e as sinapsinas, é investigada em várias formas do ciclo de vida. Quando comparados com outros representantes de Spiralia, conclui-se que a neuroanatomia geral dos ciclióforos se assemelha mais às formas larvares do que aos adultos. Apesar de possuírem um plano corporal sofisticado, com extensas áreas ciliadas e uma mioanatomia complexa, descobrimos que o macho de ambas as espécies Symbion é composto por apenas algumas dezenas de células. Baseando-nos nestas observações, inferimos que a complexidade dos metazoários não se relaciona com o tamanho corporal nem com o número de células de um organismo. Estudos sobre a ultra-estrutura da fêmea revelaram, entre outras estruturas, um putativo poro genital, extensões citoplasmáticas do oócito e glândulas posteriores. Morfologia e implicações funcionais destas estruturas são aqui discutidas. A anatomia do protonefrídeo da larva cordóide é descrita. A arquitectura deste órgão diverge daquela presente noutros representantes de Nephrozoa, particularmente ao nível da área de filtração da célula terminal. As nossas observações são discutidas em termos filogenéticos. A maturação sexual em ciclióforos é investigada. Os nossos resultados sugerem que a transição de reprodução assexual a sexual se relacione com a idade da forma séssil, a “feeding stage”. A presença da larva Prometeus assente no tronco desta também poderá influenciar o processo, embora mais estudos sejam desejáveis para o comprovar. Os nossos resultados são discutidos integrativa e comparativamente com o conhecimento prévio sobre Cycliophora. A cumulação deste conhecimento será essencial para a compreensão da evolução e filogenia deste enigmático filo.

1948-1965: o declínio do paradigma clássico e a emergência do maneirismo de Hollywood

O presente trabalho, investiga as origens históricas e as características formais do maneirismo de Hollywood, através de uma análise da produção cinematográfica dos estúdios de Hollywood, entre o final da década de 1920 e o fim do chamado studio system (em meados da década de 1960). Tal como o maneirismo histórico, o maneirismo hollywoodiano constitui um fenómeno artístico complexo e contraditório, cujas origens remontam ao estilo germânico introduzido no cinema dos estúdios em final dos anos vinte, por cineastas europeus como F.W. Murnau, Paul Leni, Rouben Mamoulian e James Whale. Tendo eclodido no final da II Guerra Mundial, período que assinala o início crise do sistema social, cultural e industrial que havia originado o cinema clássico, o maneirismo de Hollywood nasceu de uma consciência do cinema em relação ao seu passado. Obrigado a concorrer com a chamada “modernidade” cinematográfica europeia e com a ficção televisiva (que se apropriara do modelo clássico), o cinema de Hollywood explorou novos temas e géneros cinematográficos e investiu na exibição da técnica e do aparato tecnológico, forçando os limites do paradigma clássico, através de uma retórica de efeitos maneiristas que, não implicando um corte radical com o classicismo, representou uma clara tentativa de renovação formal, levada a cabo por cineastas como Alfred Hitchcock, Vincente Minnelli, Douglas Sirk, Stanley Donen e Jerry Lewis. A coexistência de varias correntes estéticas no cinema do pós-guerra (1945-1968), período em que, a par das últimas grandes obras clássicas, o maneirismo enquanto manifestação estética emergiu como modelo dominante, permite-nos concluir que o chamado cinema “clássico” (do estúdios de Hollywood) incluiu também, desde cedo, um cinema maneirista.

New approaches for the diagnosis of human papillomavirus infection:relevance for clinical practice and cancer prevention

Tese de doutoramento, Biologia (Microbiologia), Universidade de Lisboa, Faculdade de Ciências, 2014

Activities of an aqueous plant extract against Herpes simplex virus type 2 (HSV 2): role in virus replication and progeny yield

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2015