Locust adipokinetic hormones: carrier-independent transport and differential inactivation at physiological concentrations during rest and flight.

Since concomitant release of structurally related peptide hormones with apparently similar functions seems to be a general concept in endocrinology, we have studied the dynamics of the lifetime of the three known adipokinetic hormones (AKHs) of the migratory locust, which control flight-directed mobilization of carbohydrate and lipid from fat body stores. Although the structure of the first member of the AKHs has been known for 20 years, until now, reliable data on their inactivation and removal from the hemolymph are lacking, because measurement requires AKHs with high specific radioactivity. Employing tritiated AKHs with high specific radioactivity, obtained by catalytic reduction with tritium gas of the dehydroLeu2 analogues of the AKHs synthesized by the solid-phase procedure, studies with physiological doses of as low as 1.0 pmol per locust could be conducted. The AKHs appear to be transported in the hemolymph in their free forms and not associated with a carrier protein, despite their strong hydrophobicity. Application of AKHs in their free form in in vivo and in vitro studies therefore now has been justified. We have studied the degradation of the three AKHs during rest and flight. The first cleavage step by an endopeptidase is crucial, since the resulting degradation products lack any adipokinetic activity. Half-lives for AKH-I, -II and -III were 51, 40, and 5 min, respectively, for rest conditions and 35, 37, and 3 min, respectively, during flight. The rapid and differential degradation of structurally related hormones leads to changes in the ratio in which they are released and therefore will have important consequences for concerted hormone action at the level of the target organ or organs, suggesting that each of the known AKHs may play its own biological role in the overall syndrome of insect flight.

Reconstrução facial forense: comparação entre tabelas de espessuras de tecidos moles faciais

As Ciências Forenses empregam a técnica de Reconstrução Facial buscando aumentar as possibilidades de reconhecimento humano. Após análise antropológica, a face é esculpida sobre o crânio esqueletizado e divulgada na mídia. Existem várias metodologias para a modelagem do rosto e das características da face, bem como vários dados de espessuras de tecidos moles que auxiliam no contorno facial. Com o intuito de investigar se existe uma metodologia que favoreça mais reconhecimentos e que permita uma maior semelhança com o indivíduo, este trabalho buscou comparar reconstruções faciais manuais feitas com duas abordagens para o preenchimento dos tecidos moles (métodos Americano e de Manchester) e para a predição dos olhos, nariz, boca e orelhas. Também buscou comparar reconstruções realizadas com quatro tabelas de espessuras de tecidos moles, desenvolvidas para brasileiros por estudos prévios, observando a possibilidade de unir esses dados para auxiliar na reconstrução. Um quarto objetivo foi averiguar se existe influência do sexo e do conhecimento anatômico ou forense na frequência de reconhecimentos. O estudo foi dividido em duas fases. Na primeira, duas reconstruções foram realizadas para dois indivíduos alvos (um homem e uma mulher) com os métodos Americano e de Manchester, aplicando dois guias para olhos, nariz, boca e orelhas. As reconstruções foram avaliadas por quarenta indivíduos (homens e mulheres, divididos em 4 grupos - alunos de graduação em Odontologia que não passaram pela disciplina de Odontologia Legal, alunos de graduação em Odontologia que passaram pela disciplina, especialistas em Odontologia Legal e indivíduos que não possuíam conhecimento de anatomia humana) por meio dos testes de reconhecimento e semelhança. Para o alvo feminino, as frequências de reconhecimentos foram 20% e 10% para os métodos Americano e de Manchester, respectivamente; para o alvo masculino, as frequências foram 35% e 17,5%. Em relação à semelhança, as medianas foram menores que 3 (em uma escala de 1 a 5); entretanto, foi verificada uma exceção para a escultura feita com o método Americano para o alvo masculino, a qual apresentou mediana 3. Na segunda fase, reconstruções faciais para quatro alvos (dois homens e duas mulheres) foram obtidas com o método Americano, considerando as quatro tabelas de espessuras de tecidos moles para brasileiros. Dezesseis reconstruções foram avaliadas por cento e vinte indivíduos, também pelos testes de reconhecimento e semelhança. Assim como na fase I, foram considerados o sexo e o grupo dos avaliadores. Para o alvo 1, as proporções de acertos são significativamente maiores para reconstruções feitas com as tabelas de cadáveres (44% e 38%) em relação às com os dados de exames de imagem. Para o alvo 4, as proporções de acertos com os dados de cadáveres (Tedeschi-Oliveira et al.) e com os de ressonância magnética foram significativamente maiores comparados às reconstruções com dados de tomografias computadorizadas. Em relação à semelhança, somente o alvo 1 mostrou diferenças significativas de frequências de semelhança leve entre reconstruções. Além disso, não houve influência nem do sexo, nem do conhecimento de anatomia nas frequências de reconhecimentos corretos. Espera-se que a tabela proposta possa ser empregada para a população brasileira.

Construction of Structures I in Moodle: a new teaching methodology in the Degree of Building Engineer

The subject of Construction of Structures I studies, from a constructive point of view and taking into account current legislation, reinforced concrete structures used in buildings, through the acquisition of knowledge and construction criteria required in the profession of a Technical Architect. The contents acquired in this course are essential for further professional development of technicians and are closely related to many of the subjects taught in the same or other courses of the Degree in Technical Architecture at the University of Alicante. The aim of this paper is to present, analyze and discuss the development of a new methodology proposed in the mentioned subject, as it supposed an important change in the traditional way of teaching Construction and Structures I. In order to incorporate new teaching tools in 2013-2014, the course has been implemented by using a Moodle software tool to promote blended learning with online exercises. Our Moodle community allows collaborative work within an open-source platform where teachers and students share a new and personalized learning environment. Students are easily used to the interface and the platform, value the constant connection with teachers or other fellows and completely agree with the possibility of making questions or share documents 24 hours a day. The proposed methodology consists of lectures and practical classes. In the lectures, the basics of each topic are discussed; class attendance, daily study and conducting scheduled exercises are indispensable. Practical classes allow to consolidate the knowledge gained in theory classes by solving professional exercises and actual construction problems related to structures, that shall be compulsorily delivered online. So, after the correction of the teacher and the subsequent feedback of students, practical exercises ensure lifelong learning of the student, who can download any kind of material at any time (constructive details, practical exercises and even corrected exams). Regarding the general evaluation system, goals achievement is assessed on an ongoing basis (65% of the final mark) along the course through written and graphic evidences in person and online, as well as a individual development of a workbook. In all cases, the acquisition of skills, the ability to synthesize, the capacity of logical and critical thinking are assessed. The other 35 % of the mark is evaluated by a complementary graphic exam. Participation in the computing platform is essential and the student is required to do and present, at least 90% of the practices proposed. Those who do not comply with the practices in each specific date could not be assessed continuously and may only choose the final exam. In conclusion, the subject of Construction of Structures I is essential in the development of the regulated profession of Technical Architect as they are considered, among other professional profiles, as specialists in construction of building structures. The use of a new communication platform and online teaching allows the acquisition of knowledge and constructive approaches in a continuous way, with a more direct and personal monitoring by the teacher that has been highly appreciated by almost 100% of the students. Ultimately, it is important to say that the use of Moodle in this subject is a very interesting tool, which was really well welcome by students in one of the densest and important subjects of the Degree of Technical Architecture.

Dynamics of the Developing Chick Chorioallantoic Membrane Assessed by Stereology, Allometry, Immunohistochemistry and Molecular Analysis.

The chick chorioallantoic membrane (CAM) is a widely used model for the study of angiogenesis, tumour growth, as well as drug efficacy. In spite of this, little is known about the developmental alteration from its appearance to the time of hatching. In the current study the CAM has been studied by classical stereology and allometry. Expression levels of selected angiogenesis-related molecules were estimated by RT-PCR and cell dynamics assessed by proliferation and apoptosis assays. Absolute CAM volume increased from a low of 0.47 ± 0.11 cm3 at embryonic day 8 (E8) to a high of 2.05 ± 0.27 cm3 at E18, and then decreased to 1.6 ± 0.47 cm3 at E20. On allometric analysis, three growth phases were identifiable. Between E8-13 (phase I), the CAM grew fastest; moderately in phase II (E13-18) but was regressing in phase III (E18-20). The chorion, the mesenchyme and the allantoic layers grew fastest in phase I, but moderately in phase II. The mesenchyme grew slowly in phase III while the chorion and allantois were regressing. Chorionic cell volume increased fastest in phase I and was regressing in phase III. Chorionic capillaries grew steadily in phase I and II but regressed in phase III. Both the chorion and the allantois grew by intrinsic cell proliferation as well as recruitment of cells from the mesenchyme. Cell proliferation was prominent in the allantois and chorion early during development, declined after E17 and apoptosis started mainly in the chorion from E14. VEGFR2 expression peaked at E11 and declined steadily towards E20, VEGF peaked at E13 and E20 while HIF 1α had a peak at E11 and E20. Studies targeting CAM growth and angiogenesis need to take these growth phases into consideration.

Technical report[s]

Contract no DA-44-009 Eng. 2435, Department of the Army Project no. 8-35-11-101.

Biological monitoring of pesticides, heavy metals and other contaminants at Rocky Mountain Arsenal : phase 1 /

July 1979.

Theoretical investigation of optimum methods for preparing controlled mosaics from 9 x 18-inch format, long focal length photography.

"Contract no. DA-44-009 Eng-3364, Department of the Army Project no. 8-35-03-216.j"

Cantilever extension of ground control in strips of convergent photographs by analytical triangulation.

"Contract no. DA-44-009 Eng-2986, Department of the Army Project no. 8-35-11-106."

Two-phase anaerobic digestion for enhanced stabilization and methanation of sewage sludge /

"ILENR/AE-92/02."

Bioavailability of Echinacea Constituents: Caco-2 Monolayers and Pharmacokinetics of the Alkylamides and Caffeic Acid Conjugates

Many studies have been done over the years to assess the effectiveness of Echinacea as an immunomodulator. We have assessed the potential bioavailability of alkylamides and caffeic acid conjugates using Caco-2 monolayers and compared it to their actual bioavailability in a Phase I clinical trial. The caffeic acid conjugates permeated poorly through the Caco-2 monolayers. Alkylamides were found to diffuse rapidly through Caco-2 monolayers. Differences in diffusion rates for each alkylamide correlated to structural variations, with saturation and N-terminal methylation contributing to decreases in diffusion rates. Alkylamide diffusion is not affected by the presence of other constituents and the results for a synthetic alkylamide were in line with those for alkylamides found in an ethanolic Echinacea preparation. We examined plasma from healthy volunteers for 12 hours after ingestion of Echinacea tablets manufactured from an ethanolic liquid extract. Caffeic acid conjugates could not be identified in any plasma sample at any time after tablet ingestion. Alkylamides were detected in plasma 20 minutes after tablet ingestion and for each alkylamide, pharmacokinetic profiles were devised. The data are consistent with the dosing regimen of one tablet three times daily and supports their usage as the primary markers for quality Echinacea preparations.

Concentration dependent effects of N1,N11-diethylnorspermine on melanoma cell proliferation

N-1, N-11-Diethylnorspermine (DENSPM) is a polyamine analog that is currently under investigation as a novel anticancer drug. Although it has shown promising preclinical activity, there has been large variation in responsiveness reported between different human cancers. During our studies into the causes of this variation, we observed a consistent increase in cell proliferation at low drug concentrations (

Live Attenuated Chimeric Yellow Fever Dengue Type 2 (Chimeri Vax -DEN2) Vaccine: Phase 1 clinical trial for safety and immunogenicity

A randomized double-blind Phase I Trial was conducted to evaluate safety, tolerability, and immunogenicity of a yellow fever (YF)-dengue 2 (DEN2) chimera (ChimeriVax™-DEN2) in comparison to that of YF vaccine (YF-VAX®). Forty-two healthy YF naïve adults randomly received a single dose of either ChimeriVax™-DEN2 (high dose, 5 log plaque forming units [PFU] or low dose, 3 log PFU) or YF-VAXâ by the subcutaneous route (SC). To determine the effect of YF pre-immunity on the ChimeriVaxTM-DEN2 vaccine, 14 subjects previously vaccinated against YF received a high dose of ChimeriVax™-DEN2 as an open-label vaccine. Most adverse events were similar to YF-VAX® and of mild to moderate intensity, with no serious side-effects. One hundred percent and 92.3% of YF naïve subjects inoculated with 5.0 and 3.0 log10 PFU of ChimeriVaxTM-DEN2, respectively, seroconverted to wt DEN2 (strain 16681); 92% of subjects inoculated with YF-VAX® seroconverted to YF 17D virus but none of YF naïve subjects inoculated with ChimeriVax-DEN2 seroconverted to YF 17D virus. Low seroconversion rates to heterologous DEN serotypes 1, 3, and 4 were observed in YF naïve subjects inoculated with either ChimeriVax™-DEN2 or YF-VAX®. In contrast, 100% of YF immune subjects inoculated with ChimeriVax™-DEN2 seroconverted to all 4 DEN serotypes. Surprisingly, levels of neutralizing antibodies to DEN 1, 2, and 3 viruses in YF immune subjects persisted after 1 year. These data demonstrated that 1) the safety and immunogenicity profile of the ChimeriVax™-DEN2 vaccine is consistent with that of YF-VAX®, and 2) pre-immunity to YF virus does not interfere with ChimeriVaxTM-DEN2 immunization, but induces a long lasting and cross neutralizing antibody response to all 4 DEN serotypes. The latter observation can have practical implications toward development of a dengue vaccine.

Reformulation of a thermostable broadly protective recombinant vaccine against human papilloma virus

The causal relationship between Human Papilloma Virus (HPV) infection and cervical cancer has motivated the development, and further improvement, of prophylactic vaccines against this virus. 70% of cervical cancers, 80% of which in low-resources countries, are associated to HPV16 and HPV18 infection, with 13 additional HPV types, classified as high-risk, responsible for the remaining 30% of tumors. Current vaccines, Cervarix® (GlaxoSmithKline) and Gardasil®(Merk), are based on virus-like particles (VLP) obtained by self-assembly of the major capsid protein L1. Despite their undisputable immunogenicity and safety, the fact that protection afforded by these vaccines is largely limited to the cognate serotypes included in the vaccine (HPV 16 and 18, plus five additional viral types incorporated into a newly licensed nonavalent vaccine) along with high production costs and reduced thermal stability, are pushing the development of 2nd generation HPV vaccines based on minor capsid protein L2. The increase in protection broadness afforded by the use of L2 cross-neutralizing epitopes, plus a marked reduction of production costs due to bacterial expression of the antigens and a considerable increase in thermal stability could strongly enhance vaccine distribution and usage in low-resource countries. Previous studies from our group identified three tandem repeats of the L2 aa. 20-38 peptide as a strongly immunogenic epitope if exposed on the scaffold protein thioredoxin (Trx). The aim of this thesis work is the improvement of the Trx-L2 vaccine formulation with regard to cross-protection and thermostability, in order to identify an antigen suitable for a phase I clinical trial. By testing Trx from different microorganisms, we selected P. furiosus thioredoxin (PfTrx) as the optimal scaffold because of its sustained peptide epitope constraining capacity and striking thermal stability (24 hours at 100°C). Alternative production systems, such as secretory Trx-L2 expression in the yeast P. pastoris, have also been set-up and evaluated as possible means to further increase production yields, with a concomitant reduction of production costs. Limitations in immune-responsiveness caused by MHC class II polymorphisms –as observed, for example, in different mouse strains- have been overcome by introducing promiscuous T-helper (Th) epitopes, e.g., PADRE (Pan DR Epitope), at both ends of PfTrx. This allowed us to obtain fairly strong immune responses even in mice (C57BL/6) normally unresponsive to the basic Trx-L2 vaccine. Cross-protection was not increased, however. I thus designed, produced and tested a novel multi-epitope formulation consisting of 8 and 11 L2(20-38) epitopes derived from different HPV types, tandemly joined into a single thioredoxin molecule (“concatemers”). To try to further increase immunogenicity, I also fused our 8X and 11X PfTrx-L2 concatemers to the N-terminus of an engineered complement-binding protein (C4bp), capable to spontaneously assemble into ordered hepatmeric structures, previously validated as a molecular adjuvant. Fusion to C4bp indeed improved antigen presentation, with a fairly significant increase in both immunogenicity and cross-protection. Another important issue I addressed, is the reduction of vaccine doses/treatment, which can be achieved by increasing immunogenicity, while also allowing for a delayed release of the antigen. I obtained preliminary, yet quite encouraging results in this direction with the use of a novel, solid-phase vaccine formulation, consisting of the basic PfTrx-L2 vaccine and its C4bp fusion derivative adsorbed to mesoporus silica-rods (MSR).

Design, synthesis and development of PDE5 inhibitors

This research project is concerned with the design, synthesis and development of new phosphodiesterase 5 (PDE5) inhibitors with improved selectivities and lower toxicities. Two series of a 5 member and a 6 member ring fused heterocyclic compounds were designed, and synthesized. By alteration of starting materials and fragments, two virtual libraries, each is consisted of close to hundred compounds, were obtained successfully. The screening of sexual stimulation activity with rabbits demonstrated both groups of compounds were able to stimulate rabbit penile erection significantly. The following toxicity studies revealed 2-(substituted-sulfonylphenyl)-imidazo [1,5-a]-1,3,5-triazine-4-(3H)-one group possessed an unacceptable toxicity with oral LD50 about 200mg/kg; while 2-(substituted-sulfonylphenyl)-pyrrolo[2,3-d]pyrimidin-4-one group showed an acceptable toxicity with oral LD50 over 2000mg/kg. The continued bioactivity studies showed yonkenafil, the representative of 2-(substituted-sulfonylphenyl)-pyrrolo[2,3-d]pyrimidin-4-one group, has a better selectivity towards PDE5 and PDE6 than sildenafil and a better overall profile of sexual stimulation on animals than sildenafil. Chronic toxicity studies of yonkenafil further confirmed yonkenafil did not cause any serious side effect and damage on animal models and most actions were explainable. Based on evidences of the above studies, yonkenafil were recommended to enter clinical trials by the regulation authority of China, SFDA. Currently yonkenafil has been through the Phase I clinical trials and ready to progress into Phase II. Hopefully, yonkenafil will provide an alternative to the ED patients in the future.

The health and nutritional status of multiethnic perimenopausal women

An integrated, dual-phase study design assessed the health and nutritional status and practices of African-American (A-A), Caribbean (A-C), and white non-Hispanic (W-A) women during perimenopause (40–55 years). During Phase I, four focus groups (n = 37) of male and female participants discussed the health and social implications of perimenopause. A conceptual framework for the main study (Phase II) was developed from the focus groups' findings, in concert with the main study's specific aims and objectives. ^ The main study, a cross-sectional survey, quantitatively assessed the health and nutritional status of a convenience sample of 109 women (25 A-A, 31 A-C and 53 W-A), who met specific eligibility criteria. Using seven instruments, sociodemographic, dietary, medical, reproductive health, health practice and anthropometric data were collected. ^ The groups were of comparable age, education, and socioeconomic status (SES). Despite these similarities, statistically significant interethnic nutritional status differences were found. Significantly more total energy and energy from fat were consumed by A-A than W-A and A-C women. Also, significantly more A-A and A-C than W-A women were overweight or obese with android-type weight patterning. ^ Overall, iron and calcium Recommended Dietary Allowances (RDA's) were not met by 35% and 68% of participants, respectively. Iron deficiency anemia was reported by 29% of participants while 33% reported heavier menstrual bleeding. Coupled with suboptimal iron intakes, this is likely to present a serious public health problem. Similarly, increased bone demineralization characteristic of perimenopause, coupled with suboptimal calcium intakes could precipitate another public health problem, osteoporosis. ^ Participants had different expectations about the role of medical care during perimenopause. Significantly more white (57%) than black (38% [A-A and AC]) women sought medical attention for symptoms. Whereas Hormone Replacement Therapy (HRT) was prescribed for 25% of them, only 13% were compliant at enrollment. ^ The trends and statistically significant findings of this study have huge public health policy implications. It is imperative that appropriate policies are formulated to ensure that America's ethnically diverse perimenopausal women have ready access to culturally appropriate care. This would optimize their health outcomes, and enhance their quality of life and productive capacities at this critical juncture of their lives. ^