A equoterapia como auxiliar no processo de inclusão do aluno com deficiência visual: estudo de caso

This study examines the hippotherapy related to school inclusion. Inclusive education, given the diversity of human kind, seeks to understand and meet the educational needs of all students. Visual impairment is defined as a reduction or complete loss of ability to see with the better eye and after the best possible optical correction, and may be perceived by the focus when the individual educational needs of Braille for their learning. Hippotherapy is a therapeutic and educational method that uses the horse in an interdisciplinary approach in health, education and riding, searching for the biopsychosocial development of people with disabilities and/or special needs. The general objectives were: to follow a Hippotherapy program applied to a student with visual disabilities included in regular education from the practice sessions of hippotherapy and the student's school routine, and specific: to verify changes to a student with visual impairment during their participation in an equine therapy program, to check the commitment, involvement and interest in school activities the student practicing hippotherapy inserted into the regular school system. The methodology was a qualitative research, in the form of case study. The instruments of data collection were interviews, video recordings, photographs, school information and the subject of systematic observations of the sessions of hippotherapy. Interviews were conducted at three different times during the course of the program. First we used a strategy for succeeding in increasing the involvement of practicing with the school. Later, another strategy was established, consisting of the effective presence of the researcher in the school to carry out activities on the hippotherapy within the school environment. The analysis of the first interview showed no changes in school interest, however, occurred in the family environment. In analyzing the results of the second interview was seen as a...

A equoterapia como auxiliar no processo de inclusão do aluno com deficiência visual: estudo de caso

This study examines the hippotherapy related to school inclusion. Inclusive education, given the diversity of human kind, seeks to understand and meet the educational needs of all students. Visual impairment is defined as a reduction or complete loss of ability to see with the better eye and after the best possible optical correction, and may be perceived by the focus when the individual educational needs of Braille for their learning. Hippotherapy is a therapeutic and educational method that uses the horse in an interdisciplinary approach in health, education and riding, searching for the biopsychosocial development of people with disabilities and/or special needs. The general objectives were: to follow a Hippotherapy program applied to a student with visual disabilities included in regular education from the practice sessions of hippotherapy and the student's school routine, and specific: to verify changes to a student with visual impairment during their participation in an equine therapy program, to check the commitment, involvement and interest in school activities the student practicing hippotherapy inserted into the regular school system. The methodology was a qualitative research, in the form of case study. The instruments of data collection were interviews, video recordings, photographs, school information and the subject of systematic observations of the sessions of hippotherapy. Interviews were conducted at three different times during the course of the program. First we used a strategy for succeeding in increasing the involvement of practicing with the school. Later, another strategy was established, consisting of the effective presence of the researcher in the school to carry out activities on the hippotherapy within the school environment. The analysis of the first interview showed no changes in school interest, however, occurred in the family environment. In analyzing the results of the second interview was seen as a...

A general framework for multi-compartmental analysis of drug chemotherapy dynamics in human immunodeficiency virus type-1 infected individuals

An optimal control strategy for the highly active antiretroviral therapy associated to the acquired immunodeficiency syndrome should be designed regarding a comprehensive analysis of the drug chemotherapy behavior in the host tissues, from major viral replication sites to viral sanctuary compartments. Such approach is critical in order to efficiently explore synergistic, competitive and prohibitive relationships among drugs and, hence, therapy costs and side-effect minimization. In this paper, a novel mathematical model for HIV-1 drug chemotherapy dynamics in distinct host anatomic compartments is proposed and theoretically evaluated on fifteen conventional anti-retroviral drugs. Rather than interdependence between drug type and its concentration profile in a host tissue, simulated results suggest that such profile is importantly correlated with the host tissue under consideration. Furthermore, the drug accumulative dynamics are drastically affected by low patient compliance with pharmacotherapy, even when a single dose lacks. (C) 2012 Elsevier Inc. All rights reserved.

Antibodies Against Sporothrix schenckii Enhance TNF-a Production and Killing by Macrophages

Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungus Sporothrix schenckii. The immunological mechanisms involved in the prevention and control of sporotrichosis suggest that cell-mediated immunity plays an important role in protecting the host against S. schenckii. Nonetheless, recent data strongly support the existence of protective Abs against this pathogenic fungus. In a previous study, we showed that passive Ab therapy led to a significant reduction in the number of colony forming unit in the organs of mice when the MAb was injected before and during S. schenckii infection. The ability of opsonization to enhance macrophage damage to S. schenckii and subsequent cytokine production was investigated in this work. Here we show that the fungicidal characteristics of macrophages are increased when the fungus is phagocytosed in the presence of inactivated serum from mice infected with S. schenckii or mAb anti-gp70. Additionally, we show an increase in the levels of pro-inflammatory cytokines such as TNF-a and IL-1 beta. This study provides additional support for the importance of antibodies in protecting against S. schenckii and concludes that opsonization is an important process to increase TNF-a production and fungus killing by macrophages in experimental sporotrichosis.

Family Health Support Centers: challenges and opportunities from the perspective of primary care professionals in the city of Sao Paulo, Brazil

Family Health Support Centers (NASF) were created in Brazil to increase the case-resolution capacity of primary healthcare. Prior to their implementation in the West Side of the city of Sao Paulo, Brazil, a series of workshops were held for primary healthcare professionals to prepare a proposal for such centers. Hermeneutic analysis was used to study the transcribed material. The thematic categories were: role, constitution, and functioning of the NASF, relationship with family health teams, and interdisciplinarity. The participants' expected the NASF to be an empowering device for comprehensiveness of care, intervening in an existing culture of unnecessary referrals while fostering linkage with other levels of care. The participants also expected the NASF to contribute to the discussion on health professionals' training and stimulating reflection with policy-makers on health indicators based exclusively on the number of consultations. These indicators fail to reflect the impact on the services' activities and the quality of care offered to the population in the coverage area.

Obstrução de vias lacrimais associada ao tratamento radioiodoterápico de carcinoma de tireoide

OBJETIVO: Descrever uma série de pacientes portadores de obstrução do sistema lacrimal associado à radioiodoterapia para tratamento de carcinoma de tireoide, revisar os dados clínicos e a resposta ao tratamento cirúrgico desta rara complicação. MÉTODOS: Foi realizada uma análise retrospectiva dos achados oftalmológicos de pacientes com histórico de carcinoma de tireoide previamente submetidos à tireoidectomia e à RIT que foram encaminhados para cirurgia de vias lacrimais. RESULTADOS: Dezessete pacientes com carcinoma de tireoide tratados com tireoidectomia e RIT apresentaram obstrução do ducto nasolacrimal sintomática após período médio de 13,2 meses do tratamento do câncer. Onze pacientes tiveram epífora bilateral, 8 com mucocele de saco lacrimal. A idade dos pacientes variou entre 30 e 80 anos, sendo 10 com idade menor ou igual a 49 anos. A dose cumulativa média de radioiodo administrada foi de 571 mCi (variação entre 200-1200 mCi). Sintomas de obstrução nasal e aumento de glândulas salivares ocorreram em 53% dos pacientes. Todos os pacientes foram submetidos à dacriocistorrinostomia. Observou-se ainda que nos 3 pacientes mais jovens houve maior sangramento intraoperatótio e dilatação de saco lacrimal. A resolução completa da epífora e da dacriocistite ocorreu em 82,4%, e foi parcial em 17,6% (3 pacientes mantiveram queixa unilateral após a correção da obstrução bilateralmente). O seguimento médio foi de 6 meses (intervalo: 2-24 meses). CONCLUSÕES: Alta dose cumulativa de radioiodo, disfunção nasal e de glândulas salivares estão associadas à obstrução das vias lacrimais. Observa-se uma maior porcentagem de pacientes mais jovens apresentando quadro de dacriocistite quando comparado à dacrioestenose idiopática. A absorção de iodo radioativo pela mucosa do ducto nasolacrimal com subsequente inflamação, edema e fibrose parece ter relação direta com a obstrução do ducto nasolacrimal. O conhecimento desta complicação é importante para o estudo e abordagem correta desses pacientes.

PBL and B-Learning for Civil Engineering Students in a Transportation Course

The results of a pedagogical strategy implemented at the University of Sao Paulo at Sao Carlos are presented and discussed. The initiative was conducted in a transportation course offered to Civil Engineering students. The approach is a combination of problem-based learning and project-based learning (PBL) and blended-learning (B-learning). Starting in 2006, a different problem was introduced every year. From 2009 on, however, the problem-based learning concept was expanded to project-based learning. The performance of the students was analyzed using the following elements: (1) grades in course activities; (2) answers from a questionnaire designed for course evaluation; and (3) cognitive maps made to assess the effects of PBL through the comparison of the responses provided by the students involved and those not involved in the experiment. The results showed positive aspects of the method, such as a strong involvement of several students with the subject. A gradual increase in the average scores obtained by the students in the project activities (from 6.77 in 2006 to 8.24 in 2009) was concomitant with a better evaluation of these activities and of the course as a whole (90 and 97% of options "Good" or "Very good" in 2009, respectively). A growing interest in the field of transportation engineering as an alternative for further studies was also noticed. DOI: 10.1061/(ASCE)EI.1943-5541.0000115. (C) 2012 American Society of Civil Engineers.

Expression of Pancreatic Endocrine Markers by Mesenchymal Stem Cells From Human Adipose Tissue

Mesenchymal stem cells (MSCs) from human adipose tissue have a great potential for use in cell therapy due to their ease of isolation, expansion, and differentiation, besides the relative acceptance from the ethical point of view. Our intention was to isolate and promote in vitro expansion and differentiation of MSCs from human adipose tissue into cells with a pancreatic endocrine phenotype. Human adipose tissue obtained from patients undergoing abdominal dermolipectomy was digested with type I collagenase. MSCs isolated by plastic adherence and characterized by cytochemistry and FACS were expanded in vitro. MSC differentiation into an endocrine phenotype was induced over 2 to 4 months with high glucose (25 mmol/L) media containing nicotinamide, exendin-4, and 2-mercaptoethanol. Insulin and glucagon expressions were analyzed by immunofluorescence. Cells isolated from human adipose tissue and expanded in vitro expressed MSC markers as confirmed by FACS and cytochemistry. Insulin but not glucagon production by differentiated cells was demonstrated by irnmunofluorescence. MSCs isolated from human adipose tissue were induced to differentiate in vitro into an endocrine phenotype that expressed insulin

DNA vaccine containing the mycobacterial hsp65 gene prevented insulitis in MLD-STZ diabetes

Abstract Background Our group previously demonstrated that a DNA plasmid encoding the mycobacterial 65-kDa heat shock protein (DNA-HSP65) displayed prophylactic and therapeutic effect in a mice model for tuberculosis. This protection was attributed to induction of a strong cellular immunity against HSP65. As specific immunity to HSP60 family has been detected in arthritis, multiple sclerosis and diabetes, the vaccination procedure with DNA-HSP65 could induce a cross-reactive immune response that could trigger or worsen these autoimmune diseases. Methods In this investigation was evaluated the effect of a previous vaccination with DNA-HSP65 on diabetes development induced by Streptozotocin (STZ). C57BL/6 mice received three vaccine doses or the corresponding empty vector and were then injected with multiple low doses of STZ. Results DNA-HSP65 vaccination protected mice from STZ induced insulitis and this was associated with higher production of IL-10 in spleen and also in the islets. This protective effect was also concomitant with the appearance of a regulatory cell population in the spleen and a decreased infiltration of the islets by T CD8+ lymphocytes. The vector (DNAv) also determined immunomodulation but its protective effect against insulitis was very discrete. Conclusion The data presented in this study encourages a further investigation in the regulatory potential of the DNA-HSP65 construct. Our findings have important implications for the development of new immune therapy strategies to combat autoimmune diseases.

Population biology of the barbel drum Ctenosciaena gracilicirrhus (Metzelaar, 1919) (Perciformes: Sciaenidae) in Caraguatatuba Bay, Southeastern Brazil

This study evaluated the population biology of Ctenosciaena gracilicirrhus (Perciformes: Sciaenidae) in a shallow non-estuarine coastal area of southeastern Brazil. Monthly samples were taken from October 2003 through October 2004, in two distinct areas at depths from 1 to 4 m. C. gracilicirrhus was generally among the most abundant fish species during the period. Its density was significantly higher in a single sampling month, May 2004, in the South area, which may be explained by its migratory behavior together with its preference for deeper areas. Such behavior may lead to bias in community estimates that use few or only seasonal samples. C. gracilicirrhus individuals ranged from 4.0 to 10.2 cm long, with a main mode from 7.5 to 9.0 cm and a significant decrease in mean size from June onward. The estimation of body growth parameters was compromised by this population feature. Similarly, the prey spectrum was difficult to determine because of the deteriorated condition of the stomach contents, although crustaceans were clearly the most important items ingested. Amphipoda was the only subgroup that could be identified more precisely, mainly by the construction of their tubes.

Análisis de la relación que se establece entre las expectativas de padres y profesores de alumnos con necesidades educativas especiales sobre la atención escolar que reciben los hijos/alumnos y el progreso de los mismos

Programa de Doctorado: Formación del Profesorado

Prototipo del aplicación de recomendaciones sobre la dieta mediterránea

[ES] Este prototipo pretende servir como base de una aplicación que busca mejorar el estilo de vida mediante la adaptación a la dieta mediterránea,  una de las dietas con mayor aceptación por parte de los expertos del campo de la salud. Este software consiste en una aplicación servidor en entorno Ruby on Rails que realiza la función de gestor de contenidos para la aplicación del dispositivo móvil bajo plataforma iOS usando Objective-C, comunicadas entre sí por una API REST. Entre sus funciones, se permite evaluar el nivel de adaptación a la dieta mediterránea del usuario, ofreciéndole posteriormente un menú variado siguiendo las recomendaciones de dietistas expertos. Dicho menú es individualizado, tomando en cuenta las diferentes alergias que padece el usuario para realizar un filtrado de las recetas, consultables en todo momento desde la aplicación. Se presentan además una serie de pantallas interactivas con información sobre la dieta mediterránea y la vida sana.

User interaction widgets for interactive theorem proving

Matita (that means pencil in Italian) is a new interactive theorem prover under development at the University of Bologna. When compared with state-of-the-art proof assistants, Matita presents both traditional and innovative aspects. The underlying calculus of the system, namely the Calculus of (Co)Inductive Constructions (CIC for short), is well-known and is used as the basis of another mainstream proof assistant—Coq—with which Matita is to some extent compatible. In the same spirit of several other systems, proof authoring is conducted by the user as a goal directed proof search, using a script for storing textual commands for the system. In the tradition of LCF, the proof language of Matita is procedural and relies on tactic and tacticals to proceed toward proof completion. The interaction paradigm offered to the user is based on the script management technique at the basis of the popularity of the Proof General generic interface for interactive theorem provers: while editing a script the user can move forth the execution point to deliver commands to the system, or back to retract (or “undo”) past commands. Matita has been developed from scratch in the past 8 years by several members of the Helm research group, this thesis author is one of such members. Matita is now a full-fledged proof assistant with a library of about 1.000 concepts. Several innovative solutions spun-off from this development effort. This thesis is about the design and implementation of some of those solutions, in particular those relevant for the topic of user interaction with theorem provers, and of which this thesis author was a major contributor. Joint work with other members of the research group is pointed out where needed. The main topics discussed in this thesis are briefly summarized below. Disambiguation. Most activities connected with interactive proving require the user to input mathematical formulae. Being mathematical notation ambiguous, parsing formulae typeset as mathematicians like to write down on paper is a challenging task; a challenge neglected by several theorem provers which usually prefer to fix an unambiguous input syntax. Exploiting features of the underlying calculus, Matita offers an efficient disambiguation engine which permit to type formulae in the familiar mathematical notation. Step-by-step tacticals. Tacticals are higher-order constructs used in proof scripts to combine tactics together. With tacticals scripts can be made shorter, readable, and more resilient to changes. Unfortunately they are de facto incompatible with state-of-the-art user interfaces based on script management. Such interfaces indeed do not permit to position the execution point inside complex tacticals, thus introducing a trade-off between the usefulness of structuring scripts and a tedious big step execution behavior during script replaying. In Matita we break this trade-off with tinycals: an alternative to a subset of LCF tacticals which can be evaluated in a more fine-grained manner. Extensible yet meaningful notation. Proof assistant users often face the need of creating new mathematical notation in order to ease the use of new concepts. The framework used in Matita for dealing with extensible notation both accounts for high quality bidimensional rendering of formulae (with the expressivity of MathMLPresentation) and provides meaningful notation, where presentational fragments are kept synchronized with semantic representation of terms. Using our approach interoperability with other systems can be achieved at the content level, and direct manipulation of formulae acting on their rendered forms is possible too. Publish/subscribe hints. Automation plays an important role in interactive proving as users like to delegate tedious proving sub-tasks to decision procedures or external reasoners. Exploiting the Web-friendliness of Matita we experimented with a broker and a network of web services (called tutors) which can try independently to complete open sub-goals of a proof, currently being authored in Matita. The user receives hints from the tutors on how to complete sub-goals and can interactively or automatically apply them to the current proof. Another innovative aspect of Matita, only marginally touched by this thesis, is the embedded content-based search engine Whelp which is exploited to various ends, from automatic theorem proving to avoiding duplicate work for the user. We also discuss the (potential) reusability in other systems of the widgets presented in this thesis and how we envisage the evolution of user interfaces for interactive theorem provers in the Web 2.0 era.

Impatto di rifaximina sul microbiota intestinale: selezione di bifidobatteri antibiotico resistenti

The ideal approach for the long term treatment of intestinal disorders, such as inflammatory bowel disease (IBD), is represented by a safe and well tolerated therapy able to reduce mucosal inflammation and maintain homeostasis of the intestinal microbiota. A combined therapy with antimicrobial agents, to reduce antigenic load, and immunomodulators, to ameliorate the dysregulated responses, followed by probiotic supplementation has been proposed. Because of the complementary mechanisms of action of antibiotics and probiotics, a combined therapeutic approach would give advantages in terms of enlargement of the antimicrobial spectrum, due to the barrier effect of probiotic bacteria, and limitation of some side effects of traditional chemiotherapy (i.e. indiscriminate decrease of aggressive and protective intestinal bacteria, altered absorption of nutrient elements, allergic and inflammatory reactions). Rifaximin (4-deoxy-4’-methylpyrido[1’,2’-1,2]imidazo[5,4-c]rifamycin SV) is a product of synthesis experiments designed to modify the parent compound, rifamycin, in order to achieve low gastrointestinal absorption while retaining good antibacterial activity. Both experimental and clinical pharmacology clearly show that this compound is a non systemic antibiotic with a broad spectrum of antibacterial action, covering Gram-positive and Gram-negative organisms, both aerobes and anaerobes. Being virtually non absorbed, its bioavailability within the gastrointestinal tract is rather high with intraluminal and faecal drug concentrations that largely exceed the MIC values observed in vitro against a wide range of pathogenic microorganisms. The gastrointestinal tract represents therefore the primary therapeutic target and gastrointestinal infections the main indication. The little value of rifaximin outside the enteric area minimizes both antimicrobial resistance and systemic adverse events. Fermented dairy products enriched with probiotic bacteria have developed into one of the most successful categories of functional foods. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002), and mainly include Lactobacillus and Bifidobacterium species. Probiotic bacteria exert a direct effect on the intestinal microbiota of the host and contribute to organoleptic, rheological and nutritional properties of food. Administration of pharmaceutical probiotic formula has been associated with therapeutic effects in treatment of diarrhoea, constipation, flatulence, enteropathogens colonization, gastroenteritis, hypercholesterolemia, IBD, such as ulcerative colitis (UC), Crohn’s disease, pouchitis and irritable bowel syndrome. Prerequisites for probiotics are to be effective and safe. The characteristics of an effective probiotic for gastrointestinal tract disorders are tolerance to upper gastrointestinal environment (resistance to digestion by enteric or pancreatic enzymes, gastric acid and bile), adhesion on intestinal surface to lengthen the retention time, ability to prevent the adherence, establishment and/or replication of pathogens, production of antimicrobial substances, degradation of toxic catabolites by bacterial detoxifying enzymatic activities, and modulation of the host immune responses. This study was carried out using a validated three-stage fermentative continuous system and it is aimed to investigate the effect of rifaximin on the colonic microbial flora of a healthy individual, in terms of bacterial composition and production of fermentative metabolic end products. Moreover, this is the first study that investigates in vitro the impact of the simultaneous administration of the antibiotic rifaximin and the probiotic B. lactis BI07 on the intestinal microbiota. Bacterial groups of interest were evaluated using culture-based methods and molecular culture-independent techniques (FISH, PCR-DGGE). Metabolic outputs in terms of SCFA profiles were determined by HPLC analysis. Collected data demonstrated that rifaximin as well as antibiotic and probiotic treatment did not change drastically the intestinal microflora, whereas bacteria belonging to Bifidobacterium and Lactobacillus significantly increase over the course of the treatment, suggesting a spontaneous upsurge of rifaximin resistance. These results are in agreement with a previous study, in which it has been demonstrated that rifaximin administration in patients with UC, affects the host with minor variations of the intestinal microflora, and that the microbiota is restored over a wash-out period. In particular, several Bifidobacterium rifaximin resistant mutants could be isolated during the antibiotic treatment, but they disappeared after the antibiotic suspension. Furthermore, bacteria belonging to Atopobium spp. and E. rectale/Clostridium cluster XIVa increased significantly after rifaximin and probiotic treatment. Atopobium genus and E. rectale/Clostridium cluster XIVa are saccharolytic, butyrate-producing bacteria, and for these characteristics they are widely considered health-promoting microorganisms. The absence of major variations in the intestinal microflora of a healthy individual and the significant increase in probiotic and health-promoting bacteria concentrations support the rationale of the administration of rifaximin as efficacious and non-dysbiosis promoting therapy and suggest the efficacy of an antibiotic/probiotic combined treatment in several gut pathologies, such as IBD. To assess the use of an antibiotic/probiotic combination for clinical management of intestinal disorders, genetic, proteomic and physiologic approaches were employed to elucidate molecular mechanisms determining rifaximin resistance in Bifidobacterium, and the expected interactions occurring in the gut between these bacteria and the drug. The ability of an antimicrobial agent to select resistance is a relevant factor that affects its usefulness and may diminish its useful life. Rifaximin resistance phenotype was easily acquired by all bifidobacteria analyzed [type strains of the most representative intestinal bifidobacterial species (B. infantis, B. breve, B. longum, B. adolescentis and B. bifidum) and three bifidobacteria included in a pharmaceutical probiotic preparation (B. lactis BI07, B. breve BBSF and B. longum BL04)] and persisted for more than 400 bacterial generations in the absence of selective pressure. Exclusion of any reversion phenomenon suggested two hypotheses: (i) stable and immobile genetic elements encode resistance; (ii) the drug moiety does not act as an inducer of the resistance phenotype, but enables selection of resistant mutants. Since point mutations in rpoB have been indicated as representing the principal factor determining rifampicin resistance in E. coli and M. tuberculosis, whether a similar mechanism also occurs in Bifidobacterium was verified. The analysis of a 129 bp rpoB core region of several wild-type and resistant bifidobacteria revealed five different types of miss-sense mutations in codons 513, 516, 522 and 529. Position 529 was a novel mutation site, not previously described, and position 522 appeared interesting for both the double point substitutions and the heterogeneous profile of nucleotide changes. The sequence heterogeneity of codon 522 in Bifidobacterium leads to hypothesize an indirect role of its encoded amino acid in the binding with the rifaximin moiety. These results demonstrated the chromosomal nature of rifaximin resistance in Bifidobacterium, minimizing risk factors for horizontal transmission of resistance elements between intestinal microbial species. Further proteomic and physiologic investigations were carried out using B. lactis BI07, component of a pharmaceutical probiotic preparation, as a model strain. The choice of this strain was determined based on the following elements: (i) B. lactis BI07 is able to survive and persist in the gut; (ii) a proteomic overview of this strain has been recently reported. The involvement of metabolic changes associated with rifaximin resistance was investigated by proteomic analysis performed with two-dimensional electrophoresis and mass spectrometry. Comparative proteomic mapping of BI07-wt and BI07-res revealed that most differences in protein expression patterns were genetically encoded rather than induced by antibiotic exposure. In particular, rifaximin resistance phenotype was characterized by increased expression levels of stress proteins. Overexpression of stress proteins was expected, as they represent a common non specific response by bacteria when stimulated by different shock conditions, including exposure to toxic agents like heavy metals, oxidants, acids, bile salts and antibiotics. Also, positive transcription regulators were found to be overexpressed in BI07-res, suggesting that bacteria could activate compensatory mechanisms to assist the transcription process in the presence of RNA polymerase inhibitors. Other differences in expression profiles were related to proteins involved in central metabolism; these modifications suggest metabolic disadvantages of resistant mutants in comparison with sensitive bifidobacteria in the gut environment, without selective pressure, explaining their disappearance from faeces of patients with UC after interruption of antibiotic treatment. The differences observed between BI07-wt e BI07-res proteomic patterns, as well as the high frequency of silent mutations reported for resistant mutants of Bifidobacterium could be the consequences of an increased mutation rate, mechanism which may lead to persistence of resistant bacteria in the population. However, the in vivo disappearance of resistant mutants in absence of selective pressure, allows excluding the upsurge of compensatory mutations without loss of resistance. Furthermore, the proteomic characterization of the resistant phenotype suggests that rifaximin resistance is associated with a reduced bacterial fitness in B. lactis BI07-res, supporting the hypothesis of a biological cost of antibiotic resistance in Bifidobacterium. The hypothesis of rifaximin inactivation by bacterial enzymatic activities was verified by using liquid chromatography coupled with tandem mass spectrometry. Neither chemical modifications nor degradation derivatives of the rifaximin moiety were detected. The exclusion of a biodegradation pattern for the drug was further supported by the quantitative recovery in BI07-res culture fractions of the total rifaximin amount (100 μg/ml) added to the culture medium. To confirm the main role of the mutation on the β chain of RNA polymerase in rifaximin resistance acquisition, transcription activity of crude enzymatic extracts of BI07-res cells was evaluated. Although the inhibition effects of rifaximin on in vitro transcription were definitely higher for BI07-wt than for BI07-res, a partial resistance of the mutated RNA polymerase at rifaximin concentrations > 10 μg/ml was supposed, on the basis of the calculated differences in inhibition percentages between BI07-wt and BI07-res. By considering the resistance of entire BI07-res cells to rifaximin concentrations > 100 μg/ml, supplementary resistance mechanisms may take place in vivo. A barrier for the rifaximin uptake in BI07-res cells was suggested in this study, on the basis of the major portion of the antibiotic found to be bound to the cellular pellet respect to the portion recovered in the cellular lysate. Related to this finding, a resistance mechanism involving changes of membrane permeability was supposed. A previous study supports this hypothesis, demonstrating the involvement of surface properties and permeability in natural resistance to rifampicin in mycobacteria, isolated from cases of human infection, which possessed a rifampicin-susceptible RNA polymerase. To understand the mechanism of membrane barrier, variations in percentage of saturated and unsaturated FAs and their methylation products in BI07-wt and BI07-res membranes were investigated. While saturated FAs confer rigidity to membrane and resistance to stress agents, such as antibiotics, a high level of lipid unsaturation is associated with high fluidity and susceptibility to stresses. Thus, the higher percentage of saturated FAs during the stationary phase of BI07-res could represent a defence mechanism of mutant cells to prevent the antibiotic uptake. Furthermore, the increase of CFAs such as dihydrosterculic acid during the stationary phase of BI07-res suggests that this CFA could be more suitable than its isomer lactobacillic acid to interact with and prevent the penetration of exogenous molecules including rifaximin. Finally, the impact of rifaximin on immune regulatory functions of the gut was evaluated. It has been suggested a potential anti-inflammatory effect of rifaximin, with reduced secretion of IFN-γ in a rodent model of colitis. Analogously, it has been reported a significant decrease in IL-8, MCP-1, MCP-3 e IL-10 levels in patients affected by pouchitis, treated with a combined therapy of rifaximin and ciprofloxacin. Since rifaximin enables in vivo and in vitro selection of Bifidobacterium resistant mutants with high frequency, the immunomodulation activities of rifaximin associated with a B. lactis resistant mutant were also taken into account. Data obtained from PBMC stimulation experiments suggest the following conclusions: (i) rifaximin does not exert any effect on production of IL-1β, IL-6 and IL-10, whereas it weakly stimulates production of TNF-α; (ii) B. lactis appears as a good inducer of IL-1β, IL-6 and TNF-α; (iii) combination of BI07-res and rifaximin exhibits a lower stimulation effect than BI07-res alone, especially for IL-6. These results confirm the potential anti-inflammatory effect of rifaximin, and are in agreement with several studies that report a transient pro-inflammatory response associated with probiotic administration. The understanding of the molecular factors determining rifaximin resistance in the genus Bifidobacterium assumes an applicative significance at pharmaceutical and medical level, as it represents the scientific basis to justify the simultaneous use of the antibiotic rifaximin and probiotic bifidobacteria in the clinical treatment of intestinal disorders.

Valutazione dell'impatto della farmacogenetica in ambito di Sanità Pubblica. Analisi del polimorfismo genetico dell'enzima CYP2D6 metabolizzante xenobiotici.

Pharmacogenetic testing provides an outstanding opportunity to improve prescribing safety and efficacy. In Public health policy pharmacogenetics is relevant for personalized therapy and to maximize therapeutic benefit minimizing adverse events. CYP2D6 is known to be a key enzyme responsible for the biotransformation of about 25-30% of extensively used drugs and genetic variations in genes coding for drug-metabolizing enzymes might lead to adverse drug reactions, toxicity or therapeutic failure of pharmacotherapy. Significant interethnic differences in CYP2D6 allele distribution are well established, but immigration is reshaping the genetic background due to interethnic admixture which introduces variations in individual ancestry resulting in distinct level of population structure. The present thesis deals with the genetic determination of the CYP2D6 alleles actually present in the Emilia-Romagna resident population providing insights into the admixture process. A random sample of 122 natives and 175 immigrants from Africa, Asia and South America where characterized considering the present scenario of migration and back migration events. The results are consistent with the known interethnic genetic variation, but introduction of ethnic specific variants by immigrants predicts a heterogeneous admixed population scenario requiring, for drugs prescription and pharmacogenetics studies, an interdisciplinary approach applied in a properly biogeographical and anthropological frame. To translate pharmacogenetics knowledge into clinical practice requires appropriated public health policies, possibly guiding clinicians to evaluate prospectively which patients have the greatest probability of expressing a variant genotype.