This thesis deals with identification of wine yeasts isolated during fermentation process of wine cider and grapes of Sauvignon grape variety, grown in the integrated vineyard. The identification and taxonomic classification is faster and easier due to the progress of molecular methods. In this thesis PCR-RFLP method was used for identification of yeasts. Sequences of DNA specific for each species were analysed. These sequences were amplified by means of PCR method and by using ITS1 and ITS4 primers. In following step, they were put through the restrictiction analysis with five restriction endonucleases. Fragments of DNA were separated by horizontal electrophoresis. The electrophoreograms were evaluated by BioNumerics software and final dendrogram representing genetics similarity of isolated yeasts was created by using UPGMA claster analysis. The basic information about yeasts and their identification by molecular methods are described in the theoretical part of this thesis.

The theoretical part discusses the yeasts and their taxonomic classification using traditional methods and using modern methods. Detail the work is concerned with descriptions of modern molecular-biology methods. The practical part was analyzed DNA by PCR-fingerprinting (rep-PCR) type of yeasts, which we received from the CCY and subsequent analysis of yeast samples obtained from grape musts. One of the grape must was obtained in 2009 (white grape variety) and the second in 2010 (red grape variety). Both grape musts come as integrated vineyards and organic. Grape musts samples were obtained from the winery Holánek from Ivaň. The cross-comparison of images PCR-fingerprint type yeasts and yeasts PCR-fingerprint samples using BioNumerics was to evaluate the results and conclude that the diversity of yeast flora in grape must.

This thesis deals with identification of yeasts isolated during spontaneous fermentation of grape juice. For analysis the Pinot Noir grape variety grown in the integrated vineyard was chosen. In the theoretical part of this thesis basic information about yeasts are described. Genera of yeasts that occurs during fermentation process and methods based on PCR are also described. In this thesis PCR-RFLP method was used for identification of yeasts. The amplification of the 5,8S-ITS rDNA sequence was performed by the polymerase chain reaction with use of the primers ITS1 and ITS4. The restriction analysis was performed by applying five restriction endonucleases: HaeIII, HinfI, Taq?, AluI, MseI. The amplicons were split into fragments which length and number are typical for the particular species. These fragments were identified by agarose gel electrophoresis and electrophoreograms were evaluated by BioNumerics software. Dendrograms representing genetic similarity of isolated wine yeasts were created by using UPGMA cluster analysis.

The thesis deals with monitoring of the yeast's impact to the chemical and sensorial characteristics of produced wine. The aim of experimental part was obtaining of the aroma-active substances in the fermenting must and wine Hibernal and wine Pinot Blanc. SPME-GC-MS methods were used for identify wines and must aroma. The same autochthonous yeasts were used for wine production. This yeast was isolated from surface of grapes in our laboratory. It was found that both wines have a similar aromatic profile. The next aim was the monitoring of chemical parameters of samples and their changes. For this measurement were used spectrophotometric methods and HPLC methods. The last goal of the thesis was the isolation and identification of yeasts from wine Pinot Blanc by PCR-RFLP. These yeasts were then compared with yeasts isolated from Hibernal must. Identification of yeasts were done by amplification 5,8S-ITS DNA stretches using primers ITS1 and ITS4. Restriction endonuclease HaeIII, HinfI, HhaI and TaqI. were used for restriction analysis then.

[EN] Acetic acid bacteria (AAB) belong to the Acetobacteriaceae family, within the ¿-Proteobacteria class. These Gram-negative elliptical or cylindrical microorganisms occur in isolation, in pairs or in chains. They have polar flagellar or peritrichous motility. They are non-spore forming, cathalase positive and oxidase negative. They have an obligate aerobic metabolism, with oxygen as the terminal electron acceptor. Currently, the Acetobactereaceae family includes 19 genera and 72 species. Sugar and alcohol oxidising ability result in an accumulation of organic acids as final products, which is widely used in the food industry to produce vinegar from wine and fruits. The objective of this thesis was to isolate and identify AAB in blueberry and citrus epiphyt flora from plants grown in the Salto Grande region (Entre Ríos, Argentina) in order to detect the most suitable bacteria for biotechnological processes such as vinegar. 36 AAB were isolated from these fruits using enrichment techniques and plate isolation. Enrichment broths containing ethanol and acetic acid enabled the maximum recovery of AAB due to the fact that these components promote their growth. Fermented juice yielded a larger number of AAB compared to fresh fruit or peel, due to ethanol occurrence as it is an alcohol-resistant microorganisms selecting agent. Biochemical tests allowed bacteria differentiation at genus level. Six were identified: 13 bacteria isolates were identified as Acetobacter, 5 as Gluconobacter, 7 as Asaia, 5 as Acidomonas, 1 as Gluconacetobacter and 5 as Saccharibacter. Subsequently, those bacteria (Acetobacter, Gluconobacter and Gluconacetobacter genera) that could be used for the development of a suitable starter culture for the bio-oxidation of musts alcoholics obtained from these fruits, in order to obtain vinegars, were identified to specie level. Molecular techniques, such as RFLP-PCR of the 16S rDNA and RFLP-PCR of the 16S-23S rDNA intergenic spacer, were utilised. The former enabled the identification of the 16 AAB under study. C7, C8, A80, A160 and A180 isolates were identified as G. frateurii, C1, C2, C3, C4, C5, C6, A70 and A210 isolates as A. pasteurianus, A50 and A140 isolates as A. tropicalis and C9 isolate as A. syzygii. 16S-23S PCR-RFLP technique validated identifications performed using 16S; however, C1 showed a different restriction pattern from the first identification. Genus 16S partial sequencing solved this discrepancy. Growth dynamics and acetic acid production capacity were studied in the isolates in order to determine the most suitable ones to acetify fruits musts. Growth was assessed in different ethanol and acetic acid concentrations as high cell count values (109 cells/mL) are essential for the inoculum to start vinegar production, in order to achieve a short lag phase and therefore reduce process times. Three ethanol concentrations were assayed (4%, 6% and 8%) to study acetic acid production capacity. Results showed that cultures identified as A. pasteurianus (A210 and C1), A. syzygii (C9) and G. frateurii (A80) could be used as inoculum to produce vinegars, due to their high acetic acid production capacity when ethanol concentration values are 4% and 6% v/v. Finally, the most suitable culture preservation method was determined to maintain purity and activity through time. AAB may be lyophilised with 20% w/v mannitol or 10% w/v dried milk since these lyoprotective agents demonstrated they are effective at maintaining cells viability. Nevertheless, these agents did not allow acetic acid production from ethanol. This study has demonstrated that glycerol (20% v/v) and mannitol (20% w/v) can be used as cryoprotective agents during freezing since they not only protect AAB and maintain bacteria viability but also help to preserve the functional properties, such as its ability to grow and produce acetic acid in alcoholic musts. / [ES] Las bacterias del ácido acético (BAA) pertenecen a la familia Acetobacteriaceae; están incluidas en el grupo de las ¿-Proteobacterias. Son microorganismos Gram-negativos, de forma elipsoidal o cilíndrica que pueden encontrarse aislados, en parejas o formando cadenas. Son móviles por flagelación polar o perítrica. Presentan actividad catalasa positiva, oxidasa negativa y no forman endosporas. Poseen metabolismo aeróbico estricto, con el oxígeno como aceptor final de electrones. Actualmente, la familia Acetobactereaceae está compuesta por 19 géneros y 72 especies. Es conocida la habilidad de las BAA para oxidar azúcares y alcoholes, obteniéndose como producto final una acumulación de ácidos orgánicos, capacidad que es aprovechada en la industria de alimentos para la elaboración de vinagres de vinos y de frutas. La presente Tesis Doctoral planteó el aislamiento e identificación de BAA a partir de la flora epifítica de arándanos y frutas cítricas cultivadas en la región de Salto Grande (Entre Ríos, Argentina), con el fin de encontrar las más adecuadas para ser utilizadas en procesos biotecnológicos, tales como el vinagre. Se aislaron 36 BAA a partir de estas frutas mediante técnicas de enriquecimiento y aislamiento en placas. Los caldos de enriquecimiento que contenían etanol y ácido acético permitieron recuperar el mayor número de BAA, ya que dichos componentes favorecen el crecimiento de las mismas. Del jugo fermentado de las frutas cítricas se obtuvo un mayor número de BAA respecto del jugo fresco o la cáscara, debido a la presencia de etanol, el que actuó como agente de selección para estos microorganismos alcohol-resistentes. Las pruebas bioquímicas permitieron diferenciar las bacterias a nivel de género. Se reconocieron 6 géneros: 13 aislados fueron identificados como Acetobacter, 5 como Gluconobacter, 7 como Asaia, 5 como Acidomonas, 1 como Gluconacetobacter y 5 como Saccharibacter. Posteriormente, se identificaron a nivel de especie aquellas bacterias (géneros Acetobacter, Gluconobacter y Gluconacetobacter) que podrían ser utilizadas para el desarrollo de un cultivo iniciador apto para la bioxidación de mostos alcohólicos obtenidos a partir de estos frutos, con el fin de obtener vinagres. Para esto, se emplearon técnicas moleculares, tales como PCR-RFLP del gen 16S y PCR-RFLP del espaciador intergénico 16S-23S. Con la primera, se identificaron las 16 BAA estudiadas. Los aislados C7, C8, A80, A160 y A180 fueron identificados como G. frateurii, los aislados C1, C2, C3, C4, C5, C6, A70 y A210 como A. pasteurianus, los aislados A50 y A140 como A. tropicalis y el aislado C9 como A. syzygii. Se estudió la dinámica de crecimiento y la habilidad de las BAA aisladas para producir ácido acético, con el fin de elegir las más aptas para la acetificación de mostos de frutas. El crecimiento se evaluó con distintas concentraciones de etanol y ácido acético. En cuanto a la habilidad para producir ácido acético, se ensayaron tres concentraciones de etanol, 4%, 6% y 8%, evidenciándose que los cultivos, identificados como A. pasteurianus (A210 y C1) A. syzygii (C9) y G. frateurii (A80) podrían ser utilizados como inóculo para la elaboración de vinagres, por poseer una alta capacidad de producción de ácido acético cuando la concentración de etanol es de 4% y 6% v/v. Las BAA podrían ser liofilizadas con manitol 20% p/v o leche en polvo 10% p/v, ya que estos lioprotectores demostraron ser efectivos para mantener la viabilidad de las células. Sin embargo, éstos no permitieron mantener la producción de ácido acético a partir de etanol. El presente estudio demostró que el glicerol (20% v/v) y el manitol (20% p/v) pueden ser utilizados como crioprotectores en el proceso de congelación, ya que no sólo protegen a las BAA, manteniendo su viabilidad, sino que también ayudan a conservar las propiedades funcionales de las mismas, tales como su capacidad de crecer y producir ácido / [CA] Els bacteris de l'àcid acètic (BAA) pentanyen a la família Acetobacteriaceae i estan incloses en el grup dels ¿-Proteobacteris. Són microorganismes gram-negatius, de forma elipsoidal p cilíndrica que pot trobar-se aïllada, emparellada o formant cadenes. Són mòbils per flagel¿lació polar o perítrica. Presenten activitat catalasa positiva, oxidasa negativa i no formen endospores. Posseeixen metabolisme aeròbic estricte, amb l'oxigen com aceptor final d'electrons. Actualment, la familia Acetobactereaceae està composta per 19 gèneres i 72 espècies. Es coneguda l'habilitat dels BAA per a oxidar sucres i alcohols, obtenint-se com a producte final una acumulació d'àcids orgànics, capacitat aprofitada en la industria dels aliments per a l'elaboració de vinagres de vins i fruites. La present Tesi Doctoral planteja l'aïllament i identificació dels BAA a partir de la flora epifítica dels nabius i cítrics cultivats en la regió de Salto Grande (Entre Ríos, Argentina), amb la finalitat de trobar les més adequades per ser utilitzades en processos biotecnològics, tals com el vinagre. S'aïllaren 36 BAA a partir d'aquestes frutes mitjançant tècniques d'enriquiment i aïllament en plaques. Els brous d'enriquiment que contenien E i AA permeteren recuperar el major numero de BAA, ja que tals components afavorien el seu creixement. Del suc fermentat dels citrics es va obtenir un major nombre de BAA respecte del suc fresc o la pell, degut a la presència de E, que actuà com agent de selecció per a aquestos microorganismes alcohol-resistents. Es reconegueren 6 gèneres: 13 aïllats foren identificats com Acetobacter, 5 com Gluconobacter, 7 com Asaia, 5 com Acidomonas, 1 com Gluconacetobacter i 5 com Saccharibacter. Posteriorment, s'identificaren a nivell d'espècie aquells bacteris que podríen ser utilitzats per al desenvolupament d'un cultiu indicadorstarter apte per a la biooxidació de mostos alcohòlics obtinguts a partir d'aquestos fruïts, amb la finalitat d'obtenir vinagres de fruites (gèneres Acetobacter, Gluconobacter i Gluconacetobacter). Amb aquesta finalitat, s'utilitzaren tècniques moleculars, com PCR-RFLP del gen 16S i PCR-RFLP de l'espaciador intergènic 16S-23S. Amb la primera, s'identificaren els 16 BAA estudiats. La tècnica PCR-RFLP del 16S-23S va confirmar les identificacions realitzades amb el 16S, tanmateix C1 va mostrar un patró de restricció que no es corresponia amb la primera identificació realitzada. Aquesta discrepància fou resolta per la seqüenciació parcial de gen 16S, que va confirmar el resultats obtinguts per PCR-RFLP. Els aïllats C7, C8, A80, A160 i A180 foren identificats com G. Frateurii, els aïllats C1, C2, C3, C4, C5, C6, A70 i A210 com A. pasteurianus,els aïllats A50 i A140 com A.tropicalis i l'aïllat C9 com A. syzygii. S'estudià la dinàmica de creixement i la habilitat per produirAA dels BAA aïllades, amb la finalitat de triar les més aptes per a l'acetificació de mostos de frutes. El creixement s'evaluà amb diferents concentracions de E i AA. Quan a l'habilitat per a produir AA, s'assajaren tres concentracions d'etanol, 4%, 6% i 8% v/v, evidencianse que els cultius , identificats com A. pasteurianus (A210 i C1) A. syzygii (C9) i G. frateurii (A80) podrien ser utilitzats com inòcul per a l'elaboració de vinagres, per poseïr una alta capacitat de producció de AA quan la concentraició de E és de 4% i 6% v/v. Els BAA podrien ser liofilitzats amb manitol 20% p/v o llet en pols 10% p/v, ja que aquestos lioprotectors demostraren ser efectius per a mantenir la viabilitat de les cèl¿lules. Tanmateix, aquestos no permeteren mantenir la producció de AA a partir de E. El present estudi va demostrar que el glicerol (20% v/v) i el manitol (20% p/v) poden ser utilitzats com crioprotectors en el procés de congelació, ja que no sols protegeixen els BAA, mantenint la seua viabilitat, sinò que també ajuda a conservar les seues propietats funcionals, tals com la se / Gerard, LM. (2015). Caracterización de bacterias del ácido acético destinadas a la producción de vinagres de frutas [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/59401

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INTRODUÇAO: A leishmaniose atinge milhões de indivíduos mundialmente, relacionada a mudanças ambientais, urbanização, migração e susceptibilidade do hospedeiro. O aumento de casos de leishmaniose visceral (LV) em áreas urbanas pode ser explicado, não só pela adaptação do vetor a diferentes situações ambientais, circulação do parasito e introdução de hospedeiro infectado, como também pela intersecção com áreas de transmissão do HIV. No Brasil, a distribuição dos coinfectados acompanha os grupos de risco para HIV/AIDS (adultos, sexo masculino). A coinfecção LV-HIV/AIDS é registrada com grande frequência no Estado de São Paulo, onde há aumento da prevalência desta coinfecção, assim como da recidiva e da letalidade por LV. Fatores contribuintes para esta elevação, como possíveis determinantes da gravidade da LV em pacientes HIV/AIDS, não estão claros, sejam relacionados ao hospedeiro ou ao parasito. OBJETIVOS: Avaliar o comportamento clínico, epidemiológico, terapêutico e imunológico e a variação genotípica do parasito na coinfecção LV-HIV/AIDS, comparando com pacientes HIV-negativos, em pacientes do Estado de São Paulo. MATERIAIS E MÉTODOS: Coorte retrospectiva, utilizando dados secundários de programas de rotina epidemiológica da Secretaria de Estado da Saúde de São Paulo e do Ministério da Saúde do Brasil, entre 1999-2010. Análise molecular por PCR-RFLP do kDNA de Leishmania (L.) infantum de aspirado de medula óssea para desenvolvimento de uma árvore fenética, comparando os indivíduos entre si quanto ao desfecho, sexo, idade e infecção pelo HIV. RESULTADOS: 1614 casos de LV e 117 (7,25%) de coinfectados LV-HIV/AIDS, com predomínio destes no sexo masculino, entre os 31-50 anos de idade. Tríade febre e hepatoesplenomegalia foi mais frequente no grupo HIV-negativo. Maior letalidade por LV (24,2 x 8,2 - p =0,000) e recidiva (10,5 x 1,8 - p = 0,000) nos pacientes HIV-positivos comparando aos HIVnegativos. Entre os coinfectados, observou-se maior taxa de cura quando a LV foi tratada com Antimonial Pentavalente (69,44%) e Anfotericina B lipossomal (63,82%), p=0,223. Maiores falhas (16,66%, p = 0,034) e letalidade (41,66%, p = 0,192) quando tratado com Anfotericina B deoxicolato. Maiores recidivas (14,89% - p = 0,076) e nenhuma falha com Anfotericina B lipossomal. Houve maior mediana de linfócitos T CD4+ (135) e T CD8+ (550) no grupo de cura dos pacientes LV-HIV/AIDS e houve 50% de recidivas em uso de terapia antirretroviral. A distribuição dos genótipos de Leishmania (L.) infantum não apresentou relação com nenhum dos desfechos avaliados. CONCLUSÕES: Os resultados obtidos revelam pela primeira vez a magnitude da comorbidade LV-HIV/AIDS no Estado de São Paulo, com repercussão direta na recidiva e na letalidade da LV. Há aumento do número de casos de LV e LV-HIV/AIDS nessa região, com maior prevalência de coinfectados em adultos do sexo masculino. Maior letalidade e recidiva nos HIV-positivos e com pior desfecho quando tratado com Anfotericina B deoxicolato. Recidiva elevada quando tratado com Anfotericina B lipossomal, embora sem falhas. Pouca proteção da terapia antirretroviral na proteção das recidivas. Muitos dados incompletos quanto à infecção pelo HIV. PCR-RFLP não discrimina casos HIV-positivos dos HIV-negativos, nem mostra relação direta das recidivas e óbitos com um genótipo específico do parasita, podendo a evolução do paciente estar relacionada diretamente com a resposta do hospedeiro / INTRODUCTION: Leishmaniasis affects millions of individuals worldwide, related to environmental changes, urbanization, migration, and host susceptibility. The increase in cases of visceral leishmaniasis (VL) in urban areas can be explained not only by the vector adaptation to different environmental situations, movement of the parasite and introduction infected host, as well as the intersection with areas of HIV transmission. In Brazil, the distribution of coinfected is accompanying risk groups for HIV/AIDS (adult male). Coinfection VL-HIV/AIDS is recorded with great frequency in São Paulo State, where there is an increased prevalence of co-infection, as well as relapse and lethality by VL. Factors contributing to this increase, as possible determinants of severity of VL in HIV/AIDS patients are not clear, either related to the host or the parasite. OBJECTIVES: To evaluate the clinical, epidemiological, therapeutic and immunological aspects of coinfection VL-HIV/AIDS and genotypic variation in the parasite, compared with HIV-negative patients in the State of São Paulo. MATERIALS AND METHODS: Retrospective cohort study using secondary data from epidemiological routine programs of the State Department of Health of São Paulo and the Ministry of Health of Brazil, between 1999 to 2010. Molecular analysis by PCRRFLP kDNA of Leishmania (L.) infantum from bone marrow aspirate to develop a phenetic tree, comparing individuals with each other about the outcome, gender, age and HIV infection. RESULTS: 1614 cases of VL and 117 (7.25%) of coinfected VLHIV/ AIDS, predominantly those in males, between 31-50 years old. Triad of fever and hepatosplenomegaly was more frequent among HIV-negative. Increased mortality by VL (24.2 x 8.2 - p =0,000) and recurrence (10.5 x 1.8 - p = 0,000) in HIV-positive patients compared to HIV-negative. Among coinfected, there was a higher cure rate when the VL was treated with pentavalent antimony (69.44%) and liposomal amphotericin B (63.82%), p = 0.223. Major failures (16.66%, p = 0.034) and mortality (41.66%, p = 0.192) when treated with amphotericin B deoxycholate. Major recurrences (14.89% - p = 0.076) and no failure with amphotericin B liposome. There were a higher median TCD4+ (135) and TCD8+ (550) lymphocytes in the group of cures and relapse was 50% in those using antiretroviral therapy. The genotype distribution of Leishmania (L.) infantum was not associated with any of the outcomes assessed. CONCLUSIONS: These results show for the first time the magnitude of comorbidity VL-HIV/AIDS in São Paulo State, with direct impact on recurrence and mortality of VL. There are increasing numbers of cases of VL and VL-HIV/AIDS in this region, with the highest prevalence of coinfection in adult males. There is an increased mortality and recurrence in HIV-positive and with worse outcome when treated with amphotericin B deoxycholate. High relapse when treated with liposomal amphotericin B, although flawless. There is little protection of antiretroviral therapy in relapses. There are many incomplete data regarding HIV infection. PCR-RFLP does not discriminate HIVpositive cases from HIV-negative ones or showed direct nexus from recurrences and deaths with a specific genotype of the parasite, but instead could be directly related to the host response

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-10-18T11:49:54Z No. of bitstreams: 1 2014 - Ramon de Sousa Rego.pdf: 1491511 bytes, checksum: 746021e21f53b13713de3e8e94d03121 (MD5) / Made available in DSpace on 2016-10-18T11:49:54Z (GMT). No. of bitstreams: 1 2014 - Ramon de Sousa Rego.pdf: 1491511 bytes, checksum: 746021e21f53b13713de3e8e94d03121 (MD5) Previous issue date: 2014-08-26 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / The protein quality of milk of goats and digestibility of the lipid fraction are important factors that stand out when compared to milk from cows. The ?-lactoglobulin is the higher abundant protein in whey ruminants being produced in the mammary gland during lactation. It may represent up to 12% of the total protein. We seek to present work was to evaluate the gene ?-Lactoglobulin (BLG) with their genetic polymorphisms in the promoter regions 5 'and 3' UTR and associate them the characteristics of milk production in experimental goat herd. For this 150 goats (Capra hircus) of Saanen and Alpine, genotyped for polymorphisms of the BLG gene were used. For genotyping 10 mL of blood per animal were collected. The blood was used for DNA extraction and amplification of two fragments of the BLG gene by means of polymerase chain reaction (PCR). The amplified fragments were submitted to electrophoresis on polyacrylamide gel and evaluated by by restriction fragment length polymorphism (PCR-RFLP). The SmaI and SacII enzymes were used for the promoter (promoter + exon 1 region) region and the region of exon 7 (exon 7 + region 3 '), respectively. The differences in cutting patterns were visualized on a polyacrylamide gel. In the promoter region of the single nucleotide polymorphism (SNP) at position -60 (C/T) was identified and was associated with the percentage of protein in milk. This result suggests a relationship between genotypes of the promoter region of the BLG gene and the protein level of the milk. The presence of two polymorphisms +4641 (I2/I3) and +4601 (A/G) was observed in the region of exon 7. The I2-I3 variation is characterized by a repeat sequence of 10 bp, varying in two and three times, and it being possible to identify by electrophoresis. The I3 variation was often very low, and there was no association between this polymorphism and any production trait. Polymorphism +4601 (G/A) was identified by enzyme SacII digestion. Alleles were identified frequencies close to those reported by other authors and were associated with the percentage of fat in milk goats, and the animals with the S1S2 genotype had higher fat percentage than S1S1 animals and S2S2. The increased production of lipid content may be related to the characteristic of transporting fatty acids ?-lactoglob / A qualidade proteica do leite de cabras e a digestibilidade da fra??o lip?dica s?o importantes fatores que o destacam, quando comparado ao leite de vacas. A ?-Lactoglobulina ? a prote?na de maior abundancia no soro do leite em ruminantes sendo produzida na gl?ndula mam?ria durante o per?odo de lacta??o. Ela pode representar at? 12% do total proteico. Buscamos com o presente trabalho, avaliar o gene da ?-Lactoglobulina (BLG) com seus polimorfismos gen?ticos nas regi?es promotoras 5? e 3? UTR e associ?-los as caracter?sticas de produ??o de leite no rebanho caprino experimental. Para isso foram utilizadas 150 cabras (Capra hircus) das ra?as Saanen e Alpinas, genotipadas para os polimorfismos do gene BLG. Para a genotipagem foram coletados 10 mL de sangue por animal. O sangue foi utilizado para a extra??o de DNA e amplifica??o de 2 fragmentos do gene BLG por interm?dio da rea??o em cadeia da polimerase (PCR). Os fragmentos amplificados foram ent?o submetidos ? eletroforese em gel de poliacrilamida e avaliados pelo polimorfismo no tamanho do fragmento por restri??o (PCR-RFLP). Foram utilizadas as enzimas SmaI e SacII, para a regi?o promotora (regi?o promotora + ?xon 1) e para a regi?o do ?xon 7 (?xon 7 + regi?o 3?), respectivamente. As diferen?as nos padr?es de corte foram visualizadas em gel de poliacrilamida. Na regi?o promotora o polimorfismo de base ?nica (SNP) na posi??o -60 (C/T) foi identificado e apresentou associa??o com a percentagem de prote?nas no leite. Esse resultado sugere uma rela??o entre os gen?tipos da regi?o promotora do gene BLG e o n?vel proteico do leite. Foi observado na regi?o do ?xon 7 a presen?a de dois polimorfismos +4641 (I2/I3) e +4601 (A/G). A varia??o I2-I3 ? caracterizada pela repeti??o de uma sequ?ncia de 10 pb, variando em duas e tr?s vezes, e sendo poss?vel identifica-la por eletroforese. A varia??o I3 teve frequ?ncia muito baixa, e n?o houve associa??o entre esse polimorfismo e nenhuma caracter?stica de produ??o. O polimorfismo +4601 (A/G) foi identificado por meio da digest?o da enzima SacII. Os alelos identificados tiveram frequ?ncias pr?ximas ?s relatadas por outros autores e apresentaram associa??o com a percentagem de gordura no leite cabras, sendo que os animais com o gen?tipo S1S2 apresentaram maior percentagem de gordura que animais S1S1 e S2S2. A maior produ??o de conte?do lip?dico pode estar relacionada com a caracter?stica de transporte de ?cidos graxos da ?-Lactoglobulina

INTRODUÇAO: A leishmaniose atinge milhões de indivíduos mundialmente, relacionada a mudanças ambientais, urbanização, migração e susceptibilidade do hospedeiro. O aumento de casos de leishmaniose visceral (LV) em áreas urbanas pode ser explicado, não só pela adaptação do vetor a diferentes situações ambientais, circulação do parasito e introdução de hospedeiro infectado, como também pela intersecção com áreas de transmissão do HIV. No Brasil, a distribuição dos coinfectados acompanha os grupos de risco para HIV/AIDS (adultos, sexo masculino). A coinfecção LV-HIV/AIDS é registrada com grande frequência no Estado de São Paulo, onde há aumento da prevalência desta coinfecção, assim como da recidiva e da letalidade por LV. Fatores contribuintes para esta elevação, como possíveis determinantes da gravidade da LV em pacientes HIV/AIDS, não estão claros, sejam relacionados ao hospedeiro ou ao parasito. OBJETIVOS: Avaliar o comportamento clínico, epidemiológico, terapêutico e imunológico e a variação genotípica do parasito na coinfecção LV-HIV/AIDS, comparando com pacientes HIV-negativos, em pacientes do Estado de São Paulo. MATERIAIS E MÉTODOS: Coorte retrospectiva, utilizando dados secundários de programas de rotina epidemiológica da Secretaria de Estado da Saúde de São Paulo e do Ministério da Saúde do Brasil, entre 1999-2010. Análise molecular por PCR-RFLP do kDNA de Leishmania (L.) infantum de aspirado de medula óssea para desenvolvimento de uma árvore fenética, comparando os indivíduos entre si quanto ao desfecho, sexo, idade e infecção pelo HIV. RESULTADOS: 1614 casos de LV e 117 (7,25%) de coinfectados LV-HIV/AIDS, com predomínio destes no sexo masculino, entre os 31-50 anos de idade. Tríade febre e hepatoesplenomegalia foi mais frequente no grupo HIV-negativo. Maior letalidade por LV (24,2 x 8,2 - p =0,000) e recidiva (10,5 x 1,8 - p = 0,000) nos pacientes HIV-positivos comparando aos HIVnegativos. Entre os coinfectados, observou-se maior taxa de cura quando a LV foi tratada com Antimonial Pentavalente (69,44%) e Anfotericina B lipossomal (63,82%), p=0,223. Maiores falhas (16,66%, p = 0,034) e letalidade (41,66%, p = 0,192) quando tratado com Anfotericina B deoxicolato. Maiores recidivas (14,89% - p = 0,076) e nenhuma falha com Anfotericina B lipossomal. Houve maior mediana de linfócitos T CD4+ (135) e T CD8+ (550) no grupo de cura dos pacientes LV-HIV/AIDS e houve 50% de recidivas em uso de terapia antirretroviral. A distribuição dos genótipos de Leishmania (L.) infantum não apresentou relação com nenhum dos desfechos avaliados. CONCLUSÕES: Os resultados obtidos revelam pela primeira vez a magnitude da comorbidade LV-HIV/AIDS no Estado de São Paulo, com repercussão direta na recidiva e na letalidade da LV. Há aumento do número de casos de LV e LV-HIV/AIDS nessa região, com maior prevalência de coinfectados em adultos do sexo masculino. Maior letalidade e recidiva nos HIV-positivos e com pior desfecho quando tratado com Anfotericina B deoxicolato. Recidiva elevada quando tratado com Anfotericina B lipossomal, embora sem falhas. Pouca proteção da terapia antirretroviral na proteção das recidivas. Muitos dados incompletos quanto à infecção pelo HIV. PCR-RFLP não discrimina casos HIV-positivos dos HIV-negativos, nem mostra relação direta das recidivas e óbitos com um genótipo específico do parasita, podendo a evolução do paciente estar relacionada diretamente com a resposta do hospedeiro / INTRODUCTION: Leishmaniasis affects millions of individuals worldwide, related to environmental changes, urbanization, migration, and host susceptibility. The increase in cases of visceral leishmaniasis (VL) in urban areas can be explained not only by the vector adaptation to different environmental situations, movement of the parasite and introduction infected host, as well as the intersection with areas of HIV transmission. In Brazil, the distribution of coinfected is accompanying risk groups for HIV/AIDS (adult male). Coinfection VL-HIV/AIDS is recorded with great frequency in São Paulo State, where there is an increased prevalence of co-infection, as well as relapse and lethality by VL. Factors contributing to this increase, as possible determinants of severity of VL in HIV/AIDS patients are not clear, either related to the host or the parasite. OBJECTIVES: To evaluate the clinical, epidemiological, therapeutic and immunological aspects of coinfection VL-HIV/AIDS and genotypic variation in the parasite, compared with HIV-negative patients in the State of São Paulo. MATERIALS AND METHODS: Retrospective cohort study using secondary data from epidemiological routine programs of the State Department of Health of São Paulo and the Ministry of Health of Brazil, between 1999 to 2010. Molecular analysis by PCRRFLP kDNA of Leishmania (L.) infantum from bone marrow aspirate to develop a phenetic tree, comparing individuals with each other about the outcome, gender, age and HIV infection. RESULTS: 1614 cases of VL and 117 (7.25%) of coinfected VLHIV/ AIDS, predominantly those in males, between 31-50 years old. Triad of fever and hepatosplenomegaly was more frequent among HIV-negative. Increased mortality by VL (24.2 x 8.2 - p =0,000) and recurrence (10.5 x 1.8 - p = 0,000) in HIV-positive patients compared to HIV-negative. Among coinfected, there was a higher cure rate when the VL was treated with pentavalent antimony (69.44%) and liposomal amphotericin B (63.82%), p = 0.223. Major failures (16.66%, p = 0.034) and mortality (41.66%, p = 0.192) when treated with amphotericin B deoxycholate. Major recurrences (14.89% - p = 0.076) and no failure with amphotericin B liposome. There were a higher median TCD4+ (135) and TCD8+ (550) lymphocytes in the group of cures and relapse was 50% in those using antiretroviral therapy. The genotype distribution of Leishmania (L.) infantum was not associated with any of the outcomes assessed. CONCLUSIONS: These results show for the first time the magnitude of comorbidity VL-HIV/AIDS in São Paulo State, with direct impact on recurrence and mortality of VL. There are increasing numbers of cases of VL and VL-HIV/AIDS in this region, with the highest prevalence of coinfection in adult males. There is an increased mortality and recurrence in HIV-positive and with worse outcome when treated with amphotericin B deoxycholate. High relapse when treated with liposomal amphotericin B, although flawless. There is little protection of antiretroviral therapy in relapses. There are many incomplete data regarding HIV infection. PCR-RFLP does not discriminate HIVpositive cases from HIV-negative ones or showed direct nexus from recurrences and deaths with a specific genotype of the parasite, but instead could be directly related to the host response

The overall objective of this study was to test the hypothesis that insects can vector F. tumidum conidia to infect gorse plants with the aim of developing an alternative approach to mycoherbicide delivery to control weeds. Four potential insect species (Apion ulicis, Cydia ulicetana, Epiphyas postvittana and Sericothrips staphylinus) were assessed for their ability to vector F. tumidum conidia. To achieve this, the external microflora (bacteria and fungi) and the size and location of fungal spores on the cuticle of these insect species were determined. In addition, the ability of the insects to pick up and deposit F. tumidum conidia on agar was studied. Based on the results from these experiments, E. postvittana was selected for more detailed experiments to determine transmission of F. tumidum to infect potted gorse plants. The factors promoting pathogenicity of F. tumidum against gorse and the pathogen loading required to infect and kill the weed were also determined. The external microflora of the four insect species were recovered by washing and plating techniques and identified by morphology and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and sequencing of internally transcribed spacer (ITS) and 16S rDNA. A culture-independent technique (direct PCR) was also used to assess fungal diversity by direct amplification of ITS sequences from the washings of the insects. All insect species carried Alternaria, Cladosporium, Nectria, Penicillium, Phoma, Pseudozyma spp. and entomopathogens. Ninety four per cent of the 178 cloned amplicons had ITS sequences similarity to Nectria mauritiicola. E. postvittana carried the largest fungal spores (mean surface area of 125.9 ìm2) and the most fungal CFU/insect. About 70% of the fungi isolated from the insects were also present on the host plant (gorse) and the understorey grass. The mean size of fungal spores recovered from the insect species correlated strongly with their body length (R² = 85%). Methylobacterium aquaticum and Pseudomonas lutea were common on all four insect species. Pseudomonas fluorescens was the most abundant bacterial species. In the pathogenicity trials, the effectiveness of F. tumidum in reducing root and shoot biomass of 16 and 8 wk old gorse plants was significantly increased with wounding of the plants. Older plants (32 wk old) which were wounded and inoculated were significantly shorter, more infected and developed more tip dieback (80%) than plants which were not wounded (32%). This indicates that damage caused by phytophagous insect species present on gorse through feeding and oviposition may enhance infection by F. tumidum. Wounding may release nutrients (e.g. Mg and Zn) essential for conidia germination and germ tube elongation and also provide easier access for germ tube penetration. Conidial germination and germ tube length were increased by 50 and 877%, respectively when incubated in 0.2% of gorse extract solution for 24 h compared with incubation in water. Inoculum suspensions amended with 0.2% of gorse extract caused more infection and significantly reduced biomass production of 24 wk old gorse plants than suspensions without gorse extract. A minimum number of about 900 viable conidia/infection site of F. tumidum were required to infect gorse leaves. However, incorporation of amendments (which can injure the leaf cuticle) or provision of nutrients (i.e. gorse extract or glucose) in the formulation might decrease the number of conidia required for lesion formation. Scanning electron micrographs showed that germ tube penetration of gorse tissue was limited to open stomata which partly explain the large number of conidia required for infection. The flowers and leaves were more susceptible to F. tumidum infection than the spines, stems and pods. An experiment to determine the number of infection sites required to cause plant mortality showed that the entire plant needs to be inoculated in order for the pathogen to kill 10 wk old plants as F. tumidum is a non systemic pathogen. The number of infection sites correlated strongly with disease severity (R² = 99.3%). At least 50% of the plant was required to be inoculated to cause a significant reduction in shoot dry weight. F. tumidum, applied as soil inoculant using inoculated wheat grains in three separate experiments, significantly suppressed gorse seedling emergence and biomass production. In experiments to determine the loading capacity of the insect species, E. postvittana, the largest insect species studied, carried significantly more (68) and deposited significantly more (29) F. tumidum conidia than the other species. Each E. postvittana, loaded with 5,000 conidia of F. tumidum, transmitted approximately 310 conidia onto gorse plants but this did not cause any infection or affect plant growth as determined by shoot fresh weight and shoot height. E. postvittana on its own did not cause any significant damage to gorse and did not enhance F. tumidum infection. It also failed to spread the pathogen from infected plants to the healthy ones. There was no evidence of synergism between the two agents and damage caused by the combination of both E. postvittana and F. tumidum was equivalent to that caused by F. tumidum alone. This study has shown that E. postvittana has the greatest capacity to vector F. tumidum since it naturally carried the largest and the most fungal spores (429 CFU/insect). Moreover, it naturally carried Fusarium spp. such as F. lateritium, F. tricinctum and Gibberella pulicaris (anamorph Fusarium sambucinum) and was capable of carrying and depositing most F. tumidum conidia on agar. Coupled with the availability of pheromone for attracting the male insects, E. postvittana may be a suitable insect vector for delivering F. tumidum conidia on gorse using this novel biocontrol strategy. Although it is a polyphagous insect, and may visit non-target plants, F. tumidum is a very specific pathogen of gorse, broom and a few closely related plant species. Hence, using this insect species to vector F. tumidum in a biological control programme, should not pose a significant threat to plants of economic importance. However, successful control of gorse using this "lure-load-infect" concept would depend, to a large extent on the virulence of the pathogen as insects, due to the large size of F. tumidum macroconidia, can carry only a small number of it.