Orientador: Jose Wilson Magalhães Bassani / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de Computação / Made available in DSpace on 2018-08-14T22:04:59Z (GMT). No. of bitstreams: 1 Vilela_MarianaZacchi_M.pdf: 658413 bytes, checksum: 6f3a00651b1e8932faed96b1f700db9e (MD5) Previous issue date: 2009 / Resumo: Neste trabalho foi descrito o desenvolvimento e a aplicação de um indicador tridimensional de atividades produtivas (ICEB). O indicador foi aplicado para auxiliar o gerenciamento de tecnologia medica no Centro de Engenharia Biomédica (CEB) da Universidade Estadual de Campinas (UNICAMP). As três dimensões consideradas foram desempenho ( D CEB I ), custo ( C CEB I ) e satisfação ( S CEB I ). ICEB foi calculado com dados obtidos de ordens de serviço (OS) executadas pela equipe de Engenharia Clinica (EC) do CEB. O indicador foi usado para analisar: a) diferentes setores do hospital da UNICAMP; b) diferentes grupos da equipe de manutenção; c) técnicos da equipe de EC; e d) diferentes classes de equipamento. A analise foi realizada no período de 2001 a 2006. Os resultados mostraram diferencas no atendimento da manutenção entre as áreas estudadas. Diferenças importantes no desempenho entre os membros da equipe também foram encontradas. A analise de variância bifatorial mostrou diferencas significativas entre os setores do hospital, no entanto não tiveram relação com o ano da analise. Tendo em vista a diversidade de locais e equipamentos envolvidos e que os custos com pecas e serviços externos tem se elevado ao longo dos anos o C CEB I manteve-se estável e não apresentou diferencas significativas de 2001 a 2006. Diferenças nos D CEB I entre alguns técnicos do CEB chamam a atenção para o que pode ser necessidade de treinamento e qualificação. A aplicação de ICEB mostrou também que quanto maior a complexidade dos equipamentos, maior o tempo e os custos com manutenção. Alem das dimensões mais clássicas (desempenho e custo), a dimensão referente a satisfação do cliente ( S CEB I ) mostrou correlação com o desempenho ( D CEB I ). No entanto, analises adicionais devem ser feitas para explorar melhor este aspecto. As informações obtidas da analise tridimensional possibilitam a conclusão de que determinados setores do hospital requerem atenção especial em pelo menos uma das três dimensões. / Abstract: This work describes the development and application of a general tri-dimensional indicator of productive activities (ICEB). This indicator was applied for helping management of health technology at the Center for Biomedical Engineering (CEB), University of Campinas (UNICAMP). The three dimensions taken into account were performance ( D CEB I ), cost ( C CEB I ) and satisfaction ( S CEB I ). ICEB calculation was based on data obtained from the service orders (CEB database) undertaken by the Clinical Engineering (CE) staff of CEB. The indicator was used to analyze: a) support to different sectors of UNICAMP hospitals; b) different groups within the CE staff; c) individual technicians of the CE staff; and d) different types of equipment. The analysis was performed for the period from 2001 to 2006. The results indicate that the maintenance support to the different sectors is not homogeneous. Important differences were also detected in the performance of the staff members. Two-way analysis of variance detected significant differences among sectors of the hospital, which, were not related to the year of analysis. It is quite surprising that although prices for equipment parts, as well as for third-party service contracts, have markedly increased during the years, C CEB I did not change appreciably from 2001 to 2006. Differences in D CEB I among technicians of the CEB staff may indicate the need for further training and qualification. Application of ICEB also made it clear that, as the complexity of the medical device increases, time to repair and maintenance cost also increase. In addition to the more classical dimensions (performance and cost), the client satisfaction dimension ( S CEB I ) was showed to correlate with performance ( D CEB I ). Further quantitative analysis must be carried on in the future to better explore this aspect. The information obtained from the three-dimensional analysis made it possible to conclude that some hospital areas require special attention, for one or more of the analyzed dimensions. / Mestrado / Engenharia Biomedica / Mestre em Engenharia Elétrica

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-10-29T14:01:15Z No. of bitstreams: 2 Dissertação - Priscilla Santos Ferreira Ream - 2014.pdf: 1809988 bytes, checksum: e8bbea19753fbdf4001543d169a14bea (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-10-30T10:10:48Z (GMT) No. of bitstreams: 2 Dissertação - Priscilla Santos Ferreira Ream - 2014.pdf: 1809988 bytes, checksum: e8bbea19753fbdf4001543d169a14bea (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-30T10:10:48Z (GMT). No. of bitstreams: 2 Dissertação - Priscilla Santos Ferreira Ream - 2014.pdf: 1809988 bytes, checksum: e8bbea19753fbdf4001543d169a14bea (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-04-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Hospital housekeepers (HH) in healthcare services, while not working in direct patient care situations, are at risk of suffering accidents with biological material (ABM) as they are responsible for the management of waste. Objectives of this study were: to identify the frequency and profile of accidents with biological material among hospital housekeepers, characterize conduct pre and post-accidents, and compare the profile between the first and the last accidents among workers with repeated exposure. Epidemiological retrospective study of records of ABM with HH in the State of Goias, Brazil, between 1989 and June, 2012. Two sources of information were used to generate a database: medical records of all professionals who suffered ABM treated at the Hospital for Tropical Diseases, and the state database for notifications of ABM from the Information System for Notifiable Accidents referencing all records of labor ABM reported. For probabilistic record linkage of these databases, LinkPlus® was used. After selecting records of interest, referring to the HH, data were analyzed using Statistical Package for the Social Sciences, with descriptive and inferential statistics. Ethical guidelines were followed in all stages of the study (Approved in Ethics Committee, case: 033/2010 e 414.258/2013). The study examined 8,568 records of ABM, 996 (11.6%) occurring among HH. In 57 (6.1%) workers, more than one record was found, totalling 938 accidents. Among these HH, (65.6%) were aged 21-40 years, with an average of 35.6 years of age. Most common injuries were percutaneous, (98.5%), involved blood (85.5%) caused by hypodermic needles (75.1%), and due to improper sharps disposal (70.8%). In 48.2% of cases, the HH completes a vaccination program against hepatitis B (HBV) and of those, 81.7% were immune; 39.1% indicated no records of this vaccine. The difference between the number of HBV in the vaccinated HH first and last accidents was statistically significant, however, 24.5% were not vaccinated before both accidents. In most records, post-exposure prophylaxis and immunoprophylaxis were not recommended. In 86.4%, records had no accompanying clinical and laboratory information, or indicated abandonment of the HH. Given the profile and circumstances of accidents, it can be inferred that health services share responsibility for mismanagement of these injuries with this injury in terms of waste management, especially in proper segregation of waste by health professionals, the generators of the waste. Measures that can make waste management safer are presented as strategies for the prevention of accidents in this population: proper management, safety devices, and continuing education for all professionals involved. Greater accountability of health services in the management of biological risk is required for policies establishing monitoring of vaccinations as well as clinical and laboratory status is considered due to high rates of non-immunization and abandonment of monitoring found post-accident. Some analyses were limited by lack of records, reinforcing the need for improved management of biohazard waste in health services. / Os Trabalhadores da Limpeza e Conservação de Serviços de Saúde (TLCSS) mesmo não atuando na assistência direta a pacientes, também correm o risco de sofrerem acidentes com material biológico (AMB), pois são responsáveis pelo manejo dos resíduos de serviços de saúde (RSS). Foram objetivos deste estudo: identificar a frequência e o perfil dos acidentes com material biológico entre os Trabalhadores da Limpeza e Conservação de Serviços de Saúde, caracterizar condutas pré e pós-acidentes e comparar o perfil entre o primeiro e o último acidentes entre trabalhadores reincidentes. Estudo epidemiológico retrospectivo, realizado em registros de AMB ocorridos entre TLCSS no Estado de Goiás de 1989 a junho de 2012. Utilizaram-se duas fontes de informação para a geração do banco de dados (BD): registros de prontuários de todos os profissionais que sofreram AMB atendidos no Hospital de Doenças Tropicais e BD estadual do Sistema de Informação de Agravos de Notificação referente a todos os registros de AMB laboral notificados. Utilizou-se o LinkPlus® para o linkage probabilístico desses BD. Após seleção dos registros de interesse, referentes aos TLCSS, os dados foram analisados no Statistical Package for the Social Science® com estatística descritiva e inferencial. Preceitos éticos foram seguidos em todas as etapas do estudo (Aprovações em Comitês de Ética: 033/2010 e 414.258/2013). De 8.568 registros de AMB, 996 (11,6%) ocorreram entre TLCSS. Identificaram-se mais de um registro de AMB em 57 (6,1%) trabalhadores, sendo 938 o número de acidentados. Entre esses TLCSS, 65,6% tinham idade entre 21 a 40 anos com média de 35,6 anos. Predominaram acidentes percutâneos (98,5%), com sangue (85,5%) causados por agulhas hipodérmicas (75,1%) devido ao descarte inadequado de perfurocortantes (70,8%). Em 48,2% dos casos, os TLCSS apresentaram esquema vacinal completo contra vírus da hepatite B (HBV) e desses, 81,7% eram imunes; 39,1% dos registros indicavam ausência dessa vacina. A diferença entre a quantidade de TLCSS vacinados contra HBV no primeiro e no último acidentes foi estatisticamente significante, entretanto 24,5% não eram vacinados em ambos os acidentes. Na maioria dos registros, a profilaxia pós-exposição e imunoprofilaxia não foram recomendadas. Em 86,4% dos registros, não havia informação referente ao acompanhamento clínico-laboratorial ou indicava o abandono pelos TLCSS. O perfil e as circunstâncias dos AMB permitem inferir que existe responsabilidade compartilhada dos serviços de saúde com esse agravo por deficiências no gerenciamento dos RSS, especialmente, na segregação feita pelos profissionais de saúde, seus geradores. Apresentam-se como estratégias para a prevenção dos AMB nos TLCSS todas as medidas que podem tornar os RSS e o seu manejo mais seguros: o correto gerenciamento, os dispositivos de segurança e os processos de educação permanentes de todos os profissionais envolvidos. Considera-se necessária maior responsabilização dos serviços de saúde na gestão do risco biológico para os TLCSS, estabelecendo políticas de acompanhamento do status vacinal e clínico-laboratorial, devido às altas taxas de não imunização e abandono do acompanhamento pós-acidente encontradas. Algumas análises foram limitadas pela falta de registros, reforçando a necessidade de melhoria da gestão do risco biológico em serviços de saúde.

Understanding protein function is fundamental to the fields of protein engineering and drug design. While most of the previous efforts in this direction have focused on the sequence-structure-function paradigm, recent studies have pointed to protein dynamics as being integral to its activity. The work in the current thesis follows this overall theme of obtaining insights into protein function from its structure and dynamics. It can be broadly divided into two sections. In the first section, the thesis candidate has tried to elucidate the residues modulating the substrate specificity of a particular family of enzymes, known as sortases, through structural and computational studies (including dynamics simulations) on a novel member in the family. This work has been carried out in collaboration with Dr. R.P. Roy, National Institute of Immunology, New Delhi (biochemical characterization was performed by Mr. Vijay Pawale at Dr. Roy‟s laboratory). In the second half of this thesis, the candidate has described a structure-based method involving the use of cis-peptide containing segments for the function annotation of proteins. The incorporation of dynamics information leads to an improvement of our annotation approach, which is also demonstrated. This part of the work has been carried out in collaboration with Dr. Debnath Pal, Department of Computational and Data Sciences, Indian Institute of Science. Following is a chapter-wise description of the overall layout of the thesis. Section I: Insight into substrate specificity in sortase enzymes from structural studies on a novel housekeeping sortase of class E (SrtE) Chapter 1| A brief account of sortases: This chapter provides a brief survey of the literature on sortases and the scope of the work presented in the thesis. Many surface proteins in Gram-positive bacteria are incorporated into the cell wall through covalent ligation by a class of cysteine transpeptidases known as Sortase. These surface proteins contain a cell wall sorting signal (CWSS) which is recognized by sortase, enzymatically cleaved and subsequently joined covalently to the pentaglycine branch of lipid II (a peptidoglycan precursor) in general, which is finally incorporated into the peptidoglycan cell wall. Six classes of sortases have been identified on the basis of their sequence. These sortases differ in the substrate motif that they recognize and the function performed. The class A sortase (SrtA) is expressed ubiquitously in Gram-positive bacteria. It is involved in the cell surface anchoring of a large number of functionally distinct proteins which contain an LPXTG recognition motif in their CWSS, and is referred to as the „house-keeping‟ sortase. Sortases of other types are not ubiquitous and are meant to perform specialized functions. Sortase B is involved in iron acquisition, sortase C in pilus formation and sortase D in sporulation. The substrate motifs recognized by these sortases are, in general, different from the recognition motif in SrtA substrates. Several Gram-positive bacteria with a high GC content in their genome have been suggested to use a sortase E (SrtE) instead of SrtA to perform the housekeeping activity. These sortase sequences share low identity with sortases of classes A-D. The substrates of SrtE have been proposed to contain an LAXTG recognition motif instead of LPXTG based on genomic analyses. Class F consists of sortases from several Actinobacteria. However, the biological function of these sortases is not well understood. To date, structures of sortases from classes A-D have been determined, all of which display an eight-stranded beta barrel fold (termed the sortase fold), a conserved catalytic triad of His-Cys-Arg and a TLXTC motif at the active site (C: catalytic Cysteine; X varies across the different classes of sortases). Sortase B and C are augmented by additional secondary structure features which are absent in sortase A. SrtA from Staphylococcus aureus is the most well studied among sortases of known structure. Several of the surface proteins attached by sortases are responsible for bacterial virulence. SrtA deletion mutants have been found to exhibit reduced virulence without affecting cell viability. Moreover, the localization of sortase in the cell membrane and the absence of eukaryotic homologs have made sortase an attractive target for the development of novel therapeutics. In addition, the transpeptidase activity of sortase has found extensive applications in biotechnology. The prototype SrtA from Staphylococcus aureus is commonly used for these applications; however, its use is limited by its obligate Ca2+ ion-dependent activity and the stringent preference for an LPXTG motif. Hence, characterization of new sortases with altered substrate recognition profiles and rational modification of known sortases has tremendous potential for biotechnological applications and advancements. While sortases of classes A-D have been studied extensively to date and their structures determined, no structural data is available for a class E sortase. The thesis candidate has solved the first high resolution crystal structure of a putative housekeeping Sortase E in Streptomyces avermitilis (SavSrtE), a bacterium with a GC rich genome. Biochemical experiments performed by our collaborator on this protein have demonstrated Ca2+ independent transpeptidase activity and a preference for LAXTG-containing peptides as its cognate substrate over the LPXTG motif that is recognized by sortase A. Moreover, the protein exhibits a preference for small uncharged residues in the position succeeding the penta-peptide motif. This thesis documents the results of crystal structure analyses, molecular docking studies and dynamics simulations to understand the structural basis for these experimental findings. Finally, sequence analyses were performed to detect possible residues which modulate substrate specificity. Based on these analyses, mutations were performed. The thesis also documents the crystal structure solution and analysis of an active site mutant (residue T196 at the position X in the TLXTC motif). Chapter 2| Methods for the analyses of Sortase E from S. avermitilis (SavSrtE): This chapter provides a description of the procedures used to carry out the thesis work. An N-terminus truncated construct (∆N50) of wild type SavSrtE and its mutant T196V were cloned, expressed and purified in the laboratory of our collaborator, Dr. R.P. Roy (NII, New Delhi), and provided to us for structure and sequence analyses. Initially, crystallization trials were carried out on the wild type protein using commercially available screening kits and the sitting drop vapor diffusion method. The condition which gave crystals was optimized further. Finally, diffraction quality crystals were obtained in a drop containing 1μL of protein (4 mg/mL in 10 mM Tris-HCl buffer pH 7.2, 100 mM NaCl and 2 mM beta-mercaptoethanol) mixed with 1μL solution of the crystallization condition containing 1.6 M ammonium sulfate, 0.1 M citric acid at pH 3.75 using the hanging drop vapor diffusion method. The crystals were cryo-protected in a 10% sucrose solution and diffraction data collected at the European Synchrotron Radiation Facility (BM-14, ESRF). The crystals diffracted to 1.65Å. The protein crystallized in the P3221 space group with unit cell parameters a = b = 85.84Å, c = 48.20Å, α = β = 90°, γ = 120°. Calculation of Matthews coefficient indicated the presence of one molecule in the asymmetric unit. T196V mutant protein yielded diffraction quality crystals in the same condition as the wild type protein. The crystals were cryo-protected using sucrose and diffraction data were collected at the BM-14 beamline. The mutant crystals diffracted to 1.70Å. The protein crystallized in the P3221 space group with unit cell parameters a = b = 84.98Å, c = 48.00Å, α = β = 90°, γ = 120° and one molecule in the asymmetric unit. The quality of the datasets was assessed by SFCHECK and data were found to be of appropriate quality for structure solution. SavSrtE has low sequence identity (25 – 34%) to other class A sortases of known structure. Hence the scaled data, sequence information and model coordinates (sortase A from Streptococcus agalactiae, PDB ID: 3rcc) were submitted to the MR (molecular replacement) phasing option in the EMBL-Hamburg AutoRickshaw pipeline. The model generated from the server was used as input to PHASER for MR. The MR solution was subjected to one cycle of rigid body refinement followed by several cycles of restrained refinement using REFMAC from the CCP4 suite, with alternate rounds of inspection and manual model building in COOT for model improvement. The convergence of the refinement procedure was checked from the reduction in R-factors. The most essential refinement statistics for the final models of the wild type protein and T196V mutant are tabulated below. Table 1 Wild type (5GO5) T196V (5GO6) Resolution 1.65 Å 1.70 Å Rwork / Rfree (%) 16.11 / 19.05 17.31 / 20.82 R.M.S. bond lengths (Å) 0.012 0.019 R.M.S. bond angles (°) 1.53 1.89 Average B-factors (Å2) Protein 19.1 32.5 Water 32.6 42.4 SO42- 58.7 60.8 Gly 36.0 - Ramachandran map statistics Most favoured region (%) 86.8 89.8 Additional allowed region (%) 13.2 10.2 Generously allowed region (%) 0.0 0.0 Outliers (%) 0.0 0.0 The genome of S. avermitilis was searched using the ScanProsite tool to identify putative substrates, details of which are also documented in this chapter. Additionally, the thesis candidate performed Mutual Information analysis on an alignment of 1569 sortase sequences from different classes to identify the residues possibly regulating substrate specificity. Based on this analysis, mutations were performed of which the T196V mutant has been studied in this thesis. Finally, this chapter describes the protocol used to perform protein peptide docking and subsequent molecular dynamics simulations to understand how dynamics may influence substrate specificity. Chapter 3| Analyses of SavSrtE sequence and structure: This chapter provides a description of the analyses on the wild type SavSrtE and the T196V mutant. The overall fold of SavSrtE is very similar to that observed in the structures of other sortases, although the sequence similarity to other classes is low. Variations are observed in the loop regions (longer β1/β2 and β6/β7 loops). The active site is comprised by residues from the β2/H1 loop, β3/β4 loop, β4 strand, β6/β7 loop, β7 strand, β7/β8 loop and β8 strand. It also does not carry any cluster of electronegative residues close to the active site and therefore, is expected to have Ca2+ ion independent activity, which is observed in biochemical experiments (Dr. R.P. Roy‟s lab). Comparison with other housekeeping sortases showed that the β6/β7 loop in SavSrtE is in a closed conformation, indicating the presence of a preformed binding pocket for the LAXTG substrate binding, contrary to the prototype SrtA from Staphylococcus aureus which requires a Ca2+ ion to stabilize the closed conformation. Moreover, a small pocket is observed adjacent to the catalytic triad which contained electron density fitting a Gly molecule. This pocket is proposed to be the binding site for the second substrate that resolves the protein-peptide intermediate through a nucleophilic attack. Our docking simulations showed that a Gly of a triglycine moiety can be positioned in this pocket. Biochemical experiments established that SavSrtE recognizes the substrate motif LAXTG instead of LPXTG which is preferred by class A sortases. It also prefers Gly based nucleophiles as the second substrate. Additionally, the protein is found to prefer neutral residues over charged residues in the position succeeding the Gly of the LAXTG motif. Structure analyses showed the presence of a bulky Tyr residue (Y112) at the active site pocket which, according to molecular docking studies, hinders the productive binding of Pro-containing peptides (LPXTG) over Ala-containing ones (LAXTG). The OH group of Y112 is involved in a hydrogen bond with the backbone nitrogen of the second Ala in the ALANT peptide but not in the Pro-containing peptide. Y112 is held rigidly in place via interactions with neighbouring residues and a network of hydrogen-bonded water molecules in the crystal structure. A Tyr residue is found to be present in an equivalent position in several sortase sequences of Class E, and may be a general feature responsible for the specificity of sortase Es to putative LAXTG-containing substrates in their genomes. It may be mentioned that class D sortases, which contain a Phe residue at the equivalent position, recognize the LPXTA substrate motif. The side chain of this Phe displays different rotamers in the NMR structure of Bacillus anthracis SrtD, pointing to its flexibility, whereas Y112 in S. avermitilis SrtE is rigid. In addition, molecular dynamics simulations on the models of protein-peptide complex (obtained from docking) showed that the two peptides have similar backbone dynamics, unlike the case of S. aureus SrtA where the Ala-containing peptide does not maintain a kinked conformation similar to the Pro-containing cognate peptide. Hence the Tyr at the active site appears to be the main factor behind the discrimination of the two peptides. Substrate sequences in the S. avermitilis genome contain small neutral residues in the position succeeding the Thr-Gly peptide bond in the substrate. This preference is also observed in biochemical assays. Docking calculations showed that the protein cannot accommodate large side chains in the site where this residue is positioned. To detect the residues involved in altering the substrate specificity of SavSrtE, we performed a multiple sequence alignment using 1569 sortase sequences and carried out mutual information (MI) analysis on this data. Our analysis implicated several residue pairs lining the active site pocket in modulating substrate specificity. These included the aforementioned Tyr residue as well as the position X (T196 in SavSrtE) in the TLXTC motif at the active site. Mutations were performed at these positions and crystallization trials performed. We could successfully crystallize and solve the structure of the T196V mutant, which has been documented in this thesis. The mutant protein has the same overall structure as the wild type. Moreover, the catalytic Cys residue was observed to be unmodified in this structure, compared to the wild type which was presumably altered by β-mercaptoethanol added during protein purification. The mutated residue (Val) was found to have a different side chain rotamer than T196. Moreover, the absence of any polar atom in the side chain of V196 disrupted the hydrogen-bonded network of water molecules observed at the active site in the wild type structure. Experiments on the mutant showed a reduction in activity, implying that T196 is important for substrate recognition. The altered side chain orientation of V196 is expected to be responsible for the reduction in activity, though a peptide-bound crystal structure would be necessary to clearly understand the mechanism. In this respect, future crystallization trials may be performed with modified peptides that bind covalently to the active site Cys residue, similar to the strategy employed for S. aureus SrtA and Bacillus anthracis SrtA. Our structure and sequence analyses have pointed to some residue positions responsible for the modified substrate specificity. While only one mutant has been characterized, the other mutants also need to be studied (through biochemical asssays and structure analysis) to understand how they contribute to substrate recognition. In this context, double mutants may also be generated to understand the combined effect. For example, single mutations of E105 and E108 were found to reduce the activity of Staphylococcus aureus SrtA, while the double mutant resulted in Ca2+ ion independent activity. Additional structure and sequence analysis coupled with experiments are necessary to detect residues which may be mutated to enhance the activity of SavSrtE, similar to what has been performed for S. aureus SrtA. To summarize, our studies show that the substrate specificity of SavSrtE is different from that of class A sortases, and provide an explanation for it using structure analyses and computation. This altered specificity profile, orthogonal to that of S. aureus SrtA, and Ca2+ ion independent activity make it a potential candidate for use in simultaneous conjugation of multiple peptide substrates to their target. Moreover, this structure may be used firstly as a model to design inhibitors for housekeeping srtEs from pathogenic organisms like Corynebacterium diphtheriae and Tropheryma whipplei. Secondly, most of the previous studies on inhibitor design for sortases documented small molecules or peptidomimetics binding to the pocket of the first substrate. Since distinct binding pockets have been observed for the two substrates in SavSrtE, this information may be used to build inhibitors targeting the second pocket or spanning both the pockets. Section II: Identifying functionally important cis-peptide containing segments in proteins and their utility in molecular function annotation Chapter 4| Functionally important cis-peptide fragments in proteins: detection and relevance: This chapter describes the relevance of cis-peptides to protein function and a method to detect such functionally important cis-peptides in proteins. Cis-peptide bonds are comparatively rare in proteins due to the steric strain associated with the 1,4-atomic clash in the peptide chain. Consequently, only about 0.03% of Xaa-Xnp (Xaa: any amino acid; Xnp: any amino acid other than Pro) peptide bonds occur in the cis conformation; the occurrence is somewhat higher (5%) for imino peptide-containing Xaa-Pro cases. Despite their low occurrence, cis-peptides have been found to be evolutionarily conserved, pointing to their important role in structure and function. Cis-Xnp peptide bonds exhibit a significant disposition towards ligand-binding sites and dimerization interfaces, whereas cis-Pro bonds have been found to occur in a rare „touch-turn‟ motif at functional sites. Cis-trans isomerization is expected to play a regulatory role in many cellular processes. Non-conservation of these peptides is implicated in the evolution of different function among similar protein folds. Hence, there has been a renewed interest in detecting cis-peptides from residue patterns and linking them to molecular function. The importance of proteins as molecular 'workhorses' makes it imperative to understand how they function. However, a vast majority of the proteins catalogued in public sequence and structure databases do not have experimentally verified functional annotation. Experimental approaches are inadequate to manually curate these large numbers of un-annotated proteins. This necessitates the use of computational function prediction tools. The simplest prediction methods involve the assessment of similarity in sequence and three-dimensional structure with homologous proteins of known function. The presence of high overall similarity, however, does not predict function unambiguously since certain protein folds are associated with multiple functions while proteins with different folds may share functional traits. Often proteins with different global structure are found to have structural similarity at the local level of segments of residues that are responsible for the similarity in function. This has given rise to fragment-based (FB) function annotation methods. FB methods may involve locating functionally relevant surface patches or cavities formed by sequentially distant residues, or the presence of structurally conserved, contiguous residue fragments with proven relevance to function. The direct relevance of the cis-peptide bond to protein function suggests its use for the purpose of function annotation in a FB approach, yet no method exists to exploit it. This chapter describes a method using geometric clustering and level-specific Gene Ontology (GO) molecular-function (MF) terms to identify, in a statistically significant manner, cis-peptide embedded fragments (henceforth referred to as cis-fragments) in a protein linked to its molecular function. Such fragments were associated with GO MF based propensity value ≥ 20 at p-value ≤ 0.05, indicating the statistical significance of our results. The relevance of the identified cis-fragments to protein function was further verified through a literature survey. The features of these fragments are discussed in this chapter. Some of these fragments do not overlap with known PROSITE patterns, depicting the utility of these fragments as sequence patterns. Moreover, the thesis candidate identified contiguous stretches of functionally important trans-peptide fragments and cis-fragments forming extended structure-based functional signatures. Chapter 5| Use of functionally important cis-fragments in annotation: In this chapter, the candidate describes how a library of cis-peptide embedded fragments with proven association to molecular function can be useful for annotating proteins with known structure (and having cis-peptide) but unknown function. The functionally important fragments detected in the previous chapter were searched for exact matches in sequence and cis-peptide in a test set of PDB entries of known function at different thresholds of sequence redundancy and p-value. Additionally, the match or mis-match in GO MF term between the functionally important fragment and the test protein was also evaluated. To assess the efficiency of our method in annotation, true positive rate (TPR) and false positive rate (FPR) were calculated at each threshold as follows: TPR  TP and FPR  FP TP  FN FP TN The following table explains how the numbers of cases with TP, FP, etc. were assigned. Cases with match in Match in cis-peptide No match in cis- sequence peptide Match in GO MF TP FN No match in GO MF FP TN The cis-fragments alone were sufficient to identify other proteins with similar function. Over different thresholds, TPR >0.91 and FPR <0.23 were observed. Annotation recall benchmarks interpreted using receiver-operator-characteristic-plot returned >0.9 area-under-curve, corroborating the utility of the annotation method. Further, the applicability of our method in fragment-based function annotation is illustrated for cases where homology-based annotation transfer is not possible. The work presented here adds to the repertoire of function annotation approaches and also facilitates engineering, design and allied studies around the cis-peptide neighbourhood of proteins. The results presented in chapters 4 and 5 have already been published (reprint enclosed) with the thesis candidate as the first author. Chapter 6| Molecular dynamics information improves cis-peptide based function annotation of proteins: The preceding chapters have demonstrated the use of functionally relevant cis-peptide segments in a homology-independent, fragment match-based protein function annotation method. However, proteins are not static molecules; their dynamics is integral to their activity. Hence we have incorporated the dynamics (obtained using an in-house coarse-grained forcefield) of functionally important cis-peptide segments in our annotation method. This is the first study to include both static and dynamics information to improve the prediction of protein molecular function. To ascertain the improvement upon incorporating dynamics, the ACV-based dynamics profiles (details in chapter) were compared in a dataset consisting of 102 pairs each of positive data (PDB entries with match in fragment sequence and cis-peptide) and negative data (PDB entries with match in fragment sequence but no match in cis-peptide). Our analyses depicted that using only cis-peptide information gave less false positives and a low FPR (0.11), which is desirable, but also a relatively low TPR (0.72). This is due to large FN (trans-peptide with matching GO MF), which can arise when the cis-fragment undergoes cis-trans isomerization to accomplish its function and coordinates have been obtained for the segment in the test data in the trans-state, or if there is an error in assignment of the omega angle during structure solution. On the other hand, using only dynamics information increases the numbers of both true and false positives and hence the TPR (0.95) and FPR (0.51). This is due to false-positive matches for cases where fragments with similar secondary structure show similar dynamics, but the proteins do not share a common function. Combining the predictions from the two methods reduces errors while detecting the true matches, thereby enhancing the utility of our method in function annotation (TPR: 0.95 and FPR: 0.07). Subsequently, we have combined static and dynamics information to annotate proteins of unknown function. A combined approach, therefore, opens up new avenues of improving existing automated function annotation methodologies. The work described in this chapter has been submitted to a peer reviewed journal. Future prospects include the development of a web server to facilitate the application of our method by a wide research community. A possible improvement includes identification and comparison of the dynamics of additional sites close to the identified cis-fragment, in an automated manner, to improve the accuracy of our annotation. Appendix 1 gives a description of the results of biochemical experiments performed in the laboratory of our collaborator Dr. R.P. Roy, NII, New Delhi. Appendix 2 contains additional data supplementary to chapter 4. Appendix 3 provides additional data supplementary to chapter 5. Appendix 4 provides additional data supplementary to chapter 6. Appendix 5 contains reprints of publications.

This study aimed to analyze factors associated to occupational accidents with workers from the Hospital Housekeeping of a public university hospital in Rio Grande do Sul, Brazil. It is a cross-sectional study, involving 157 workers. It was used a form for socio-demographic, labor, habit, health and occupational accidents characterization, as well as an Effort-Reward Imbalance scale. Data colection was developed between March and June 2013. This study is part of the matrix project Evaluation of work and health conditions of hospital housekeeping workers , inserted in the Research Group Work, Health, Education and Nursing of Nursing Department of Federal University of Santa Maria, in the Research line Work and Management in Nursing and Health. Epiinfo 6.04 and PASW 18.0 were used on data preparation and analysis. To verify associations, it was adopted Chi-square or Fisher s Exact Test, Prevalence ration and its respective trust intervals (TI95%). Multivariate regressions were used to identify the association adjusted for confounders (p<0,25). As a result, it was evidences a prevalence of female, mean age of 39.9 years (DP=9,8), with self-referred white race and complete high school education. We observed a 33.1% prevalence of exposure to occupational stress and 17.8% of accidents at work. It was predominant sharps injuries and falls. The hands and fingers were the body parts most affected. Younger workers, those with no time for leisure, the day and those who reported insufficient number of workers at work scale were significantly associated with greater exposure to stress. After adjusting for confounding factors, it was found that workers who reported not having in-service training had higher prevalence of work injury. This research will contribute a perspective of health promotion and continuing health education, providing benefits to employees of the SHL in general. / Este estudo teve por objetivo analisar os fatores associados à ocorrência dos acidentes de trabalho com trabalhadores do Serviço Hospitalar de Limpeza de um hospital universitário público do Rio Grande do Sul, Brasil. Trata-se de um estudo transversal, envolvendo 157 trabalhadores. Utilizou-se um formulário para a caracterização sociodemográfica, laboral, de hábitos, saúde, acidentes de trabalho, e a versão brasileira da escala Desequilíbrio Esforço-Recompensa. A coleta de dados foi realizada entre os meses de março e junho de 2013. Este estudo faz parte do projeto matricial Avaliação das condições de trabalho e saúde dos trabalhadores do serviço hospitalar de limpeza , inserido no Grupo de Pesquisa Trabalho, Saúde, Educação e Enfermagem, do Departamento de Enfermagem da Universidade Federal de Santa Maria, na Linha de Pesquisa Trabalho e Gestão em Enfermagem e Saúde. Os programas Epiinfo 6.04 e PASW 18.0 foram utilizados na preparação e análise dos dados. Para verificar associações, adotou-se o teste Qui-quadrado ou Exato de Fisher, Razão de Prevalência e seus respectivos intervalos de confiança (IC95%). Regressões multivariadas foram utilizadas para identificar a associação ajustada por fatores de confundimento (p<0,25). Como resultados, Evidenciou-se predomínio do sexo feminino, idade média de 39,9 anos (DP=9,8), raça autorreferida branca e ensino médio completo. Observaram-se prevalências de 33,1% de exposição ao estresse ocupacional e 17,8% de ocorrência de acidentes de trabalho. Predominaram os acidentes com perfurocortantes e quedas. As mãos e os dedos foram as partes do corpo mais atingidas. Os trabalhadores mais jovens, os sem tempo para o lazer, os do diurno e os que referiram número insuficiente de trabalhadores na escala de trabalho apresentaram associação significativa com maior exposição ao estresse. Após ajustes por fatores de confundimento, verificou-se que os trabalhadores que referiam não ter treinamento no serviço tiveram maior prevalência de acidente de trabalho. Esta pesquisa poderá contribuir numa perspectiva de promoção da saúde e de educação permanente em saúde, proporcionando benefícios aos trabalhadores do SHL de uma maneira geral.

This study aimed to evaluate the relation between healthy habits and work ability of hospital cleaning staff in a university hospital from Rio Grande do Sul. It is a cross-sectional research with quantitative approach developed with 157 workers, from March to April 2013. It was used the Perception of Healthy Habits Questionnaire (QPHAS), and the Brazilian version of Index of Work Capacity (ICT). Data analysis was developed on the software PASW 18.0, through descriptive and multivariate statistics. It was found 87.9% of workers to be female, aged average 39,9 years old (±9,8); 38,9% with high school education; average of family income per capita lower than the national minimum wage (average 0.86 (±0.47), minimum of 0.20 and maximum 2.95 wages); 65.6% cleaning servants; 4.4%, Material cleaning helpers; with an average of 32.5 months (±48.9) of working at the institution; 58.6% needed medical assistance last year, 6.4%, of psychological following and 51% used medication. Regarding to perception of healthy habits, 67.5% of the workers had high perception. Dimensions in body weight control, diet and physical activity QPHAS, respectively, 70.7%, 73.9% and 68.8% showed high awareness of healthy habits. With respect to capacity for the current job, 36.6% identified as 10 score perceived , in relation to the physical and mental demands 44.4% and 63.5% respectively showed how good the current capacity to work, 29.9% showed no disease diagnosed by a physician, 56.1% reported not having impediment to his current work in relation to their illness or injury, 56.7% showed no absence from work; 77.7% reported the ability to be good two years from now they can play their current job, 54.8% reported being always satisfied with the daily activities of their work, 71% reported feeling constantly full of hope for the future; 79.6% had good/great capacity for work, and 20.4% low/moderate. Revealed a low positive correlation between ICT and power dimensions (r = 0.174, p = 0.029) and physical activity (r = 0.207, p = 0.009) and overall score QPHAS (r = 0.179, p = 0.025). After adjustment for confounding factors (p <0.25) factors, not having time for leisure was associated with reduced work ability (OR = 2.26, 95% CI 1.268 to 4.015, p = 0.006). These results can inform the planning of promotion and prevention of health hazards to workers Housekeeping, Hospital, contributing to the maintenance of work ability. / Este estudo objetivou avaliar a associação entre hábitos saudáveis e capacidade para o trabalho nos trabalhadores do Serviço Hospitalar de Limpeza de um hospital universitário público do Rio Grande do Sul. Trata-se de uma pesquisa transversal com abordagem quantitativa realizada com 157 trabalhadores, no período de março a abril de 2013. Utilizaram-se o Questionário de Percepção de Hábitos Saudáveis (QPHAS) e a versão brasileira do Índice de Capacidade para o Trabalho (ICT). A análise dos dados foi realizada no programa PASW 18.0, por meio da estatística descritiva e multivariada. Encontraram-se 87,9% de trabalhadores do sexo feminino, com idade média de 39,9 anos (±9,8); 38,9% com ensino médio completo; renda familiar per capita média menor que um salário mínimo nacional (média 0,86 (±0,47), mínimo de 0,20 e máximo de 2,95 salários); 65,6% Serventes de Limpeza; 34,4%, Auxiliares de Limpeza de Materiais; com tempo médio de trabalho na instituição de 32,5 meses (±48,9); 58,6% necessitaram de atendimento médico no último ano; 6,4% de acompanhamento psicológico e 51% faziam uso de medicação. Em relação à percepção de hábitos saudáveis, 67,5% dos trabalhadores apresentaram alta percepção. Nas dimensões controle do peso corporal, alimentação e atividade física do QPHAS, respectivamente 70,7%, 73,9% e 68,8% apresentaram alta percepção de hábitos saudáveis. Em relação à capacidade para o trabalho atual, 36,6% apontaram 10 como pontuação percebida; em relação às exigências físicas e mentais 44,4% e 63,5% respectivamente apontaram como boa a capacidade atual para o trabalho; 29,9% não apresentaram nenhuma doença diagnosticada pelo médico; 56,1% referiram não ter impedimento para o seu trabalho atual em relação às suas doenças ou lesões; 56,7% não apresentaram nenhuma falta ao trabalho; 77,7% referiram ser boa a possibilidade de daqui a dois anos conseguirem desempenhar o seu trabalho atual; 54,8% referiram estarem sempre satisfeitos com as atividades diárias de seu trabalho; 71% referiram sentir-se continuamente cheios de esperança para o futuro; 79,6% apresentaram boa/ótima capacidade para o trabalho, e 20,4%, baixa/moderada. Evidenciou-se correlação positiva baixa entre o ICT e as dimensões alimentação (r=0,174; p=0,029) e atividade física (r=0,207; p=0,009) e no escore geral do QPHAS (r=0,179; p=0,025). Após ajustes por fatores de confundimento (p<0,25), não ter tempo para o lazer se mostrou associado à redução da capacidade para o trabalho (OR=2,26; IC95%=1,268 4,015; p=0,006). Estes resultados podem subsidiar o planejamento de ações de promoção e de prevenção de agravos à saúde dos trabalhadores do Serviço Hospitalar de Limpeza, contribuindo para a manutenção da capacidade para o trabalho.

The thesis handles the topic of families and households from the point of view of EU-SILC, Statistika rodinných účtů and also Sčítání lidu, domů a bytů, especially from the year 2011. It discribes kinds, types of families and households and what is possible on basis of gained information about each family and household see on national as well as international level. Another part of the thesis is focused on definition of poverty limit and it´s impact risks on particular family types mainly in Czech households. On the base of previous development, the future development tendency of number and structure of households and families is mentioned and is expressed by three projections concerning the household development.

Actualmente, la pandemia ha generado un cambio inesperado en la vida cotidiana de las personas a nivel mundial. A raíz de ello, en el Perú los pobladores se vieron obligados a adaptarse a la utilización de las plataformas digitales, puesto que muchos negocios cerraron debido a los nuevos protocolos de salubridad, por lo que tuvieron que modificar sus planes estratégicos. Es por ello por lo que, nos encontramos en una situación en la cual debemos adaptarnos al mundo digital. En este presente trabajo nos enfocaremos en el servicio de limpieza, en el cual consideramos como público objetivo a los clientes que soliciten este servicio a través de un aplicativo móvil, en el cual se implementará inteligencia artificial. Estará dirigido al NSE A y B + que están compuestos de hogares y personas que residen solas; y que estén dispuestos a adquirir este tipo de servicio. También, estará dirigido a personas que ofrecen el servicio de limpieza, ya que muchos de ellos fueron afectados gravemente por la pandemia y se busca la forma de ayudarlos a que tengan un mayor alcance hacia sus clientes. Los experimentos que se realizarán nos ayudarán a validar los problemas que existen hoy en día en este rubro, como por ejemplo la falta de tiempo en realizar actividades domésticas. Además, el contenido de este trabajo de investigación abarcará el desarrollo del plan financiero para validar la viabilidad de este proyecto en base a datos reales. / Currently, the pandemic has generated an unexpected change in the daily lives of people worldwide. As a result, in Peru the inhabitants were forced to adapt to the use of digital platforms, since many businesses closed due to the new health protocols, so they had to modify their strategic plans. That is why we find ourselves in a situation in which we must adapt to the digital world. In this present work, we will focus on the cleaning service, in which customers who request this service through a mobile application are considered as the target audience, in which artificial intelligence will be implemented. It will be aimed at NSE A and B + that are made up of households and people who reside alone; and that they are available to purchase this type of service. It will also be aimed at people who offer cleaning services, since many of them were seriously affected by the pandemic and a way is being sought to help them have a greater reach towards their clients. The experiments that will be carried out will help us to validate the problems that exist today in this area, such as the lack of time to carry out domestic activities. In addition, the content of this research work will include the development of the financial plan to validate the viability of this project based on real data. / Trabajo de investigación

Understanding the mechanisms of transcriptional regulation and the constraints that operate in gene promoter sequences is the key step in understanding the evolutionary conservation of transcriptional regulation. It is well known that regulatory regions with the same expression outputs do not have to share the sequence similarity. The most important elements in regulatory sequences are transcription factor binding sites and their position relocation does not usually influence the expression output. The least complex transcriptional regulation is characteristic for housekeeping genes. For their expression they require only basal core promoter elements (sometimes only CpG islands are sufficient) and general transcription factors, so they can be transcribed easily and immediately whenever they are needed. In this study we focused on transcriptional regulation of invertebrate amphioxus (Branchiostoma floridae) housekeeping genes in vertebrate systems. We prepared a set of constructs with amphioxus regulatory regions for testing their activity in different mammalian cell lines and a set of constructs with the same amphioxus regulatory regions for observing their spatial recognition in developing medaka fish embryo. We found that half of investigated amphioxus regulatory regions are recognized by...

Background: The pain in general, located in different areas of the body, presents with higher prevalence in adolescents. At the national level it is estimated that three out of four children between 6 and 17 years of age dedicate approximately 11 hours a week to activities within their home, which implies repetitive mechanics, muscle overloads and as a consequence the presence of pain. Objectives: Determine the association between the number of hours an adolescent does household activities and the presence of pain. Design: Cross sectional analytical observational that analyzes a secondary population data base of the INEI in urban and rural areas of the 24 departments of Peru and in the Constitutional Province of Callao. The data collection period was from October to December 2015. Patients/Materials: The secondary base of the INEI was included for children and adolescents from 10 to 17 years old. The dependent variable was defined as the presence of pain in the back, head or bones. The independent variable was defined as the number of hours dedicated to domestic activities and was categorized. The descriptive analysis shows absolute and relative frequencies for the categorical variables and in the bivariate, the Chi2 test was used. Finally, in the multivariate analysis, the Poisson model was used considering the 95% confidence intervals. Results: The results obtained according to our objectives show that adolescents who perform domestic activities for a time greater than 3 to 13 hours have a 10% lower risk of presenting pain compared to those of 1 to 3 hours (CI = 0.84 - 0.97) and a p value = 0.007. In a multivariate analysis adjusted to area, region, sex, age (years) -categorized, reason for which they carry out domestic work, adolescents who carry out dangerous tasks and work outside the home; adolescents who perform domestic activities in a time greater than 3 to 13 hours have a 5% lower risk of presenting pain compared to those of 1 to 3 hours (CI = 0.87 - 1.04) and a value of p = 0.320 Conclusion: Our study was able to determine that there is an association between the number of hours an adolescent performs domestic activities and the presence of pain. / Introducción: El dolor en general, localizado en diferentes zonas del cuerpo se presenta con mayor prevalencia en los adolescentes con un rango de 20% a 46% a nivel mundial. A nivel nacional se estima que tres de cada cuatro niños entre los 6 y 17 años dedican aproximadamente 11 horas semanales a las actividades dentro de su hogar, lo que implica mecánicas repetitivas, sobrecargas musculares y como consecuencia la presencia de dolor. Objetivos: Determinar la asociación entre el número de horas que un adolescente de 10 a 17 años realiza actividades domésticas y la presencia de dolor. Diseño: Estudio observacional analítico de corte transversal que analiza una base secundaria poblacional del INEI en zona urbana y rural de los 24 departamentos del Perú y en la Provincia Constitucional del Callao. El periodo de recolección de la data fue en los meses de octubre a diciembre del 2015. Participantes/Materiales: Se incluyó de la base secundaria del INEI a niños y adolescentes de 10 a 17 años. La variable dependiente se definió como presencia de dolor en espalda, cabeza o huesos. La variable independiente se definió como el número de horas dedicadas a actividades domésticas y fue categorizada. En el análisis descriptivo se muestran frecuencias absolutas y relativas para las variables categóricas y en el bivariado se utilizó la prueba de Chi2. Finalmente, en el análisis multivariado se utilizó el modelo de Poisson considerando los intervalos de confianza al 95%. Resultados: En el análisis multivariado ajustado a la variable Área, Región, Sexo, Edad (años) categorizada, Motivo por el cual realiza la labor doméstica y trabajo fuera del hogar se encontró que los adolescentes que realizan Actividades domésticas por un tiempo mayor de 3 hasta 13 horas tienen 8% menor probabilidad de presentar dolor de espalda cabeza o huesos en comparación a los adolescentes que realizan las actividades del hogar durante 1 a 3 horas y un valor estadísticamente significativo de p= 0.029 (IC= 0.85 – 0.99), Conclusión: Nuestro estudio logró determinar que sí hay asociación entre la cantidad de horas que un adolescente realiza actividades domésticas y la presencia de dolor. / Tesis

Since the beginning of the early 20th century bed rugs have been interesting research objects, but only in passing researchers have paid attention to rugs woven with rags. Noone has taken a closer look upon the reasons why people have woven these rugs. What does the materials of the rag – like recycled garments and interior textiles in the form of clothing rags – have to tell about the times when these rugs were woven? Did the rag rugs have any specific function or were the materials available and therefore used? The main sources of information are 21 rag rugs from Roslagen in Uppland, with a varied amount of rags. They were woven during the latter half of 19th century, and estate inventories from Väddö- and Häverö Ship District have altogether given some answers to the primary question of this paper: in wich way can the examined bed rugs bear witness to the use and value of recycled textile materials and the view of these in the context of the community where they were manufactured and used? This research doesn´t give an answer to whether these rugs have any particular function or not. Instead these rag rugs can be looked upon as representing a general development of the society towards an increasing amount of textiles surplus material. This being due an increasing consumption of factory-made clothing and textiles, manufactured in factories, as well as the paper mills development from producing paper made of cellulose rather than textile waste. To this we can add a principle lingering on from the 19th century, of domestic production and a thrift of resources. This resulted in an obvious recycling of discarded textiles. The home weaving of interior textiles was still strong by the end of the 19th century, and in Rosagen there was also a long tradition of weaving and of using rugs in the beds. At the same time there was, in the coastal regions of Roslagen, a local need for warming covers in boats and boat houses. This demand was related to the shooting of seals and other hunting in the coastal areas, as well as in the fishing- and maritime trades.