Resumo: A falta de um kit otimizado para avaliação da resistência aos inibidores de integrase no Brasil conduziu à necessidade da padronização de uma técnica inhouse para o estudo de mutações no gene da integrase do HIV-1, possibilitando, assim, a avaliação da resistência a esta classe de antirretrovirais recentemente liberada no país. RNA viral plasmático foi utilizado como fonte para amplificação por RT- Nested PCR e sequencimento do gene da integrase do HIV-1 de 60 pacientes para análise do padrão de resistência, o qual foi avaliado utilizando o algoritmo proposto pela Universidade de Stanford. Cinquenta e quatro (90%) amostras obtiveram sucesso na amplificação e sequenciamento, sendo que destas dez foram amplificadas por protocolos alternativos que envolveram a utilização de enhancer, de enzimas com maior especificidade, RT-PCR one-step e alterações nas condições de ciclagem, o que possibilitou com sucesso o estudo da região de interesse e a análise de resistência. A metodologia in-house mostrou ser uma alternativa metodológica viável para avaliação da resistência aos inibidores de integrase, principalmente nos casos em que há falha terapêutica a esta nova classe de medicamento, uma vez que a técnica permite o estudo completo da região de interesse / Abstract: The absence of an optimized kit to evaluate the resistance to integrase inhibitors in Brazil led to the necessity for standardization of an in-house technique to study the mutations in the HIV-1 integrase gene, thus enabling the evaluation of resistance to these novel antiretroviral agents recently released in the country. Plasma viral RNA was obtained from 60 patients and used as source for RT- Nested PCR amplification and automatic sequencing of the HIV-1 IN gene to analysis of the pattern of drug resistance, which was evaluated using the algorithm proposed by Stanford University. Fifty-four (90%) samples were successfully amplified and sequenced, and ten of these samples were amplified using alternative protocols involving the use of enhancers, enzymes with more specificity, RT-PCR one step and changes in the conditions of cycling, which allowed the successful study of the region of interest and analysis of resistance. The in-house methodology showed to be a viable alternative to assess resistance to integrase inhibitors, especially in cases when there is therapeutic failure to this new class of antiretroviral, since the technique allows the complete study of the region of interest / Orientador: Maria Inês de Moura Campos Pardini / Coorientador: Rejane Maria Tommasini Grotto / Banca: Paulo Inácio da Costa / Banca: João Manuel Grisi Candeias / Mestre

The main purpose of this thesis is to encourage the development of bioeffect planning as an experimental tool by which means bioeffect plans may be compared with standard isodose plans. This thesis also addresses the fundamental problems of the derivation of useful biological models for clinical application and the description of tumour and normal tissue parameter values and their variability. Particular emphasis has been placed on comparing the predictive value of the models and parameters against clinical results of fractionated and continuous irradiation either alone or combined.

The main purpose of this thesis is to encourage the development of bioeffect planning as an experimental tool by which means bioeffect plans may be compared with standard isodose plans. This thesis also addresses the fundamental problems of the derivation of useful biological models for clinical application and the description of tumour and normal tissue parameter values and their variability. Particular emphasis has been placed on comparing the predictive value of the models and parameters against clinical results of fractionated and continuous irradiation either alone or combined.

Made available in DSpace on 2014-06-11T19:23:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-15Bitstream added on 2014-06-13T18:09:23Z : No. of bitstreams: 1 tanikawa_aa_me_botfm.pdf: 940370 bytes, checksum: 202b79e681e44fdd7980b5e940c2ade9 (MD5) / Ministério da Saúde / A falta de um kit otimizado para avaliação da resistência aos inibidores de integrase no Brasil conduziu à necessidade da padronização de uma técnica inhouse para o estudo de mutações no gene da integrase do HIV-1, possibilitando, assim, a avaliação da resistência a esta classe de antirretrovirais recentemente liberada no país. RNA viral plasmático foi utilizado como fonte para amplificação por RT- Nested PCR e sequencimento do gene da integrase do HIV-1 de 60 pacientes para análise do padrão de resistência, o qual foi avaliado utilizando o algoritmo proposto pela Universidade de Stanford. Cinquenta e quatro (90%) amostras obtiveram sucesso na amplificação e sequenciamento, sendo que destas dez foram amplificadas por protocolos alternativos que envolveram a utilização de enhancer, de enzimas com maior especificidade, RT-PCR one-step e alterações nas condições de ciclagem, o que possibilitou com sucesso o estudo da região de interesse e a análise de resistência. A metodologia in-house mostrou ser uma alternativa metodológica viável para avaliação da resistência aos inibidores de integrase, principalmente nos casos em que há falha terapêutica a esta nova classe de medicamento, uma vez que a técnica permite o estudo completo da região de interesse / The absence of an optimized kit to evaluate the resistance to integrase inhibitors in Brazil led to the necessity for standardization of an in-house technique to study the mutations in the HIV-1 integrase gene, thus enabling the evaluation of resistance to these novel antiretroviral agents recently released in the country. Plasma viral RNA was obtained from 60 patients and used as source for RT- Nested PCR amplification and automatic sequencing of the HIV-1 IN gene to analysis of the pattern of drug resistance, which was evaluated using the algorithm proposed by Stanford University. Fifty-four (90%) samples were successfully amplified and sequenced, and ten of these samples were amplified using alternative protocols involving the use of enhancers, enzymes with more specificity, RT-PCR one step and changes in the conditions of cycling, which allowed the successful study of the region of interest and analysis of resistance. The in-house methodology showed to be a viable alternative to assess resistance to integrase inhibitors, especially in cases when there is therapeutic failure to this new class of antiretroviral, since the technique allows the complete study of the region of interest

In recent years non-vitamin K dependent oral anticoagulants (NOACs) have started to replace warfarin for treatment and prevention of deep venous thrombosis (DVT), pulmonary embolism (PE) and stroke in patients with and without atrial fibrillation. There is a need for a simple and rapid method to detect the presence of these drugs in patient plasma. To meet these new demands, MediRox is developing a screening assay based on a novel prothrombin time (PT) method for rapid detection of NOACs in plasma. The assay is semi-quantitative and by dividing the International Normalised Index (INR) from a NOAC sensitive PT method with the INR from a NOAC insensitive PT method, NOAC containing samples be detected while plasma from normal donors and with warfarin are excluded.   The purpose of this project is to develop prototypes of assay quality controls for detection of NOACs in plasma.   The results show that the method used for the NOAC control prototypes is applicable and the PT ratio is comparable to patient samples for the low, medium and high concentrations of NOAC. The effect of lyophilisation indicates that the PT ratios for the NOAC control prototypes were nearly unaffected by the lyophilisation. The in-use stability at room temperature (20-25oC) for all NOAC control prototypes were at least 24 hours.   The methodology for production needs to be further optimised to increase the commutability to patient samples with very high concentrations of NOAC. The data indicates that the effect of lyophilisation is minimal and the stability of the NOAC control prototypes are satisfying, which is promising for future product development of NOAC controls.

Viral infections are constantly increasing and impose a large threat to the public health. Alphaviruses are responsible for several animal and human diseases and have a large medical importance with few treatments available today. Alphaviruses are small, spherical single stranded RNA viruses, and are most often transmitted by mosquito vectors. Alphaviruses contains a domain of nonstructural proteins that compose the replication machinery. The domain is crucial for viral replication to occur and is therefore an interesting target for antiviral therapy. With the focus on Chikungunya and Semliki Forest Virus this work investigates the events in the cells on molecular level during infections. To do this a panel of Camelid derived single domain antibodies are developed to target the nonstructural proteins of Chikungunya and Semliki Forest Virus. Binding of the produced nanobodies to the viral proteins was investigated by biochemical methods including immunoprecipitations, western blot, and ELISA. Cell lines that express nsP-specific nanobodies in the cytosol were employed for infection- and plaque assays with Semliki Forest Virus in order to determine the antiviral potential of the new nanobodies. Three of the nanobodies proved to bind two different nonstructural proteins of the viruses, providing opportunities for further investigations and a possible use of these nanobodies to identify viral vulnerabilities that could be exploited for antiviral intervention.

An unexpected biological removal of phosphorous in IVL Hammarby Sjöstadsverket pilot membrane bioreactor (MBR) wastewater treatment process was investigated by three distinct methods. a) Investigation and quantification of the biological phosphorous uptake (bio-P) capacity by phosphate release tests (PRT), b) validation of bio-P occurrence by granular polyphosphate (poly-P) staining and microscopy and c) metagenomic community structure analysis to map the sludge habitat. Validation of phosphate accumulating organisms (PAO) was successfully performed using the Neisser Methylene Blue Metachromatic staining protocol. Quantification by a novel staining protocol gave indications of the relative activity of bio-P in the bioreactor process line. The bio-P activity by PRT showed high capacity of phosphate accumulation in the treatment process. Furthermore, the use of ethylenediaminetetraacetic acid (EDTA) to induce stressful conditions was successfully shown to increase the release rate and depletion of intracellular poly-P of PAO. Finally, the impact of temperature dependency in PRT was investigated.A metagenomic community structure analysis by 16S rRNA successfully prepared 28 samples for sequencing and analysis. The project has successfully validated and quantified bio-P with an improved methodology as a foundation for upcoming studies of the treatment process. / En oväntad biologisk fosforrening (bio-P) i IVL Hammarby Sjöstadsverkets membranreaktorpilot undersöktes med tre distinkta metoder. Kvantifiering av det biologiska fosforupptaget undersöktes med fosforsläppstest (PRT). Validering av förekomsten av fosfatackumulerande organismer (PAO) via infärgning av polyfosfatgranuler med Neissers protokoll för infärgning med metylenblå. Ett nytt protokoll för kvantifiering av polyfosfatgranulkluster gav indikationer på relativ aktivitet av bio-P i reningsprocessens bioreaktorer. Bio-P aktiviteten som uppmättes med PRT visade på hög kapacitet av biologisk fosforackumulation. Vidare visades framgångsrikt att stresspåslag av PAO med etylendiamintetraättiksyra (EDTA) ökade fosfatsläppshastigheten och uttömningen av intracellulär poly-P. Slutligen undersöktes temperaturberoendet i PRT. En 16S rRNA metagenomikstudie gav 28 st prover redo att sekvensieras och analyseras. Projektet har framgångsrikt validerat och kvantifierat bio-P med en utvecklad metodologi som kan utgöra grunden för kommande studier vid reningsverket.

One of the major challenges in cancer treatment is delivering high enough doses of active substance specifically to cancer cells without accumulation in healthy organs. Pretargeting has emerged as a potential solution, where the delivery of a cancer recognizing (primary) agent and a cancer killing (secondary) agent are separated. Pretargeted cancer therapy utilizing PNA probes has proved to be a promising approach to selectively deliver toxic payloads to cancer cells while minimizing accumulation in healthy organs. The aim of this project was to develop a new set of secondary PNA probes specifically designed for PNA pretargeted delivery of cytotoxic drugs. A HER2-specific Affibody molecule, ZHER2:2891-SR-H6, was recombinantly produced in E. coli before being conjugated to a primary PNA hybridization probe, HP9, through sortase A-mediated ligation, to produce the primary agent, ZHER2:2891-SR-HP9. Circular dichroism (CD) spectroscopy confirmed the stability of the constructs with high melting temperatures of 71.2 and 73.7 °C. Surface plasmon resonance (SPR) analysis demonstrated high binding affinity to HER2, slightly affected by PNA conjugation. Three new secondary PNA hybridization probes were designed, differing mainly in prevalence and position of a hydrophilic PEG molecule. The probes were produced by solid phase peptide synthesis and conjugated to the cytotoxic drug DM1 through maleimide-cysteine coupling. Analytical RP-HPLC evaluation revealed a slightly higher apparent hydrophobicity for the probe with PEG in the main chain. All three secondary probes displayed high affinity to the primary probe with KD values between 498–505 pM. In vitro cytotoxicity studies on HER2-overexpressing cells demonstrated comparable potent cytotoxic activity for pre-incubated primary and secondary probes with IC50 values of 10–14 nM. These results indicate the successful development of three PNA-drug conjugates for pretargeted delivery of cytotoxic drugs.

Surface plasmon resonance (SPR) is a label-free, versatile and highly sensitive method for studying molecular interactions in real time. It is widely used by industry and academia alike in fields ranging from Alzheimer’s disease research to detection of heavy metals. In this review, studies published during the last 10 years using Biacore or other SPR instruments were compiled and compared. Trends were also identified in the field. Amine coupling was found to be the most common ligand strategy for proteins, and most SPR research related to the field of medicine. Furthermore, three main purposes of an SPR experiment were identified: To determine the affinity between a pair of molecules, kinetics between a pair of molecules or to detect a certain molecule in a solution. The results presented are often related to these three purposes, and are most often presented and evaluated in terms of kinetic, affinity and sensitivity constants. SPR can be used for studying a broad range of molecular interactions, and an overview was obtained by dividing up the field into different parts based on molecular interactions and SPR methods. The study of molecular interactions using SPR was divided into protein-protein interactions (PPIs), antibody-antigen, protein-biomolecule interactions, interactions between proteins and small molecules, and non-conventional SPR methods. Non-conventional SPR methods include localized surface plasmon resonance (LSPR) and SPR imaging (SPRi), which are both based on the same optical sensing principles as SPR.

Porosity is one of the most critical functional attributes regarding non-woven cellulose-based nanofiber mats. It is of crucial importance when it comes to filtration because it is one of the factors that determine at which rate the fluids will pass through the filters and particle rejection cut-off. In this thesis a method with systematic studies on controlling porosity in non-woven cellulose-based nanofiber mats is performed. This is done through statistical process controls and empirical verifications using two batches of cellulose materials. Cellulose A and cellulose B were extracted from green filamentous algae that were sourced at different locations and time by different vendors. By choosing a DoE matrix from a full factorial design with an optimum set of input factors and then proceeding to manufacture the non-woven cellulose-based nanofiber mats and analysing the output parameters this could be achieved. The input parameters were the size of the chamber and number of passes and the output response was the fibre length. Every experiment was done in triplicates and non-woven cellulose filters were made from each experiment. The pore size, flux, thickness, and basis weight were then measured to observe how the different cellulose dispersions affected the non-woven cellulose filters. The results of this work showed that controlling porosity of non-woven mats is not trivial and depends on complex relationships between several parameters. Cellulose A filters showed lower pore size values compared to cellulose B. By careful processing of the materials, the batch-to-batch variability between different raw materials could be significantly decreased.