Polymeric nanoparticles are potential candidates as carriers for pharmaceutical agents. Development of such nanoparticles generally requires molecules immobilized on the particle surfaces to ensure biocompatibility and/or targeting abilities. Following particle preparation and surface modification, excess reagents must be removed. Ultracentrifugation, which is the most widely used purification technique as per today, is not feasible in industrial applications. In this diploma work, tangential flow filtration is studied as an alternative purification method which is better suited for implementation in a large-scale process. Comparison of ultracentrifugation and tangential flow filtration in diafiltration mode for purification of nanoparticles, indicate that they are comparable with respect to particle stability and the removal of the surfactant SDS from methacrylic anhydride nanoparticles. The purification efficiency of tangential flow filtration is superior to that of ultracentrifugation. Conductivity measurements of filtrates and supernatant liquids show that a stable conductivity value can be reached 6 times faster in filtration than in centrifugation with equipment and settings used. This conductivity arises from several types of molecules, and the contribution from surfactant molecules alone is not known. However, protein adsorption on the particles indicates successful removal of surfactant. Conductivity and tensiometry were evaluated as potential methods to quantify surfactant in solutions, but both proved unsatisfactory. Using bovine serum albumin as a model protein, the extent of immobilization to nanoparticles is evaluated at different pH. A maximum amount of 6,8 mg/m2 is immobilized, whereof an unknown part is covalently bound. This coverage is achieved at pH 4,0 and is probably partly due to low electrostatic repulsion between particle and protein. An estimation of 2,0 µmol covalently bound BSA per gram of nanoparticles corresponds to 5,3 mg/m2 and a surface coverage of 76%. Removal of excess reagents after surface modification is done with ultracentrifugation instead of filtration, as particle aggregates present after the immobilization reaction might foul the membrane.

<p>Polymeric nanoparticles are potential candidates as carriers for pharmaceutical agents. Development of such nanoparticles generally requires molecules immobilized on the particle surfaces to ensure biocompatibility and/or targeting abilities. Following particle preparation and surface modification, excess reagents must be removed. Ultracentrifugation, which is the most widely used purification technique as per today, is not feasible in industrial applications. In this diploma work, tangential flow filtration is studied as an alternative purification method which is better suited for implementation in a large-scale process.</p><p>Comparison of ultracentrifugation and tangential flow filtration in diafiltration mode for purification of nanoparticles, indicate that they are comparable with respect to particle stability and the removal of the surfactant SDS from methacrylic anhydride nanoparticles. The purification efficiency of tangential flow filtration is superior to that of ultracentrifugation. Conductivity measurements of filtrates and supernatant liquids show that a stable conductivity value can be reached 6 times faster in filtration than in centrifugation with equipment and settings used. This conductivity arises from several types of molecules, and the contribution from surfactant molecules alone is not known. However, protein adsorption on the particles indicates successful removal of surfactant. Conductivity and tensiometry were evaluated as potential methods to quantify surfactant in solutions, but both proved unsatisfactory.</p><p>Using bovine serum albumin as a model protein, the extent of immobilization to nanoparticles is evaluated at different pH. A maximum amount of 6,8 mg/m2 is immobilized, whereof an unknown part is covalently bound. This coverage is achieved at pH 4,0 and is probably partly due to low electrostatic repulsion between particle and protein. An estimation of 2,0 µmol covalently bound BSA per gram of nanoparticles corresponds to 5,3 mg/m2 and a surface coverage of 76%. Removal of excess reagents after surface modification is done with ultracentrifugation instead of filtration, as particle aggregates present after the immobilization reaction might foul the membrane.</p>

Σκοπός της ακτινοθεραπείας του μαστού μετά το χειρουργείο είναι να μειωθεί η πιθανότητα της τοπικής επανεμφάνισης της νόσου παρέχοντας ταυτόχρονα κατά το δυνατό μία ανώτερη αισθητική στον μαστό. Ο στόχος αυτός μπορεί να επιτευχθεί με να δοθεί η κατά το δυνατό μέγιστη δόση σε ολόκληρο το μαστό ενώ ταυτόχρονα η δόση στα γειτονικά από το μαστό όργανα (πνεύμονας, καρδιά και ο δίπλα μαστός) να είναι η μικρότερη δυνατή. Ως εκ τούτου, είναι πολύ σημαντική η ακριβής καθημερινή τοποθέτηση της ασθενούς σε θέση θεραπείας καθώς ακόμη και μικρές αποκλίσεις από την προτιθέμενη τοποθέτηση μπορεί να έχει σημαντικές δοσιμετρικές συνέπειες. Αλλαγές στη δοσιμετρία έχουν σημαντικές συνέπειες στην καλή κάλυψη του όγκου στόχου άρα και στην πιθανότητα ελέγχου του όγκου. Τα γεωμετρικά λάθη εμφανίζονται σαν αποκλίσεις μεταξύ της γεωμετρίας που επιθυμείτε να εφαρμοστεί σύμφωνα με το πλάνο της θεραπείας και της πραγματικής γεωμετρίας που εφαρμόζεται κατά την θεραπεία. Tα συστηματικά λάθη παριστάνουν την διαφορά μεταξύ του επιθυμητού πλάνου θεραπείας και του πλάνου που εφαρμόζεται ενώ οι αποκλίσεις που εμφανίζονται μεταξύ των συνεδριών αφορούν τα τυχαία λάθη. Οι αποκλίσεις αυτές μετρώνται καθημερινά με ένα σύστημα ηλεκτρονικής κινητής απεικόνισης και εν συνεχεία με κατάλληλη μέθοδο προβλέπονται, ελαχιστοποιούνται και ελέγχονται. Στην παρούσα εργασία σκοπός ήταν να μετρηθούν τα γεωμετρικά λάθη που εμφανίζονται κατά την τοποθέτηση της ασθενούς και να εκτιμηθεί η μεταβολή στη δόση που προκύπτει και οφείλεται στα λάθη της τοποθέτησης και ταυτόχρονα να εξαχθούν από τις μεταβολές αυτές φυσικές και βιολογικές παράμετροι. / The aim of breast radiotherapy after surgery is to reduce the probability of local recurrence while providing a superior breast cosmesis. The main purpose of radiotherapy is to give the maximum prescribed dose to the whole breast while at the same time minimizing the dose to the organs placed at the vicinity of breast (lung, heart, contralateral breast). Therefore, accuracy of daily set up for treatments is important because small spatial deviations from the intended set up may have important dosimetric consequences. Changes in dosimetry have implications for coverage of the target volume and hence for tumor control probability. Geometrical errors are presented as deviation between intended geometry of radiotherapy plan and real geometry of radiotherapy treatment. . Total set up error consists of systematic (Σ set up) and random component (σ set up). Systematic errors represent the recurring difference between intended and actual geometry while fraction-to-fraction variations are called random errors. Errors’ measuring for patients undergoing breast radiotherapy with electronic portal imaging device and afterwards a proper correction strategy enables to predict, minimize, and keep under control the amount for most of geometrical errors. The aim of this work was to determine these geometrical uncertainties and to evaluate dose perturbation due to setup error in tangential breast irradiation and as well as extract physical and biological parameters.

Diplominiame darbe nagrinėjamas specialusis atraminių elementų erdvės atvejis, kai atraminis elementas yra II – os eilės viršvektorius ( p = 2 ). Pirmieji du darbo paragrafai yra referatyvinio pobūdžio. Pirmajame paragrafe gautos diferencijuojamos daugdaros struktūrinės lygtys, pateikiamos kai kurių diferencialinių geometrinių objektų ( skaliarinės funkcijos, tenzorinio lauko, viršvektorinio lauko ir afiniosios sieties objekto ) diferencialinės lygtis. Antrajame paragrafe afiniųjų ir tenzorinių siečių pagalba apibrėžta tenzoriaus invariantinio diferencijavimo operacija ir surastos tenzoriaus kovariantinės išvestinės. Surastos sąlygos kada tenzoriaus kovariantinės išvestinės, apibrėžto afiniosios sieties pagalba sutampa su šio tenzoriaus kovariantinėmis išvestinėmis, apibrėžtomis tenzorinės sieties pagalba. Trečiajame paragrafe apibrėžiama antros eilės atraminių viršvektorių erdvė, surastos šios erdvės struktūrinės lygtys ir ištirta liečiamosios ir koliečiamosios erdvių struktūra. Ketvirtajame paragrafe nagrinėjamos koliečiamosios erdvės normalizacijos. Tiesinės sieties pagalba kolie���iamojoje erdvėje išskiriami du invariantiniai poerdviai. Iš tiesinės sieties komponenčių ir jų pirmojo tęsinio sukonstruotos šios erdvės nepilna afinioji ir nepilna tenzorinė sietys, surasti šių siečių tarpusavio ryšiai, surastos pakankamos sąlygos, kad nepilnoji afinioji sietis sutaptų su nepilnosios tenzorinės sieties indukuota afiniąja sietimi. Čia tai pat surastos erdvės su nepilnąja... [toliau žr. visą tekstą] / In work it is examined special case of spaces support elements, when support elements are the super – vectors of the second order ( p = 2 ). The space of the second order support super – vectors is certain, the structural equations of this space are found and the structure tangential and cotangential spaces is researched. Further it is examined to space tangential spaces normalization. By means of linear connectivity in tangential space are detach two invariant subspaces. By means of components of linear connectivity and their first continuation are designed incomplete affine and incomplete tensors connectivity of this space, also connection between them is found. Further by means of linear connectivity natural normalization of space is examined, it is proved that the object of linear connectivity and its first continuation induces incomplete super – vector connectivity of this space. The structural equations of space with incomplete super – vector and complete super – vector connectivity are found. As operation of invariant differentiation of a super - vector field is certain, analogues of Richi and Bianca identities are received.

Understanding the specificity of homeobox genes has been hampered by the lack of verified direct transcriptional targets. The Dlx family of homeobox genes is expressed in the ganglionic eminences of the developing forebrain. Dlx1/Dlx2 double knockout (DKO) mice die at birth. Phenotypic analyses demonstrate abnormal development of the basal telencephalon, including defects in neuronal differentiation in the basal ganglia, reduced expression of GABA in the basal telencephalon, and loss of migration of GABAergic inhibitory interneurons to the neocortex. The mechanisms underlying DLX protein regulation of differentiation and migration of GABAergic interneurons are poorly defined. We have successfully applied chromatin immunoprecipitation to identify potential direct transcriptional targets of DLX homeoproteins from embryonic tissues in vivo. Reporter gene assays demonstrated the transcriptional significance of the binding of DLX proteins to different downstream regulatory elements, which were confirmed in vitro by electrophoretic mobility shift assay and site-directed mutagenesis. The functional significance of DLX mediated transcriptional regulation of these targets was further elaborated through several series of loss-of-function assays including gene expression in Dlx1/2 knockout embryonic forebrain tissues, as well as siRNA or Lentiviral mediated shRNA knockdown experiments with primary forebrain cultures. Quantitative analysis of the regulatory effect of Dlx genes on various forebrain markers of differentiation and migration was performed using in situ hybridization, high-performance liquid chromatography coupled with cell counting. Neuronal migration was assessed by forebrain explants and diI labelling of migratory cells from ganglionic eminence to neocortex. We have demonstrated that DLX1 and DLX2 can transcriptionally activate (Gad1, Gad2) or repress (Nrp2) different downstream targets. In the Dlx1/2 DKO, reduction of GABA expression and failure of GABAergic interneurons to migrate to the neocortex is partly due to loss or aberrant expression of these DLX downstream targets. In the triple Dlx1/2; Nrp2KO, partial restoration of tangential migration of GABAergic interneurons from basal ganglia to the neocortex was successfully established signifying the importance of DLX regulation of Semaphorin-Neuropilin signalling during forebrain development.

During the teeming process of molten steel from a ladle, a bathtub-type vortex may be formed in the ladle. The vortex entrains undesired slag on the surface into the tundish, lowering the quality. The formation of such vortices has been studied using two different scale models. Since the kinematic viscosity of water is similar to that of molten steel, the molten steel was simulated by water in the experiments. The Particle Image Velocimetry (PIV) technique was used to measure water flow patterns. Results show that the initial tangential velocity of water is responsible for the vortex formation. The effects of Reynolds and Froude numbers on the vortex formation were investigated and Froude number was found to be the dominant pi-number. A Computational Fluid Dynamics (CFD) modeling was also conducted to simulate the vortex formation with good agreement with the experiments.

Flies are highly visually guided animals. In this thesis, I have used hoverflies as a model for studying motion vision. Flies process motion vision in three visual ganglia: the lamina, the medulla, and the lobula complex. In the posterior part of lobula complex, there are around 60 lobula plate tangential cells (LPTCs). Most of LPTCs have large receptive fields where the local direction sensitivity suggests that they function as matched filters to specific types of optic flow. LPTCs connect to descending or neck motor neurons that control wing and head movements, respectively. Therefore, in this thesis I have focused on the electrophysiological responses of LPTCs to gain understanding of visual behaviors in flies. The elementary motion detector (EMD) is a model that can explain the formation of local motion sensitivity. However, responses to higher order motion, where the direction of luminance change is uncorrelated with the direction of movement, cannot be predicted by classic EMDs. Nevertheless, behavior shows that flies can see and track bars with higher order motion cues. I showed (Paper I) that several LPTCs also respond to higher order motion. Many insects, including flies, release octopamine during flight. Therefore, adding octopamine receptor agonists can mimic physical activity. Our study (Paper II) investigated the effect of octopamine on three adaptation components. We found that the contrast gain reduction showed a frequency dependent increase after octopamine stimulation. Since the contrast gain is non-directional, it is likely presynaptic to the LPTC. We therefore believe that octopamine acts on the delay filter in the EMD. In the third paper we describe a novel LPTC. The centrifugal stationary inhibited flicker excited (cSIFE) is excited by flicker and inhibited by stationary patterns. Neither of these responses can be predicted by EMD models. Therefore, we provide a new type of motion detector that can explain cSIFE’s responses (Paper III). During bar tracking, self-generated optic flow may counteract the steering effect by inducing a contradictory optomotor response. Behavior shows that during bar fixation, flies ignore background optic flow. Our study (Paper IV) focus on the different receptive fields of two LPTCs, and relate these to the bar fixation behavior. In the neuron with a small and fronto-dorsal receptive field, we find a higher correlation with bar motion than with background motion. In contrast, the neuron with a larger receptive field shows a higher correlation with background motion.

Understanding the specificity of homeobox genes has been hampered by the lack of verified direct transcriptional targets. The Dlx family of homeobox genes is expressed in the ganglionic eminences of the developing forebrain. Dlx1/Dlx2 double knockout (DKO) mice die at birth. Phenotypic analyses demonstrate abnormal development of the basal telencephalon, including defects in neuronal differentiation in the basal ganglia, reduced expression of GABA in the basal telencephalon, and loss of migration of GABAergic inhibitory interneurons to the neocortex. The mechanisms underlying DLX protein regulation of differentiation and migration of GABAergic interneurons are poorly defined. We have successfully applied chromatin immunoprecipitation to identify potential direct transcriptional targets of DLX homeoproteins from embryonic tissues in vivo. Reporter gene assays demonstrated the transcriptional significance of the binding of DLX proteins to different downstream regulatory elements, which were confirmed in vitro by electrophoretic mobility shift assay and site-directed mutagenesis. The functional significance of DLX mediated transcriptional regulation of these targets was further elaborated through several series of loss-of-function assays including gene expression in Dlx1/2 knockout embryonic forebrain tissues, as well as siRNA or Lentiviral mediated shRNA knockdown experiments with primary forebrain cultures. Quantitative analysis of the regulatory effect of Dlx genes on various forebrain markers of differentiation and migration was performed using in situ hybridization, high-performance liquid chromatography coupled with cell counting. Neuronal migration was assessed by forebrain explants and diI labelling of migratory cells from ganglionic eminence to neocortex. We have demonstrated that DLX1 and DLX2 can transcriptionally activate (Gad1, Gad2) or repress (Nrp2) different downstream targets. In the Dlx1/2 DKO, reduction of GABA expression and failure of GABAergic interneurons to migrate to the neocortex is partly due to loss or aberrant expression of these DLX downstream targets. In the triple Dlx1/2; Nrp2KO, partial restoration of tangential migration of GABAergic interneurons from basal ganglia to the neocortex was successfully established signifying the importance of DLX regulation of Semaphorin-Neuropilin signalling during forebrain development.

Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins. Conventional purification processes are lengthy, technically complicated, product specific and time-consuming. To address this issue, herein we develop a one step purification system that due to the nature of the non-selective secretion system and the versatility of ion-exchange membrane chromatography can be widely applied to the production of many recombinant proteins. This was achieved through the integration of the intrinsically coupled upstream, midstream and downstream processes, a connection that is rarely exploited. A bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography medium while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange medium, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level of 487 U/L/OD600, with more than 90% was localized in the extracellular medium. Both, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. Furthermore, formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. iv At harvest, the whole non-clarified culture broth was applied directly to a tangential flow filtration anion-exchange membrane chromatography system. One-step purification of recombinant PAC was achieved based on the dual nature of membrane chromatography (i.e. microfiltration-sized pores and anion-exchange chemistry). Due to their size, cells remained in the retentate while the extracellular medium penetrated the membrane. Most contaminate proteins were captured by the anion-exchange membrane, whereas the purified PAC was collected in the filtrate. The batch time for both cultivation and purification was less than 24 h and recombinant PAC with high purity (19 U/mg), process yield (74%), and productivity (41 mg/L) was obtained.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-08-03T13:51:22Z No. of bitstreams: 1 Graziela Kopinits de Oliveira.pdf: 764547 bytes, checksum: 2a8e9f311235bc2f8df978f6a19f926d (MD5) / Made available in DSpace on 2016-08-03T13:51:22Z (GMT). No. of bitstreams: 1 Graziela Kopinits de Oliveira.pdf: 764547 bytes, checksum: 2a8e9f311235bc2f8df978f6a19f926d (MD5) Previous issue date: 2016-01-22 / Medial patellar dislocation is a common condition in small dogs and should be diagnosed and treated as early as possible in order to prevent further deformities and secondary osteoarthritis. The objective of this article was to study the measures and the anatomic relationships of the trochlear groove and patella in radiographic examinations of healthy dogs predisposed to medial patellar dislocation breeds, analyzing and comparing with the trochlear groove and patellar of patients with such disease. 40 knees of predisposed pure breeds dogs were used which were divided into two groups: group 1 (G1) consisting of 18 knees that do not show patellar dislocation and group 2 (G2) consisting of 22 knees showing patella in the medial grade II or III. Depth and width trochlear groove, height and thickness of the patella, a percentage of the patella in the groove, groove angle troclear were measured and calculated the ratios of patellar width and trochlear groove and depth of the trochlear groove and thickness of the patella. All parameters studied showed statistically significant differences, in comparison between healthy and affected dogs, compared to being evaluated by Student's t test. Through evaluation and measurements on radiographs G1 dogs, it were developed predictor formulas of broadening (Lsd = Lpp/0,89 – Lsp) and deepening (Psd = 0,65Ep –Psp) required the trochlear groove to perform trochleoplasty block. / Luxação medial da patela é uma afecção comum em cães de pequeno porte, devendo ser diagnosticada e tratada o mais cedo possível com objetivo de evitar maiores deformações e alterações articulares secundárias. O objetivo deste trabalho foi estudar as medidas e as relações anatômicas do sulco troclear e da patela através de exames radiográficos de cães sadios de raças predispostas à luxação patelar medial, analisando e comparando com o sulco troclear e patela de pacientes portadores desta enfermidade. Foram utilizados 40 joelhos de cães de raças predispostas que foram divididos em dois grupos, sendo o grupo 1 (G1) composto de 18 joelhos que não apresentavam luxação de patela e o grupo 2 (G2) composto de 22 joelhos que apresentem luxação de patela medial em graus II ou III. Foram realizadas mensurações de profundidade e largura do sulco troclear, altura e espessura da patela, percentual da patela dentro do sulco, ângulo do sulco troclear, sendo calculadas as razões entre largura patelar e do sulco troclear e profundidade do sulco troclear e espessura da patela. Todos os parâmetros estudados demonstraram diferença estatística significativa, na comparação entre cães sadios e portadores da afecção, ao serem avaliadas pelo teste t de Student. Através dessas medições realizadas nas radiografias dos cães do G1 foram desenvolvidas fórmulas preditivas do alargamento (Lsd = Lpp/0,89 – Lsp) e aprofundamento (Psd = 0,65Ep –Psp) do sulco troclear, para realização de trocleoplastia em bloco.