Advanced analysis and design methods for preparative chromatographic separation processes

Preparative liquid chromatography is one of the most selective separation techniques in the fine chemical, pharmaceutical, and food industries. Several process concepts have been developed and applied for improving the performance of classical batch chromatography. The most powerful approaches include various single-column recycling schemes, counter-current and cross-current multi-column setups, and hybrid processes where chromatography is coupled with other unit operations such as crystallization, chemical reactor, and/or solvent removal unit. To fully utilize the potential of stand-alone and integrated chromatographic processes, efficient methods for selecting the best process alternative as well as optimal operating conditions are needed. In this thesis, a unified method is developed for analysis and design of the following singlecolumn fixed bed processes and corresponding cross-current schemes: (1) batch chromatography, (2) batch chromatography with an integrated solvent removal unit, (3) mixed-recycle steady state recycling chromatography (SSR), and (4) mixed-recycle steady state recycling chromatography with solvent removal from fresh feed, recycle fraction, or column feed (SSR–SR). The method is based on the equilibrium theory of chromatography with an assumption of negligible mass transfer resistance and axial dispersion. The design criteria are given in general, dimensionless form that is formally analogous to that applied widely in the so called triangle theory of counter-current multi-column chromatography. Analytical design equations are derived for binary systems that follow competitive Langmuir adsorption isotherm model. For this purpose, the existing analytic solution of the ideal model of chromatography for binary Langmuir mixtures is completed by deriving missing explicit equations for the height and location of the pure first component shock in the case of a small feed pulse. It is thus shown that the entire chromatographic cycle at the column outlet can be expressed in closed-form. The developed design method allows predicting the feasible range of operating parameters that lead to desired product purities. It can be applied for the calculation of first estimates of optimal operating conditions, the analysis of process robustness, and the early-stage evaluation of different process alternatives. The design method is utilized to analyse the possibility to enhance the performance of conventional SSR chromatography by integrating it with a solvent removal unit. It is shown that the amount of fresh feed processed during a chromatographic cycle and thus the productivity of SSR process can be improved by removing solvent. The maximum solvent removal capacity depends on the location of the solvent removal unit and the physical solvent removal constraints, such as solubility, viscosity, and/or osmotic pressure limits. Usually, the most flexible option is to remove solvent from the column feed. Applicability of the equilibrium design for real, non-ideal separation problems is evaluated by means of numerical simulations. Due to assumption of infinite column efficiency, the developed design method is most applicable for high performance systems where thermodynamic effects are predominant, while significant deviations are observed under highly non-ideal conditions. The findings based on the equilibrium theory are applied to develop a shortcut approach for the design of chromatographic separation processes under strongly non-ideal conditions with significant dispersive effects. The method is based on a simple procedure applied to a single conventional chromatogram. Applicability of the approach for the design of batch and counter-current simulated moving bed processes is evaluated with case studies. It is shown that the shortcut approach works the better the higher the column efficiency and the lower the purity constraints are.

Limited-sampling strategy models for estimating the pharmacokinetic parameters of 4-methylaminoantipyrine, an active metabolite of dipyrone

Bioanalytical data from a bioequivalence study were used to develop limited-sampling strategy (LSS) models for estimating the area under the plasma concentration versus time curve (AUC) and the peak plasma concentration (Cmax) of 4-methylaminoantipyrine (MAA), an active metabolite of dipyrone. Twelve healthy adult male volunteers received single 600 mg oral doses of dipyrone in two formulations at a 7-day interval in a randomized, crossover protocol. Plasma concentrations of MAA (N = 336), measured by HPLC, were used to develop LSS models. Linear regression analysis and a "jack-knife" validation procedure revealed that the AUC0-¥ and the Cmax of MAA can be accurately predicted (R²>0.95, bias <1.5%, precision between 3.1 and 8.3%) by LSS models based on two sampling times. Validation tests indicate that the most informative 2-point LSS models developed for one formulation provide good estimates (R²>0.85) of the AUC0-¥ or Cmax for the other formulation. LSS models based on three sampling points (1.5, 4 and 24 h), but using different coefficients for AUC0-¥ and Cmax, predicted the individual values of both parameters for the enrolled volunteers (R²>0.88, bias = -0.65 and -0.37%, precision = 4.3 and 7.4%) as well as for plasma concentration data sets generated by simulation (R²>0.88, bias = -1.9 and 8.5%, precision = 5.2 and 8.7%). Bioequivalence assessment of the dipyrone formulations based on the 90% confidence interval of log-transformed AUC0-¥ and Cmax provided similar results when either the best-estimated or the LSS-derived metrics were used.

Proteinase activity regulation by glycosaminoglycans

There are few reports concerning the biological role and the mechanisms of interaction between proteinases and carbohydrates other than those involved in clotting. It has been shown that the interplay of enzymes and glycosaminoglycans is able to modulate the activity of different proteases and also to affect their structures. From the large number of proteases belonging to the well-known protease families and also the variety of carbohydrates described as widely distributed, only few events have been analyzed more deeply. The term "family" is used to describe a group of proteases in which every member shows an evolutionary relationship to at least one other protease. This relationship may be evident throughout the entire sequence, or at least in that part of the sequence responsible for catalytic activity. The majority of proteases belong to the serine, cysteine, aspartic or metalloprotease families. By considering the existing limited proteolysis process, in addition to the initial idea that the proteinases participate only in digestive processes, it is possible to conclude that the function of the enzymes is strictly limited to the cleavage of intended substrates since the destruction of functional proteins would result in normal tissue damage. In addition, the location as well as the eventual regulation of protease activity promoted by glycosaminoglycans can play an essential role in the development of several physiopathological conditions.

Fluvio-morphological processes of meander bends - Combining conventional field measurements, close-range remote sensing and computational modelling

Meandering rivers have been perceived to evolve rather similarly around the world independently of the location or size of the river. Despite the many consistent processes and characteristics they have also been noted to show complex and unique sets of fluviomorphological processes in which local factors play important role. These complex interactions of flow and morphology affect notably the development of the river. Comprehensive and fundamental field, flume and theoretically based studies of fluviomorphological processes in meandering rivers have been carried out especially during the latter part of the 20th century. However, as these studies have been carried out with traditional field measurements techniques their spatial and temporal resolution is not competitive to the level achievable today. The hypothesis of this study is that, by exploiting e increased spatial and temporal resolution of the data, achieved by combining conventional field measurements with a range of modern technologies, will provide new insights to the spatial patterns of the flow-sediment interaction in meandering streams, which have perceived to show notable variation in space and time. This thesis shows how the modern technologies can be combined to derive very high spatial and temporal resolution data on fluvio-morphological processes over meander bends. The flow structure over the bends is recorded in situ using acoustic Doppler current profiler (ADCP) and the spatial and temporal resolution of the flow data is enhanced using 2D and 3D CFD over various meander bends. The CFD are also exploited to simulate sediment transport. Multi-temporal terrestrial laser scanning (TLS), mobile laser scanning (MLS) and echo sounding data are used to measure the flow-based changes and formations over meander bends and to build the computational models. The spatial patterns of erosion and deposition over meander bends are analysed relative to the measured and modelled flow field and sediment transport. The results are compared with the classic theories of the processes in meander bends. Mainly, the results of this study follow well the existing theories and results of previous studies. However, some new insights regarding to the spatial and temporal patterns of the flow-sediment interaction in a natural sand-bed meander bend are provided. The results of this study show the advantages of the rapid and detailed measurements techniques and the achieved spatial and temporal resolution provided by CFD, unachievable with field measurements. The thesis also discusses the limitations which remain in the measurement and modelling methods and in understanding of fluvial geomorphology of meander bends. Further, the hydro- and morphodynamic models’ sensitivity to user-defined parameters is tested, and the modelling results are assessed against detailed field measurement. The study is implemented in the meandering sub-Arctic Pulmanki River in Finland. The river is unregulated and sand-bed and major morphological changes occur annually on the meander point bars, which are inundated only during the snow-melt-induced spring floods. The outcome of this study applies to sandbed meandering rivers in regions where normally one significant flood event occurs annually, such as Arctic areas with snow-melt induced spring floods, and where the point bars of the meander bends are inundated only during the flood events.

Predicting bankruptcy of Finnish limited liability companies from historical financial statements

This master’s thesis studies the probability of bankruptcy of Finnish limited liability companies as a part of credit risk assessment. The main idea of this thesis is to build and test bankruptcy prediction models for Finnish limited liability companies that can be utilized in credit decision making. The data used in this thesis consists of historical financial statements from 2112 Finnish limited liability companies, half of which have filed for bankruptcy. A total of four models are developed, two with logistic regression and two with multivariate discriminant analysis (MDA). The time horizon of the models varies from 1 to 2 years prior to the bankruptcy, and 14 different financial variables are used in the model formation. The results show that the prediction accuracy of the models ranges between 81.7% and 88.9%, and the best prediction accuracy is achieved with the one year prior the bankruptcy logistic regression model. However the difference between the best logistic model and the best MDA model is minimal. Overall based on the results of this thesis it can be concluded that predicting bankruptcy is possible to some extent, but naturally the results are not perfect.

Energy and raw material consumption analysis of powder bed fusion: case study: CNC machining and laser additive manufacturing

Laser additive manufacturing (LAM), known also as 3D printing, is a powder bed fusion (PBF) type of additive manufacturing (AM) technology used to manufacture metal parts layer by layer by assist of laser beam. The development of the technology from building just prototype parts to functional parts is due to design flexibility. And also possibility to manufacture tailored and optimised components in terms of performance and strength to weight ratio of final parts. The study of energy and raw material consumption in LAM is essential as it might facilitate the adoption and usage of the technique in manufacturing industries. The objective this thesis was find the impact of LAM on environmental and economic aspects and to conduct life cycle inventory of CNC machining and LAM in terms of energy and raw material consumption at production phases. Literature overview in this thesis include sustainability issues in manufacturing industries with focus on environmental and economic aspects. Also life cycle assessment and its applicability in manufacturing industry were studied. UPLCI-CO2PE! Initiative was identified as mostly applied exiting methodology to conduct LCI analysis in discrete manufacturing process like LAM. Many of the reviewed literature had focused to PBF of polymeric material and only few had considered metallic materials. The studies that had included metallic materials had only measured input and output energy or materials of the process and compared to different AM systems without comparing to any competitive process. Neither did any include effect of process variation when building metallic parts with LAM. Experimental testing were carried out to make dissimilar samples with CNC machining and LAM in this thesis. Test samples were designed to include part complexity and weight reductions. PUMA 2500Y lathe machine was used in the CNC machining whereas a modified research machine representing EOSINT M-series was used for the LAM. The raw material used for making the test pieces were stainless steel 316L bar (CNC machined parts) and stainless steel 316L powder (LAM built parts). An analysis of power, time, and the energy consumed in each of the manufacturing processes on production phase showed that LAM utilises more energy than CNC machining. The high energy consumption was as result of duration of production. Energy consumption profiles in CNC machining showed fluctuations with high and low power ranges. LAM energy usage within specific mode (standby, heating, process, sawing) remained relatively constant through the production. CNC machining was limited in terms of manufacturing freedom as it was not possible to manufacture all the designed sample by machining. And the one which was possible was aided with large amount of material removed as waste. Planning phase in LAM was shorter than in CNC machining as the latter required many preparation steps. Specific energy consumption (SEC) were estimated in LAM based on the practical results and assumed platform utilisation. The estimated platform utilisation showed SEC could reduce when more parts were placed in one build than it was in with the empirical results in this thesis (six parts).

Spatial modeling techniques for mapping and characterization of acid sulfate soils

Acid sulfate (a.s.) soils constitute a major environmental issue. Severe ecological damage results from the considerable amounts of acidity and metals leached by these soils in the recipient watercourses. As even small hot spots may affect large areas of coastal waters, mapping represents a fundamental step in the management and mitigation of a.s. soil environmental risks (i.e. to target strategic areas). Traditional mapping in the field is time-consuming and therefore expensive. Additional more cost-effective techniques have, thus, to be developed in order to narrow down and define in detail the areas of interest. The primary aim of this thesis was to assess different spatial modeling techniques for a.s. soil mapping, and the characterization of soil properties relevant for a.s. soil environmental risk management, using all available data: soil and water samples, as well as datalayers (e.g. geological and geophysical). Different spatial modeling techniques were applied at catchment or regional scale. Two artificial neural networks were assessed on the Sirppujoki River catchment (c. 440 km2) located in southwestern Finland, while fuzzy logic was assessed on several areas along the Finnish coast. Quaternary geology, aerogeophysics and slope data (derived from a digital elevation model) were utilized as evidential datalayers. The methods also required the use of point datasets (i.e. soil profiles corresponding to known a.s. or non-a.s. soil occurrences) for training and/or validation within the modeling processes. Applying these methods, various maps were generated: probability maps for a.s. soil occurrence, as well as predictive maps for different soil properties (sulfur content, organic matter content and critical sulfide depth). The two assessed artificial neural networks (ANNs) demonstrated good classification abilities for a.s. soil probability mapping at catchment scale. Slightly better results were achieved using a Radial Basis Function (RBF) -based ANN than a Radial Basis Functional Link Net (RBFLN) method, narrowing down more accurately the most probable areas for a.s. soil occurrence and defining more properly the least probable areas. The RBF-based ANN also demonstrated promising results for the characterization of different soil properties in the most probable a.s. soil areas at catchment scale. Since a.s. soil areas constitute highly productive lands for agricultural purpose, the combination of a probability map with more specific soil property predictive maps offers a valuable toolset to more precisely target strategic areas for subsequent environmental risk management. Notably, the use of laser scanning (i.e. Light Detection And Ranging, LiDAR) data enabled a more precise definition of a.s. soil probability areas, as well as the soil property modeling classes for sulfur content and the critical sulfide depth. Given suitable training/validation points, ANNs can be trained to yield a more precise modeling of the occurrence of a.s. soils and their properties. By contrast, fuzzy logic represents a simple, fast and objective alternative to carry out preliminary surveys, at catchment or regional scale, in areas offering a limited amount of data. This method enables delimiting and prioritizing the most probable areas for a.s soil occurrence, which can be particularly useful in the field. Being easily transferable from area to area, fuzzy logic modeling can be carried out at regional scale. Mapping at this scale would be extremely time-consuming through manual assessment. The use of spatial modeling techniques enables the creation of valid and comparable maps, which represents an important development within the a.s. soil mapping process. The a.s. soil mapping was also assessed using water chemistry data for 24 different catchments along the Finnish coast (in all, covering c. 21,300 km2) which were mapped with different methods (i.e. conventional mapping, fuzzy logic and an artificial neural network). Two a.s. soil related indicators measured in the river water (sulfate content and sulfate/chloride ratio) were compared to the extent of the most probable areas for a.s. soils in the surveyed catchments. High sulfate contents and sulfate/chloride ratios measured in most of the rivers demonstrated the presence of a.s. soils in the corresponding catchments. The calculated extent of the most probable a.s. soil areas is supported by independent data on water chemistry, suggesting that the a.s. soil probability maps created with different methods are reliable and comparable.

Toward including the effect of manufacturing processes in the pre-estimated losses of the switched reluctance motor

The estimation of losses plays a key role in the process of building any electrical machine. How to estimate those losses while designing any machine; by obtaining the characteristic of the electrical steel from the catalogue and calculate the losses. However, this way is inaccurate since the electrical steel performs several manufacturing processes during the process of building any machine, which affects directly the magnetic property of the electrical steel and accordingly the characteristic of the electrical steel will be affected. That means the B–H curve of the steel that was obtained from the catalogue will be changed. Moreover, during loading and rotating the machine, some important changes occur to the B–H characteristic of the electrical steel such as the stress on the laminated iron. Accordingly, the pre-estimated losses are completely far from the actual losses because they were estimated based on the data of the electrical steel obtained from the catalogue. So in order to estimate the losses precisely significant factors of the manufacturing processes must be included. The paper introduces the systematic estimation of the losses including the effect of one of the manufacturing factors. Similarly, any other manufacturing factor can be included in the pre-designed losses estimations.

Metal additive manufacturing of internal flow channels with powder bed fusion processes

This thesis studies the advantages, disadvantages and possibilities of additive manufacturing in making components with internal flow channels. These include hydraulic components, components with cooling channels and heat exchangers. Processes studied in this work are selective laser sintering and selective laser melting of metallic materials. The basic principles of processes and parameters involved in the process are presented and different possibilities of internal channel manufacturing and flow improvement are introduced

Rekrytointimarkkinointi Y-sukupolvelle, miten tavoitamme parhaat osaajat? Case IBM Finland OY

Pro Gradu- tutkimuksen keskeisin tavoite oli selvittää tehokkaimmat rekrytointimarkkinointikanavat ja tyylit Y-sukupolveen kohdistuen. Y-sukupolvi on isoin ja uusin sukupolvi, joka on siirtymässä työelämään. Tällä hetkellä teoreettinen pohja on vielä ohut liittyen y-sukupolven rekrytointiin ja työnantajamielikuvaan ja tämän tutkimuksen oli tarkoitus selvittää tätä tutkimusaukkoa. Tutkimus toteutettiin kvalitatiivisena case-tutkimuksena. Aineistonkeruutapana oli kysely. Kysely toteutettiin sähköpostilla lähetettävällä kyselyllä IBM Suomen nuorille harjoittelijoille ja työntekijöille. Kysely lähetettiin noin 150:lle työntekijälle. Tutkimustulokset osoittivat, että IBM Suomella ovat perusprosessit ja nykyisten harjoittelijoiden tyytyväisyys hyvällä tasolla. Kuitenkin tutkimuskyselyn kautta saatiin selville osa-alueita, joilla rekrytointimarkkinoinnin tehokkuutta voidaan lisätä ja hyväksikäyttää vieläkin paremmin. Tutkimustulosten perusteella Y-sukupolven arvot ja odotukset erosivat muista sukupolvista oletettua vähemmän.

Tuotantoympäristön jatkuva parantaminen

Tutkimuksen tavoitteena oli luoda kohdeyritykseen toimintamalli, jonka avulla saadaan osallistettua työntekijät, tiiminvetäjät ja työnjohto tuotannon jatkuvaan parantamiseen sekä parannettua tuotannon takaisinkytkentää tiimitasolla. Tutkimus rajattiin pilottitiimiin sekä tiimissä olevien työpisteiden kautta reititettyihin tuotteisiin. Ennen tutkimuksen aloitusta yrityksellä oli jo olemassa sähköinen aloitejärjestelmä, mutta sen käyttö oli organisaation uudelleen järjestelyjen myötä vähentynyt. Tutkimuksen teoriaosassa tutustuttiin jatkuvan parantamisen kulttuuriin ja työkaluihin. Lisäksi tutustuttiin laadunhallinnan sisältöön, käsitteistöön ja laadunvalvontatyökaluihin sekä tuotannon mittareihin. Teorian pohjalta tutkimuksessa luotiin jatkuvan parantamisen toimintamalli, joka tunnistaa ja eliminoi prosessissa olevaa hukkaa osallistamalla pilottitiimin työntekijöitä hukkakorttien avulla. Lisäksi tutkimuksessa luotiin toimintamalli tuotannon kehitysideoiden raportointiin ja käsittelyyn. Tuotannon takaisinkytkentää kehitettiin luomalla pilottitiimiin tuloskortti sekä perustamalla yritykseen päiväkatsauskäytäntö. Tutkimuksessa suoritettiin myös toimihenkilötason kehitysprojekteja käyttäen apuna teoriassa esiteltyjä malleja ja työkaluja. Tuloksena saatiin toimintamalli, joka tuottaa työntekijämäärään suhteutettuna enemmän kehitysideoita sekä käsittelee ne tehokkaammin kuin sähköinen aloitejärjestelmä. Hukkakorteilla toteutetun hukan raportoinnin kautta tunnistettiin ja raportoitiin seitsemän viikon tarkasteluajanjakson aikana yhteensä 23,6 tuntia hukka-aikaa. Tiimin tuloskortin avulla tiimin työntekijät pystyivät viikkotasolla seuraamaan oman tiiminsä suorituskykyä tavoitearvoihin verrattuna. Tämä näkyi muun muassa tiimin suoritustason nousuna. Kehitysprojektien avulla saatiin parannettua pilottitiimin toiminnan ja tuotteiden laatua. Päiväkatsauskäytännön avulla saatiin osallistettua tiiminvetäjät ongelmaratkaisuun sekä tuotannon suorituskyvyn varmistamiseen.

A new model for the population pharmacokinetics of didanosine in healthy subjects

Didanosine (ddI) is a component of highly active antiretroviral therapy drug combinations, used especially in resource-limited settings and in zidovudine-resistant patients. The population pharmacokinetics of ddI was evaluated in 48 healthy volunteers enrolled in two bioequivalence studies. These data, along with a set of co-variates, were the subject of a nonlinear mixed-effect modeling analysis using the NONMEM program. A two-compartment model with first order absorption (ADVAN3 TRANS3) was fitted to the serum ddI concentration data. Final pharmacokinetic parameters, expressed as functions of the co-variates gender and creatinine clearance (CL CR), were: oral clearance (CL = 55.1 + 240 x CL CR + 16.6 L/h for males and CL = 55.1 + 240 x CL CR for females), central volume (V2 = 9.8 L), intercompartmental clearance (Q = 40.9 L/h), peripheral volume (V3 = 62.7 + 22.9 L for males and V3 = 62.7 L for females), absorption rate constant (Ka = 1.51/h), and dissolution time of the tablet (D = 0.43 h). The intraindividual (residual) variability expressed as coefficient of variation was 13.0%, whereas the interindividual variability of CL, Q, V3, Ka, and D was 20.1, 75.8, 20.6, 18.9, and 38.2%, respectively. The relatively high (>30%) interindividual variability for some of these parameters, observed under the controlled experimental settings of bioequivalence trials in healthy volunteers, may result from genetic variability of the processes involved in ddI absorption and disposition.

Study of the comprehension of the scientific method by members of a university health research laboratory

In Brazil, scientific research is carried out mainly at universities, where professors coordinate research projects with the active participation of undergraduate and graduate students. However, there is no formal program for the teaching/learning of the scientific method. The objective of the present study was to evaluate the comprehension of the scientific method by students of health sciences who participate in scientific projects in an academic research laboratory. An observational descriptive cross-sectional study was conducted using Edgar Morin complexity as theoretical reference. In a semi-structured interview, students were asked to solve an abstract logical puzzle - TanGram. The collected data were analyzed using the hermeneutic-dialectic analysis method proposed by Minayo and discussed in terms of the theoretical reference of complexity. The students’ concept of the scientific method is limited to participation in projects, stressing the execution of practical procedures as opposed to scientific thinking. The solving of the TanGram puzzle revealed that the students had difficulties in understanding questions and activities focused on subjects and their processes. Objective answers, even when dealing with personal issues, were also reflected on the students’ opinions about the characteristics of a successful researcher. Students’ difficulties concerning these issues may affect their scientific performance and result in poorly designed experiments. This is a preliminary study that should be extended to other centers of scientific research.

The influence of stress on fear memory processes

It is well recognized that stressful experiences promote robust emotional memories, which are well remembered. The amygdaloid complex, principally the basolateral complex (BLA), plays a pivotal role in fear memory and in the modulation of stress-induced emotional responses. A large number of reports have revealed that GABAergic interneurons provide a powerful inhibitory control of the activity of projecting glutamatergic neurons in the BLA. Indeed, a reduced GABAergic control in the BLA is essential for the stress-induced influence on the emergence of associative fear memory and on the generation of long-term potentiation (LTP) in BLA neurons. The extracellular signal-regulated kinase (ERK) subfamily of the mitogen-activated protein kinase (MAPK) signaling pathway in the BLA plays a central role in the consolidation process and synaptic plasticity. In support of the view that stress facilitates long-term fear memory, stressed animals exhibited a phospho-ERK2 (pERK2) increase in the BLA, suggesting the involvement of this mechanism in the promoting influence of threatening stimuli on the consolidation fear memory. Moreover, the occurrence of reactivation-induced lability is prevented when fear memory is encoded under intense stressful conditions since the memory trace remains immune to disruption after recall in previously stressed animals. Thus, the underlying mechanism in retrieval-induced instability seems not to be functional in memories formed under stress. All these findings are indicative that stress influences both the consolidation and reconsolidation fear memory processes. Thus, it seems reasonable to propose that the emotional state generated by an environmental challenge critically modulates the formation and maintenance of long-term fear memory.

Tissue transglutaminase (TG2) activity regulates osteoblast differentiation and mineralization in the SAOS-2 cell line

Tissue transglutaminase (type II, TG2) has long been postulated to directly promote skeletal matrix calcification and play an important role in ossification. However, limited information is available on the expression, function and modulating mechanism of TG2 during osteoblast differentiation and mineralization. To address these issues, we cultured the well-established human osteosarcoma cell line SAOS-2 with osteo-inductive conditioned medium and set up three time points (culture days 4, 7, and 14) to represent different stages of SAOS-2 differentiation. Osteoblast markers, mineralization, as well as TG2 expression and activity, were then assayed in each stage. Furthermore, we inhibited TG activity with cystamine and then checked SAOS-2 differentiation and mineralization in each stage. The results showed that during the progression of osteoblast differentiation SAOS-2 cells presented significantly high levels of osteocalcin (OC) mRNA, bone morphogenetic protein-2 (BMP-2) and collagen I, significantly high alkaline phosphatase (ALP) activity, and the increased formation of calcified matrix. With the same tendency, TG2 expression and activity were up-regulated. Furthermore, inhibition of TG activity resulted in a significant decrease of OC, collagen I, and BMP-2 mRNA and of ALP activity and mineralization. This study demonstrated that TG2 is involved in osteoblast differentiation and may play a role in the initiation and regulation of the mineralization processes. Moreover, the modulating effects of TG2 on osteoblasts may be related to BMP-2.