A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D) models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer.

Intensity and physiological responses to the 6-minute walk test in middle-aged and older adults: a comparison with cardiopulmonary exercise testing

The 6-minute walk test (6MWT) is a simple field test that is widely used in clinical settings to assess functional exercise capacity. However, studies with healthy subjects are scarce. We hypothesized that the 6MWT might be useful to assess exercise capacity in healthy subjects. The purpose of this study was to evaluate 6MWT intensity in middle-aged and older adults, as well as to develop a simple equation to predict oxygen uptake ( V ˙ O 2 ) from the 6-min walk distance (6MWD). Eighty-six participants, 40 men and 46 women, 40-74 years of age and with a mean body mass index of 28±6 kg/m2, performed the 6MWT according to American Thoracic Society guidelines. Physiological responses were evaluated during the 6MWT using a K4b2 Cosmed telemetry gas analyzer. On a different occasion, the subjects performed ramp protocol cardiopulmonary exercise testing (CPET) on a treadmill. Peak V ˙ O 2 in the 6MWT corresponded to 78±13% of the peak V ˙ O 2 during CPET, and the maximum heart rate corresponded to 80±23% of that obtained in CPET. Peak V ˙ O 2 in CPET was adequately predicted by the 6MWD by a linear regression equation: V ˙ O 2 mL·min-1·kg-1 = -2.863 + (0.0563×6MWDm) (R2=0.76). The 6MWT represents a moderate-to-high intensity activity in middle-aged and older adults and proved to be useful for predicting cardiorespiratory fitness in the present study. Our results suggest that the 6MWT may also be useful in asymptomatic individuals, and its use in walk-based conditioning programs should be encouraged.

Transtracheal puncture: a forgotten procedure

Transtracheal puncture has long been known as a safe, low-cost procedure. However, with the advent of bronchoscopy, it has largely been forgotten. Two researchers have suggested the use of α-amylase activity to diagnose salivary aspiration, but the normal values of this enzyme in tracheobronchial secretions are unknown. We aimed to define the normal values of α-amylase activity in tracheobronchial secretions and verify the rate of major complications of transtracheal puncture. From October 2009 to June 2011, we prospectively evaluated 118 patients without clinical or radiological signs of salivary aspiration who underwent transtracheal puncture before bronchoscopy. The patients were sedated with a solution of lidocaine and diazepam until they reached a Ramsay sedation score of 2 or 3. We then cleaned the cervical region and anesthetized the superficial planes with lidocaine. Next, we injected 10 mL of 2% lidocaine into the tracheobronchial tree. Finally, we injected 10 mL of normal saline into the tracheobronchial tree and immediately aspirated the saline with maximum vacuum pressure to collect samples for measurement of the α-amylase level. The α-amylase level mean ± SE, median, and range were 1914 ± 240, 1056, and 24-10,000 IU/L, respectively. No major complications (peripheral desaturation, subcutaneous emphysema, cardiac arrhythmia, or hemoptysis) occurred among 118 patients who underwent this procedure. Transtracheal aspiration is a safe, low-cost procedure. We herein define for the first time the normal α-amylase levels in the tracheobronchial secretions of humans.

Model-based testing of software systems : functionality and performance

Software is a key component in many of our devices and products that we use every day. Most customers demand not only that their devices should function as expected but also that the software should be of high quality, reliable, fault tolerant, efficient, etc. In short, it is not enough that a calculator gives the correct result of a calculation, we want the result instantly, in the right form, with minimal use of battery, etc. One of the key aspects for succeeding in today's industry is delivering high quality. In most software development projects, high-quality software is achieved by rigorous testing and good quality assurance practices. However, today, customers are asking for these high quality software products at an ever-increasing pace. This leaves the companies with less time for development. Software testing is an expensive activity, because it requires much manual work. Testing, debugging, and verification are estimated to consume 50 to 75 per cent of the total development cost of complex software projects. Further, the most expensive software defects are those which have to be fixed after the product is released. One of the main challenges in software development is reducing the associated cost and time of software testing without sacrificing the quality of the developed software. It is often not enough to only demonstrate that a piece of software is functioning correctly. Usually, many other aspects of the software, such as performance, security, scalability, usability, etc., need also to be verified. Testing these aspects of the software is traditionally referred to as nonfunctional testing. One of the major challenges with non-functional testing is that it is usually carried out at the end of the software development process when most of the functionality is implemented. This is due to the fact that non-functional aspects, such as performance or security, apply to the software as a whole. In this thesis, we study the use of model-based testing. We present approaches to automatically generate tests from behavioral models for solving some of these challenges. We show that model-based testing is not only applicable to functional testing but also to non-functional testing. In its simplest form, performance testing is performed by executing multiple test sequences at once while observing the software in terms of responsiveness and stability, rather than the output. The main contribution of the thesis is a coherent model-based testing approach for testing functional and performance related issues in software systems. We show how we go from system models, expressed in the Unified Modeling Language, to test cases and back to models again. The system requirements are traced throughout the entire testing process. Requirements traceability facilitates finding faults in the design and implementation of the software. In the research field of model-based testing, many new proposed approaches suffer from poor or the lack of tool support. Therefore, the second contribution of this thesis is proper tool support for the proposed approach that is integrated with leading industry tools. We o er independent tools, tools that are integrated with other industry leading tools, and complete tool-chains when necessary. Many model-based testing approaches proposed by the research community suffer from poor empirical validation in an industrial context. In order to demonstrate the applicability of our proposed approach, we apply our research to several systems, including industrial ones.

Spectrally Tuned Lanthanide Photoluminescence for Point-of-Care Testing

Point-of-care (POC) –diagnostics is a field with rapidly growing market share. As these applications become more widely used, there is an increasing pressure to improve their performance to match the one of a central laboratory tests. Lanthanide luminescence has been widely utilized in diagnostics because of the numerous advantages gained by the utilization of time-resolved or anti-Stokes detection. So far the use of lanthanide labels in POC has been scarce due to limitations set by the instrumentation required for their detection and the shortcomings, e.g. low brightness, of these labels. Along with the advances in the research of lanthanide luminescence, and in the field of semiconductors, these materials are becoming a feasible alternative for the signal generation also in the future POC assays. The aim of this thesis was to explore ways of utilizing time-resolved detection or anti-Stokes detection in POC applications. The long-lived fluorescence for the time-resolved measurement can be produced with lanthanide chelates. The ultraviolet (UV) excitation required by these chelates is cumbersome to produce with POC compatible fluorescence readers. In this thesis the use of a novel light-harvesting ligand was studied. This molecule can be used to excite Eu(III)-ions at wavelengths extending up to visible part of the spectrum. An enhancement solution based on this ligand showed a good performance in a proof-of-concept -bioaffinity assay and produced a bright signal upon 365 nm excitation thanks to the high molar absorptivity of the chelate. These features are crucial when developing miniaturized readers for the time-resolved detection of fluorescence. Upconverting phosphors (UCPs) were studied as an internal light source in glucose-sensing dry chemistry test strips and ways of utilizing their various emission wavelengths and near-infrared excitation were explored. The use of nanosized NaYF :Yb3+,Tm3+-particles enabled the replacement of an external UV-light source with a NIR-laser and gave an additional degree of freedom in the optical setup of the detector instrument. The new method enabled a blood glucose measurement with results comparable to a current standard method of measuring reflectance. Microsized visible emitting UCPs were used in a similar manner, but with a broad absorbing indicator compound filtering the excitation and emission wavelengths of the UCP. This approach resulted in a novel way of benefitting from the non-linear relationship between the excitation power and emission intensity of the UCPs, and enabled the amplification of the signal response from the indicator dye.

Kuparin hitsaus kattavilla laatuvaatimuksilla

Kuparin ja kupariseosten hitsaus eroaa merkittävästi esimerkiksi terästen hitsauksesta. Suuri lämmönjohtavuus, lämpölaajeneminen, pehmeneminen ja kuparin taipumus liuottaa kaasuja sulaan asettavat hitsaukselle haasteita. Kuparia on perinteisesti hitsattu kaasuhitsaamalla ja kaasukaarihitsausprosesseilla, mutta uudemmat menetelmät kuten laserhitsaus, elektronisuihkuhitsaus ja FSW-hitsaus tarjoavat uudenlaisia käyttökohteita korkealla laadulla. ISO 3834-2 asettaa noudatettavat vaatimukset hitsaustoiminnalle laatuvaatimusten ollessa kattavia. Ydinvoimalaitoksella hitsauksessa tulee lisäksi noudattaa Säteilyturvakeskuksen YVL-ohjeita, joissa on määritetty lisävaatimuksia liitosten materiaalivalinnoille, pätevöittämiselle ja tarkastamiselle. Tässä työssä tutkittiin kuparimetallien hitsauksen mahdollisuutta Loviisan ydinvoimalaitoksella juottamisen sijasta siten, että kattavat laatuvaatimukset täyttyisivät. Hitsauskokeissa ja laboratoriotutkimuksissa testattiin hitsausta erilaisilla hitsausaineilla ja hitsausprosesseilla. Koetulosten pohjalta toteutettiin hitsausmenetelmä deoksidoidun kupariputken ja tinapronssilaipan TIG-hitsaukselle.

A novel procedure to measure the antioxidant capacity of Yerba maté extracts

Yerba maté extracts have in vitro antioxidant capacity attributed to the presence of polyphenolic compounds, mainly chlorogenic acids and dicaffeoylquinic acid derivatives. DPPH is one of the most used assays to measure the antioxidant capacity of pure compounds and plant extracts. It is difficult to compare the results between studies because this assay is applied in too many different conditions by the different research groups. Thus, in order to assess the antioxidant capacity of yerba maté extracts, the following procedure is proposed: 100 µL of an aqueous dilution of the extracts is mixed in duplicate with 3.0 mL of a DPPH 'work solution in absolute methanol (100 µM.L-1), with an incubation time of 120 minutes in darkness at 37 ± 1 °C, and then absorbance is read at 517 nm against absolute methanol. The results should be expressed as ascorbic acid equivalents or Trolox equivalents in mass percentage (g% dm, dry matter) in order to facilitate comparisons. The AOC of the ethanolic extracts ranged between 12.8 and 23.1 g TE % dm and from 9.1 to 16.4 g AAE % dm. The AOC determined by the DPPH assay proposed in the present study can be related to the total polyphenolic content determined by the Folin-Ciocalteu assay.

Plantability and moisture content of naked and pelleted seeds of supersweet (Sh2) corn during cold storage conditions

The supersweet (Sh2) corn seeds have a thin tegument and an irregular shape, which hinder the sowing procedure. As a function of those factors, the significance of using the pelleting process to improve sowing and standardize the stand of plants in the field without the need of thinning within the row is emphasized. Although this technique has already been known for many years, little has been studied on the effect of such procedure on the plantability and on the moisture content of the supersweet corn seeds. Therefore, this research work aimed at evaluating the effects of pelleting on the moisture content of the seeds along the storing process, on the occurrence of gaps on seed deposition and on the dropping of double seeds, with the aid of a testing seeder. Other physical attributes of seeds and pellets were also evaluated through tests of fragmentation, screen retention, mass of a thousand seeds and apparent volume. Results have shown an increase on the mass and on the volume of the pelleted seeds that presented highly resistance to cracks and breaking. The pelleting was efficient on the reduction of gaps and on the deposition of double seeds at sowing, thus providing highly satisfactory results for these attributes. An increase on the seed moisture content was also observed as a function of storage with a significant reduction on this factor after the pelleting procedure. Results allow concluding that the pelleting process reduces seed moisture content and improves plantability, positively affecting the efficiency of the sowing process.

Orbitaali-TIG-hitsauksen kehittäminen compoundputkien hitsaamisessa

Tässä työssä tutkittiin orbitaali-TIG-hitsauksen hyödyntämistä paineenalaisten compoundputkien jatkohitsien hitsaamiseen. Tutkimus tehtiin Warkaus Works Oy:n omistamalle konepajalle. Tutkimuksen tavoitteena oli laatia standardin SFS-EN ISO 15613 mukaan hyväksytty hitsausmenetelmä. Työn kirjallisuuskatsauksessa esiteltiin orbitaalihitsauksessa käytettävät hitsausprosessit ja laitteistot eri liitosmuodoille. Työssä esiteltiin myös kuumavedettyjen compoundputkien valmistusprosessia ja rakennetta. Tämän lisäksi kerrottiin putkien hitsattavuudesta ja hitsauksen esivalmisteluista, sekä käyttökohteista voimalaitoskattilassa. Ennen menetelmäkokeen hitsausta suoritettiin suuri määrä koehitsauksia yrityksen orbitaali-TIG-laitteistolla. Onnistuneiden koehitsausten perusteella laadittiin hitsausohjelma, jolla hitsattiin testiputket menetelmäkokeeseen. Hitsausmenetelmää varten valittiin Sandvikin valmistama compoundputki. Testihitseille suoritettiin rikkomattomia ja rikkovia tarkastuksia. Hyväksyttyjen tarkastusten pohjalta laadittiin hitsausmenetelmän hyväksymispöytäkirjan. Lisäksi diplomityössä verrattiin mekanisoidun hitsauksen hitsausaikaa käsinhitsauksen hitsausaikaa. Orbitaali-TIG-hitsauksen käyttäminen edellyttää hitsattavalta railolta aina mahdollisimman samanlaisen lähtötilanteen, jotta sen suoritusvarmuus saadaan mahdollisimman korkeaksi. Railon valmistamisen tarkkuudella on mahdollista saada riittävän tarkkoja railoja hitsien laadukkaaseen hitsaukseen. Hitsaus edellyttää laitteiston langansyötön ja elektrodin tarkkoja asetuksia, jotta hitsaus on laadukasta. Lisäaineen jähmeä sula ja hitsin tunkeumavaatimus hankaloittavat orbitaalihitsausta.

Development of techniques for testing the effect of the E1 region on adenovirus integration /

Adenoviral vectors are currently the most widely used gene therapeutic vectors, but their inability to integrate into host chromosomal DNA shortened their transgene expression and limited their use in clinical trials. In this project, we initially planned to develop a technique to test the effect of the early region 1 (E1) on adenovirus integration by comparing the integration efficiencies between an E1-deleted adenoviral vector (SubE1) and an Elcontaining vector (SubE3). However, we did not harvest any SubE3 virus, even if we repeated the transfection and successfully rescued the SubE1 virus (2/4 transfections generated viruses) and positive control virus (6/6). The failure of rescuing SubE3 could be caused by the instability of the genomic plasmid pFG173, as it had frequent intemal deletions when we were purifying It. Therefore, we developed techniques to test the effect of E1 on homologous recombination (HR) since literature suggested that adenovirus integration is initiated by HR. We attempted to silence the E1 in 293 cells by transfecting E1A/B-specific small interfering RNA (siRNA). However, no silenced phenotype was observed, even if we varied the concentrations of E1A/B siRNA (from 30 nM to 270 nM) and checked the silencing effects at different time points (48, 72, 96 h). One possible explanation would be that the E1A/B siRNA sequences are not potent enough to Induce the silenced phenotype. For evaluating HR efficiencies, an HR assay system based on bacterial transfonmatJon was designed. We constmcted two plasmids ( designated as pUC19-dl1 and pUC19-dl2) containing different defective lacZa cassettes (forming white colonies after transformation) that can generate a functional lacZa cassette (forming blue colonies) through HR after transfecting into 293 cells. The HR efficiencies would be expressed as the percentages of the blue colonies among all the colonies. Unfortunately, after transfonnation of plasmid isolated from 293 cells, no colony was found, even at a transformation efficiency of 1.8x10^ colonies/pg pUC19, suggesting the sensitivity of this system was low. To enhance the sensitivity, PCR was used. We designed a set of primers that can only amplify the recombinant plasmid fomied through HR. Therefore, the HR efficiencies among different treatments can be evaluated by the amplification results, and this system could be used to test the effect of E1 region on adenovirus integration. In addition, to our knowledge there was no previous studies using PCR/ Realtime PCR to evaluate HR efficiency, so this system also provides a PCR-based method to carry out the HR assays.

The generation effect and word learning: a test of the effect of the language experience approach versus the text approach in the acquisition of new reading vocabulary

A class of twenty-two grade one children was tested to determine their reading levels using the Stanford Diagnostic Reading Achievement Test. Based on these results and teacher input the students were paired according to reading ability. The students ages ranged from six years four months to seven years four months at the commencement of the study. Eleven children were assigned to the language experience group and their partners became the text group. Each member of the language experience group generated a list of eight to be learned words. The treatment consisted of exposing the student to a given word three times per session for ten sessions, over a period of five days. The dependent variables consisted of word identification speed, word identification accuracy, and word recognition accuracy. Each member of the text group followed the same procedure using his/her partner's list of words. Upon completion of this training, the entire process was repeated with members of the text group from the first part becoming members of the language experience group and vice versa. The results suggest that generally speaking language experience words are identified faster than text words but that there is no difference in the rate at which these words are learned. Language experience words may be identified faster because the auditory-semantic information is more readily available in them than in text words. The rate of learning in both types of words, however, may be dictated by the orthography of the to be learned word.

Attachment and alcohol : testing a motivational model of problem drinking /

The purpose of this study was to replicate and extend a motivational model of problem drinking (Cooper, Frone, Russel, & Mudar, 1995; Read, Wood, Kahler, Maddock & Tibor, 2003), testing the notion that attachment is a common antecedent for both the affective and social paths to problem drinking. The model was tested with data from three samples, first-year university students (N=679), students about to graduate from university (N=206), and first-time clients at an addiction treatment facility (N=21 1). Participants completed a battery of questionnaires assessing alcohol use, alcohol-related consequences, drinking motives, peer models of alcohol use, positive and negative affect, attachment anxiety and attachment avoidance. Results underscored the importance of the affective path to problem drinking, while putting the social path to problem drinking into question. While drinking to cope was most prominent among the clinical sample, coping motives served as a risk factor for problem drinking for both individuals identified as problem drinkers and university students. Moreover, drinking for enhancement purposes appeared to be the strongest overall predictor of alcohol use. Results of the present study also supported the notion that attachment anxiety and avoidance are antecedents for the affective path to problem drinking, such that those with higher levels of attachment anxiety and avoidance were more vulnerable to experiencing adverse consequences related to their drinking, explained in terms of diminished affect regulation. Evidence that nonsecure attachment is a potent predictor of problem drinking was also demonstrated by the finding that attachment anxiety was directly related to alcohol-related consequences over and above its indirect relationship through affect regulation. However, results failed to show that attachment anxiety or attachment avoidance increased the risk of problem drinking via social influence.

The reliability of an isometric test based on constant perception of effort

To date there is no documented procedure to extrapolate findings of an isometric nature to a whole body performance setting. The purpose of this study was to quantify the reliability of perceived exertion to control neuromuscular output during an isometric contraction. 21 varsity athletes completed a maximal voluntary contraction and a 2 min constant force contraction at both the start and end of the study. Between pre and post testing all participants completed a 2 min constant perceived exertion contraction once a day for 4 days. Intra-class correlation coefficient (R=O.949) and standard error of measurement (SEM=5.12 Nm) concluded that the isometric contraction was reliable. Limits of agreement demonstrated only moderate initial reliability, yet with smaller limits towards the end of 4 training sessions. In conclusion, athlete's na"ive to a constant effort isometric contraction will produce reliable and acceptably stable results after 1 familiarization sessions has been completed.