Discovery of a 29-gene panel in peripheral blood mononuclear cells for the detection of colorectal cancer and adenomas using high throughput real-time PCR.

Colorectal cancer (CRC) is the second leading cause of cancer-related death in developed countries. Early detection of CRC leads to decreased CRC mortality. A blood-based CRC screening test is highly desirable due to limited invasiveness and high acceptance rate among patients compared to currently used fecal occult blood testing and colonoscopy. Here we describe the discovery and validation of a 29-gene panel in peripheral blood mononuclear cells (PBMC) for the detection of CRC and adenomatous polyps (AP). Blood samples were prospectively collected from a multicenter, case-control clinical study. First, we profiled 93 samples with 667 candidate and 3 reference genes by high throughput real-time PCR (OpenArray system). After analysis, 160 genes were retained and tested again on 51 additional samples. Low expressed and unstable genes were discarded resulting in a final dataset of 144 samples profiled with 140 genes. To define which genes, alone or in combinations had the highest potential to discriminate AP and/or CRC from controls, data were analyzed by a combination of univariate and multivariate methods. A list of 29 potentially discriminant genes was compiled and evaluated for its predictive accuracy by penalized logistic regression and bootstrap. This method discriminated AP >1cm and CRC from controls with a sensitivity of 59% and 75%, respectively, with 91% specificity. The behavior of the 29-gene panel was validated with a LightCycler 480 real-time PCR platform, commonly adopted by clinical laboratories. In this work we identified a 29-gene panel expressed in PBMC that can be used for developing a novel minimally-invasive test for accurate detection of AP and CRC using a standard real-time PCR platform.

Selection of internal control genes for real-time quantitative PCR in ovary and uterus of sows across pregnancy

Reproductive traits play a key role in pig production in order to reduce costs and increase economic returns. Among others, gene expression analyses represent a useful approach to study genetic mechanisms underlying reproductive traits in pigs. The application of reverse-transcription quantitative PCR requires the selection of appropriate reference genes, whose expression levels should not be affected by the experimental conditions, especially when comparing gene expression across different physiological stages.

Methods of Design and Testing of Real Time Systems

Teollusuussovelluksissa vaaditaan nykyisin yhä useammin reaaliaikaista tiedon käsittelyä. Luotettavuus on yksi tärkeimmistä reaaliaikaiseen tiedonkäsittelyyn kykenevän järjestelmän ominaisuuksista. Sen saavuttamiseksi on sekä laitteisto, että ohjelmisto testattava. Tämän työn päätavoitteena on laitteiston testaaminen ja laitteiston testattavuus, koska luotettava laitteistoalusta on perusta tulevaisuuden reaaliaikajärjestelmille. Diplomityössä esitetään digitaaliseen signaalinkäsittelyyn soveltuvan prosessorikortin suunnittelu. Prosessorikortti on tarkoitettu sähkökoneiden ennakoivaa kunnonvalvontaa varten. Uusimmat DFT (Desing for Testability) menetelmät esitellään ja niitä sovelletaan prosessorikortin sunnittelussa yhdessä vanhempien menetelmien kanssa. Kokemukset ja huomiot menetelmien soveltuvuudesta raportoidaan työn lopussa. Työn tavoitteena on kehittää osakomponentti web -pohjaiseen valvontajärjestelmään, jota on kehitetty Sähkötekniikan osastolla Lappeenrannan teknillisellä korkeakoululla.

Beaming into the rat world: enabling real-time intereaction between rat and human each at their own scale

Immersive virtual reality (IVR) typically generates the illusion in participants that they are in the displayed virtual scene where they can experience and interact in events as if they were really happening. Teleoperator (TO) systems place people at a remote physical destination embodied as a robotic device, and where typically participants have the sensation of being at the destination, with the ability to interact with entities there. In this paper, we show how to combine IVR and TO to allow a new class of application. The participant in the IVR is represented in the destination by a physical robot (TO) and simultaneously the remote place and entities within it are represented to the participant in the IVR. Hence, the IVR participant has a normal virtual reality experience, but where his or her actions and behaviour control the remote robot and can therefore have physical consequences. Here, we show how such a system can be deployed to allow a human and a rat to operate together, but the human interacting with the rat on a human scale, and the rat interacting with the human on the rat scale. The human is represented in a rat arena by a small robot that is slaved to the human"s movements, whereas the tracked rat is represented to the human in the virtual reality by a humanoid avatar. We describe the system and also a study that was designed to test whether humans can successfully play a game with the rat. The results show that the system functioned well and that the humans were able to interact with the rat to fulfil the tasks of the game. This system opens up the possibility of new applications in the life sciences involving participant observation of and interaction with animals but at human scale.

Beaming into the rat world: enabling real-time intereaction between rat and human each at their own scale

Immersive virtual reality (IVR) typically generates the illusion in participants that they are in the displayed virtual scene where they can experience and interact in events as if they were really happening. Teleoperator (TO) systems place people at a remote physical destination embodied as a robotic device, and where typically participants have the sensation of being at the destination, with the ability to interact with entities there. In this paper, we show how to combine IVR and TO to allow a new class of application. The participant in the IVR is represented in the destination by a physical robot (TO) and simultaneously the remote place and entities within it are represented to the participant in the IVR. Hence, the IVR participant has a normal virtual reality experience, but where his or her actions and behaviour control the remote robot and can therefore have physical consequences. Here, we show how such a system can be deployed to allow a human and a rat to operate together, but the human interacting with the rat on a human scale, and the rat interacting with the human on the rat scale. The human is represented in a rat arena by a small robot that is slaved to the human"s movements, whereas the tracked rat is represented to the human in the virtual reality by a humanoid avatar. We describe the system and also a study that was designed to test whether humans can successfully play a game with the rat. The results show that the system functioned well and that the humans were able to interact with the rat to fulfil the tasks of the game. This system opens up the possibility of new applications in the life sciences involving participant observation of and interaction with animals but at human scale.

Improving the molecular diagnosis of Chlamydia psittaci and Chlamydia abortus infection with a species-specific duplex real-time PCR.

Chlamydia psittaci and Chlamydia abortus are closely related intracellular bacteria exhibiting different tissue tropism that may cause severe but distinct infection in humans. C. psittaci causes psittacosis, a respiratory zoonotic infection transmitted by birds. C. abortus is an abortigenic agent in small ruminants, which can also colonize the human placenta and lead to foetal death and miscarriage. Infections caused by C. psittaci and C. abortus are underestimated mainly due to diagnosis difficulties resulting from their strict intracellular growth. We developed a duplex real-time PCR to detect and distinguish these two bacteria in clinical samples. The first PCR (PCR1) targeted a sequence of the 16S-23S rRNA operon allowing the detection of both C. psittaci and C. abortus. The second PCR (PCR2) targeted the coding DNA sequence CPSIT_0607 unique to C. psittaci. The two PCRs showed 100 % detection for ≥ 10 DNA copies per reaction (1000 copies ml- 1). Using a set of 120 samples, including bacterial reference strains, clinical specimens and infected cell culture material, we monitored 100 % sensitivity and 100 % specificity for the detection of C. psittaci and C. abortus for PCR1. When PCR1 was positive, PCR2 could discriminate C. psittaci from C. abortus with a positive predictive value of 100 % and a negative predictive value of 88 %. In conclusion, this new duplex PCR represents a low-cost and time-saving method with high-throughput potential, expected to improve the routine diagnosis of psittacosis and pregnancy complication in large-scale screening programs and also during outbreaks.

Capillary thermal desorption unit for near real-time analysis of VOCs at sub-trace levels. Application to the analysis of environmental air contamination and breath samples

A capillary microtrap thermal desorption module is developed for near real-time analysis of volatile organic compounds (VOCs) at sub-ppbv levels in air samples. The device allows the direct injection of the thermally desorbed VOCs into a chromatographic column. It does not use a second cryotrap to focalize the adsorbed compounds before entering the separation column so reducing the formation of artifacts. The connection of the microtrap to a GC–MS allows the quantitative determination of VOCs in less than 40 min with detection limits of between 5 and 10 pptv (25 °C and 760 mmHg), which correspond to 19–43 ng m−3, using sampling volumes of 775 cm3. The microtrap is applied to the analysis of environmental air contamination in different laboratories of our faculty. The results obtained indicate that most volatile compounds are easily diffused through the air and that they also may contaminate the surrounding areas when the habitual safety precautions (e.g., working under fume hoods) are used during the manipulation of solvents. The application of the microtrap to the analysis of VOCs in breath samples suggest that 2,5-dimethylfuran may be a strong indicator of a person's smoking status

Integration of Real-Time Simulator, Motion Platform and Haptics

This thesis describes the process of the integration of a real-time simulator environment with a motion platform and a haptic device as a part of the Kvalive project. Several programs running on two computers were made to control the different devices of the environment. User tests were made to obtain information of needed improvements to make the simulator more realistic. Also new ideas for improving the simulator and directions of further research were obtained with the help of this research.

Real-Time Simulation of Mobile and Industrial Machines Using the Multibody Simulation Approach

This thesis introduces a real-time simulation environment based on the multibody simulation approach. The environment consists of components that are used in conventional product development, including computer aided drawing, visualization, dynamic simulation and finite element software architecture, data transfer and haptics. These components are combined to perform as a coupled system on one platform. The environment is used to simulate mobile and industrial machines at different stages of a product life time. Consequently, the demands of the simulated scenarios vary. In this thesis, a real-time simulation environment based on the multibody approach is used to study a reel mechanism of a paper machine and a gantry crane. These case systems are used to demonstrate the usability of the real-time simulation environment for fault detection purposes and in the context of a training simulator. In order to describe the dynamical performance of a mobile or industrial machine, the nonlinear equations of motion must be defined. In this thesis, the dynamical behaviour of machines is modelled using the multibody simulation approach. A multibody system may consist of rigid and flexible bodies which are joined using kinematic joint constraints while force components are used to describe the actuators. The strength of multibody dynamics relies upon its ability to describe nonlinearities arising from wearing of the components, friction, large rotations or contact forces in a systematic manner. For this reason, the interfaces between subsystems such as mechanics, hydraulics and control systems of the mechatronic machine can be defined and analyzed in a straightforward manner.

Detection and dynamics of porcine circovirus 2 shedding in semen using conventional and real-time PCR

The dynamics of porcine circovirus type 2 (PCV2) shedding in semen of naturally infected boars was studied. Semen was collected serially each 15 or 20 days during 62 days from 5 boars from a herd and from 11 boars from an artificial insemination center. All boars were positive for PCV2 DNA by nested polymerase chain reaction of raw semen in at least two sampling dates, and most of them had detectable shedding in all sampling dates. Real-time quantitative PCR was performed in 23 samples. All samples showed low amounts of PCV2 DNA, ranging from 98 to 652 PCV2 copies/mL. No differences between the frequencies of PCV2 DNA shed in semen were found considering herds and age of boars. PCV2 shedding in the semen can occur continuously or intermittently up to 60 days in naturally infected boars at 12 to 42 months old in absence of PCV2 clinical signs. These results demonstrate sporadic and long-term shedding patterns of low amounts of PCV2 DNA in semen from naturally infected boars.

Comparison between dSPACE and NI systems based on real-time intelligent control of a teleoperated hydraulic servo system

The Laboratory of Intelligent Machine researches and develops energy-efficient power transmissions and automation for mobile construction machines and industrial processes. The laboratory's particular areas of expertise include mechatronic machine design using virtual technologies and simulators and demanding industrial robotics. The laboratory has collaborated extensively with industrial actors and it has participated in significant international research projects, particularly in the field of robotics. For years, dSPACE tools were the lonely hardware which was used in the lab to develop different control algorithms in real-time. dSPACE's hardware systems are in widespread use in the automotive industry and are also employed in drives, aerospace, and industrial automation. But new competitors are developing new sophisticated systems and their features convinced the laboratory to test new products. One of these competitors is National Instrument (NI). In order to get to know the specifications and capabilities of NI tools, an agreement was made to test a NI evolutionary system. This system is used to control a 1-D hydraulic slider. The objective of this research project is to develop a control scheme for the teleoperation of a hydraulically driven manipulator, and to implement a control algorithm between human and machine interaction, and machine and task environment interaction both on NI and dSPACE systems simultaneously and to compare the results.

Evaluation of stunned and infarcted canine myocardium by real time myocardial contrast echocardiography

Differentiation between stunned and infarcted myocardium in the setting of acute ischemia is challenging. Real time myocardial contrast echocardiography allows the simultaneous assessment of myocardial perfusion and function. In the present study we evaluated infarcted and stunned myocardium in an experimental model using real time myocardial contrast echocardiography. Sixteen dogs underwent 180 min of coronary occlusion followed by reperfusion (infarct model) and seven other dogs were submitted to 20 min of coronary occlusion followed by reperfusion (stunned model). Wall motion abnormality and perfusional myocardial defect areas were measured by planimetry. Risk and infarct areas were determined by tissue staining. In the infarct model, the wall motion abnormality area during coronary occlusion (5.52 ± 1.14 cm²) was larger than the perfusional myocardial defect area (3.71 ± 1.45 cm²; P < 0.001). Reperfusion resulted in maintenance of wall motion abnormality (5.45 ± 1.41 cm²; P = 0.43 versus occlusion) and reduction of perfusional myocardial defect (1.51 ± 1.29 cm²; P = 0.004 versus occlusion). Infarct size determined by contrast echocardiography correlated with tissue staining (r = 0.71; P = 0.002). In the stunned model, the wall motion abnormality area was 5.49 ± 0.68 cm² during occlusion and remained 5.1 ± 0.63 cm² after reperfusion (P = 0.07). Perfusional defect area was 2.43 ± 0.79 cm² during occlusion and was reduced to 0.2 ± 0.53 cm² after reperfusion (P = 0.04). 2,3,5-Triphenyl tetrazolium chloride staining confirmed the absence of necrotic myocardium in all dogs in the stunned model. Real time myocardial contrast echocardiography is a noninvasive technique capable of distinguishing between stunned and infarcted myocardium after acute ischemia.

Novel virtual environment and real-time simulation based methods for improving life-cycle efficiency of non-road mobile machinery

Virtual environments and real-time simulators (VERS) are becoming more and more important tools in research and development (R&D) process of non-road mobile machinery (NRMM). The virtual prototyping techniques enable faster and more cost-efficient development of machines compared to use of real life prototypes. High energy efficiency has become an important topic in the world of NRMM because of environmental and economic demands. The objective of this thesis is to develop VERS based methods for research and development of NRMM. A process using VERS for assessing effects of human operators on the life-cycle efficiency of NRMM was developed. Human in the loop simulations are ran using an underground mining loader to study the developed process. The simulations were ran in the virtual environment of the Laboratory of Intelligent Machines of Lappeenranta University of Technology. A physically adequate real-time simulation model of NRMM was shown to be reliable and cost effective in testing of hardware components by the means of hardware-in-the-loop (HIL) simulations. A control interface connecting integrated electro-hydraulic energy converter (IEHEC) with virtual simulation model of log crane was developed. IEHEC consists of a hydraulic pump-motor and an integrated electrical permanent magnet synchronous motorgenerator. The results show that state of the art real-time NRMM simulators are capable to solve factors related to energy consumption and productivity of the NRMM. A significant variation between the test drivers is found. The results show that VERS can be used for assessing human effects on the life-cycle efficiency of NRMM. HIL simulation responses compared to that achieved with conventional simulation method demonstrate the advances and drawbacks of various possible interfaces between the simulator and hardware part of the system under study. Novel ideas for arranging the interface are successfully tested and compared with the more traditional one. The proposed process for assessing the effects of operators on the life-cycle efficiency will be applied for wider group of operators in the future. Driving styles of the operators can be analysed statistically from sufficient large result data. The statistical analysis can find the most life-cycle efficient driving style for the specific environment and machinery. The proposed control interface for HIL simulation need to be further studied. The robustness and the adaptation of the interface in different situations must be verified. The future work will also include studying the suitability of the IEHEC for different working machines using the proposed HIL simulation method.

Bioeffects of albumin-encapsulated microbubbles and real-time myocardial contrast echocardiography in an experimental canine model

Myocardial contrast echocardiography has been used for assessing myocardial perfusion. Some concerns regarding its safety still remain, mainly regarding the induction of microvascular alterations. We sought to determine the bioeffects of microbubbles and real-time myocardial contrast echocardiography (RTMCE) in a closed-chest canine model. Eighteen mongrel dogs were randomly assigned to two groups. Nine were submitted to continuous intravenous infusion of perfluorocarbon-exposed sonicated dextrose albumin (PESDA) plus continuous imaging using power pulse inversion RTMCE for 180 min, associated with manually deflagrated high-mechanical index impulses. The control group consisted of 3 dogs submitted to continuous imaging using RTMCE without PESDA, 3 dogs received PESDA alone, and 3 dogs were sham-operated. Hemodynamics and cardiac rhythm were monitored continuously. Histological analysis was performed on cardiac and pulmonary tissues. No hemodynamic changes or cardiac arrhythmias were observed in any group. Normal left ventricular ejection fraction and myocardial perfusion were maintained throughout the protocol. Frequency of mild and focal microhemorrhage areas in myocardial and pulmonary tissue was similar in PESDA plus RTMCE and control groups. The percentages of positive microscopical fields in the myocardium were 0.4 and 0.7% (P = NS) in the PESDA plus RTMCE and control groups, respectively, and in the lungs they were 2.1 and 1.1%, respectively (P = NS). In this canine model, myocardial perfusion imaging obtained with PESDA and RTMCE was safe, with no alteration in cardiac rhythm or left ventricular function. Mild and focal myocardial and pulmonary microhemorrhages were observed in both groups, and may be attributed to surgical tissue manipulation.