Analysis of testicular cell populations using flow cytometry

Studying testis is complex, because the tissue has a very heterogeneous cell composition and its structure changes dynamically during development. In reproductive field, the cell composition is traditionally studied by morphometric methods such as immunohistochemistry and immunofluorescence. These techniques provide accurate quantitative information about cell composition, cell-cell association and localization of the cells of interest. However, the sample preparation, processing, staining and data analysis are laborious and may take several working days. Flow cytometry protocols coupled with DNA stains have played an important role in providing quantitative information of testicular cells populations ex vivo and in vitro studies. Nevertheless, the addition of specific cells markers such as intracellular antibodies would allow the more specific identification of cells of crucial interest during spermatogenesis. For this study, adult rat Sprague-Dawley rats were used for optimization of the flow cytometry protocol. Specific steps within the protocol were optimized to obtain a singlecell suspension representative of the cell composition of the starting material. Fixation and permeabilization procedure were optimized to be compatible with DNA stains and fluorescent intracellular antibodies. Optimization was achieved by quantitative analysis of specific parameters such as recovery of meiotic cells, amount of debris and comparison of the proportions of the various cell populations with already published data. As a result, a new and fast flow cytometry method coupled with DNA stain and intracellular antigen detection was developed. This new technique is suitable for analysis of population behavior and specific cells during postnatal testis development and spermatogenesis in rodents. This rapid protocol recapitulated the known vimentin and γH2AX protein expression patterns during rodent testis ontogenesis. Moreover, the assay was applicable for phenotype characterization of SCRbKO and E2F1KO mouse models.

Family rivalry in the testis: Retinoblastoma protein and E2F transcription factors in testicular development and adult spermatogenesis

Disorders of male reproductive health are becoming increasingly prevalent globally. These defects, ranging from decreasing sperm counts to an increasing rate of infertility and testicular cancer, have a common origin in the early phases of testicular development, but the exact mechanisms that cause them remain unknown. Testicular development and adult spermatogenesis are complex processes in which different cell types undergo mitosis, meiosis, differentiation and apoptosis. The retinoblastoma protein family and its associated E2F transcription factors are key regulators of these cellular events. In the present study, the functions of these factors in postnatal testicular development and adult spermatogenesis were explored using different animal models. In addition, a new application of flow cytometry to study testicular cell dynamics was developed. An ablation of retinoblastoma protein in mouse Sertoli cells resulted in their cell cycle re-entry in adult testes, dedifferentiation and a severe spermatogenic defect. We showed that deregulated E2F3 contributed to these changes. Our results indicated that the E2F1 transcription factor is critical for the control of apoptosis in the developing postnatal testis. In the adult testis, E2F1 controls the maintenance of the spermatogonial stem cell pool, in addition to inhibiting apoptosis of spermatocytes. In summary, this study elucidated the complex interdependencies of the RB and E2F transcription factor families in the control of postnatal testicular development and adult spermatogenesis. Furthermore, this study provided a new methodology for the analysis of testicular cells.

Entry mode choice of Finnish startups entering Chinese market in tablet industry

Since the implement of opening policy, the overall economy of China has maintained rapid and stable development, which has now makes China become the world's second largest economy. China, it is to become the largest overseas market for many large global enterprises from various industries, this naturally also includes the Tablet PC industry that raised in recent years. The purpose of this thesis is to analyze different internal and external factors that influence the entry mode choices of Finnish SMEs in tablet industry entering Chinese market. The goal is to find out the suitable entry modes for the Finnish tablet or other relevant SMEs entering Chinese market. Qualitative analysis is the main research method in empirical part of this study. The interviews were carried out with the case company and other two Finnish business organizations in China. The result of the study indicated that the internal resource and external business environment affect the entry modes choices much more than other factors for SMES. The exporting mode and sales subsidiary could be a better choice for SMEs entering Chinese market. Furthermore, firms should fully learn the Chinese market combine with its own background before making decisions.

Flow injection analysis (FIA) determination of lead by hydride generation in banded coral skeletons /

A flow injection hydride generation direct current plasma atomic emission spectrometric (FI-HG-DCP-AES) method was developed for the determination of lead at ng.ml-l level. Potassium ferricyanide (K3Fe(CN)6) was used along with sodium tetrahydroborate(III) (NaBH4) to produce plumbane (PbH4) in an acid medium. The design of a gas-liquid separator (hydride generator) was tested and the parameters of the flow injection system were optimized to achieve a good detection limit and sample throughput. The technique developed gave a detection limit of 0.7 ng.ml-l(3ob). The precision at 20 ng.ml"* level was 1.6 % RSD with 1 1 measurements (n=l 1). Volume of sample loop was 500 |J.l. A sample throughput of 120 h"^ was achieved. The transition elements, Fe(II), FeOH), Cd(n), Co(II), Mn(n), Ni(II) and Zn(n) do not interfere in this method but 1 mg,l'l Cu(II) will suppress 50 % of the signal from a sample containing 20 ng.ml'l Pb. This method was successfully applied to determine lead in a calcium carbonate (CaC03) matrix of banded coral skeletons from Si-Chang Island in Thailand.

Does altering brachial artery tone with lower-body negative pressure and flow-mediated dilation affect arterial stiffness?

Although medium sized, muscular vessels normally respond to sympathetic stimulation by reducing compliance, it is unclear whether the large brachial artery is similarly affected by sympathetic stimulation induced via lower-body negative pressure (LBNP). Similarly, the impact of flow-mediated dilation (FMD) on brachial artery compliance and distensibility remains unresolved, hi addition, before such measures can be used as prognostic tools, it is important to investigate the reliability and repeatability of both techniques. Using a randomized order design, the effects of LBNP and FMD on the mechanical properties of the brachial artery were examined in nine healthy male subjects (mean age 24y). Non-invasive Doppler ultrasound and a Finometer were used to measure simultaneously the variation in systolic and diastolic diameter, and brachial blood pressure, respectively. These values were used to calculate compliance and distensibility values at baseline, and during both LBNP and FMD. The within-day and between-day repeatability of arterial diameter, compliance, distensibility, and FMD measures were assessed using the error coefficient and intra-class correlation coefficient (ICC). While heart rate (P<0.01) and peripheral resistance increased during LBNP (P<0.05), forearm blood flow and pulse pressure decreased (P<0.01). hi terms of mechanical properties, vessel diameters decreased (P<0.05), but both compliance and distensibility were not changed. On the other hand, FMD resulted in a significant increase in diameter (P<0.001), with no change in compliance or distensibility. hi summary, LBNP and FMD do not appear to alter brachial artery compliance or distensibility in young, healthy males. Whereas measures ofFMD were not found to be repeatable between days, the ICC indicated that compliance and distensibility were repeatable only within-day.

Seasonal changes in behavioural and thermoregulatory responses to hypoxia in the Eastern Chipmunk (Tamias striatus) /

Mammalian heterotherms, such as hibemators, are known to be more tolerant of low oxygen tensions than their homeothermic counterparts. It has been suggested that this relative hypoxia tolerance is related to their ability to deal with dramatic changes in body temperature during entry to and arousal from torpor. However, hibemators demonstrate dramatic seasonality in both daily heterothermy and overall torpor expression. It was of interest to test if seasonal comparisons of normothermic individuals within a single species with the capacity to hibernate produce changes in the response to hypoxia that would reflect a seasonal change in tolerance to low oxygen. In particular, the species studied, the Eastern chipmunk {Tamias striatus), is known to enter into torpor exclusively in the winter. To test for seasonal differences in the metabolic and thermoregulatory responses to hypoxia, flow-through respirometry was used to compare metabolic rate, minimum thermal conductance, body temperature, and a thermal gradient used to assess selected ambient temperature in response to hypoxia in both summer and winter acclimated animals. Although the animals periodically expressed torpor throughout the winter, no differences between season in resting metabolic rate, body temperature or minimum thermal conductance were observed in normoxia. The metabolic trials indicated that chipmunks are less responsive to hypoxia in the winter than they are in the summer. Although body temperature dropped in response to hypoxia in both seasons, the decrease was less in the winter, and there was no corresponding decrease in metabolic rate. Providing the animals with a choice of ambient temperatures in hypoxia resulted in a blunting of the drop in body temperature in both seasons, suggesting that the reported fall in body temperature set point in hypoxia is not fully manifested in the behavioural pathways responsible for thermoregulation in chipmunks. Instead, body temperature in hypoxia appears to be highly dependent on ambient temperature and oxygen concentration. The results of this study suggest that the season in which the responses to hypoxia are measured is important, especially in a heterotherm where seasonality can affect the degree to 1 which the animal is tolerant of hypoxia. Winter-acclimated chipmunks appear more capable of defending metabolic heat production in hypoxia, a response consistent with the increased thermogenic capacity observed in animals that must periodically enter and arouse from torpor during hibernation.

The relation of attentional, emotional, and social regulation to cognitive competence, from infancy to school entry

Recent research on the sources of cognitive competence in infancy and early childhood has highlighted the role of social and emotional factors (for example, Lewis, 1993b). Exploring the roots of competence requires a longitudinal and multivariate approach. To deal with the resulting complexity, potentially integrative theoretical constructs are required. One logical candidate is self-regulation. Three key developmental questions were the focus of this investigation. 1) Does infant self-regulation (attentional, emotional, and social) predict preschool cognitive competence? 2) Does infant self-regulation predict preschool self-regulation? 3) Does preschool self-regulation predict concurrent preschool cognitive competence? One hundred preschoolers (46 females, 54 males; mean age = 5 years, 11 months) who had participated at 9- and/ or 12-months of age in an object permanence task were recruited to participate in this longitudinal investigation. Each subject completed four scales of the WPPSI-R and two social cognitive tasks. Parents completed questionnaires about their preschoolers' regulatory behaviours (Achenbach's Child Behavior Checklist [1991] and selected items from Eisenberg et ale [1993] and Derryberry & Rothbart [1988]). Separate behavioural coding systems were developed to capture regulatory capabilities in infancy (from the object permanence task) and preschool (from the WPPSIR Block Design). Overall, correlational and multiple regression results offered strong affirmative answers to the three key questions (R's = .30 to .38), using the behavioural observations of self-regulation. Behavioural regulation at preschool substantially predicted parental reports of regulation, but the latter variables did not predict preschool competence. Infant selfregulation and preschool regulation made statistically independent contributions to competence, even though regulation at Time 1 and Time 2 ii were substantially related. The results are interpreted as supporting a developmental pathway in which well-regulated infants more readily acquire both expertise and more sophisticated regulatory skills. Future research should address the origins of these skills earlier in infancy, and the social contexts that generate them and support them during the intervening years.

The effects of plants(Typha Latifolia)and root-bed medium on the treatment of domestic sewage within a vertical flow constructed wetland /

The effect that plants {Typha latifolia) as well as root-bed medium physical and chemical characteristics have on the treatment of primary treated domestic wastewater within a vertical flow constructed wetland system was investigated. Five sets of cells, with two cells in each set, were used. Each cell was made of concrete and measured 1 .0 m X 1 .0 m and was 1.3 m deep. Four different root-bed media were tested : Queenston Shale, Fonthill Sand, Niagara Shale and a Michigan Sand. Four of the sets contained plants and a single type of root-bed medium. The influence of plants was tested by operating a Queenston Shale set without plants. Due to budget constraints no replicates were constructed. All of the sets were operated independently and identically for twenty-eight months. Twelve months of data are presented here, collected after 16 months of continuous operation. Root-bed medium type did not influence BOD5 removal. All of the sets consistently met Ontario Ministry of Environment (MOE) requirements (<25 mg/L) for BOD5 throughout the year. The 12 month average BOD5 concentration from all sets with plants was below 2.36 mg/L. All of the sets were within MOE discharge requirements (< 25 mg/L) for suspended solids with set effluent concentrations ranging from 1.53 to 14.80 mg/L. The Queenston Shale and Fonthill Sand media removed the most suspended solids while the Niagara Shale set produced suspended solids. The set containing Fonthill Sand was the only series to meet MOE discharge requirements (< Img/L) for total phosphorus year-round with a twelve month mean effluent concentration of 0.23 mg/L. Year-round all of the root-bed media were well below MOE discharge requirements (< 20mg/L in winter and < 10 mg/L in sumnner) for ammonium. The Queenston Shale and Fonthill Sand sets removed the most total nitrogen. Plants had no effect on total nitrogen removal, but did influence how nitrogen was cycled within the system. Plants increased the removal of suspended solids by 14%, BOD5 by 10% and total phosphorus by 22%. Plants also increased the amount of dissolved oxygen that entered the system. During the plant growing season removal of total phosphorus was better in all sets with plants regardless of media type. The sets containing Queenston Shale and Fonthill Sand media achieved the best results and plants in the Queenston Shale set increased treatment efficiency for every parameter except nitrogen. Vertical flow wetland sewage treatment systems can be designed and built to consistently meet MOE discharge requirements year-round for BOD5, suspended solids, total phosphorus and ammonium. This system Is generally superior to the free water systems and sub-surface horizontal flow systems in cold climate situations.

Investigations in flow injection analysis (FIA): conventional FIA determination of chloride in water; sequential injection (SI)-FIA determination of mercury in water

Flow injection analysis (FIA) was applied to the determination of both chloride ion and mercury in water. Conventional FIA was employed for the chloride study. Investigations of the Fe3 +/Hg(SCN)2/CI-,450 nm spectrophotometric system for chloride determination led to the discovery of an absorbance in the 250-260 nm region when Hg(SCN)2 and CI- are combined in solution, in the absence of iron(III). Employing an in-house FIA system, absorbance observed at 254 nm exhibited a linear relation from essentially 0 - 2000 Jlg ml- 1 injected chloride. This linear range spanning three orders of magnitude is superior to the Fe3+/Hg(SCN)2/CI- system currently employed by laboratories worldwide. The detection limit obtainable with the proposed method was determin~d to be 0.16 Jlg ml- 1 and the relative standard deviation was determined to be 3.5 % over the concentration range of 0-200 Jig ml- 1. Other halogen ions were found to interfere with chloride determination at 254 nm whereas cations did not interfere. This system was successfully applied to the determination of chloride ion in laboratory water. Sequential injection (SI)-FIA was employed for mercury determination in water with the PSA Galahad mercury amalgamation, and Merlin mercury fluorescence detection systems. Initial mercury in air determinations involved injections of mercury saturated air directly into the Galahad whereas mercury in water determinations involved solution delivery via peristaltic pump to a gas/liquid separator, after reduction by stannous chloride. A series of changes were made to the internal hardware and valving systems of the Galahad mercury preconcentrator. Sequential injection solution delivery replaced the continuous peristaltic pump system and computer control was implemented to control and integrate all aspects of solution delivery, sample preconcentration and signal processing. Detection limits currently obtainable with this system are 0.1 ng ml-1 HgO.