Comparison of promoter-specific events during transcription initiation in mycobacteria

DNA protein interactions that occur during transcription initiation play an important role in regulating gene expression. To initiate transcription, RNA polymerase (RNAP) binds to promoters in a sequence-specific fashion. This is followed by a series of steps governed by the equilibrium binding and kinetic rate constants, which in turn determine the overall efficiency of the transcription process. We present here the first detailed kinetic analysis of promoter RNAP interactions during transcription initiation in the sigma(A)-dependent promoters P-rrnAPCL1, P-rrnB and P-gyr of Mycobacterium smegmatis. The promoters show comparable equilibrium binding affinity but differ significantly in open complex formation, kinetics of isomerization and promoter clearance. Furthermore, the two rrn promoters exhibit varied kinetic properties during transcription initiation and appear to be subjected to different modes of regulation. In addition to distinct kinetic patterns, each one of the housekeeping promoters studied has its own rate-limiting step in the initiation pathway, indicating the differences in their regulation.

Early biochemical events during adventitious root initiation in the hypocotyl of Phaseolus vulgaris

Indole butyric acid (IBA) initiates roots in the hypocotyl tissue of Phaseolus vulgaris (French bean). The response is dependent on the concentration of IBA and the duration of exposure to the hormone. IBA enhances the rate of total protein synthesis in ca 30 min after exposure of the hypocotyl segments to the hormone. There is no detectable change in total or poly(A)-containing RNA synthesis in this period although significant increases are seen 2 hr after hormone pre-treatment. The early IBA-mediated increase in protein synthesis (30 min) is not sensitive to Actinomycin D but the antibiotic blocks the increase manifested 2 hr after hormone pre-treatment. Inhibition of early protein synthesis by cycloheximide depresses and delays root initiation. Cytosol prepared from IBA-treated hypocotyl tissue stimulates protein synthesis in vitro to a greater extent than that of the control.

Opportunity Exploration and Exploitation in International New Ventures: A Study of Relationships’ Involvement in Early Entrepreneurial and Internationalisation Events

International new ventures (INVs) are firms that engage very early after their foundation, if not immediately, in inter-national activities. INVs are a relatively recent phenomenon that deviates from earlier theories on international business. In order to develop our understanding of the emergence and early internationalisation of INVs three different research areas are built upon in the dissertation: International Entrepreneurship, Entrepreneurship and Networks. Net-works have been identified as important for INVs. However, there is a lack of more profound studies regarding the way different types of relationships influence INVs. Few studies are concerned with exploration and exploitation of opportunities and research on the benefits and drawbacks of entrepreneurs’ relationships for the international opportunity recognition process has been called for. By taking a network approach to opportunity exploration and exploitation, the dissertation develops our under-standing of how entrepreneurs’ relationships are involved in exploring and exploiting opportunities during an INV’s early and critical entrepreneurial and internationalisation events. The critical events are studied during three phases: pre-founding, start-up and early internationalisation. Since internationalisation is present from the very beginning, the early internationalisation phase may be parallel to both the pre-founding and the start-up phase. The dissertation contributes to international entrepreneur-ship research in mainly two ways. First, by offering a deep insight into which opportunity exploration and exploitation activities entrepreneurs’ relationships are involved. Second, by adding to our understanding of what the relationships contribute to these activities, mainly in the sense of benefits gained through the relationships. Studying micro firms in real time in their early development towards INVs is considered a unique contribution of the study as it offers valuable insights into pre-founding, start-up, pre-internationalisation as well as early internationalisation. The study shows that in order to understand the development of INVs, it is beneficial to go back to times when there was no thought of starting the INV. By focusing on the entrepreneurs’ background and relationships a more complete picture of the INV is gained. Relationships created at former workplaces or during school time might be the ones that develop business opportunities and set off internationalisation. By focusing on the pre-founding phase, the study also contributes to entrepreneurship literature as this stage has often been neglected or assumed obvious in earlier research. This dissertation shows that an important and mostly lengthy pre-founding phase precedes the decision to start a f rm. In addition, the integration of entrepreneurs’ real experiences with existing theory to develop a continuum for the strength of relationships allows for contributions to network theory.

Doing marriage and parenthood: The division of household labour across two life course events

The current study is a longitudinal investigation into changes in the division of household labour across transitions to marriage and parenthood in the UK. Previous research has noted a more traditional division of household labour, with women performing the majority of housework, amongst spouses and couples with children. However, the bulk of this work has been cross-sectional in nature. The few longitudinal studies that have been carried out have been rather ambiguous about the effect of marriage and parenthood on the division of housework. Theoretically, this study draws on gender construction theory. The key premise of this theory is that gender is something that is performed and created in interaction, and, as a result, something fluid and flexible rather than fixed and stable. The idea that couples 'do gender' through housework has been a major theoretical breakthrough. Gender-neutral explanations of the division of household labour, positing rational acting individuals, have failed to explicate why women continue to perform an unequal share of housework, regardless of socioeconomic status. Contrastingly, gender construction theory situates gender as the key process in dividing household labour. By performing and avoiding certain housework chores, couples fulfill social norms of what it means to be a man and a woman although, given the emphasis on human agency in producing and contesting gender, couples are able to negotiate alternative gender roles which, in turn, feed back into the structure of social norms in an ever-changing societal landscape. This study adds extra depth to the doing gender approach by testing whether or not couples negotiate specific conjugal and parent roles in terms of the division of household labour. Both transitions hypothesise a more traditional division of household labour. Data comes from the British Household Panel Survey, a large, nationally representative quantitative survey that has been carried out annually since 1991. Here, data tracks the same 776 couples at two separate time points - 1996 and 2005. OLS regression is used to test whether or not transitions to marriage and parenthood have a significant impact on the division of household labour whilst controlling for host of relevant socio-economic factors. Results indicate that marriage has no significant effect on how couples partition housework. Those couples making the transition from cohabitation to marriage do not show significant changes in housework arrangements from those couples who remain cohabiting in both waves. On the other hand, becoming parents does lead to a more traditional division of household labour whilst controlling for socio-economic factors which accompany the move to parenthood. There is then some evidence that couples use the site of household labour to 'do parenthood' and generate identities which both use and inform socially prescribed notions of what it means to be a mother and a father. Support for socio-economic explanations of the division of household labour was mixed although it remains clear that they, alone, cannot explain how households divide housework.

Functional outcome and health-related quality of life after traumatic brain injury in the framework of the International Classification of Functioning, Disability and Health (ICF)

Traumatic brain injury (TBI) affects people of all ages and is a cause of long-term disability. In recent years, the epidemiological patterns of TBI have been changing. TBI is a heterogeneous disorder with different forms of presentation and highly individual outcome regarding functioning and health-related quality of life (HRQoL). The meaning of disability differs from person to person based on the individual s personality, value system, past experience, and the purpose he or she sees in life. Understanding of all these viewpoints is needed in comprehensive rehabilitation. This study examines the epidemiology of TBI in Finland as well as functioning and HRQoL after TBI, and compares the subjective and objective assessments of outcome. The frame of reference is the International Classification of Functioning, Disability and Health (ICF). The subjects of Study I represent the population of Finnish TBI patients who experienced their first TBI between 1991 and 2005. The 55 Finnish subjects of Studies II and IV participated in the first wave of the international Quality of life after brain injury (QOLIBRI) validation study. The 795 subjects from six language areas of Study III formed the second wave of the QOLIBRI validation study. The average annual incidence of Finnish hospitalised TBI patients during the years 1991-2005 was 101:100 000 in patients who had TBI as the primary diagnosis and did not have a previous TBI in their medical history. Males (59.2%) were at considerably higher risk of getting a TBI than females. The most common external cause of the injury was falls in all age groups. The number of TBI patients ≥ 70 years of age increased by 59.4% while the number of inhabitants older than 70 years increased by 30.3% in the population of Finland during the same time period. The functioning of a sample of 55 persons with TBI was assessed by extracting information from the patients medical documents using the ICF checklist. The most common problems were found in the ICF components of Body Functions (b) and Activities and Participation (d). HRQoL was assessed with the QOLIBRI which showed the highest level of satisfaction on the Emotions, Physical Problems and Daily Life and Autonomy scales. The highest scores were obtained by the youngest participants and participants living independently without the help of other people, and by people who were working. The relationship between the functional outcome and HRQoL was not straightforward. The procedure of linking the QOLIBRI and the GOSE to the ICF showed that these two outcome measures cover the relevant domains of TBI patients functioning. The QOLIBRI provides the patients subjective view, while the GOSE summarises the objective elements of functioning. Our study indicates that there are certain domains of functioning that are not traditionally sufficiently documented but are important for the HRQoL of persons with TBI. This was the finding especially in the domains of interpersonal relationships, social and leisure activities, self, and the environment. Rehabilitation aims to optimize functioning and to minimize the experience of disability among people with health conditions, and it needs to be based on a comprehensive understanding of human functioning. As an integrative model, the ICF may serve as a frame of reference in achieving such an understanding.

The variants of the protein toxins abrin and ricin A useful guide to understanding the processing events in the toxin transport

Kinetic data on inhibition of protein synthesis in thymocyte by three abrins and ricin have been obtained. The intrinsic efficiencies of A chains of four toxins to inactivate ribosomes, as analyzed by k1-versus-concentration plots were abrin II, III > ricin > abrin I. The lag times were 90, 66, 75 and 105 min at a 0.0744 nM concentration of each of abrin I, II, III and ricin, respectively. To account for the observed differences in the dose-dependent lag time, functional and structural variables of toxins such as binding efficiency of B chains to receptors and low-pH-induced structural alterations have been analyzed. The association constants obtained by stopped flow studies showed that abrin-I (4.13 × 105 M−1 s−1) association with putative receptor (4-methylumbelliferyl-α-D-galactoside) is nearly two times more often than abrin III (2.6 × 105 M−1 s−1) at 20°C. Equillibrium binding constants of abrin I and II to thymocyte at 37°C were 2.26 × 107 M−1 and 2.8 × 107 M−1 respectively. pH-induced structural alterations as studied by a parallel enhancement in 8-anilino-L-naphthalene sulfonate fluorescence revealed a high degree of qualitative similarity. These results taken with a nearly identical concentration-independent lag time (minimum lag of 41–42 min) indicated that the binding efficiencies and internalization efficiencies of these toxins are the same and that the observed difference in the dose-dependent lag time is causally related to the proposed processing event. The rates of reduction of inter-subunit disulfide bond, an obligatory step in the intoxication process, have been measured and compared under a variety of conditions. Intersubunit disulfide reduction of abrin I is fourfold faster than that of abrin II at pH 7.2. The rate of disulfide reduction in abrin I could be decreased 1 I-fold by adding lactose, compared to that without lactose. The observed differences in the efficiencies of A chains, the dose-dependent lag period, the modulating effect of lactose on the rates of disulfide reduction and similarity in binding properties make the variants a valuable tool to probe the processing events in toxin transport in detail.

Gene Expression Profiling of Preovulatory Follicle in the Buffalo Cow: Effects of Increased IGF-I Concentration on Periovulatory Events

The preovulatory follicle in response to gonadotropin surge undergoes dramatic biochemical, and morphological changes orchestrated by expression changes in hundreds of genes. Employing well characterized bovine preovulatory follicle model, granulosa cells (GCs) and follicle wall were collected from the preovulatory follicle before, 1, 10 and 22 h post peak LH surge. Microarray analysis performed on GCs revealed that 450 and 111 genes were differentially expressed at 1 and 22 h post peak LH surge, respectively. For validation, qPCR and immunocytochemistry analyses were carried out for some of the differentially expressed genes. Expression analysis of many of these genes showed distinct expression patterns in GCs and the follicle wall. To study molecular functions and genetic networks, microarray data was analyzed using Ingenuity Pathway Analysis which revealed majority of the differentially expressed genes to cluster within processes like steroidogenesis, cell survival and cell differentiation. In the ovarian follicle, IGF-I is established to be an important regulator of the above mentioned molecular functions. Thus, further experiments were conducted to verify the effects of increased intrafollicular IGF-I levels on the expression of genes associated with the above mentioned processes. For this purpose, buffalo cows were administered with exogenous bGH to transiently increase circulating and intrafollicular concentrations of IGF-I. The results indicated that increased intrafollicular concentrations of IGF-I caused changes in expression of genes associated with steroidogenesis (StAR, SRF) and apoptosis (BCL-2, FKHR, PAWR). These results taken together suggest that onset of gonadotropin surge triggers activation of various biological pathways and that the effects of growth factors and peptides on gonadotropin actions could be examined during preovulatory follicle development.

Indian Ocean dipole mode events in an ocean general circulation model

The evolution of the dipole mode (DM) events in the Indian Ocean is examined using an ocean model that is driven by the NCEP fluxes for the period 1975-1998. The positive DM events during 1997, 1994 and 1982 and negative DM events during 1996 and 1984-1985 are captured by the model and it reproduces both the surface and subsurface features associated with these events. In its positive phase, the DM is characterized by warmer than normal SST in the western Indian Ocean and cooler than normal SST in the eastern Indian Ocean. The DM events are accompanied by easterly wind anomalies along the equatorial Indian Ocean and upwelling-favorable alongshore wind anomalies along the coast of Sumatra. The Wyrtki jets are weak during positive DM events, and the thermocline is shallower than normal in the eastern Indian Ocean and deeper in the west. This anomaly pattern reverses during negative DM events. During the positive phase of the DM easterly wind anomalies excite an upwelling equatorial Kelvin wave. This Kelvin wave reflects from the eastern boundary as an upwelling Rossby wave which propagates westward across the equatorial Indian Ocean. The anomalies in the eastern Indian Ocean weaken after the Rossby wave passes. A similar process excites a downwelling Rossby wave during the negative phase. This Rossby wave is much weaker but wind forcing in the central equatorial Indian Ocean amplifies the downwelling and increases its westward phase speed. This Rossby wave initiates the deepening of the thermocline in the western Indian Ocean during the following positive phase of the DM. Rossby wave generated in the southern tropical Indian Ocean by Ekman pumping contributes to this warming. Concurrently, the temperature equation of the model shows upwelling and downwelling to be the most important mechanism during both positive events of 1994 and 1997. (C) 2002 Elsevier Science Ltd. All rights reserved.

A study of epileptogenic network structures in rat hippocampal cultures using first spike latencies during synchronization events

Study of hypersynchronous activity is of prime importance for combating epilepsy. Studies on network structure typically reconstruct the network by measuring various aspects of the interaction between neurons and subsequently measure the properties of the reconstructed network. In sub-sampled networks such methods lead to significant errors in reconstruction. Using rat hippocampal neurons cultured on a multi-electrode array dish and a glutamate injury model of epilepsy in vitro, we studied synchronous activity in neuronal networks. Using the first spike latencies in various neurons during a network burst, we extract various recurring spatio-temporal onset patterns in the networks. Comparing the patterns seen in control and injured networks, we observe that injured networks express a wide diversity in their foci (origin) and activation pattern, while control networks show limited diversity. Furthermore, we note that onset patterns in glutamate injured networks show a positive correlation between synchronization delay and physical distance between neurons, while control networks do not.

Signatures of Indian ocean Dipole and El Nino-Southern Oscillation events in sea level variations in the Bay of Bengal

We investigate the impact of the Indian Ocean Dipole (IOD) and El Nino and the Southern Oscillation (ENSO) on sea level variations in the North Indian Ocean during 1957-2008. Using tide-gauge and altimeter data, we show that IOD and ENSO leave characteristic signatures in the sea level anomalies (SLAs) in the Bay of Bengal. During a positive IOD event, negative SLAs are observed during April-December, with the SLAs decreasing continuously to a peak during September-November. During El Nino, negative SLAs are observed twice (April-December and November-July), with a relaxation between the two peaks. SLA signatures during negative IOD and La Nina events are much weaker. We use a linear, continuously stratified model of the Indian Ocean to simulate their sea level patterns of IOD and ENSO events. We then separate solutions into parts that correspond to specific processes: coastal alongshore winds, remote forcing from the equator via reflected Rossby waves, and direct forcing by interior winds within the bay. During pure IOD events, the SLAs are forced both from the equator and by direct wind forcing. During ENSO events, they are primarily equatorially forced, with only a minor contribution from direct wind forcing. Using a lead/lag covariance analysis between the Nino-3.4 SST index and Indian Ocean wind stress, we derive a composite wind field for a typical El Nino event: the resulting solution has two negative SLA peaks. The IOD and ENSO signatures are not evident off the west coast of India.