Plectin interacts with the rod domain of type III intermediate filament proteins desmin and vimentin

Plectin is a versatile cytolinker protein critically involved in the organization of the cytoskeletal filamentous system. The muscle-specific intermediate filament (IF) protein desmin, which progressively replaces vimentin during differentiation of myoblasts, is one of the important binding partners of plectin in mature muscle. Defects of either plectin or desmin cause muscular dystrophies. By cell transfection studies, yeast two-hybrid, overlay and pull-down assays for binding analysis, we have characterized the functionally important sequences for the interaction of plectin with desmin and vimentin. The association of plectin with both desmin and vimentin predominantly depended on its fifth plakin repeat domain and downstream linker region. Conversely, the interaction of desmin and vimentin with plectin required sequences contained within the segments 1A-2A of their central coiled-coil rod domain. This study furthers our knowledge of the interaction between plectin and IF proteins important for maintenance of cytoarchitecture in skeletal muscle. Moreover, binding of plectin to the conserved rod domain of IF proteins could well explain its broad interaction with most types of IFs.

Comparison of objectively measured motor behavior with ratings of the motor behavior domain of the Bern Psychopathology Scale (BPS) in schizophrenia

Motor symptoms in schizophrenia occur frequently and are relevant to diagnosis and antipsychotic therapy. To date motor symptoms are difficult to assess and their pathobiology is a widely unresolved issue. The Bern Psychopathology Scale for the assessment of system-specific psychotic symptoms (BPS) was designed to identify homogenous patient groups by focusing on three domains: language, affectivity and motor behavior. The present study aimed to validate the motor behavior domain of the BPS using wrist actigraphy. In total, 106 patients were rated with the BPS and underwent 24 h continuous actigraphy recording. The ratings of the global severity of the motor behavior domain (GSM) as well as the quantitative and the subjective items of the motor behavior domain of the BPS were significantly associated with actigraphic variables. In contrast, the qualitative items of the motor domain failed to show an association with actigraphy. Likewise, scores of the language and the affectivity domains were not related to actigraphic measures. In conclusion, we provided substantial external validity for global, quantitative and subjective ratings of the BPS motor behavior domain. Thus, the BPS is suitable to assess the dimension of quantitative motor behavior in the schizophrenia spectrum.

Restructuring Justice: Moving Towards Gender-Responsive Programming and Alternatives to Incarceration for Non-Violent Female Offenders

The United States¿ Federal and State laws differentiate between acceptable (or, legal) and unacceptable (illegal) behavior by prescribing restrictive punishment to citizens and/or groups that violate these established rules. These regulations are written to treat every person equally and to fairly serve justice; furthermore, the sanctions placed on offenders seek to reform illegal behavior through limitations on freedoms and rehabilitative programs. Despite the effort to treat all offenders fairly regardless of social identity categories (e.g., sex, race, ethnicity, socioeconomic status, age, ability, and gender and sexual orientation) and to humanely eliminate illegal behavior, the American penal system perpetuates de facto discrimination against a multitude of peoples. Furthermore, soaring recidivism rates caused by unsuccessful re-entry of incarcerated offenders puts economic stress on Federal and State budgets. For these reasons, offenders, policy-makers, and law-abiding citizens should all have a vested interest in reforming the prison system. This thesis focuses on the failure of the United States corrections system to adequately address the gender-specific needs of non-violent female offenders. Several factors contribute to the gender-specific discrimination that women experience in the criminal justice system: 1) Trends in female criminality that skew women¿s crime towards drug-related crimes, prostitution, and property offenses; 2) Mandatory minimum sentences for drug crimes that are disproportionate to the crime committed; 3) So-called ¿gender-neutral¿ educational, vocational, substance abuse, and mental health programming that intends to equally rehabilitate men and women, but in fact favors men; and 4) The isolating nature of prison structures that inhibits smooth re-entry into society. I argue that a shift in the placement and treatment of non-violent female offenders is necessary for effective rehabilitation and for reducing recidivism rates. The first component of this shift is the design and implementation of gender- responsive treatment (GRT) rather than gender-neutral approaches in rehabilitative programming. The second shift is the utilization of alternatives to incarceration, which provide both more humane treatment of offenders and smoother reintegration to society. Drawing on recent scholarship, information from prison advocacy organizations, and research with men in an alternative program, I provide a critical analysis of current policies and alternative programs, and suggest several proposals for future gender- responsive programs in prisons and in place of incarceration. I argue that the expansion of gender-responsive programming and alternatives to incarceration respond to the marginalization of female offenders, address concerns about the financial sustainability of the United States criminal justice system, and tackle high recidivism rates.

Cyclic nucleotide specific phosphodiesterases of Leishmania major

Background Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. Results This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs) from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range. Several PDE inhibitors were found to be active against these PDEs in vitro, and to inhibit cell proliferation. Conclusion The genome of L. major contains only PDE genes that are predicted to code for class I PDEs, and none for class II PDEs. This is more similar to what is found in higher eukaryotes than it is to the situation in Dictyostelium or the fungi that concomitantly express class I and class II PDEs. Functional complementation demonstrated that LmjPDEA, LmjPDEB1 and LmjPDEB2 are capable of hydrolyzing cAMP. In vitro studies with recombinant LmjPDEB1 and LmjPDEB2 confirmed this, and they demonstrated that both are completely cAMP-specific. Both enzymes are inhibited by several commercially available PDE inhibitors. The observation that these inhibitors also interfere with cell growth in culture indicates that inhibition of the PDEs is fatal for the cell, suggesting an important role of cAMP signalling for the maintenance of cellular integrity and proliferation.

Native EEG and treatment effects in neuroleptic-naïve schizophrenic patients: time and frequency domain approaches

Time domain analysis of electroencephalography (EEG) can identify subsecond periods of quasi-stable brain states. These so-called microstates assumingly correspond to basic units of cognition and emotion. On the other hand, Global Field Synchronization (GFS) is a frequency domain measure to estimate functional synchronization of brain processes on a global level for each EEG frequency band [Koenig, T., Lehmann, D., Saito, N., Kuginuki, T., Kinoshita, T., Koukkou, M., 2001. Decreased functional connectivity of EEG theta-frequency activity in first-episode, neuroleptic-naive patients with schizophrenia: preliminary results. Schizophr Res. 50, 55-60.]. Using these time and frequency domain analyzes, several previous studies reported shortened microstate duration in specific microstate classes and decreased GFS in theta band in drug naïve schizophrenia compared to controls. The purpose of this study was to investigate changes of these EEG parameters after drug treatment in drug naïve schizophrenia. EEG analysis was performed in 21 drug-naive patients and 21 healthy controls. 14 patients were reevaluated 2-8 weeks (mean 4.3) after the initiation of drug administration. The results extended findings of treatment effect on brain functions in schizophrenia, and imply that shortened duration of specific microstate classes seems a state marker especially in patients with later neuroleptic responsive, while lower theta GFS seems a state-related phenomenon and that higher gamma GFS is a trait like phenomenon.

Adapting health-risk communication to the specific cultural contexts of diverse populations : an assessment of malaria-treatment programs in Liberia

Drawing on theories of technical communication, rhetoric, literacy, language and culture, and medical anthropology, this dissertation explores how local culture and traditions can be incorporated into health-risk-communication-program design and implementation, including the design and dissemination of health-risk messages. In a modern world with increasing global economic partnerships, mounting health and environmental risks, and cross-cultural collaborations, those who interact with people of different cultures have “a moral obligation to take those cultures seriously, including their social organization and values” (Hahn and Inhorn 10). Paradoxically, at the same time as we must carefully adapt health, safety, and environmental-risk messages to diverse cultures and populations, we must also recognize the increasing extent to which we are all becoming part of one, vast, interrelated global village. This, too, has a significant impact on the ways in which healthcare plans should be designed, communicated, and implemented. Because communicating across diverse cultures requires a system for “bridging the gap between individual differences and negotiating individual realities” (Kim and Gudykunst 50), both administrators and beneficiaries of malaria-treatment-and-control programs (MTCPs) in Liberia were targeted to participate in this study. A total of 105 people participated in this study: 21 MTCP administrators (including designers and implementers) completed survey questionnaires on program design, implementation, and outcomes; and 84 MTCP beneficiaries (e.g., traditional leaders and young adults) were interviewed about their knowledge of malaria and methods for communicating health risks in their tribe or culture. All participants showed a tremendous sense of courage, commitment, resilience, and pragmatism, especially in light of the fact that many of them live and work under dire socioeconomic conditions (e.g., no electricity and poor communication networks). Although many MTCP beneficiaries interviewed for this study had bed nets in their homes, a majority (46.34 percent) used a combination of traditional herbal medicine and Western medicine to treat malaria. MTCP administrators who participated in this study rated the impacts of their programs on reducing malaria in Liberia as moderately successful (61.90 percent) or greatly successful (38.10 percent), and they offered a variety of insights on what they might do differently in the future to incorporate local culture and traditions into program design and implementation. Participating MTCP administrators and beneficiaries differed in their understanding of what “cultural incorporation” meant, but they agreed that using local indigenous languages to communicate health-risk messages was essential for effective health-risk communication. They also suggested that understanding the literacy practices and linguistic cultures of the local people is essential to communicating health risks across diverse cultures and populations.

Computational fluids domain reduction to a simplified fluid network

The primary goal of this project is to demonstrate the practical use of data mining algorithms to cluster a solved steady-state computational fluids simulation (CFD) flow domain into a simplified lumped-parameter network. A commercial-quality code, “cfdMine” was created using a volume-weighted k-means clustering that that can accomplish the clustering of a 20 million cell CFD domain on a single CPU in several hours or less. Additionally agglomeration and k-means Mahalanobis were added as optional post-processing steps to further enhance the separation of the clusters. The resultant nodal network is considered a reduced-order model and can be solved transiently at a very minimal computational cost. The reduced order network is then instantiated in the commercial thermal solver MuSES to perform transient conjugate heat transfer using convection predicted using a lumped network (based on steady-state CFD). When inserting the lumped nodal network into a MuSES model, the potential for developing a “localized heat transfer coefficient” is shown to be an improvement over existing techniques. Also, it was found that the use of the clustering created a new flow visualization technique. Finally, fixing clusters near equipment newly demonstrates a capability to track temperatures near specific objects (such as equipment in vehicles).

PHYLOGENETIC ANALYSES, GENE DOSAGE EFFECT AND SELECTION IN MEMBERS OF THE LATERAL BOUNDARY DOMAIN (LBD) GENES TRANSCRIPTION FACTOR FAMILY

Wood formation is an economically and environmentally important process and has played a significant role in the evolution of terrestrial plants. Despite its significance, the molecular underpinnings of the process are still poorly understood. We have previously shown that four Lateral Boundary Domain (LBD) transcription factors have important roles in the regulation of wood formation with two (LBD1 and LBD4) involved in secondary phloem and ray cell development and two (LBD15 and LBD18) in secondary xylem formation. Here, we used comparative phylogenetic analyses to test potential roles of the four LBD genes in the evolution of woodiness. We studied the copy number and variation in DNA and amino acid sequences of the four LBDs in a wide range of woody and herbaceous plant taxa with fully sequenced and annotated genomes. LBD1 showed the highest gene copy number across the studied species, and LBD1 gene copy number was strongly and significantly correlated with the level of ray seriation. The lianas, cucumber and grape, with multiseriate ray cells showed the highest gene copy number (12 and 11, respectively). Because lianas’ growth habit requires significant twisting and bending, the less lignified ray parenchyma cells likely facilitate stem flexibility and maintenance of xylem conductivity. We further demonstrate conservation of amino acids in the LBD18 protein sequences that are specific to woody taxa. Neutrality tests showed evidence for strong purifying selection on these gene regions across various orders, indicating adaptive convergent evolution of LBD18. Structural modeling demonstrates that the conserved amino acids have a significant impact on the tertiary protein structure and thus are likely of significant functional importance.

CONVERSION OF DOMAIN TYPE ENFORCEMENT LANGUAGE TO THE JAVA SECURITY MANAGER

With today's prevalence of Internet-connected systems storing sensitive data and the omnipresent threat of technically skilled malicious users, computer security remains a critically important field. Because of today's multitude of vulnerable systems and security threats, it is vital that computer science students be taught techniques for programming secure systems, especially since many of them will work on systems with sensitive data after graduation. Teaching computer science students proper design, implementation, and maintenance of secure systems is a challenging task that calls for the use of novel pedagogical tools. This report describes the implementation of a compiler that converts mandatory access control specification Domain-Type Enforcement Language to the Java Security Manager, primarily for pedagogical purposes. The implementation of the Java Security Manager was explored in depth, and various techniques to work around its inherent limitations were explored and partially implemented, although some of these workarounds do not appear in the current version of the compiler because they would have compromised cross-platform compatibility. The current version of the compiler and implementation details of the Java Security Manager are discussed in depth.

Using Dynamic Programming Decomposition for Revenue Management with Opaque Products

Opaque products enable service providers to hide specific characteristics of their service fulfillment from the customer until after purchase. Prominent examples include internet-based service providers selling airline tickets without defining details, such as departure time or operating airline, until the booking has been made. Owing to the resulting flexibility in resource utilization, the traditional revenue management process needs to be modified. In this paper, we extend dynamic programming decomposition techniques widely used for traditional revenue management to develop an intuitive capacity control approach that allows for the incorporation of opaque products. In a simulation study, we show that the developed approach significantly outperforms other well-known capacity control approaches adapted to the opaque product setting. Based on the approach, we also provide computational examples of how the share of opaque products as well as the degree of opacity can influence the results.

The SAKK cancer-specific geriatric assessment (C-SGA): a pilot study of a brief tool for clinical decision-making in older cancer patients

BACKGROUND Recommendations from international task forces on geriatric assessment emphasize the need for research including validation of cancer-specific geriatric assessment (C-SGA) tools in oncological settings. The objective of this study was to evaluate the feasibility of the SAKK Cancer-Specific Geriatric Assessment (C-SGA) in clinical practice. METHODS A cross sectional study of cancer patients >=65 years old (N = 51) with pathologically confirmed cancer presenting for initiation of chemotherapy treatment (07/01/2009-03/31/2011) at two oncology departments in Swiss canton hospitals: Kantonsspital Graubunden (KSGR N = 25), Kantonsspital St. Gallen (KSSG N = 26). Data was collected using three instruments, the SAKK C-SGA plus physician and patient evaluation forms. The SAKK C-SGA includes six measures covering five geriatric assessment domains (comorbidity, function, psychosocial, nutrition, cognition) using a mix of medical record abstraction (MRA) and patient interview. Five individual domains and one overall SAKK C-SGA score were calculated and dichotomized as below/above literature-based cut-offs. The SAKK C-SGA was evaluated by: patient and physician estimated time to complete, ease of completing, and difficult or unanswered questions. RESULTS Time to complete the patient questionnaire was considered acceptable by almost all (>=96%) patients and physicians. Patients reported slightly shorter times to complete the questionnaire than physicians (17.33 +/- 7.34 vs. 20.59 +/- 6.53 minutes, p = 0.02). Both groups rated the patient questionnaire as easy/fairly easy to complete (91% vs. 84% respectively, p = 0.14) with few difficult or unanswered questions. The MRA took on average 8.32 +/- 4.72 minutes to complete. Physicians (100%) considered time to complete MRA acceptable, 96% rated it as easy/fairly easy to complete. Individual study site populations differed on health-related characteristics (excellent/good physician-rated general health KSGR 71% vs. KSSG 32%, p = 0.007). The overall mean C-SGA score was 2.4 +/- 1.12. Patients at KSGR had lower C-SGA scores (2.00 +/- 1.19 vs. 2.81 +/- 0.90, p = 0.009) and a smaller proportion (28% vs.65%, p = 0.008) was above the C-SGA cut-off score compared to KSSG. CONCLUSIONS These results suggest the SAKK C-SGA is a feasible practical tool for use in clinical practice. It demonstrated discriminative ability based on objective geriatric assessment measures, but additional investigations on use for clinical decision-making are warranted. The SAKK C-SGA also provides important usable domain information for intervention to optimize outcomes in older cancer patients.

Inhibition of Fas-associated death domain-containing protein (FADD) protects against myocardial ischemia/reperfusion injury in a heart failure mouse model

AIM As technological interventions treating acute myocardial infarction (MI) improve, post-ischemic heart failure increasingly threatens patient health. The aim of the current study was to test whether FADD could be a potential target of gene therapy in the treatment of heart failure. METHODS Cardiomyocyte-specific FADD knockout mice along with non-transgenic littermates (NLC) were subjected to 30 minutes myocardial ischemia followed by 7 days of reperfusion or 6 weeks of permanent myocardial ischemia via the ligation of left main descending coronary artery. Cardiac function were evaluated by echocardiography and left ventricular (LV) catheterization and cardiomyocyte death was measured by Evans blue-TTC staining, TUNEL staining, and caspase-3, -8, and -9 activities. In vitro, H9C2 cells transfected with ether scramble siRNA or FADD siRNA were stressed with chelerythrin for 30 min and cleaved caspase-3 was assessed. RESULTS FADD expression was significantly decreased in FADD knockout mice compared to NLC. Ischemia/reperfusion (I/R) upregulated FADD expression in NLC mice, but not in FADD knockout mice at the early time. FADD deletion significantly attenuated I/R-induced cardiac dysfunction, decreased myocardial necrosis, and inhibited cardiomyocyte apoptosis. Furthermore, in 6 weeks long term permanent ischemia model, FADD deletion significantly reduced the infarct size (from 41.20 ± 3.90% in NLC to 26.83 ± 4.17% in FADD deletion), attenuated myocardial remodeling, improved cardiac function and improved survival. In vitro, FADD knockdown significantly reduced chelerythrin-induced the level of cleaved caspase-3. CONCLUSION Taken together, our results suggest FADD plays a critical role in post-ischemic heart failure. Inhibition of FADD retards heart failure progression. Our data supports the further investigation of FADD as a potential target for genetic manipulation in the treatment of heart failure.

AMPA RECEPTOR ALLOSTERISM: MEASUREMENT OF THE CONFORMATIONAL CHANGES IN THE LIGAND BINDING DOMAIN OF A FUNCTIONAL RECEPTOR

Ionotropic glutamate receptors are important excitatory neurotransmitter receptors in the mammalian central nervous system that have been implicated in a number of neuropathologies such as epilepsy, ischemia, and amyotrophic lateral sclerosis. Glutamate binding to an extracellular ligand binding domain initiates a series of structural changes that leads to the formation of a cation selective transmembrane channel, which consequently closes due to desensitization of the receptor. The crystal structures of the AMPA subtype of the glutamate receptor have been particularly useful in providing initial insight into the conformational changes in the ligand binding domain; however, these structures are limited by crystallographic constraint. To gain a clear picture of how agonist binding is coupled to channel activation and desensitization, it is essential to study changes in the ligand binding domain in a dynamic, physiological state. In this dissertation, a technique called Luminescence Resonance Energy Transfer was used to determine the conformational changes associated with activation and desensitization in a functional AMPA receptor (ÄN*-AMPA) that contains the ligand binding domain and transmembrane segments; ÄN*-AMPA has been modified such that fluorophores can be introduced at specific sites to serve as a readout of cleft closure or to establish intersubunit distances. Previous structural studies of cleft closure of the isolated ligand binding domain in conjunction with functional studies of the full receptor suggest that extent of cleft closure correlates with extent of activation. Here, LRET has been used to show that a similar relationship between cleft closure and activation is observed in the “full length” receptor showing that the isolated ligand binding domain is a good model of the domain in the full length receptor for changes within a subunit. Similar LRET investigations were used to study intersubunit distances specifically to probe conformational changes between subunits within a dimer in the tetrameric receptor. These studies show that the dimer interface is coupled in the open state, and decoupled in the desensitized state, similar to the isolated ligand binding domain crystal structure studies. However, we show that the apo state dimer interface is not pre-formed as in the crystal structure, hence suggesting a mechanism for functional transitions within the receptor based on LRET distances obtained.

Syntaxin 3b is a t-SNARE specific for ribbon synapses of the retina.

Previous studies have demonstrated that ribbon synapses in the retina do not contain the t-SNARE (target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 1A that is found in conventional synapses of the nervous system. In contrast, ribbon synapses of the retina contain the related isoform syntaxin 3. In addition to its localization in ribbon synapses, syntaxin 3 is also found in nonneuronal cells, where it has been implicated in the trafficking of transport vesicles to the apical plasma membrane of polarized cells. The syntaxin 3 gene codes for four different splice forms, syntaxins 3A, 3B, 3C, and 3D. We demonstrate here by using analysis of EST databases, RT-PCR, in situ hybridization, and Northern blot analysis that cells in the mouse retina express only syntaxin 3B. In contrast, nonneuronal tissues, such as kidney, express only syntaxin 3A. The two major syntaxin isoforms (3A and 3B) have an identical N-terminal domain but differ in the C-terminal half of the SNARE domain and the C-terminal transmembrane domain. These two domains are thought to be directly involved in synaptic vesicle fusion. The interaction of syntaxin 1A and syntaxin 3B with other synaptic proteins was examined. We found that both proteins bind Munc18/N-sec1 with similar affinity. In contrast, syntaxin 3B had a much lower binding affinity for the t-SNARE SNAP25 compared with syntaxin 1A. By using an in vitro fusion assay, we could demonstrate that vesicles containing syntaxin 3B and SNAP25 could fuse with vesicles containing synaptobrevin2/VAMP2, demonstrating that syntaxin 3B can function as a t-SNARE.