The fabrication of biomimetic heart muscle suitable for pharmaceutical compound evaluation and disease modeling is hindered by limitations in our understanding of how to guide and assess the maturity of engineered myocardium in vitro. We hypothesized that tissue architecture serves as an important cue for directing the maturation of engineered heart tissues and that reliable assessment of maturity could be performed using a multi-parametric rubric utilizing cardiomyocytes of known developmental state as a basis for comparison. Physical micro-environmental cues are recognized to play a fundamental role in normal heart development, therefore we used micro-patterned extracellular matrix to direct isolated cardiac myocytes to self-assemble into anisotropic sheets reminiscent of the architecture observed in the laminar musculature of the heart. Comparison of global sarcomere alignment, gene expression, and contractile stress in engineered anisotropic myocardium to isotropic monolayers, as well as, adult ventricular tissue revealed that anisotropic engineered myocardium more closely matched the characteristics of adult ventricular tissue, than isotropic cultures of randomly organized cardiomyocytes. These findings support the notion that tissue architecture is an important cue for building mature engineered myocardium. Next, we sought to develop a quality assessment strategy that utilizes a core set of 64 experimental measurements representative of 4 major categories (i.e. gene expression, myofibril structure, electrical activity, and contractility) to provide a numeric score of how closely stem cell-derived cardiac myocytes match the physiological characteristics of mature, post-natal cardiomyocytes. The efficacy of this rubric was assessed by comparing anisotropic engineered tissues fabricated from commercially-available murine ES- (mES) and iPS- (miPS) derived myocytes against neonatal mouse ventricular myocytes. The quality index scores calculated for these cells revealed that the miPS-derived myocytes more closely resembled the neonate ventricular myocytes than the mES-derived myocytes. Taken together, the results of these studies provide valuable insight into the fabrication and validation of engineered myocardium that faithfully recapitulate the characteristics of mature ventricular myocardium found in vivo. These engineered tissue design and quality validation strategies may prove useful in developing heart muscle analogs from human stem cell-derived myocytes that more accurately predict patient response than currently used animal models. / Engineering and Applied Sciences

The work in this thesis addresses the general problem of how ribosomal subunits are exported from the nucleus to mature in the cytoplasm. There are three parts in this dissertation. In the first part, I asked questions about the specificity for export receptors in the nuclear export of the large (60S) ribosomal subunit in yeast. In principle, I tethered different export receptors that are known to work in various unrelated export pathways to the ribosome by fusing them to the trans-acting factor Nmd3. Interestingly, all the chimeric receptors were able to support export, although to different degrees. Moreover, 60S export driven by these chimeric receptors was independent of Crm1, an export receptor that is essential for 60S export in wild-type cells. The second question I addressed in this project was whether or not a nuclear export signal could be provided in cis on ribosomal proteins (Rpls) rather than in trans by a transacting factor. The nuclear export signal (NES) of Nmd3 was fused to different ribosomal proteins and tested for support of 60S export. Several Rpl-NES fusion constructs worked to promote 60S export. Rpl3 gave the best efficiency. In conclusion, these results imply unexpected flexibility in the 60S export pathway. This may help explain how different export receptors could have evolved in different eukaryotic lineages. In the second part of my thesis, I identified the assembly pathway for the base of the ribosome stalk. The stalk is an important functional domain of the large ribosomal subunit because of its requirement for interaction with translation factors. Mrt4 is a nuclear paralog of P0, which is an essential part of the stalk. Here, I identified Yvh1 a novel ribosome biogenesis factor that is required for the release of Mrt4. Yvh1 is a conserved dual phosphatase, but the C-terminal zinc-binding domain rather than the phosphatase function was required for its activity to release Mrt4. Mrt4 localizes in the nucleus and nucleolus in the wild-type cells, but was persistent on cytoplasmic 60S subunits in yvh1[Delta] cells. The persistence of Mrt4 on the 60S subunits blocked the loading of P0 and assembly of the stalk. I also found the binding of Yvh1 depended on Rpl12, a protein that binds together with P0 to form the base of the stalk. Deletion of Rpl12 phenocopied yvh1[Delta]. These data identified the function of Yvh1 as a release factor of Mrt4. I also showed that the function of Yvh1 is conserved in human cells. In my final project, I analyzed the interdependence and order of the known cytoplasmic maturation events of the 60S subunit. 60S subunits require several maturation steps in the cytoplasm before they become competent in translation. There are four major steps involving two ATPases, Drg1 and Ssa1, and two GTPases, Efl1 and Lsg1. In my study, I ordered these steps into one serial pathway. Drg1 releases Rlp24 in the earliest step of 60S maturation in the cytoplasm. Truncation of the C-terminus of Rlp24 blocked cytoplasmic maturation of the large subunit by preventing the recruitment of Drg1 and led to a secondary defect in the release of Arx1 because of a failure to recruit Rei1. Deletion of REI1 mislocalized Tif6 from the nucleus and nucleolus to the cytoplasm and deletion of ARX1 suppressed the Tif6 mislocalization, indicating that the release of Arx1 was required for Tif6 release downstream. I found that mutation of efl1 or sdo1, the known release factors for Tif6, also blocked Nmd3 release. Tif6-V192F, which could bypass the growth defects of efl1 or sdo1 mutants, suppressed the defect of Nmd3 recycling. These results showed that the release of Tif6 was a prerequisite for Nmd3 release. Thus, the release of Nmd3 is downstream of the Tif6 release step. In conclusion, I have ordered the events of cytoplasmic maturation with Drg1 as the first step after ribosome export, followed by Rei1/Jji1 and then Sdo1/Efl1. The release of Nmd3 by Lsg1 appears to be the last step of ribosome maturation in the cytoplasm. Thus, the two ATPases Drg1 and Ssa work first and then the two GTPases Efl1 and Lsg1 work in a linear pathway of 60S maturation in the cytoplasm. / text

<p>This study identifies and elaborates on a theme in the Hebrew Bible (HB) that has largely gone unnoticed by scholars: the transition of a male adolescent from boyhood to manhood. Beyond identifying the coming-of-age theme in different HB texts, the project also describes how the theme is employed by biblical narrators and redactors to highlight broader messages and transitions in the historical narratives of the HB. It also considers how these stories provide insight into the varying representations of biblical masculinity.</p><p> The project begins by showing how the recent discussions on masculinity in the HB and biblical rites of passage are incomplete without an analysis of how a boy becomes a man in the biblical text. It then establishes important principles for recognizing the maturation theme in a given narrative. More foundational work is done in chapter 2, which describes the characteristic features of manhood and boyhood as depicted in the HB to facilitate the identification of narratives where a transition is made from boyhood to manhood. </p><p> The next two chapters identify five case studies of coming-of-age: David in 1 Sam 17; Solomon in 1 Kgs 1-2; an alternative tale of Solomon's maturation in 1 Kgs 3; Moses in Exod 2; and Samuel in 1 Sam 3. Chapter 5 discusses the converse of the coming-of-age theme by presenting stories of boys who fail to mature: Jether in Judg 8, and Samson in Judg 13-16. In each case study, the narrator's techniques for highlighting the maturation theme are identified. The ways that the narrator employs the theme to point to other significant plot points or narrative transitions are also identified. Most notably, the failure-to-mature theme in the Samson narratives typifies Israel's political immaturity in Judges, and the two alternative tales of Solomon's maturation highlight an important transition in the Deuteronomistic History from the uncertain and often bloody years of the monarchy's establishment to the peaceful, prosperous reign of Solomon. </p><p> The seven case studies are also examined for the image of masculinity that they present, and that presentation is compared to the general view of manhood in the HB. Five of the seven offer quite similar images of masculinity; and these also cohere to the general picture of biblical manhood. However, two narratives (Samuel's maturation in 1 Sam 3 and Solomon's in 1 Kgs 3) depart from this conception of masculinity, each in the same way: both depict a masculinity free of violence and the need for the constant, forceful defense of manhood and honor. Since these two texts have often been ascribed to the same author, the Deuteronomistic Historian, the study suggests that he may be offering a new view of masculinity more suited to his historical context. </p><p> The project ultimately proves that the theme of male coming-of-age, heretofore virtually unrecognized, is found in several biblical texts. Moreover, this theme is often used to indicate other important messages and transitions in Israel's historical narrative and can provide unique insight into biblical constructions of masculinity.</p> / Dissertation

Die Transkriptionsfaktoren der p53 Familie besitzen diverse Aufgaben sowohl während der Tumorentstehung als auch während der Entwicklung. In seiner ursprünglichen, evolutionär konservierten Rolle hat p63, ein Mitglied der p53 Familie, die Aufgabe die genetische Stabilität von Keimzellen zu gewährleisten und beeinflusst zudem die Keimbahnentwicklung. Mit Hilfe von „knockout“ (KO) Mausmodellen wurde bereits der Einfluss der 3 Transkriptionsfaktoren p53, p63 und p73 auf die weibliche und männliche Keimbahn untersucht. Während p53KO und p63KO Mäuse fruchtbar sind, führt der gemeinsame Verlust aller p73 Isoformen oder der Verlust der transkriptionell aktiven Isoform TAp73 hingegen zur Infertilität der Tiere. Weibliche TAp73KO Tiere sind deshalb infertil, da es bei ihnen zu einer Inhibition der Ovulation kommt, die Blastozystenentwicklung gestört ist und Spindeldefekte in den Oozyten auftreten. Eine Erklärung für die Infertilität von männlichen TAp73KO Mäusen wurde hingegen noch nicht gegeben. In der vorliegenden wissenschaftlichen Studie beschreiben wir eine bisher unbekannte Funktion des Transkriptionsfaktors TAp73: seinen Einfluss auf die Entwicklung und den Erhalt der adulten männlichen Keimbahn. Untersuchungen an Mausmodellen zeigen, dass „total“ p73KO und Isoform-spezifische TAp73KO Mäuse im Alter von 6 Wochen oder älter einen starken Verlust von sich entwickelnden Keimzellen in den testikulären Tubuli aufweisen. ΔNp73KO Tiere zeigen diesen Phänotyp nicht und ihre Testis Morphologie entspricht der der Wildtyp (WT) Tiere. Der Phänotyp der p73KO und TAp73KO Testes lässt sich durch eine starke Reduktion in der Zahl der späten Stadien der Spermatozyten und Spermatiden charakterisieren, wohingegen die Zahl der basalen Spermatogonien und pachytänen Spermatozyten des Keimepithels nicht betroffen ist (reguläre Zellzahl und Proliferation). Die Depletion von p73 oder TAp73 führt zu einer Anhäufung von apoptotischen und unreifen Keimzellen im Lumen des Epididymis, was auf eine atypische, verfrühte Ablösung der Keimzellen aus dem Keimepithel schließen lässt. Diese Beobachtung wird dadurch bestätigt, dass die Sertoli-Zellen, welche die Keimzellen stützen und ernähren, im Elektronenmikroskop verkürzte cytoplasmatische Arme sowie eine verstärkte Vakuolisierung aufweisen. Außerdem zeigt das Keimepithel große strukturelle Veränderungen, die dadurch gekennzeichnet sind, dass die Keimzellen nicht mehr dicht zu einem Epithel gepackt sind und die Sertoli-Keimzell-Verbindung gestört ist. Der Verlust der Epithelstruktur begleitet von der Reduktion der Keimzellen scheint das Ergebnis eines Defekts der Blut-Testis-Schranke (BTS) zu sein. Dies wird durch einen in vivo BTS Permeabilitätstest und die gestörte Morphologie der Sertoli-Sertoli „tight junctions“ deutlich. Der funktionelle Verlust der BTS führt zur Zerstörung der epithelialen Polarität und der Umgebung der sich entwickelnden Keimzellen. Somit wird auch die basal-luminal gerichtete Migration der Keimzellen entlang der Keimzelltaschen der Sertoli-Zellen verhindert. Der molekulare Hintergrund für das Ungleichgewicht in der Reorganisation der Zell-Zell Verbindungen wurde mit Hilfe der quantitativen Expressionsanalyse des Gesamtgenoms untersucht. Dabei wurde Testis Gewebe von TAp73KO mit WT Mäusen verglichen, um TAp73-Zielgene zu finden. Die Depletion von TAp73 führt zur Induktion von Genen, die an der Adhäsion und Zellmigration beteiligt sind; Integrine und Proteaseinhibitoren wie Timp1 und die Serpine zeigen eine Hochregulation in der Expression. Es ist bekannt, dass diese Proteine bei der Reorganisation von Zell-Zell Verbindungen im Testis involviert sind. Die Studie zeigt, dass TAp73 vorwiegend in den Keimzellen exprimiert wird und dass diese parakrin auf die Sertoli-Zellen zu wirken scheinen, da die Überexpression von TAp73 Zielgenen in isolierten Sertoli-Zellen in der Langzeitkultur zurückgeht. Zusammenfassend konnte zum ersten Mal gezeigt werden, dass die Funktion von TAp73 für die adulte Spermatogenese unerlässlich ist. TAp73 reguliert im Testis ein Transkriptionsprogramm von Genen, die an Adhäsion und Migration beteiligt sind, sichert den Zusammenhalt des Keimepithels und verhindert den frühzeitigen Verlust von Keimzellen. Somit wird die ungestörte Keimzellentwicklung ermöglicht. Umgekehrt führt die Depletion von TAp73 zu starken Defekten in Zell-Zell Verbindungen von sich entwickelnden Keimzellen im Keimepithel, was die Infertilität von p73KO und TAp73KO Mäusen erklärt.

Laws in South Africa, such as the Children’s Amendment Act 41 of 2007(Government Gazette, Act 38 of 2005), is developed with good intentions of promoting prevention and intervention on various health-related issues. Laws also dictate, based on developmental and evolving capabilities, chronological ages at which children and adolescents may access certain healthcare services without parental consent, whilst limiting them in other areas such as decision-making for research participation. Of interest to this study is how specialists in health care, conceptualise, understand and apply “sufficient maturity” in their encounters with minors presenting for treatment, in order to identify key concepts of sufficient maturity. From the interviews conducted, themes were identified that were relevant to the construct of “sufficient maturity.”Results indicated that there were two primary perspectives participants used to assess “sufficient maturity” when minors presented for treatment.Health care practitioners, depending on the health care context, assess minors’ sufficient maturity in relation to, either a competency based or a deficiency model. / Thesis (M.A.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Vaiko brandinimo mokyklai kokybės vadyba priešmokyklinio ugdymo programas teikiančiose mokyklose Darbo autorė: Lina Zolubienė Darbo vadovas: doc. Dr. Ramutė Bruzgelevičienė Šiame darbe pasirenkama tiriamuoju laikotarpiu (2011 m.) švietimo dokumentų oficialiai įtvirtinta vaiko brandos mokyklai ir vaiko brandinimo mokyklai samprata ir tiriama, kaip praktiškai suprasta brandinimo mokyklai kokybė ir kaip ji įtvirtinama vadybos veiksmais priešmokyklinio ugdymo programas teikiančiose Rokiškio savivaldybės mokyklose. Tyrimo problema Kaip suvokta ir praktiškai valdoma vaikų brandinimo mokyklai kokybė Rokiškio savivaldybės mokyklose, teikiančiose priešmokyklinio ugdymo programas. Tyrimo objektas Vaikų brandinimo mokyklai kokybės vadyba Rokiškio savivaldybės mokyklose, teikiančiose priešmokyklinio ugdymo programas. Tyrimo tikslas Atskleisti vaikų brandinimo mokyklai kokybės vadybos poreikį įgyvendinant priešmokyklinį ugdymą. Tyrimo uždaviniai • Išanalizavus mokslinę literatūrą ir švietimo dokumentus teoriškai apibrėžti vaikų brandos mokyklai kaip siekio ir vaikų brandinimo mokyklai kaip proceso skirtumus bei šio proceso kokybės vadybos sampratą. • Empiriniu tyrimu atskleisti vaikų brandinimo mokyklai kokybės suvoktį, kokybės vadybos būklę ir galimybes Rokiškio savivaldybės mokyklose, teikiančiose priešmokyklinio ugdymo programas. Tyrimo metodai Darbe remiamasi empirinio tyrimo rūšimi – atvejo tyrimu (Bitinas B., 2006, p. 90), atveju laikomos Rokiškio savivaldybės mokyklos, teikiančios... [toliau žr. visą tekstą] / Quality management of child’s maturation for school at schools providing pre-school education programs The Author: Lina Zolubienė Writing leader: Assoc. dr. Ramute Bruzgelevičienė In this work there is taken conception of child mature and maturation for school in official certified in education documents during the time 2011 and there is established practical understanding of quality of child’s maturation for school and management actions at local schools providing pre-school education programs in Rokiskis district. The problem of investigation What is practical understanding and control of the quality of school children maturation in Rokiskis local schools providing pre-school education programs. The object of investigation Quality management of children's maturation for school at Rokiskis local schools providing pre-school education programs. The aim of investigation To reveal the need of quality management of child maturation for school implementing pre-primary education . The goals of investigation • To analyze the nonfiction literature, education documents and to define the mature of children as objective, the maturation of children for school as differences of process and conception of management of this process quality. • Using Empirical research reveal the quality of seeing children’s maturation for school, the state and possibilities of quality management in Rokiskis local schools providing pre-school education programs. The Methods of investigation Work is based on... [to full text]

Cystatin Related Epididymal Spermatogenic protein (CRES) is expressed in both the testis and epididymis and found associated with spermatozoa. It appears as non-glycosylated (14 and 12 kDa) and glycosylated isoforms (19 and 17 kDa). The role of CRES is enigmatic and dependent on localization of its isoforms, which is the objective of this study. The initial approach was to investigate testicular and epididymal origins of these isoforms by immunohistochemistry and immunogold cytochemistry. To further pinpoint CRES localization we then selectively extracted and fractionated epididymal spermatozoa in order to find by immunoblotting which sperm fractions contained CRES isoforms. Immunohistochemical analysis of mouse spermatogenesis showed that CRES was expressed in the tail cytoplasm of elongating spermatids from step 9-16, with a pattern reminiscent of outer dense fibre (ODF) proteins. Ultrastructural immunocytochemistry revealed that the immunogold label was concentrated over growing ODFs and mitochondrial sheath in the testes which persisted in spermatozoa through the epididymis. Sequential extractions of isolated sperm tails with Triton X-100-dithiothreitol (DTT) to remove the mitochondrial sheath, whose extract contained an unrelated 66 kDa immunoreactive band, followed by either sodium dodecyl sulfate (SDS)-DTT or urea-DTT to solubilise accessory fibres of the tail revealed a 14 kDa immunoreactive band associated with the ODF. In addition, Western blots revealed glycosylated and non-glycosylated CRES isoforms in nonyl phenoxylpolyethoxylethanol (NP40) extracts of the caput, but not cauda, sperm. Immunohistochemical analysis of the caput and cauda epithelium showed that CRES is secreted by the Golgi apparatus of the ii initial segment, fills the proximal caput lumen, and disappears by mid caput. Western blots of caput and cauda tissue and luminal fluid revealed 14 and 19 kDa immunoreactive bands in caput tissues and luminal fluid, but not in the cauda. This study concludes that there are two origins of CRES, one arising in the testis and the other in the epididymis. Testicular CRES is ionically and covalently associated with the ODF while epididymal CRES is detergent soluble and is most likely associated temporarily with the surface of caput epididymal sperm. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2010-09-03 14:22:01.913

Seed maturation is an important process that is evolutionarily advantageous, allowing for seed dispersal and germination under favourable growth conditions. The B3-domain transcription factor FUSCA3 (FUS3) is a master regulator of seed maturation and controls developmental phase transitions through hormonal regulation in Arabidopsis thaliana. The aim of this study was to determine the post-translational regulation of FUS3 during embryonic and vegetative development. Here, FUS3 was found to interact with the E3 ubiquitin ligase ABI3-INTERACTING PROTEIN2 (AIP2) in yeast two-hybrid, in vitro, and in planta assays. Analysis of transcriptional and translational reporters also showed overlapping spatial and temporal expression patterns of AIP2 and FUS3. Furthermore, in vitro FUS3 degradation was delayed in aip2-1 mutant and increased FUS3-GFP levels were observed during mid-embryogenesis in aip2-1. Finally, double transgenic plants overexpressing AIP2 and FUS3 showed reduced FUS3 levels and reversion of the gain-of-function FUS3 phenotypes back to WT. Together, these results indicate that AIP2 is a negative regulator of FUS3.

Feeding problems in preterm neonates stem from complications of early delivery. Attainment of independent feeding is a prerequisite for Neonatal Intensive Care Unit (NICU) discharges. Some quantitative studies of infant feeding involve excessive amounts of time for data processing. Multivariate spectral analysis was used to minimize time for investigation of variability in these rhythms. Auto and Cross-spectral parameters of the rhythms were correlated with Gestational Age (GA), Postmenstrual Age (PMA), Birthweight (BW), Days of Life (DOL), and Time Since First Nipple feeding (TSFN). Auto-spectral analysis showed 25.55% increase in Bandwidth of suck (bw-su) for a 2-week increase in GA (DOL fixed) and 8.99% increase in bw-su for a 10-day increase in DOL (GA fixed). Crossspectral analysis showed a decrease of 0.158Hz of Bandwidth of Suck-Swallow (bw-SS) for a 2-week increase in GA for GA later than 28 weeks. For GA earlier than 28 weeks, peak coherence decreased by 0.774 for a 2-week increase in GA (PMA fixed) and decreased by 0.126 for a 2-week increase in PMA (GA fixed). The method describes the progression of feeding rhythms through correlations with clinical indexes, thus providing clinicians with an understanding of the development of infant feeding and helps predict long-term developmental outcomes.

Leptin is a peripheral adiposity signal and a key hormone in energy balance regulation in mammals, acting as a link between nutritional status and the endocrine reproductive axis. If this is also the role of leptin in fish is not fully understood. This thesis investigates how different components of the leptin system are affected by sexual maturation and seasonal changes in energy balance in male Atlantic salmon (Salmo salar L.) parr under fully fed and feed-restricted conditions. Moreover, the role of sex steroids as being one of the possible mechanisms by which sexual maturation interacts with leptin is explored. The salmon leptin-a genes, lepa1 and lepa2, were expressed mainly in liver and the leptin receptor (lepr) in brain and ubiquitously in peripheral tissues. Seasonal characterization of the lepa genes and lepr during the growth and reproductive season in one-year old males showed that hepatic lepa1 and lepa2 mRNA levels and plasma leptin levels were down-regulated concomitantly with an increase in weight and body fat. Feed restriction up-regulated hepatic leptin, and pituitary lepr expression as well as plasma leptin levels. Correlation between leptin levels and body lipid stores were either lacking or negative. These findings show that leptin and lepr are sensitive to changes in energy balance, but that leptin might not reflect adiposity in juvenile salmon. Hepatic lepa1 and lepa2, and testicular lepr expression increased during mid- to late spermatogenesis in early maturing males. This up-regulation was preceded by rapid gonadal growth and elevated pituitary follicle-stimulating hormone gene expression levels, whereas peak leptin levels coincided with peak pituitary luteinizing hormone expression and the presence of running milt in the testes. The sex steroids testosterone (T), 11-ketotestosterone and 17-β estradiol stimulated lepa1 and lepa2 gene expression in Atlantic salmon hepatocytes in vitro differentially depending on developmental stage. T was also able to stimulate hepatic lepa1 and pituitary lepa1 and lepr gene expression in immature male salmon in vivo. These results suggest that leptin plays a role in male fish reproduction during later stages of the maturational process and that the elevation of leptin expression during spermatogenesis could be caused by androgen stimulation.