Vesicular traffic along the biosynthetic and endocytic pathways is essential for homeostasis of eukaryotic cells. However, it raised the question of how the proteins characteristic for each compartment are transported to their destination (Bonifacino and Glick, 2004). This study is especially focusing on the connection between the Golgi apparatus and the endosomal compartment, mediated by two parallel trafficking pathways regulated by the clathrin adaptors AP-1A and AP-3 (Owen et al., 2004). Typical cargo molecules sorted along the AP-1A regulated pathway are mannose 6-phosphate receptors (MPRs) (Ghosh et al., 2003) or the gpI envelop glycoprotein of the Vesicular Zoster virus (Alconada et al., 1996), while sorting of lysosomal membrane proteins like Lamp-1 and LimpII is AP-3 regulated (Eskelinen et al., 2003). To study how AP-1A and AP-3 coats are stabilized on membranes and to identify the protein networks involved, a liposome based in vitro assay that recapitulates the fidelity of protein sorting in vivo was developed and combined with proteomic screens. Therefore, liposomes carrying cytoplasmic domains of gpI or Lamp-1/LimpII were used as affinity matrix to recruit selectively AP-1A or AP-3 and associated protein machineries. The coated liposomes were then analyzed by mass spectrometry. Using the in vitro recruitment assay, it was possible to demonstrate that efficient and selective recruitment of AP-1A and AP-3 coats depends on the presence of several low affinity binding sites on membranes. Thus, AP-1A and AP-3 recognize their target membranes by activated Arf1 GTPases, organelle specific phosphoinositides, PI-4P and PI-3P respectively, and distinct cargo molecules carrying intact signals in their cytoplasmic domains. The implication of PI-3P in AP-3 recruitment was further supported by in vivo experiments. During the biochemical characterization of the assay, several lines of evidence indicated that cargo tails containing intact sorting signals stabilize not only AP-1A and AP-3 coats on membranes but also influence the membrane recruitment of Arf1. It is possible that cargo molecules indirectly drive an Arf1 amplification loop, thereby ensuring efficient AP coat assembly. The proteomic screens identified protein networks of ≈40 proteins selectively recruited on AP-1A coated structures. The most appealing result of the analysis was the presence of two additional protein machineries, one involved in actin nucleation the other involved membrane fusion. More precisely, the AP-1A analysis identified the selective recruitment of the AP-1A subunits and interacting molecules (clathrin, g-synergin), Arf1 and Arf1 effectors (Big2, Git1), Rac1 including Rac1 effectors (b-PIX, RhoGEF7) and a Rac1 dependent actin nucleation machinery (Wave/Scar complex, Arp2/3 complex, associated effectors) as well as members of a Rab machinery (Rab11, Rab14). This finding was further supported by in vivo colocalization studies of the AP-1A cargo CI-MPR with CYFIP2, a protein of the Wave/Scar complex, and the localization of Big2 and Git1 on Rab11 positive membranes (Matafora et al., 2001; Shin et al., 2004). The biochemical characterization revealed that the stabilization of AP-1A coats, most probably driven by cargo molecules that stabilize AP-1A and Arf1 on membranes, leads as well to the stabilization of the two other machineries. Thus, the results support the notion that cargo sorting, vesicular movement and membrane fusion are coordinated during early steps of vesicular traffic. In analogy, the proteomic screens on AP-3 coated structures identified as well ≈40 selectively recruited proteins, which constituted a similar supramolecular network of protein machineries involved in coat formation, action nucleation and membrane fusion via Rab proteins. Thus, beside the AP-3 coat including the AP-3 subunits, Arf1 and Arf effectors (Big1, ARAP1, AGAP1), members of the septin family involved in actin rearrangements and most of the already described effectors of Rab5 microdomains (EEA1, Rabaptin-5, Rabex-5, Vps45) involved in early endosomal dynamics were selectively recruited together with Rab5 and Rab7. Thus, the proteomic analysis of AP-1A and AP-3 coated structures suggest that both AP coats use similar principles - coats, actin nucleation devices and Rab fusion machineries - to assemble supramolecular structures needed for membrane traffic. Although we do not have the ultimate proves yet, it seems as AP-1A and AP-3 use different members of subcomplexes, hence different GTPase effectors, different actin nucleation machineries and different Rab GTPases, to regulate their specific transport pathways and to link the different protein machineries. The proteomic analysis revealed for example that they probably use different Arf and Rho GTPase effectors to link the coat with actin nucleation. However, this has to be proven experimentally. In order to understand the networks of protein interactions, bioinformatic tools were used as a first approach. Even though some clues about the overall organization of the supramolecular protein complexes were provided, the direct links to the Rab machinery are still elusive. Maybe the proteins with thus far unknown functions could be involved. The biochemical analysis, especially the role of PIPs, and the Rab GTPases identified in the context of AP-1A and AP-3, provide indications about AP-1A and AP-3 function in vivo. The results could be interpreted in a way that AP-1A functions either in traffic from PI-4P positive membranes towards Rab11/Rab14 positive membranes or AP-1A coats assemble on PI-4P and Rab11 or Rab14 positive membranes, hence, TGN to endosomes traffic. The same holds true for AP-3, the results either suggest AP-3 mediates traffic from PI-3P positive towards Rab5/Rab7 positive membranes or they could be interpreted in a way that AP-3 assembles on PI-3P and Rab5 positive membranes for subsequent transport to Rab7 positive membranes, thus traffic from early to late endosomes. Overall, the results of this thesis research provided important insight into the formation of AP-1A and AP-3 coated structures and the potential interconnection between AP coats, actin nucleation and membrane fusion machineries. Alconada, A., U. Bauer, and B. Hoflack. 1996. A tyrosine-based motif and a casein kinase II phosphorylation site regulate the intracellular trafficking of the varicella-zoster virus glycoprotein I, a protein localized in the trans-Golgi network. Embo J. 15:6096-110. Bonifacino, J.S., and B.S. Glick. 2004. The mechanisms of vesicle budding and fusion. Cell. 116:153-66. Eskelinen, E.L., Y. Tanaka, and P. Saftig. 2003. At the acidic edge: emerging functions for lysosomal membrane proteins. Trends Cell Biol. 13:137-45. Ghosh, P., N.M. Dahms, and S. Kornfeld. 2003. Mannose 6-phosphate receptors: new twists in the tale. Nat Rev Mol Cell Biol. 4:202-12. Matafora, V., S. Paris, S. Dariozzi, and I. de Curtis. 2001. Molecular mechanisms regulating the subcellular localization of p95-APP1 between the endosomal recycling compartment and sites of actin organization at the cell surface. J Cell Sci. 114:4509-20. Owen, D.J., B.M. Collins, and P.R. Evans. 2004. Adaptors for clathrin coats: structure and function. Annu Rev Cell Dev Biol. 20:153-91. Shin, H.W., N. Morinaga, M. Noda, and K. Nakayama. 2004. BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity. Mol Biol Cell. 15:5283-94.

Magister Scientiae Dentium - MSc(Dent) / Restorative systems classified as polyacid modified composite resins, or compomers, have appeared on the market. An example of this is Dyract AP. Dyract AP must be used with the Prime & Bond NT bonding system. Prime & Bond NT can be applied without any form of prior etching, or it can be applied after application of Non Rinse Conditioner, or it can be applied after etching with 36% phosphoric acid. The purpose of the study was to determine the qualitative microleakage of Dyract AP and its bonding systems, as well as the influence of saliva contamination there of. Freshly extracted, non-carious, human premolars were randomly divided into 8 groups of 18 teeth each. Apiseetomies coated with Polivar varnish and restored with amalgam were performed on all teeth. Class V type cavities of 3 mm diameter and 1,5 mm depth were prepared on the CEJ junction on the buccal side of all teeth. Dyract AP restorations were placed using the bonding procedures indicated: Group 1: P&B NT (Prime & Bond NT); group 2: acid (36% phosphoric acid) + P&B NT; group 3: NRC (Non Rinse Conditioner) + P&B NT; group 4: P&B NT + Saliva; group 5: acid + Saliva + P&B NT; group 6: acid + P&B NT + Saliva; group 7: NRC + Saliva + P&B NT; group 8: NRC + P&B NT + Saliva. Restorations were finished with Sof-Lex discs. After 24 hours storage in distilled water the teeth were removed and coated with two layers of nail varnish, except for 1 mm around the restorations. The teeth were then thermocycled in a 0.5% basic fuchsin solution for 500 complete cycles between 8°C and 50°C, with a dwell time of 15 seconds.

Planes frequently used to identify radiologic and abdominal ultrasono‐graphic images such as transverse, coronal, and sagittal are generally not anatomically correct when applied to transvaginal ultrasonographic planes and images. More appropriate terminology specific for the planes imaged during transvaginal ultra‐sonography, such as TRANS‐pelvic and AP‐pelvic planes, are suggested. A TRANS‐pelvic plane refers to a plane imaged when the sound beam is directed across or from side to side in the pelvis. An AP‐pelvic plane refers to an image obtained when the sound beam is directed anteriorly and posteriorly.

No description available.

Thesis (Ed.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / This study examined the effect of three schedule types on student achievement in Advanced Placement Biology classes. AP Biology test scores from students on three types of full-year schedules were analyzed to assess the impact schedule type had on student achievement. The three schedules included the block and traditional schedules, and the rotating/hybrid, a blend of the former two schedules. The results indicated the variable most closely associated with success on the AP Biology exam was the length of experience the teachers had teaching the course, regardless of schedule type. Although significant differences were seen in mean AP Biology test scores among the three schedule types, this could be explained by the relationship between instructors' experience and schedule type. Regression analysis determined the two strongest predictors of successful performance on the AP Biology exam were instructors' experience and perceived teaching style, regardless of schedule type. It appears that the economically developed suburbs, had teachers with the largest amount of experience teaching AP Biology, and these teachers reported using a direct approach to teaching, using lecture greater than 50% of the time. The results of this study also suggest when restructuring to improve student achievement, educators should examine other variables in addition to the high school schedule. Restructuring the day to allow for longer classes must be accompanied by professional staff development to allow teachers to develop new teaching methods. Most of the teachers in the suNey reported using lecture a great deal of the time, regardless of schedule type. Comments from the teachers from the various schedules revealed that the ability to add student centered, inquiry based activities and labs were dependent on adequate class time. No information on whether or not the teachers were given professional development to expand their repertoire of teaching methods when the school adopted a block or rotating hybrid schedule was obtained. Limitations to this study include the fact that there was no independent verification of teaching style as reported by the teachers in this study. This study involved only Advanced Placement Biology classes, so no generalizations can be made to other science classes. / 2999-01-01

Since the release of A Nation at Risk in 1983 student performance on a variety of high stakes tests have become increasingly important in educational settings. The results of this type of assessment are quantifiable, and are intended to indicate certain levels of academic performance and achievement. Advanced Placement (AP) Exams are one example of high stakes tests. With the rapid growth of Advanced Placement (AP) courses and the corresponding popularity of the AP Exams there is a need in the research to identify specific variables that may be influential to AP Exam score performance. Course schedule type, either in the 4⨉4 block or traditional yearlong format, has been examined as a variable that influences student AP Exam score performance. In some studies the implementation of a 4⨉4 block in place of a traditional yearlong course schedule type resulted in increased AP Exam score performance, while in other studies replacement of a traditional yearlong course schedule by a 4⨉4 block course schedule type resulted in decreased AP Exam score performance. The limitations in the existing research present a need for more controlled studies using multiple years of data to further examine the relationship between clearly identified course schedule types and AP Exam score performance. In response to the need, this research performed a controlled study and examined the relationship between three specific course schedule types and AP Exam performance over time. This study analyzed sample data using participants (N=428) from a single institution in a southeastern state in the United States enrolled in the same AP course subject, taught by the same instructor, and who completed the same subject AP Exam over multiple years (2008-2012). / Ph. D.

Yes / The activity-regulated cytoskeleton-associated (Arc) protein control synaptic strength by facilitating AMPA receptor (AMPAR) endocytosis. Here we demonstrate that Arc targets AMPAR to be internalized through a direct interaction with the clathrin-adaptor protein 2 (AP-2). We show that Arc overexpression overexpression in dissociated hippocampal neurons obtained from C57BL/6 mouse reduces the density of AMPAR GluA1 subunits at the cell surface and reduces the amplitude and rectification of AMPAR-mediated miniature-excitatory postsynaptic currents (mEPSC). Mutations of Arc, that prevent the AP-2 interaction reduce Arc-mediated endocytosis of GluA1 and abolish the reduction in AMPAR-mediated mEPSC amplitude and rectification. Depletion of the AP-2 subunit µ2 blocks the Arc-mediated reduction in mEPSC amplitude, effect that is restored by re-introducing µ2. The Arc/AP-2 interaction plays an important role in homeostatic synaptic scaling as the Arc-dependent decrease in mEPSC amplitude, induced by a chronic increase in neuronal activity, is inhibited by AP-2 depletion. This data provides a mechanism to explain how activity-dependent expression of Arc decisively controls the fate of AMPAR at the cell surface and modulates synaptic strength, via the direct interaction with the endocytic clathrin adaptor AP-2. / This work was supported by the BBSRC_FAPPA BB/J02127X/1 and BBSRC-BB/H018344/1 to SALC and by the FAPESP_RCUK_FAPPA 2012/50147-5 and FAPESP_Young Investigator’s grant 2009/50650-6 to LLdS. SCW was a PhD Student supported be the BBSRC/GSK PhD-CASE Studentship, LPdA is a postdoc fellow supported by FAPESP, YCJ was supported by a FAPESP scientific initiation scholarship.

The cornea, which is the outermost part of the anterior segment of the eye, is composed of an outer stratified epithelium, an inner endothelial monolayer, and a collagen-rich, avascular stroma. The corneal endothelium and stroma are derived from neural crest cells (NCCs), while the corneal epithelium develops from surface ectoderm (SE). Our lab has developed a mutant mouse model lacking expression of activating protein 2β (AP-2β), a transcription factor critical in corneal development, in NCC-derived tissues utilizing the Wnt1Cre transgene. In this mutant mouse model, we have observed an absent endothelium, a vascularized stroma, and a non-stratified epithelium. The lack of proper corneal epithelial stratification is interesting to note as AP-2β expression is unaltered in the SE-derived corneal epithelium. A possible mechanism for the observed lack of stratification may be due to altered Wnt/β-catenin/bone morphological protein 4 (BMP4) signalling between the corneal stroma and the epithelium. Thus, the purpose of this thesis was to investigate changes in Wnt/β-catenin/BMP4 signaling in the absence of stromal AP-2β expression and determine whether the lack of corneal epithelial stratification can be rescued. Immunohistochemical staining of corneal sections revealed that compared to controls, AP-2β NCC KO mutant mice experienced elevated Wnt/β-catenin signaling and reduced BMP4 signaling at later timepoints. Due to the decrease in BMP4 signaling, we hypothesized that administration of exogenous BMP4 delivered via subconjunctival injections would increase BMP4 downstream signaling and trigger corneal epithelial stratification in mutant mice. Haematoxylin and eosin staining of BMP4 treated mice revealed a change in corneal epithelial stratification in control mice compared to untreated control mice. However, no change in corneal epithelial stratification was observed between untreated and BMP4 treated mutant eyes. Ultimately, these results indicate that AP-2β expression in NCC-derived tissues are critical for corneal epithelial stratification. / Thesis / Master of Science (MSc) / Limbal stem cell deficiency is a corneal disorder caused by the death or dysfunction of limbal epithelial stem cells that reside in the limbal epithelium. Due to the importance of these stem cells in corneal health and maintenance, patients suffering from this condition are at risk for corneal ulceration, neovascularization, scarring, and ultimately, corneal blindness. Our lab has developed a mutant mouse model that displays a similar phenotype to patients suffering from limbal stem cell deficiency. In this thesis, we explored the importance of cell signaling in corneal development and identified changes to signaling in our mutant mouse model. Furthermore, we investigated whether a potential treatment could rescue the phenotype observed in our mutant mouse model, allowing them to return to a normal phenotype. Results from this thesis allowed us to gain a better insight into the pathology of limbal stem cell deficiency, which can help guide future treatments, procedures, and therapies.

Esta dissertação baseia-se em observações etnográficas e em entrevistas realizadas entre 2016 e 2018, principalmente com policiais do Batalhão de Operações Especiais (Bope) de Macapá/ AP. O trabalho se voltou para a compreensão que eles têm de seu processo de formação e para a construção de um ethos bopeano. Foram analisadas as suas percepções a respeito do uso da força e do que entendem por violência policial, além de símbolos identificadores da corporação, como a farda preta e a caveira. Uma das principais conclusões é que a formação de um bopeano implica a construção de um Corpo-Arma coletivo a partir de Corpos-Armas individuais, sendo que, para a maioria deles, a violência policial ou excessos respondem ao amplo contexto de violência social em que se inserem. / This dissertation is based on ethnographic observations and interviews conducted between 2016 and 2018, mainly with police officers from the Special Operations Unit (Bope) of Macapá/AP, in the North of Brazil. The work has focused on the understanding these officers have of their training process and to the construction of a bopean ethos. Their perceptions regarding the use of force and what they understood as police violence were analyzed, as well as symbols that identify the corporation, such as the black uniform and the skull. One of the main conclusions is that the formation of a bopean implies the construction of a collective Body-Weapon that is a result of singular body-weapons and, for most of them, police violence or \"excessive force\" respond to the broad context of social violence in which they live in.

A proteína jun é um dos principais integrantes do complexo AP-1 e está envolvido nos processos inflamatórios, diferenciação, apoptose e migração celular. Esta proteína pode formar homodímeros e heterodímeros por meio da dimerização que ocorre pelo sítio de sequências de leucinas. Existem evidências de que a proteína jun pode ser inibida pela proteína RPL10 mediante a ligação destas proteínas, no mesmo sítio de sequências de leucinas no núcleo celular, parando a progressão de tumores. O objetivo deste trabalho foi expressar, isolar e caracterizar a região de ligação das sequências de leucinas (região AP-1), para estudos posteriores de ligação com a proteína RPL10. O cDNA para proteína jun foi amplificado por PCR e clonado nos vetores de expressão pET 26b(+), pET 28a-c(+) e p1813 e expressa em E.coli BL21 (DE3). A proteína expressa em vetor pET28_AP1 foi eficientemente purificada pela técnica de cromatografia de afinidade a íons metálicos, por possuir uma sequência (poli)histidina que facilitou a purificação, apresentando um excelente grau de pureza. A identidade da proteína foi confirmada através de análise feita por western blotting e dot blotting e também por analise por espectrometria de massas. / The jun protein is one of the main AP-1 complex members and is involved in the inflammatory process, differentiation, apoptosis and cell migration. The Jun protein may form homodimers and heterodimers by dimerization in the leucines sequences site. There are evidences that jun protein can be inhibited by RPL10 protein through these protein binding in the same leucine sequences site, in the cell nucleus, stopping the tumor progress. The objective of this study was to express, isolate and characterize the binding region of the leucine sequences (AP-1 region) for subsequent binding studies with RPL10 protein. The jun protein cDNA was amplified by PCR and cloned into pET 26b(+), pET 28a-c(+) and p1813 expression vectors, and was expressed in E.coli BL21 (DE3). The protein expressed in pET28_AP1 vector was efficiently purified by the affinity chromatography technique to metal ions because have a (poly)histidine sequence which facilitate the protein purification, showing an excellent high purity. The protein identity was confirmed by western blotting and dot blotting and also by mass spectrometry analysis.