Photoreceptors are highly compartmentalized neurons in the retina, and they function by detecting light and initiating signaling through the visual network. The photoreceptor contains several compartments including the outer segment (OS) which is a sensory cilium for detecting photons and the inner segment (IS) that carries out important modulatory functions via its resident channels and transporters. Those proteins are membrane proteins that function together to shape electrical properties of the cell membrane during both rest and active states. Therefore it is essential to maintain proper function of the membrane proteins in the IS. One important way to regulate the function of a membrane protein is via controlling its trafficking to ensure a proper amount of the protein in the proper cellular compartment. To date, little is known about how IS membrane protein trafficking is controlled in photoreceptors. In this study, our goal is to understand those mechanisms using cell biology and biochemistry approaches. To achieve the goal, we investigated trafficking of two unrelated IS resident proteins: the hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) that mediates a feedback current in photoreceptors, and the sodium potassium ATPase (NKA) which maintains the basic electrochemical property of the cell. In order to study trafficking of HCN1, we first investigated the dependence of HCN1 trafficking in photoreceptors on TRIP8b, an accessory subunit that influences trafficking of HCN1 in hippocampal neurons. By studying TRIP8b knockout mice we found that TRIP8b is dispensable for HCN1 trafficking in photoreceptors but required for maintaining the maximal expression level of HCN1. Since we revealed that HCN1 trafficking can be regulated in a cell-type specific manner, we subsequently focused on the amino acid sequence of HCN1 to identify novel trafficking signals that function in photoreceptors. By examining localization of a series of HCN1 mutants in transgenic Xenopus photoreceptors, we discovered a di-arginine ER retention motif and a leucine-based ER export motif. These two sequence motifs must function together to maintain equilibrium of HCN1 level between the endomembrane system and the cell surface. The study of HCN1 uncovered a mechanism for the photoreceptor to control membrane protein trafficking via the early secretory pathways. To reveal additional trafficking machineries in photoreceptors, we investigated trafficking of NKA. We first tested for an interaction with ankyrin, an adaptor protein that regulates NKA trafficking in epithelial cells, and found these proteins do not co-localize in photoreceptors. We then aimed to identify novel trafficking signals by studying the trafficking behavior of two NKA isozymes: NKA-α 3 and NKA-α 4. When expressed in transgenic Xenopus photoreceptors, these two proteins localize to the IS and the OS respectively. By studying localization of multiple chimeras and truncation mutants, we found that the distinct localization pattern is due to a VxP OS/ciliary targeting motif present in NKA-α 4. Since NKA-α 4 is naturally expressed in the ciliary compartment of the sperm, our finding in the photoreceptor suggests a mechanism for NKA-alpha 4 trafficking in its native environment. Overall, our studies of HCN1 and NKA together provide new insights into controlling membrane protein trafficking in photoreceptors and help establish the basics for future therapeutic intervention targeting trafficking pathways that are linked to about one third of proteins reported in retinal diseases.

Ion signaling plays fundamental role in cell survival. Na+ and Ca2+ are critical players in ion signaling. Cells spend the major amount of energy to maintain and regulate Na+ and Ca2+ gradients across the cell membrane. Any disruption in cellular energy consumption by plasma membrane ATPases affects ion signaling and vice versa. This thesis is a combination of four separate research studies. In the first study, We measured ATP consumption dynamics of Na+/K+-ATPase using a genetically encoded fluorescent indicator called Perceval HR. we demonstrate that PercevalHR is an excellent tool to visualize ATP:ADP in mammalian cells. In the second study, We studied the role of calcium signaling and TRP channels in angiotensin II type 1 receptor  signaling cascade. We prove that low inhibition of CaV1.2 with physiological and therapeutically relevant concentration of Angiotensin II up regulate AT1R signaling. In the third study, We studied the role of the TRPM5 channel in regulating insulin secretion, and cytoplasmic free calcium concentration in the rat β-cells by usingtriphenyl phosphine oxide, a selective inhibitor of the channel. In the fourth study, We tested whether, the genetically engineered human β-cell line (EndoC-BH1) could be used as models for studying Ca2+signaling in the context of Type II Diabetes. We found that the EndoC-BH1 cells could be a relevant model to study stimulus-secretion coupling and Ca2+ signaling in the human β-cells. / <p>QC 20170328</p>

Inanga (Galaxias maculatus) is an amphidromous fish species that is able to successfully inhabit a variety of salinities. Using an integrated approach this thesis has characterised for the first time the physiological characteristics that facilitate acclimation in inanga. Structural studies using scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM) revealed freshwater-acclimated inanga have a high density of apical pits and freshwater-type mitochondria-rich cells (MRCs) that can facilitate ion absorption from the hypo-osmotic environment. In seawater, inanga remodel their gills by increased proliferation of seawater-type MRCs to facilitate ion secretion in the hyper-osmotic environment. Concentration-dependent sodium (Na+) kinetic analysis revealed that at a whole body level, inanga regulate Na+ using a saturable, high affinity, low capacity uptake system which makes them extremely adept at extracting Na+ from very dilute freshwater environments. In fact inanga displayed an uptake affinity (Km) of 52 ± 29 µM, which is one of the lowest ever recorded in freshwater fish. The sodium/potassium ATPase transporter (NKA) is central to Na+ regulation within the gill. In high salinties inanga displayed increased NKA activity (6.42 ± 0.51 µmol ADP mg protein-1 h-1) in an effort to excrete the excess Na+, diffusively gained from the hyper-osmotic environment. This increase in NKA was most likely a reflection of the proliferation of NKA-containing MRCs. The NKA activities seen in freshwater- and 50% seawater-acclimated inanga were similar (2.54 ± 0.19 and 2.07 ± 0.22 µmol ADP mg protein-1 h-1 respectively) to each other suggesting the inanga gill is capable of supporting ion regulation in brackish waters without a significant increase in NKA activities, and the energetically-expensive changes in gill structure and function that accompany such a change. Molecular investigation of NKA isoform expression using quantitative PCR (qPCR) showed that inanga displayed salinity-induced changes in the expression of the three α NKA isoform variants investigated. Isoform α1a exhibited a pattern consistent with an important role in freshwater, confirming results from other fish species. While it is generally accepted that α1b isoform is the predominant NKA isoform in seawater, inanga did not display this pattern with a freshwater dominance seen. None of the salinity-induced changes could quantitatively explain the increased NKA activity in seawater suggesting that different isoforms may convey different activities, that there is also regulation of NKA at a post-transcriptional level, and/or other isoforms or subunits may have a significant role. The importance of the osmoregulatory hormone cortisol and prolactin is widely accepted and inanga were treated with cortisol, prolactin and a combination of the two in an effort to further elucidate their role. NKA activity and NKA isoform expression were assessed but no specific patterns were deduced, except for a decrease in both NKA activity and isoform expression in 100% seawater-acclimated inanga treated with cortisol and prolactin. The reasons for this decrease were not evident, although the impact of stress induced by the injection protocol was likely to be a confounding factor. The development of a new confocal-based technique in this study was able to describe, for the first time, intracellular sodium levels ([Na+]i) as a function of salinity in an intact euryhaline fish gill cell. Using the fluorescent Na+ indicator dye CoroNa Green this study demonstrated the ability of inanga gill cells to maintain [Na+]i in the face of environmental change. Freshwater-acclimated inanga displayed basal [Na+]i of 5.2 ± 1.8 mM, with 12 ± 2.3 mM and 16.2 ± 3.0 mM recorded in 50% seawater- and 100% seawater-acclimated cells, respectively. Low [Na+]i is advantageous in hypo-osmotic environments as it provides a gradient between the cell and the blood which is essential for generating electrochemical gradients cell volume regulation and other cellular homeostatic mechanisms. A slightly elevated [Na+]i seen at the higher sanities would help minimise the diffusive gradient for passive influx from the environment which would be of benefit in hyper-osmotic environments. Upon salinity challenge 50% seawater cells were equally adept at maintaining a constant [Na+]i at any salinity, suggesting these cells are have the necessary constituents to regulate Na+ in both lower and higher salinities. This novel LSCM approach is advantageous relative to existing transport models as it will allow the observation of cellular ion transport in real time, within a native filament structure displaying functional interaction of different cell types. The extreme ion uptake characteristics of the inanga and their amenability to in situ confocal-based studies demonstrated in this study, confirm inanga as a valuable model species for future research.

No description available.

Does the active membrane transporter, Na+,K+-ATPase dimerize? If it does, whatis the functional benefit? Does it increase or decrease the turnover rate? Theseare still unanswered questions and current research topics. Previous studies havedemonstrated dimerizations in closely related proteins of the P-type ATPase family.For the Na+, K+-ATPase a first indication of dimerization has been shown viaFluorescence lifetime imaging microscopy (FLIM) or Fluorescence resonance energytransfer - Fluorescence correlation spectroscopy (FRET-FCS) experiments. Theprecise dimer structure, dimerization process, and its ultimate functional effecthowever, remain to be found. This master thesis approaches those questions froma co-evolutionary standpoint. It predicts a possible dimer structure by starting with amultiple sequence alignment, direct coupling analysis, and structural contact filteringalgorithm. This model would strengthen the dimerization model of a decreasedturnover rate due to a competitive behavior of two Na+, K+-ATPases for its energysource ATP. / Dimeriserar den aktiva membrantransporten Na+,K+-ATPas? Om den gör det,vad är den funktionella nyttan? Ökar eller minskar det omsättningshastigheten?Dessa är obesvarade frågor och rådande forskningsämnen. Tidigare studier hardemonstrerat dimeriseringar i nära relaterade proteiner av P-typ ATPas-familjen.För Na+, K+-ATPas har en första indikation av dimerisering visats via ”Flourescencelifetime imaging microscopy (FLIM)” eller ”Flourescence resonance energy transfer- Flourescence correlation spectroscopy (FRET-FCS)”. Den precisa dimerstrukturen,dimeriseringsprocessen och dess slutgiltiga funktionella effekt emellertid, återståratt ses. Detta examensarbete på masternivå närmar sig dessa frågor från ettsamevolutionärt perspektiv. Det förutser en möjlig dimerstruktur genom att utgåfrån en flersekvenslinjering, direkt kopplingsanalys och en strukturell kontaktfiltreringsalgoritm. Denna modell skulle stärka dimeriseringsmodellen av minskadomsättningshastighet till följd av tävlingsbeteende mellan två Na+,K+-ATPaser fördess energikälla ATP.

Nondeterministic finite automaton is an important tool, which is used to process strings in many different areas of programming. It is important to try to reduce its size for increasing programs' effectiveness. However, this problem is computationally hard, so we need to search for new techniques. Basics of finite automata are described in this work. Some methods for their reduction are then introduced. Usable reduction algorithms are described in greater detail. Then they are implemented and tested. The test results are finally evaluated.

Abstract in English, Afrikaans and Sesotho / The study was inspired by the global phenomenon of failure rates of outsourcing ventures; yet, the outsourcing value proposition of 3PL providers is promising and potentially lucrative. The approach of the outsourcing venture was thus investigated, specifically how to align and integrate outsourcing parameters appropriately at the inception of the outsourcing venture in order to attain the benefits brought about by the outsourcing decision over the projected life of the outsourcing venture. To this effect, a strategic decision-making model, with the objective of ascertaining optimal alignment and integration was developed. The model comprises a dualistic development followed by a methodological process path to operationalise the strategic decision-making model. A services continuum (development one) is established, which is a permutation matrix that classifies four aspects of importance (service type, category of 3PL provider, strategic alignment and investment), in order to align the aspects optimally and enable appropriate model application. The services continuum is a conglomeration of nine models:  a capabilities matrix for 3PL provider services;  a framework for evaluating the supply chain role of 3PL providers;  a mathematical model;  a supplier selection and evaluation process;  a presentation of the Hayes–Wheelwright framework;  an outsourcing variables differentiated model;  four categories of the 3PL providers model;  an estimated path model; and  a process integration via survey data collection model. The services continuum was extended for utilisation with risk management practices, and an outsourcing risk matrix (development two) was established. The enablement of the outsource risk matrix is an element review, consisting of three categories: collaboration and integrated planning systems; performance measurement; and broad-based black economic empowerment. The strategic decision-making methodology process path was developed as having three phases, which operationalise the strategic decision-making model. The research was primarily focussed on literature reviews, with the models classified according to the services continuum. To a lesser extent, the research focussed on primary data, which served as model application input specifically for application requirements related to Sasol. The study established a universally applicable strategic decision-making model, as well as the application of the model for Sasol’s outbound (final packaged product) supply chain. / Thuto e ile ya kgothaletswa ke tshebetso ya lefatshe mabapi le maemo a ho hloleha ha ho nyaolwa/ntshuwa ha diprojeke kgwebong; leha ho le jwalo, tlhahiso ya boleng ba ditshebeletso tsa 3PL e a tshepisa ebile e ka ba le hlahiso ya tjhelete e ngata. Ka tsela eo, mokgwa wa ho tswa kgwebong o ile wa phenyekollwa, haholoholo mabapi le ho tsamaisana le ho kenyelletsa maemo a ho ntshetsa pele maruo ka mokgwa o nepahetseng ha ho thehwa kgwebo ya ho ntshetsa pele ho fumana melemo e tliswang ke qeto ya ho ntshetsa pele ka bophelo ba morero wa ho nyaolwa kgwebong. Ka lebaka lena, ho ntshetswa pele mokgwa/motlolo o motle wa ho etsa diqeto, ka sepheo sa ho netefatsa hore ho na le boemo bo nepahetseng le ho kopanngwa ho tsitsitseng. Motlolo ona o na le ntshetso pele e habedi e latelwang ke tshebetso ya mokgwa wa ho kenya tshebetsong tsela ya ho nka diqeto. Tatellano ya ditshebeletso e tswelang pele (ntshetsopele ya pele) e a thehwa, e leng phethoho ya tikoloho e kgethollang dikarolo tse nne tsa bohlokwa (mofuta oa tshebeletso, mokga wa mofani wa 3PL, kemiso ya moralo le matsete), e le ho dumellana le dintlha ka tsela e nepahetseng le ho etsa hore ho sebediswe motlolo hantle. Ditshebeletso tse tswelang pele ke kopano ya dimotlolo tse robong:  bokgoni ba maemo a tikoloho bakeng sa ditshebeletso tsa bafani ba 3PL;  sebopeho sa ho hlahloba phepelo ya tatellano ya ho nka karolo ha bafani ba 3PL;  mokgwa/motlolo wa mathemathiks;  kgetho ya mofani le mokgwa wa o hlahloba;  tlhaloso ya moralo wa Hayes-Wheelwright;  mefuta e fapaneng ya ho nyaola dikarolo tse fapaneng;  ntho tse mene tsa bafani ba 3PL;  mohlala wa tsela ya dimotlolo; le  mokgwa wa ho kopanya ka mokgwa wa ho bokella lesedi la dipatlisiso. Dishebeletso tse tswelang pele di ile tsa atoloswa bakeng sa tshebediso ya mekgwa ya taolo ya dikotsi, mme maemo a tikoloho a kotsing ya boipheliso (ntlafatso ya bobeli) a thehwa. Ho kengwa tshebetsong ha kotsi ya maemo a tikoloho ke ntho e shejwang botjha e nang le mekga e meraro: mekgwa ya ho sebedisana le meralo e kopanetsweng; tekanyo ya tshebetso; le ho matlafatsa bofuma ba batho batsho lehlakoreng la moruo. Mokgwa wa ho etsa diqeto o ile wa ntlafatswa e le o nang le mekgahlelo e meraro, e sebetsang ka mokgwa o motle wa ho etsa diqeto. Phuputso e ne e lebisitswe haholo ditabeng tsa tlhahlobo ya dingodilweng, le dimotlolo tse kgethollwang ho latela ditshebeletso tse tswelang pele. Ho ya ka tekanyo e nyenyane, dipatlisiso di lebisitse tlhokomelong ya lesedi la mantlha, le neng le sebetsa e le mokgwa wa ho kenya letsoho bakeng sa ditlhoko tsa kopo tse amanang le Sasol. Phuputso ena e thehile mokgwa wa ho etsa diqeto tse amohelehang lefatsheng ka bophara, hammoho le ho sebediswa ha setshwantsho sa phepelo ya tlhahiso ya Sasol (thlahiso ya ho qetela). / Dié studie is deur die globale verskynsel van die falingstempo van uitkontrakteringsondernemings geïnspireer; ten spyte hiervan lyk die uitkontrakteringswaardevoorstel van 3PL-verskaffers belowend en is dit potensieel winsgewend. Die benadering wat die uitkontrakteringonderneming volg, is derhalwe ondersoek en in die besonder hoe om die uitkontrakteringparameters by die aanvang van die uitkontrakteringonderneming toepaslik in lyn te stel en te integreer om die voordele te benut van die uitkontrakteringsbesluit oor die geprojekteerde lewe van die uitkontrakteringsonderneming. ’n Strategiesebesluitnemingsmodel wat ten doel het om optimale inlynstelling en integrasie te verseker, is dus ontwikkel. Die model bestaan uit ’n dualistiese ontwikkeling, gevolg deur ’n metodologiese prosesbaan om die strategiese besluitnemingsmodel te operasionaliseer. ’n Dienstekontinuum (ontwikkeling een) is op die been gebring, wat ’n permutasiematriks is wat vier aspekte van belangrikheid (dienssoort, kategorie van die 3PL-verskaffer, strategiese inlynstelling en investering) klassifiseer, ten einde die aspekte optimaal in lyn te stel en toepaslike modeltoepassing moontlik te maak. Die dienstekontinuum bestaan uit ’n versameling van nege modelle:  ’n bekwaamheidsmatriks vir 3PL-diensverskafferdienste;  ’n raamwerk om die voorsieningskettingsrol van 3PL-verskaffers te evalueer;  ’n wiskundige model;  ’n verskafferseleksie en evalueringsproses;  ’n aanbieding van die Hayes-Wheelwright-raamwerk;  ’n uitkontrakteringsveranderlike-gedifferensieerde model;  vier kategorieë van die 3PL-verskaffersmodel;  ’n geraamde baanmodel; en  prosesintegrasie deur middel van ’n ondersoekdata-insamelingsmodel. Die dienstekontinuum is uitgebrei vir gebruik by risikobestuurspraktyke en ’n uitkontrakteringrisikomatriks (ontwikkeling twee) is op die been gebring. Die bemagtiging van die uitkontrakteringsrisikomatriks is ’n elementhersiening, wat uit drie kategorieë bestaan: samewerkings- en geïntegreerdebeplanningstelsels; prestasiemeting; en breë swart ekonomiese bemagtiging. Die strategiese besluitnemingsmetodologieprosesbaan is met drie fases ontwikkel, wat die strategiese besluitnemingsmodel operasionaliseer. Die navorsing het hoofsaaklik op literatuuroorsigte gefokus, met die modelle wat volgens die dienstekontinuum geklassifiseer is. Die navorsing het in ’n mindere mata op die primêre data gekonsentreer, wat as die modeltoepassingsinset gedien het, in die besonder vir die toepassingsvereistes wat op Sasol betrekking het. Die studie het ’n universele, toepaslike strategiesebesluitnemingsmodel daargestel, asook die aanwending van die model op Sasol se uitgaande voorsieningsketting (finaal verpakte produk). / Business Management / D. Phil. (Management Studies)