It has been proposed that the various 14-3-3 isotypes and isoforms present in all eukaryotes are largely functionally equivalent. However, this is not consistent with the conservation of multiple isoforms and isotypes, especially in vertebrates with seven 14-3-3 encoding genes and nine isotypes. The hypothesis tested in this thesis is that both isoform-specific and overlapping functions are likely mediated through tissue specific expression, colocalization and dimerization of 14-3-3 proteins occur in vivo. Drosophila melanogaster was selected because it offers a simple, but representative system to study these proteins functionally. This thesis focuses primarily on D14-3-3?, although the expression pattern and phenotypes associated with all three Drosophila 14-3-3s were determined. I first determined the expression pattern of the three different 14-3-3 isotypes (leoI, leoII and D14-3-3?) and described developmental phenotypes associated with mutations in 14-3-3 isotypes in Drosophila. I found that there is partial redundancy with respect to lethality. Both LEO and D14-3-3? appear required for normal germ-line and somatic gonadal development. However, they do not appear to be functionally equivalent with respect to this phenotype since LEO is unable to compensate for the loss of D14-3-3?. I also determined that D14-3-3? mutants have unique phenotypes including deficits in adult cross-vein formation and rapid habituation to olfactory and footshock stimuli. To further understand the unique role that D14-3-3? plays in the adult CNS, I mapped the areas in the brain involved in olfactory and footshock habituation. I found that although the mushroom bodies (MBs) are necessary to inhibit premature habituation such as that exhibited by D14-3-3? mutants, D14-3-3? expression specifically in the MBs is not sufficient to rescue premature habituation. Although the loss of either LEO or D14-3-3? appears to cause a deficit in olfactory associative learning, premature habituation is the cause of the deficit seen in D14-3-3? mutants. As leo mutants do not exhibit a premature habituation phenotype, it appears that within the MBs LEO and D14-3-3? are not functionally equivalent. Therefore, the data supports the hypothesis that 14-3-3s have functional specificity and redundancy likely to represent use of homo and heterodimers in different processes within the tissues of an organism.

It has been proposed that the various 14-3-3 isotypes and isoforms present in all eukaryotes are largely functionally equivalent. However, this is not consistent with the conservation of multiple isoforms and isotypes, especially in vertebrates with seven 14-3-3 encoding genes and nine isotypes. The hypothesis tested in this thesis is that both isoform-specific and overlapping functions are likely mediated through tissue specific expression, colocalization and dimerization of 14-3-3 proteins occur in vivo. Drosophila melanogaster was selected because it offers a simple, but representative system to study these proteins functionally. This thesis focuses primarily on D14-3-3?, although the expression pattern and phenotypes associated with all three Drosophila 14-3-3s were determined. I first determined the expression pattern of the three different 14-3-3 isotypes (leoI, leoII and D14-3-3?) and described developmental phenotypes associated with mutations in 14-3-3 isotypes in Drosophila. I found that there is partial redundancy with respect to lethality. Both LEO and D14-3-3? appear required for normal germ-line and somatic gonadal development. However, they do not appear to be functionally equivalent with respect to this phenotype since LEO is unable to compensate for the loss of D14-3-3?. I also determined that D14-3-3? mutants have unique phenotypes including deficits in adult cross-vein formation and rapid habituation to olfactory and footshock stimuli. To further understand the unique role that D14-3-3? plays in the adult CNS, I mapped the areas in the brain involved in olfactory and footshock habituation. I found that although the mushroom bodies (MBs) are necessary to inhibit premature habituation such as that exhibited by D14-3-3? mutants, D14-3-3? expression specifically in the MBs is not sufficient to rescue premature habituation. Although the loss of either LEO or D14-3-3? appears to cause a deficit in olfactory associative learning, premature habituation is the cause of the deficit seen in D14-3-3? mutants. As leo mutants do not exhibit a premature habituation phenotype, it appears that within the MBs LEO and D14-3-3? are not functionally equivalent. Therefore, the data supports the hypothesis that 14-3-3s have functional specificity and redundancy likely to represent use of homo and heterodimers in different processes within the tissues of an organism.

Práce se zabývá zarovnáváním excitabilních buněk na multielektrodových polích. Nejprve bylo analyzováno zarovnávání excitabilních buněk. Byly použity embryonální neurony z hippocampusu potkanů a HL-1 buňky, které jsou odvozeny z AT-1 linie nádorových myších atriálních kardiomyocytů. Zarovnávání bylo testováno na drážkovaných površích a na površích s materiály s různou buněčnou afinitou. Bylo prokázáno, že na drážkových površích se ve směru drážek zarovnávají neurony i HL-1 buňky, ale na površích s různou chemickou affinitou se zarovnávají pouze neurony. Dále byly vyrobeny vlastní multielektrodová pole, na těchto multielektrodových polích byly kultivovány HL-1 buňky a byl změřen a analyzován akčních potenciál HL-1 buněk. Cílem bylo prokázat, že je možné měřit akční potenciál na vyrobených multielektrodových polích. Pro zarovnání buněk na multielektrodovém poli bylo vyrobeno speciální multieletrodové pole s uniformním povrchem. Toto multielektrodové pole je nazýváno planární multielektrodové pole. Planární multielektrodové pole bylo vyrobeno speciálním vyrobním procesem. Vrstvy planárního multielektrodového pole byly deponovány na pomocný substrát v opačném pořadí. Pomocný substrátem pro depozici byla křemíková deska, na který byla nadeponována další pomocná vrstva zlata. Horní izolační vrstva planárního multielektrodové pole byla deponována jako první a nejspodnější vrstva substrátu byla nadeponována jako poslední. Planární multielektrodové pole i s pomocnou zlatou vrstvou bylo strhnuto s křemíku díky nízké adhezi zlata ke křemíku a planární multielektrodové pole se otočilo vzhůru nohama. Pomocná zlatá vrstva byla odstraněna mokrým leptadlem a tím bylo planární multielektrodové pole dokončeno. Na planárním multielektrodovém poli byly zarovnány HL-1 buňky do pruhů chemickou metodou pomocí kombinace otisku adhezní látky a následným potažení neotisklých ploch anti-adhezní látkou. Elektrofyziologické vlastnosti zarovnaných HL-1 buněk byly změřeny pomocí planárního multielektrodového pole. Tímto experimentem byla představena výrobní technologie pro výrobu planárních multielektrodových polí a toto planární multielektrodové pole bylo úspěšně testováno pro zarovnání HL-1 buněk na jeho povrchu kombinací otisku adhezní látky a potahování antiadhezivním činidlem.