Biofilms are a common mode of yeast growth in which cells adhere to each other and adhere to abiotic surfaces to form complex multicellular structures. Living together in biofilms provides cells with several beneficial features compared to planktonic cells. Undoubtedly, protection and resistance are advantages of life inside colony biofilms. Biofilms are found in many environments and play many important roles in commercial industries. However, biofilms can also be extremely dangerous in clinical settings. There is thus great interest in studying biofilms and how to eliminate them. In this study, we used wild yeast Saccharomyces cerevisiae colony biofilm as an ideal system to investigate potential functions of the yeast Cyc8-Tup1 transcriptional corepressor complex in the regulation of yeast adhesion, and biofilm formation on agar and at solid-liquid interfaces. Unexpectedly, we have found that Cyc8p and Tup1p antagonistically control the formation of structured biofilm colonies on agar and FLO11 expression. Cyc8p itself acts as a key repressor of FLO11, whereas Tup1p promotes the formation of biofilm colonies and induces FLO11 expression by inhibiting the repressive function of Cyc8p and preventing Flo11p degradation possibly by inhibiting an extracellular protease. In addition, other features...

Two major roadblocks in synthetic biology are the difficulties associated with the physical assembly of gene regulatory networks (GRNs) and the lack of characterized biological parts. In this work we aimed to address both of these issues. We developed a novel method for the assembly of GRNs called Brick- Mason assembly. We have shown that the method can assemble a 6 part network in a single day and provides significant advancements over traditional cloning methods. We used BrickMason to assemble GRNs that would allow us to compare natural yeast mechanisms of repression to the steric hindrance based mechanisms that are commonly used in synthetic GRNs in yeast. Our results show that the two mechansisms of repression are not equivalent. This finding opens possibilities for using a new class of repressor in a synthetic context in yeast.

Two major roadblocks in synthetic biology are the difficulties associated with the physical assembly of gene regulatory networks (GRNs) and the lack of characterized biological parts. In this work we aimed to address both of these issues. We developed a novel method for the assembly of GRNs called Brick- Mason assembly. We have shown that the method can assemble a 6 part network in a single day and provides significant advancements over traditional cloning methods. We used BrickMason to assemble GRNs that would allow us to compare natural yeast mechanisms of repression to the steric hindrance based mechanisms that are commonly used in synthetic GRNs in yeast. Our results show that the two mechansisms of repression are not equivalent. This finding opens possibilities for using a new class of repressor in a synthetic context in yeast.

Two major roadblocks in synthetic biology are the difficulties associated with the physical assembly of gene regulatory networks (GRNs) and the lack of characterized biological parts. In this work we aimed to address both of these issues. We developed a novel method for the assembly of GRNs called Brick- Mason assembly. We have shown that the method can assemble a 6 part network in a single day and provides significant advancements over traditional cloning methods. We used BrickMason to assemble GRNs that would allow us to compare natural yeast mechanisms of repression to the steric hindrance based mechanisms that are commonly used in synthetic GRNs in yeast. Our results show that the two mechansisms of repression are not equivalent. This finding opens possibilities for using a new class of repressor in a synthetic context in yeast.

The CCAAT binding-factor (CBF) is a transcriptional activator conserved in eukaryotes. The CBF in Saccharomyces cerevisiae is a multimeric heteromer termed the Hap2/3/4/5 complex. Hap4, which contains the activation domain of the complex, is also the regulatory subunit and is known to be transcriptionally controlled by carbon sources. However, little is known about Hap4 regulation. In this report, I identify mechanisms by which Hap4 is regulated, including: (1) transcriptional regulation via two short upstream open reading frames (uORFs) in the 5' leader sequence of HAP4 mRNA; (2) proteasome-dependent degradation of Hap4; and (3) identification of two negative regulators of HAP4 expression, CYC8 and SIN4. I also report differential patterns of Hap4 cellular localization which depends on (1) carbon sources, (2) abundance of Hap4 protein, and (3) presence or absence of mitochondrial DNA (mtDNA).

5 Abstract Although yeasts are unicellular microorganisms, they form complex multicellular formations such as biofilms and colonies under natural conditions. Within these structures, processes such as cell differentiation, specialization by particular cell populations and cell signalling, which are typical of multicellular organisms, take place. The literature introduction to this thesis summarizes current knowledge regarding the development of biofilms and colonies, in particular those of the model organism, Saccharomyces cerevisiae, and some selected regulations that are important for the formation of multicellular structures. In the results section, I focus on two lines of research. The first is directed towards mechanisms, involved in the formation of multicellular structures. In studying the formation of SLI biofilms (biofilms at the solid/liquid interface), we have documented the antagonistic role of the regulators CYC8 and TUP1 in their formation and have also described the effect of the presence of glucose on the development and stability of SLI biofilms of strain BR-F. During this study we[D1] have developed an imaging method that allows us to prepare and observe the internal structure (vertical cross-section) of SLI biofilms, as well as the growth of unattached cells, under physiological...